Bio 349: Final Final

Front 1

. You dissect the animal and vegetal portions of a Xenopus blastula stage embryo and recombine them in an explant assay. After incubating for several days, describe the cell identity portions derived from the animal and vegetal tissues.

Back 1

The animal cap will be converted to mesoderm. The endoderm will give rise to endodermal derivatives only.

Front 2

You experimentally, deplete embryos for b-catenin, and repeat the assay from #1 (don't worry about possible secondary effects from losing cadherin-based cell adhesion). What will be the cell identity of the animal cap and vegetal portions? Why?

Back 2

A loss of Beta-catenin in the vegetal endoderm means there will be no dorsalizing signal secreted from the endoderm to the mesoderm. This means that the vegetal region of the embryo will give rise only to ventral endodermal tissue types. There is a caveat / trick to this question. The beta-catenin would have to be inhibited before the end of the first cell division and would more realistically have to be done in the oocyte (looking at the lecture slides, compare phenotype of beta-catenin depleted embryos from oocytes verses 1 hour after fertilization).

Front 3

List two of the general mesodermal inducing factors?

Back 3

Any of the following: Vg1 and VegT, TGF-beta/nodal related genes (Xnr, activin). Remember that nodal signaling is a subset of the TGF-beta superfamily.

Front 4

What is the primary purpose of the Nieuwkoop center?

Back 4

To induce the organizer cells (remember that organizer cells are mesodermal, but are induced by the vegetal-specific Nieuwkoop center. The combination of beta-catenin and Vg1/nodal also directly induces gene expression of some of the BMP antagonists expressed by the organizer cells. Remember, B-catenin induces dorsal tissue identity in both the mesoderm and ectoderm (becomes neural) but does not directly induce mesoderm.

Front 5

Describe the role of Dishevelled in the Wnt pathway. Can it be used to determine the difference between canonical and non-canonical signaling?

Back 5

Wnt ligand binding to frizzled activates disheveled (Dsh). In canonical signaling, Dsh causes the stabilization of Beta catenin and the activation of Wnt signaling. Dsh is also involved in non-canonical Wnt signaling, which is indendent of B-catenin stabilization. So Dsh cannot be used to distinguish different types of Wnt signaling. In contrast, a dominant negative GSK3B construct would result in activation of beta-catenin. So comparing experiments in which each pathway was disrupted allowed Wallingford et al to conclude that the planar cell polarity (PCP) pathway was required for convergent extension during frog gastrulation.

Front 6

List the four morphogenetic processes involved in Xenopus gastrulation. For each, write a sentence that summarizes the specific area of gastrulation that each is involved with.

Back 6

A) Invagination
B) Involution
C) Convergent extension
D) Epiboly
Invagination, through the bottle cells, causes the emergence of the blastopore lip
As the marginal cells reach the dorsal lip, they involute over the inner layer of the animal cap cells.
The mesoderm undergoes convergent extension, which acts as a driving force to push the mesoderm further into the embryo (remember not all mesoderm undergoes this process (NIMZ = head mesoderm and also pharyngeal endoderm)..
The animal cap (and some of the marginal zone that does not involute) then cover the entire outer layer of the embryo by epiboly; this covers the vegetal cells (think of pulling a cap down from your head to completely cover your face).

Front 7

What is one major molecules that is secreted by the dorsal lip (organizer region) of Xenopus embryos. What is its biochemical function? What happens if it is also expressed in the ventral marginal zone?

Back 7

Chordin, Noggin, Follistatin are all BMP inhibitors. By blocking BMP signals, the ectoderm is induced to become neural tissue and also causes mesoderm to become dorsal. By expressing these molecules at the ventral marginal zone (instead of their normal dorsal region), a second axis is formed, and you get a twin embryo.

Front 8

You experimentally generate a Keller explant. What is this? If you culture this in isolation, what structures do you predict will be specified? Will all the tissues undergo convergent extension?

Back 8

This is an explant that includes the mesoderm that will migrate into the dorsal side of the embryo during gastrulation. By removing it from the embryo, we can study some of the morhpological processes. It will make a dorsal mesodermal structures (only dorsal since you take explant from dorsal lip side at inception of gastrulation). Essentially, it makes tissues that undergo convergent extension (mainly notohord); at the most anterior end it has the NIMZ that makes the head mesoderm. Thus you ultimately see a stick like structure (notochord) with a knob at the most rostral end (very anterior head mesoderm; remember Cereberus and Lim from lecture).

Front 9

What is the archenteron?

Back 9

The embryonic gut; it is formed during gastrulation

Front 10

In the following image, increasing amounts of a certain protein are added to a neural plate-like tissue that are all stained for the presence of Nkx2.2. The top has no protein, the middle has a low amount of protein, while the bottom image has a higher amount. What protein do you think this is? Why do you need a lot of it?

Back 10

Sonic hedgehog (Shh). You need a lot because Nkx2.2 is one of the most ventral targets of Shh (V3 interneurons). Remember that Shh activates targets in different classes of the ventral neurons in a dose-dependent fashion. V3 interneurons lie right above the floorplate and require the second highest doses of Shh protein.

Front 11

What is an inhibitory signal in the somite that helps guide trunk neural cell migration? Where is it expressed?

Back 11

EphrinB1 which is expressed in the posterior somite.

Front 12

You want to study neural tube closture in mouse embryos. Design an experimental setup that would allow you to monitor this process occurring in real time development.

Back 12

You could use the strategy outline in class lecture, which was to generate a transgenic mouse that has myristoylated YFP (yellow fluorescent protein that is specifically expressed in the cell membrane). You could then culture mouse embryos and take movies of the process in real time under a fluorescent microscope.

Front 13

What happens when you electroporate chick embryos with Sox9 in the neural tube?

Back 13

You get the expression of neural crest markers on the electroporated side of the neural tube, but these cells are unable to migrate out of the neurepithelium.

Front 14

Where do neural crest cells form in relation to the neural plate of the epidermis?

Back 14

The form at the boundary of the neural plate and epidermis.

Front 15

What are the human birth defects corresponding to neural tube closure defects in the a) anterior neural tube and b) in the posterior neural tube?

Back 15

a) anencephaly b) spinal bifida

Front 16

Describe the 3 primary vesicles of the brain. What will each of them become?

Back 16

prosencephalon = forebrain
mescencephalon = midbrain
rhombencephalon = hindbrain

Front 17

In classic experiments, embryologists took naive neural plate and cultured it with notochord tissue to form ventral neuronal fates (including floorplate and motor neurons). What do you predict would happen if you performed these same experiments, substituting notochord tissue from a) Shh -/- embryos? b) Ptch -/- embryos? What if you incubated wt notochord with neural plate tissue from Smo -/- embryos?

Back 17

The active component from the notochord is secreted Shh. In a) there is no Shh so the neural explants will not become ventralized. b) Ptch1 is a gain-of-function mutation in the Shh pathway, but the notochord will still make Shh so you will get ventral tissue. c) The notochord will still make Shh, but the neural explant tissue can't receive it (Smo is required for signal transduction of the hedgehog pathway). Therefore, you will not get ventralized neuronal tissue (it will be dorsal).

Front 18

You want to misexpress Dbx2 throughout the right side (and only the right side) of a chick neural tube. How do you do this? What type of gene is Dbx2? Would you still form motor neurons? Why or why not?

Back 18

You need to electroporate a plasmid that is expressing Dbx2 into the neural tube (electroporation hits only one side of the neural tube). Dbx2 is a type 1 Shh target that represses the type 2 Shh target Nkx6.1. Misexpressing Dbx2 will cause a loss of Nkx6.1 expression on the right side of the neural tube. Loss of Nikx6.1 will result in a failure to generate motor neurons on that side of the neural tube.

Front 19

You want to further characterize the switch that tells trunk neural crest cells to start becoming melanocytes. a) Which protein, essential for making all neural crest cells, needs to be downregulated for this to happen?

Back 19

FoxD3 needs to be downregulated.

Front 20

You remain unimpressed by the supporting data from lecture (which is all about cell culture) and decide to test the model by performing an experiment where you forcibly maintain expression of this protein in neural crest cells. How do you do this? How could you determine if this supported the model?

Back 20

The key is that you need to maintain FoxD3 expression in neural crest cells. If the model is true, all trunk neural crest cells should become sensory neurons/glia or at the very least should not express Mitf1. One strategy would be to electroporate the neural tube at an early stage before the neural crest cells migrate with a vector driving FoxD3 expression as well as some other marker (such as GFP). This marker is important because it is unlikely that all cells will get the electroporated construct. This expression would be maintainted well after neural crest migrationYou would to monitor for a general NCC antibody that is not specific to either lineage (eg HNK) as well as for your fluorescent maker (GFP). If the GFP+ cells all migrate through the ventral somites, this suggests they are forming glial cells (could confirm that they do not express Mitf1 with a second immunostaining). If most cells are electroporated, you would predict that you would also have a strong depletion of melanocyte cells.
Another way is to misexpress FoxD3 throughout most NCCs. You could give the embryo a virus or you could make the embryo express FoxD3 ubiquitously and see it with GFP and transplant pre-migratory NCCs to a recipient embryo where controls would be NCC transplants from donor embryos only expressing GFP.

Front 21

You wish to generate a deletion of Pax6 in mice
A) Draw a schematic showing the targeting vector below the relevant region of the HoxD cluster.

Back 21

-Should have a homology arm of roughly >=2Kb before the Pax6 coding region and a second homology arm (roughly >=2Kb) immediately after HoxD12. The region in between should contain a positive selection marker such as Neomycin resistance and may contain additional add-ons (like a reporter gene).

Front 22

You wish to generate a deletion of Pax6 in mice. B) Once you have generated ES cells containing the targeting vector, describe the steps you would take to obtain homozygous null embryos for experimental analysis.

Back 22

The ES cells are injected into a blastula stage mouse embryo, and this is then reimplanted into the uterus of a host mother. If the ES cells contribute to the forming embryo, mother will give birth to chimeric embryos, which can be seen with a different coat color (usually ES cells make a brown color while the blastocysts give rise to embryos with a black coat color. So chimeric embryos are black and brown - the more brown the better). Cross the chimera with wild-type mice to generate F1 heterozygous animals. Intercross F1 hets to make F2s. Mendelian genetics predicts that 25% of these should be homozygous for the null allele.
FYI, Pax6 not only has roles in neural tube development (e.g. the antagonistic role between Pax6 and Nkx2.2 that establishes one of the progenitor boundaries in the neural tube) but is also important for eye development (recall the second lecture of class).

Front 23

You knock out a novel gene and are disappointed to discover that it has no embryonic phenotype. What are some reasons why there is no phenotype?

Back 23

Perhaps you did not create a null allele. Could check this by Western blot with an antibody against a region of the protein not deleted; could also check by RT-PCR. Perhaps the gene is not required during embryogenesis. Is it expressed in the embryo (hopefully you did this before making the knockout!), perhaps there is redundancy with other genes. Are their closely related genes?

Front 24

Velocardial facial syndrome (also called DiGeorge Syndrome) is a genetic disorder with a complex range of symptoms. Patients typically have cardiac problems, facial defects, and cleft palates, and may also have immune system problems. The syndrome is caused by a deletion of 1.5 - 3 million base pairs on chromosome 22. This large deletion removes many genes, and it has been challenging to determine which ones are causing the syndrome. Using your knowledge of developmental biology, speculate on what might be the embryonic origin of this phenotype (i.e. cell type). Using this speculation, devise an set of experiments using a model organism that will allow you to identify candidate gene(s) in the deleted region that might be responsible for the disorder.

Back 24

Pharyngeal arch-derived neural crest cells generate the vast majority of the face and the aortic arches, but the thymus, which is responsible for T-cell maturation in the immune system, is derived from the 3rd and 4th pharyngeal arch (not mentioned in class). Cranial neural crest defects, as mentioned in class, also cause cleft palate. Defects in pharyngeal-arch derived crest cells could explain this complex set of symptoms. One experiment to explore this would be to identify all the candidate genes in the deleted region, perform in situ hybridization on a vertebrate model system (probably mouse; fish, frog and chick would work as well), and look for genes that are expressed in pharyngeal arch neural crest cells.

Front 25

What would you predict would be the phenotype of a netrin mutant mouse?

Back 25

Netrin acts as an intermediate-range chemoattractant that causes commissural axons (upon blocking Robo1 signaling via Robo3) to migrate towards the floorplate of the neural tube. In mouse embryos lacking functional netrin gene expression, commissural axons should still migrate ventrally down the neural tube but would not attracted to the floorplate. As a result, netrin deficient commissural axonal projections would fail to cross over to the opposite side of the neural tube.

Front 26

You are so impressed with the Brainbow mouse that you decide to use it to study cranial neural crest migration. A) Describe an experimental strategy that would allow you to adapt the Brainbow approach to studying cranial neural crest (hint: we described a method for general genetic fate mapping of neural crest cells in the Feb 28th lecture). B) Would such an approach be useful for studying cranial neural crest - why or why not?

Back 26

First, you need to convert the Brainbow construct from a neural specific promoter (Thy1) to one that will be active in neural crest. A couple of ways to do this. The easiest is to just use the ubiquitous Rosa26 promoter used in the genetic fate mapping. Using standard molecular techniques, insert the Brainbow cassette downstream of this promoter, where LaZ was previously. It is now in an OFF state, since the LoxP-STOP-LoxP cassette is in place. It could then be crossed to the Wnt1Cre construct used previously (acceptable answer). Alternatively, you could generate a Wnt1CreER mouse that allowed for transient activation of Cre in the neural crest cells of the embryos at a specific timepoint only via Tamoxifen injection (also acceptable answer).
B)The advantage to the brainbow approach is that it allows specific axonal projections and connections to be cleanly mapped back to the source. The cranial neural crest cells are not interconnected with anything; they migrate as single cells, and so aside from generating a pretty picture, it would not be particularly useful.

Front 27

Describe the features of the Notch pathway that allow it to behave like a clock or oscillator. On a theoretical level, how could you modify the Notch proteins (any protein or proteins in the pathway) to speed up or slow down the oscillator?

Back 27

The Delta (or Delta-like) ligand activates the Notch receptor. This in turn activates 1) the transcription of a transcriptional repressor (Hairy aka Hey aka Hes); in amniotes (chick/mouse) this core oscillatory pathway also has the transcription of Lunatic fringe (Lfng) a negative regulator of Notch signaling. Hairy represses its own transcription and also represses the transcription of Lfng.

The keys are the time-lag between the induction of Notch signaling and the production of Hairy and Lfng proteins (which have to be transcribed then translated). During this lag, Notch signaling is active. The presence of Lfng protein causes an inhibition of Notch signaling. At the same time, the presence of Hes proteins represses the transcription of both itself and Lfng. Notch inhibition will occur until Lfng and Hes mRNA/protein decays. Once they decay, the pathway can be activated again, setting up another oscillation.

The timing components involve the half-life of Lfng and/or Hes. If you were to generate more stable forms of these proteins (or mRNAs), this would increase the amount of time between oscillations. Conversely, if you were to destabilize the mRNA/protein, the oscillator would move faster.

Front 28

LMC motor neurons are only present at specific axial levels of the spinal cord - why? In contrast, MMC motor neurons are rpesent throughout the entire spinal cord - why? Where are motor neurons found within the neural tube?

Back 28

They only innervate the limbs, and are only found at the levels of the spinal column that project to the limbs. The MMC motor neurons innervate the body wall muscles and so are needed at all axial levels. Motor neurons are found in the ventral neural tube - just dorsal to the V3 interneurons.

Front 29

What do you think would happen if you forced expression of ephrinA2 throughout the entire limb bud?

Back 29

-This experiment hasn’t been done. But one prediction would be the complete absence of dorsally projecting motor axons, which are repelled by ephrins.

Front 30

Based on material covered in lecture, what do you think would happen to neuronal migration in a Robo1 mutant embryo? Why?

Back 30

Robo1 is the receptor for the midline chemorepellant ligand, Slit. In its absence, the migrating commisural axons would not avoid the midline. At the same time, Robo1 normally inhibits the Netrin receptor, DCC (except in the more ventral neural tube where Robo3 is expressed). So the axons would still be attracted ventrally - but would likely migrate straight down. Also, they might cross over and then cross back across the ventral midline.

Front 31

What is the predicted phenotype of Robo3 null embryos? Why?

Back 31

Without Robo3, commissural axons will not cross over the midline and will be stuck at the ventral lateral side of the neural tube. This is because Robo1/2 –mediated repulsion prevents the axons from going toward the midline. In addition, Robo1/2 prevents DCC from sensing a chemotactic netrin gradient.

Front 32

Describe a way of generating genetically fate mapping the fate of the posterior sceleretome of the somites using -galactosidase activity. Mention the name of genes and how this system works. What are two tissues you might expect to see labeled? What are your controls?

Back 32

Generate a transgenic mouse line that expresses Cre in the posterior scleretome. A great marker would be to use Ephrin B1::Cre (could generate a transgenic with EphrinB1 promoter enhancer as long as you showed / knew ahead of time that the construct contained enhancer elements active in the posterior scleretome. Alternatively, you could also generate a targeted mouse where you insert Cre and Neomycin in place of the EprhinB1 coding region (so mouse is an Ephrin B1 heterezygote – with the non-active gene copy driving Cre activity in the native Ephrin B1 domain. Typically hemizygous (Tg::EprhinB1 +/-)
2. Obtain a RosaLacZ reporter line. Rosa26 drives expression of LacZ, but only after Cre-mediated excision of a ‘STOP’ cassette. Typically homozygous (RosaLacZ c/c)
3. Cross the mice from steps 1 and 2 together. Stain for -galactosidase activity and visualize expression. Should be present in many tissues: vertebrate, ribs, blood vesses. Genotype embryos. Genotype all embryos. The only ones with staining should be Tg::EphrinB1 +/-; RosaLacZ c/+. The controls are Tg::EphrinB1 -/-; RosaLacZ c/+ (no staining should be present). Important note: this strategy will not label the migratory motor neurons or neural crest cells in this region; these are not somite-derived.

Front 33

How is the gray crescent formed?

Back 33

Cortical rotation

Front 34

What are the domains of the blastula?

Back 34

Animal cap, marginal zone, and vegetal base

Front 35

What is the animal cap made of?

Back 35

Ectoderm

Front 36

What is the marginal zone made of?

Back 36

Mesoderm

Front 37

What is the vegetal base made of?

Back 37

Endoderm

Front 38

What happens when the sperm enters the frog egg?

Back 38

There's a cortical rotation of 30* which makes the gray crescent.

Front 39

What do microtubules do following sperm entry in a frog egg?

Back 39

They're negative at the centriole and grow outward with positive ends. These positive ends move up along the sides.

Front 40

What does beta-catenin do?

Back 40

It's the dorsalizing signal.

Front 41

What happens to embryos when they're severely depleted of beta catenin?

Back 41

They become severely ventralized = belly pieces.

Front 42

What's the experiment you use to find out if beta-catenin plays a role in DV patterning?

Back 42

You dissect a frog embryo into dorsal and ventral sides and perform qPCR. There is higher Wnt 11 dorsally which makes sense because it's part of the canonical Wnt pathway.

Front 43

What happens if you inject Wnt into ooctyes before fertilization?

Back 43

They become super dorsalized.

Front 44

Why should you inject Wnt before fertilization to see dorsalization?

Back 44

Wnt is a maternal factor.

Front 45

What does Wnt do when the sperm gets inside the egg?

Back 45

It rotates 30* and activates the canoncial Wnt pathway.

Front 46

Where does the wnt come from in the DV axis formation?

Back 46

From the vegetal cells.

Front 47

How many cells in the blastula?

Back 47

20K

Front 48

Name 3 mesodermal markers?

Back 48

Xbra
chordin
FGF8

Front 49

What happens when you stick a wt cap on a wt base of a blastula?

Back 49

You get an increase of FGF8 and a decrease of chordin.

Front 50

Mesoderm induction requires what two signals?

Back 50

Vg1 and VegT

Front 51

What happens in Vg1 and VegT mutants?

Back 51

You get belly pieces.

Front 52

Where is Vg1 and VegT located in the blastula?

Back 52

In the bottom vegetal portion.

Front 53

Another name for Vg1

Back 53

Nodal

Front 54

What does Vg1 do?

Back 54

It activates Smad 2,3 trxn factors.

Front 55

What happens if you stick a wt animal cap on a vg1- base?

Back 55

You get a severe loss of mesoderm markers. No chordin and reduced Xbra and fgf8.

Front 56

What does chordin do?

Back 56

It's a BMP antagonist

Front 57

What's the nieuwkoop center?

Back 57

The dorsal vegetal region of the blastula

Front 58

What factors are high in the Nieuwkoop center?

Back 58

beta-catenin and VegT/Vg1. There's also a higher level of Xnr and a gradient of BMP4.

Front 59

What specific structure is in the Nieuwkoop center?

Back 59

The organizer.

Front 60

What does beta-catenin in the Nieuwkoop center lead to? Show the pathway?

Back 60

beta-catenin w/ VegT/Vg1 --> high nodal --> organizer

Front 61

What's the pathway for VegT/Vg1 alone?

Back 61

VegT/Vg1 --> low Nodal --> Ventral mesoderm

Front 62

What happens when you transfer a doral blastopore lip onto a blastula?

Back 62

You make a two-headed monster

Front 63

What does the blastopore lip do?

Back 63

It is the organizer and induces host tissue to form the body axis.

Front 64

4 properties of the organizer:

Back 64

1. initiates the movements of gastrulation
2. differentiates into dorsal mesoderm
3. induces ventral mesoderm to become paraxial mesoderm (somites)
4. induces dorsal ectoderm and neural tube formation

Front 65

What does Noggin antagonize?

Back 65

BMP

Front 66

What does BMP do?

Back 66

It's involved in ventral patterning

Front 67

Where is noggin expressed?

Back 67

In the organizer and notochord

Front 68

What happens if you inject BMP4 into a blastula?

Back 68

You get a belly piece.

Front 69

What is the default of the organizer?

Back 69

Neural ectoderm

Front 70

What is a great neural marker?

Back 70

Sox2

Front 71

What's the initiating step of gastrulation?

Back 71

The marginal zone cells become bottle shaped.

Front 72

How does the blastopore form?

Back 72

Via invagination at the marginal zone.

Front 73

What is the archenteron?

Back 73

The beginning of the gut of the embryo.

Front 74

Name the example of epiboly in gastrulation.

Back 74

The ectoderm engulfs the endoderm via epiboly.

Front 75

What is the convergent extension?

Back 75

It's the stretching and organization of the organizer.

Front 76

Explain what a Keller explant is:

Back 76

You take off the organizer and you put it under a microscrope and you will see that it will undergo convergent extension.

Front 77

What molecules are involved in convergent extension?

Back 77

Non-canonical Wnt signaling is used to induce cell polarity by activating Dsh (Disheveled).

Front 78

What can convergent extension live without?

Back 78

Canonical Wnt signaling

Front 79

Where does the head mesoderm come from?

Back 79

The mesoderm that is pushed up from the blastopore.

Front 80

How can you make multiple heads from head mesoderm?

Back 80

You inject Cerberus which inhibits Wnt8, BMPs, Nodal and causes more head mesoderm.

Front 81

Where is Cerberus expressed?

Back 81

In the anterior endomesoderm.

Front 82

What happens if you mess with the anterior endomesoderm?

Back 82

You get headless embryos.

Front 83

What happens to Lim1 -/- mutants?

Back 83

They don't have heads.

Front 84

How common are neural tube defects?

Back 84

1/1000 live births

Front 85

What happens in primary neurulation?

Back 85

Neural plate folds to generate hollow neural tube.

Front 86

What happens in secondary neurulation?

Back 86

A solid cylinder of cells cavitates to form hollow neural tube.

Front 87

What are the 3 structures of the neural plate?

Back 87

1. The neural plate
2. Neural crest
3. Epidermis

Front 88

What is the neural plate made out of?

Back 88

Ectoderm

Front 89

What drives the folding of the neural tube?

Back 89

Microtubes and actin.

Front 90

How does the cephalic region of the neural tube close?

Back 90

It zippers toward the anterior neuropore.

Front 91

What happens if your neural tube fails to close?

Back 91

You get spinal bifida or anencephaly.

Front 92

How does the neural tube close at the midbrain?

Back 92

A process called buttoning created by cellular bridges.

Front 93

What factor is needed for neural tube closure?

Back 93

B9 (folic acid).

Front 94

Where is the folate receptor expressed?

Back 94

In the dorsal neural tube.

Front 95

How can you decrease neural tube defects?

Back 95

By taking folic acid at the beginning of the pregnancy. It decreases it by as much as 70%.

Front 96

what is Mthfd11?

Back 96

An enzyme in the pathway that makes folic acid available to the cell.

Front 97

What happens in Mthfd11 mutants?

Back 97

They have an open hindbrain. Their neural tube doesn't close.

Front 98

What is the result from the neural tube closing?

Back 98

Occlusion of the neural tube allows pressure to swell in the lumen of the developing brain.

Front 99

In zebrafish, what causes the ventricles to swell?

Back 99

The Na/K ATPase which causes an osmotic gradient.

Front 100

Name the 3 developmental structures of the brain:

Back 100

1. prosencephalon
2. mesenchephalon
3. rhombencephalon

Front 101

forebrain comes from:

Back 101

prosencephalon

Front 102

midbrain comes from:

Back 102

mesencephalon

Front 103

hindbrain comes from:

Back 103

rhombencephalon

Front 104

What happens to Otx2 mutants?

Back 104

They are missing their forebrain and midbrain.

Front 105

What happens to Gbx2 mutants?

Back 105

They're missing their metencephalon (rostral hindbrain).

Front 106

What does Gbx2 do?

Back 106

It restricts Otx2 expression

Front 107

What does the Otx/Gbx border do?

Back 107

It sets up the mid/hindbrain boundary.

Front 108

What does Gbx2 establish?

Back 108

The metencephalon

Front 109

What does Otx2 establish?

Back 109

The mesencephalon

Front 110

What is the isthumus?

Back 110

The major molecular organizing center in brain development.

Front 111

Another name for rostral?

Back 111

Anterior

Front 112

Another name for caudal?

Back 112

Posterior

Front 113

What is Hoxb8 a marker for?

Back 113

caudal (posterior) neural identity

Front 114

Where is RA expressed?

Back 114

In the caudal paraxial mesoderm

Front 115

What does RA induce?

Back 115

RA from the paraxial mesoderm induces caudal neurons.

Front 116

What is Shh a morphogen for?

Back 116

It's a ventral morphogen

Front 117

Where is the highest level of Shh?

Back 117

In the notochord.

Front 118

What is the Shh pathway?

Back 118

Shh binds --> Patched --I Smo --> Upregulation trxn

Front 119

How can you visualize the Hh gradient?

Back 119

You make a mouse with Shh::GFP

Front 120

What happens to Ptch1-/- mutants?

Back 120

They way overexpress Shh.

Front 121

What happens to Shh mutant embryos?

Back 121

They lack ventral neurons

Front 122

Name 2 factors induced by high Shh.

Back 122

Nkx6.1 and Nkx2.2

Front 123

Name two factors induced by low Shh.

Back 123

Pax7 and Dbx1

Front 124

What factor is repressed in high Shh?

Back 124

Pax6

Front 125

How do you electroporate something?

Back 125

You shock the cells and DNA will move to one side.

Front 126

What does ectopic Nkx6.1 do?

Back 126

It causes the loss of Dbx2

Front 127

What does ectopic Dbx2 do?

Back 127

Leads to loss of Nkx6.1

Front 128

What is the fourth germ layer?

Back 128

Neural crest

Front 129

Where are neural crest cells born?

Back 129

In the dorsal neural tube.

Front 130

What is EMT?

Back 130

The change that neural crest cells undergo called the epithelial to mesenchymal transition and then they migrate to the periphery.

Front 131

What is RhoB?

Back 131

It is the factor expressed during neural crest initiating migration

Front 132

When do crest cells express RhoB?

Back 132

When they undergo EMT.

Front 133

What do neural crest cells express just prior to migration?

Back 133

Sox9.

Front 134

What do Sox9 and RhoB do?

Back 134

They induce Neural Crest differentiation and EMT.

Front 135

What do neural crest cells end up generating?

Back 135

Sensory and sympathetic neurons.

Front 136

What is the sclerotome?

Back 136

They'll make sensory and sympathetic neurons.

Front 137

Trunk crest cells only migrate through...

Back 137

that anterior half of somites.

Front 138

What is expressed in the posterior half-somite?

Back 138

Ephrin B1

Front 139

What do crest cells express?

Back 139

EphB3.

Front 140

What repells crest cells from going into the posterior portion of the somite?

Back 140

Ephrin B1.

Front 141

What is the stripe assay?

Back 141

The way to show that neural crest cells are repelled by ephrin B1.

Front 142

What do path 2 crest cells generate?

Back 142

They make melanocytes.

Front 143

Where do path 2 crest cells migrate?

Back 143

Between the dermamyotome and the surface ectoderm.

Front 144

What does FoxD3 do?

Back 144

It's essential for generating NCC and says on Pathway 1.

Front 145

What does FoxD3 repress?

Back 145

A gene called Mitf, which is the master regulator of melanocyte fate.

Front 146

What does Mitf do?

Back 146

It's the regulator of melanocyte fate.

Front 147

Mitf1 is essential for...

Back 147

melanocyte stem cells.

Front 148

Why do we turn gray?

Back 148

the cells expressing mitf1 are lost.

Front 149

Where do the pharyngeal arches come from?

Back 149

The migration of specific streams of cranial neural crest cells.

Front 150

Pharyngeal arches are populated by what kind of cell?

Back 150

Neural crest cells.

Front 151

What happens to pdgfra mutants?

Back 151

They're defective for neural crest migration

Front 152

What is Pdgf?

Back 152

It's a cranial neural crest chemoattractant.

Front 153

What is the anatomy of an arch?

Back 153

Muscle in the middle, wrapped in crest cells with endoderm on top and ectoderm on the bottom.

Front 154

What does the first pharyngeal arch contribute to?

Back 154

Malleus
Incus
Mechel's cartilage

Front 155

What does the second pharyngeal arch contribute to?

Back 155

The stapes and styloid process

Front 156

What does the 3rd pharyngeal arch make?

Back 156

Hyoid bone

Front 157

What do pharyngeal arch 4 and 5 make?

Back 157

Thyroid cartilage

Front 158

When genetically fate mapping a mouse, where do the facial structures come from?

Back 158

Neural crest cells and mesodermally derived cells.

Front 159

Where do facial prominences come from?

Back 159

The first pharyngeal arch.

Front 160

3 facial structures from the 1st pharyngeal arch.

Back 160

1. midline facial skeleton
2. upper jaw and palatal skeleton
3. lower jaw

Front 161

What occurs at the border of Shh and Fgf8?

Back 161

It's the distal tip of the upper break

Front 162

What happens if you make extra Fgf8-Shh boundaries?

Back 162

You get multiple ectopic beaks in a bird.

Front 163

What makes up an axon growth cone?

Back 163

Filopodia and lamellipodia.

Front 164

What are filopodia?

Back 164

The microspike fingers of the growth cone

Front 165

What are lamellipodia?

Back 165

The webbing of the growth cone.

Front 166

What does the axon growth cone do?

Back 166

It senses the environment and connects to the appropriate targets.

Front 167

What is pathway selection?

Back 167

Axon chooses route through the environment to its target.

Front 168

What is target selection?

Back 168

Axon recognizes target and initiates synaptogenesis.

Front 169

Addess selection:

Back 169

Synapses are strengthed and lost

Front 170

Name the two structures of the neural tube that contribute to motor axon projections?

Back 170

Lateral motor column
Medial motor column

Front 171

Where does the medial motor column project to?

Back 171

The dermamyotome

Front 172

What are the subcategories of the lateral motor column?

Back 172

Lateral subdivision --> dorsal
Medial subdivision --> ventral

Front 173

Where do LMCl project to?

Back 173

Dorsal musculature

Front 174

Where do LMCm project to?

Back 174

Ventral musculature

Front 175

Ephrins are concentrated in what part of the limb bud?

Back 175

The ventral part

Front 176

EphA4 expressing axons project...

Back 176

dorsally

Front 177

EphA4 is sufficient to drive axons...

Back 177

dorsally.

Front 178

What does Lim1 do?

Back 178

It activates Eph4A.

Front 179

What does Isl1 do?

Back 179

It represses Eph4A.

Front 180

What is the pathway for LMCl axons?

Back 180

Lim1 --> Eph4A --> dorsal projection

Front 181

What must axons to at the ventral midline?

Back 181

They must project to the ventral midline and only cross once.

Front 182

What are Robo and Slit?

Back 182

Slit is a ligand in the midline of drosophila.
Robo is a receptor on axons.

Front 183

What happens to slit mutants?

Back 183

Axons never leave the midline.

Front 184

How do Slit and Robo interact?

Back 184

Robo1 is a receptor and when Slit binds to it, it inhibits DCC.

Front 185

Slit binding to Robo1 results in what?

Back 185

Chemorepulsion

Front 186

What happens when Robo3 is expressed?

Back 186

It inhibits Robo1 so now DCC can work and the axon can cross the midline.

Front 187

What does Netrin bind to?

Back 187

DCC.

Front 188

What happens after the axon crosses the midline?

Back 188

Robo1 turns back on and blocks DCC chemoattraction

Front 189

What does the brainbow help you do?

Back 189

You can see the neuronal connections based on the random turning on of GFPs.

Front 190

How does brainbow work?

Back 190

It's a Cre-base recombination resulting in expression of 3 different fluorescent proteins.

Front 191

What is the axon chemoattractant?

Back 191

Netrin binding to DCC receptor.

Front 192

What is the axon chemorepellant?

Back 192

Slit binding to Robo1.

Front 193

Where does somitic formation occur?

Back 193

In the presomitic mesoderm

Front 194

What is the somite clock?

Back 194

The time period of somite formation.

Front 195

What is the Notch pathway?

Back 195

Delta (membrane-bound ligand) --> Notch (receptor) --> Lfng --I Hairy --I trxn of Hes and Lfng

Front 196

What is the Clock and Wavefront model?

Back 196

There is an oscillator (clock) that regulates the periodic generation of somites and a wavefront that defines the region competent to respond to the clock and become a somite.

Front 197

What does the clock part of the Clock and Wavefront model do?

Back 197

It regulates the periodic generation of somites.

Front 198

What does the Wavefront part of the model do?

Back 198

It defines the region that will become a somite.

Front 199

What is the central oscillator controlled by?

Back 199

Notch/Delta signaling

Front 200

Another name for Hairy...

Back 200

Hes

Front 201

What is the oscillating expression of Hairy?

Back 201

Hairy1 expression moves from posterior plate to anterior. The posterior expression boundary matches the posterior of what will be the next somite. Oscillation happens every 90 minutes in a chick.

Front 202

How does Notch affect the Wavefront model?

Back 202

It provides signaling for the border information. Notch signaling is sufficient to cause segmentation.

Front 203

What happens to Delta-like 3 mutants?

Back 203

They have defects in somite formation, leading to rib fusions.

Front 204

What signaling is necessary for somite formation?

Back 204

Notch signaling

Front 205

What happens to MESP2 mutants?

Back 205

They have defects in ribs and vertebrae.

Front 206

What happens to Lfng mutant mice?

Back 206

They lose Notch cycling

Front 207

How does Lnfg aid in oscillating expression?

Back 207

Lnfg is a negative regulator of Notch, but is very unstable. So it leads to an oscillating signal.

Front 208

How does Wnt signaling affect somite segmentation?

Back 208

It helps position determination front.

Front 209

What does Axin2 do?

Back 209

Inhibits Wnt activity and is short-lived.

Front 210

If Notch can't oscillate, how does Wnt react?

Back 210

They still show axin2 expression oscillation so Wnt pathways target genes still oscillate.

Front 211

What happens if beta-catenin can't be degraded? This is relating to somite formation.

Back 211

There's no Wnt activity. There are no visible somites.

Front 212

What happens to somite formation when there's no beta-catenin?

Back 212

Oscillation still occurs, but it's abnormal. Wnts do not appear to be a pacemaker for oscillations.

Front 213

What regulates the size of somites?

Back 213

Fgf

Front 214

What happens if you stick an FGF-soaked bead near the undetermined zone?

Back 214

It makes a smaller somite.

Front 215

How is FGF related to the wavefront?

Back 215

It regulates segmentation size by changing the "size" of the wavefront.

Front 216

What happens at somite IV?

Back 216

Somite segmentation is determined. The receding Fgf8 marks a boundary.

Front 217

What is the determination front in a molecular definition?

Back 217

It is the posterior boundary of Mesp2 gene expression.

Front 218

What happens when the wavefront breaks?

Back 218

It's the region at which presomitic mesoderm first acquires segmental identity.

Front 219

What happens to cells anterior to the determination front?

Back 219

They are more epithelialized due to a downregulation of fgf8. They undero a mesenchyal - epithelial transformation.

Front 220

How is somitogenesis in fgf8 nulls?

Back 220

It's normal.

Front 221

Why do fgf8 nulls still have normal somitogenesis?

Back 221

fgf8 is redundant for fgf4.

Front 222

How can you knock out fgf8/4?

Back 222

You have do conditionally KO.

Front 223

What happens when you conditionally KO fgf4/8?

Back 223

They have major somite defects. They make a bunch of somites.

Front 224

What happens to the the presomitic mesoderm in fgf4/8 cKOs?

Back 224

It all undergoes somitogenesis.

Front 225

What does the wavefront prevent?

Back 225

Early differentiation.

Front 226

How is the determination front in fgf4/8 cKOs?

Back 226

It's expanded in the PSM.

Front 227

In the Clock and Wavefront model, what is keeping the somites from differentiating prematurely?

Back 227

the fgf/Wnt gradient.

Front 228

How are snakes somitogenesis different from others?

Back 228

They have slower wavefront

Front 229

A slower wavefront leads to...

Back 229

lots of somites.

Front 230

What pinches off somites?

Back 230

Eph/ephrin interactions.

Front 231

What does Eph4A do in somite formation?

Back 231

It precedes somite formation and is restricted to the anterior half of the somite.

Front 232

Where are ephrins expressed during somitogenesis?

Back 232

In the posterior somite.

Front 233

How does Eph signaling affect somitogenesis?

Back 233

It positions somite boundaries.

Front 234

How does epithelialization of a somite work?

Back 234

Integrins and N-cadherin polarize outer cells into an epithelium. It occurs immediately after somite fission.

Front 235

Which process provies the major force to extend the anterior-posterior axis of frog embryos during gastrulation?

Back 235

Convergent extension

Front 236

Name a general mesodermal inducing factor.

Back 236

Vg1

Front 237

Name 4 neural crest derivatives:

Back 237

1. The stapes
2. The peripheral neuvous system
3. The hyoid bone
4. The mandible

Front 238

What branchial arch gives rise to most of the face?

Back 238

First

Front 239

Which molecular interaction is most important for helping scult the motor neuron in the neural tube?

Back 239

Nkx2.2 -- Pax6

Front 240

Name the pathway that mediates convergent extension.

Back 240

Non-canonical Wnt signaling.

Front 241

You take the dorsal life of a gastrulating frog embryo and transplant it to the ventral side of the host embryo. What happens?

Back 241

The embryo forms another body axis.

Front 242

You electroporate a chick neural tube with Sox9. What happens to the electroporated cells?

Back 242

They become neural crest cells.

Front 243

3 things you need to make a targeting vector that can modify the genome of mouse ES cells.

Back 243

1. left homology arm
2. right homology arm
3. Positive selection cassette eg NeoR

Front 244

You express Eph4A in all motor neurons. What happens?

Back 244

All the projections will go in the dorsal part of the limb bud.

Front 245

What general route do non-melanocyte trunk neural cells take when they migrate? How could you block or inhibit this route?

Back 245

They migrate through the anterior sclerotome. You could reroute them by forcible expression of mitf (They'll now become a melanocyte). Or you could block the route by misexpressing Ephrin B1 throughout the whole somite.

Front 246

Embryologist took naive neural plate and cultured it with notochord tissue to form ventral neuronal fates. What do you predict would happen if you performed these same experiments substituting notochord tissue from Smo-/- embryos? B: Shh-/-? What if you incubated with wt notochord with neural plate tissue from Shh-/- embryos?

Back 246

A. Will not express ventral neural tube markers
B. Will not express ventral neural tube markers
C. Will express ventral neural tube markers.

Front 247

How could you use genetic fate mapping to determine the fate of somites?

Back 247

Somite-specific Cre crossed with Cre reporter construct (a construct with a ubiquitous promoter then a loxP-stop-loxP followed by reporter gene that will only be trxned when Cre removes the stop cassette).

Front 248

You treat recently fertilized frog eggs with Nocodazole. What will this do? What will the embryo look like as a result?

Back 248

This will block microtubule polymerization, preventing cortical rotation. The embryo will be a belly piece.

Front 249

You have just generated a genetically modified ES cell line in which one copy of a gene is deleted, creating a null allele. Briefly list the steps you need to take to generate and analyze homozygous null embryos.

Back 249

1. Inject ES cells into host mouse embryo and implant injected embryos into surrogate mom.
2. Mom will give birth to chimeras. You can tell chimeras by coat color contribution.
3. Breed chimeras to wt mice to test for germline transmission. These progeny are heterozygous embryos.
4. Cross heterozygous animals and collect embryos for genotyping and analysis.

Front 250

Why are pregnant women encouraged to take folic acid?

Back 250

Because folic acid supplements reduce the prevalence of neural tube defects.

Front 251

What do you think would happen to somite development if you incubated embryos in an inhibitor that reduces but does not eliminate fgf signaling.

Back 251

You would have a reduced gradient of the wavefront. This would likely give rise to fewer and bigger somites.

Front 252

In class, we discussed the general features of an oscillator. The Hh pathway has some of these same features although it does not appear to oscillate. Describe the feature of the Hh pathway that could make it act as an oscillator? How you could modify this in a way that might make it into an oscillator?

Back 252

The Hh lignad can activate the trxn of Ptch1 mRNA would make Ptch1 protein. Ptch1 inhibits Hh signaling (unless boud by more ligand) and therefore acts as a negative regulator of the Hh pathway. You could modify the Ptch protein to make it more unstable. This would allow the pathway to oscillate between off and on in the presence of the Hh ligand.

Front 253

LMC motor neurons are only present at specific axial levels of the spinal cord - why? In contrast, MMC motor neurons are present throughout the entire spinal cord - why? Where are motor neurons found within the neural tube?

Back 253

A. LMC neurons only project to the forelimb and hindlimb and are only found at these levels.
B. MMC neurons project to axial musculature of the body at all levels.
C. Motor neurons are found in the ventral neural tube.

Front 254

You take vegetal tissue from a frog embryo and culture it together with an animal cap tissue. What happens to both tissues?

Back 254

The vegetal tissue will form endoderm. The animal cap tissue will form mesoderm.

Front 255

You have just discovered a new gene that, when depleted in frog embryos with siRNA antisense oligonucleotides, gives
rise to an embryo that lacks all neuroepithelium.

A) Based on what we learned in class, propose a molecular mechanism for how this gene might work? Describe one
additional piece of experimental data that would be helpful for you to refine your model?
B) What do you think would happen if you over-expressed this molecule? Devise an experiment to test your
hypothesis. Be as specific as you can be and include appropriate controls.

Back 255

Secreted BMP antagonists from the organizer inhibit the BMP pathway, resulting in the formation of neural tissue. So the BMP pathway is activated in all ectodermal cells when the gene is lost. It could be the loss of a major BMP antagonist like noggin, chordin, or follistatin. There's a lot of redundancy though. It could also be a problem in the BMP pathway to make it constantly expressed. B. Overexpression could result in neural expression in all neural tissue. You could do insitu for neural marker like Sox2. Or overexpressing some aspect of the BMP pathway could turn off BMP.

Front 256

When are the kidneys formed?

Back 256

Kidney formation is a later developmental event, occurring after the formation of the primary A-P axis.

Front 257

What are the kidneys made up of?

Back 257

The kidneys come from intermediate mesoderm. Development relies on multiple interactions between the epithelial ureteric bud and the adjacent mesenchyme.

Front 258

What two structures interact to make the kidney?

Back 258

The epithelial ureteric bud and the adjacent mesenchyme.

Front 259

What is the functional unit of the kidney?

Back 259

The nephron.

Front 260

How many nephrons are in a kidney?

Back 260

A human kidney has 1 million nephrons.

Front 261

What are the kidneys specified from in relation to the neural tube?

Back 261

The kidneys come from the intermediate mesoderm which is inbetween the paraxial and lateral plate mesoderm on either side of the neural tube.

Front 262

How does the kidney form from the intermediate mesoderm?

Back 262

The intermediate mesoderm travels down caudally and forms the pronephic duct. On the inside of the duct are structures forming called the pronephric tubules.

Front 263

What is the pronephros?

Back 263

The pronephic duct + the pronephric tubules

Front 264

What is the 2nd stage of kidney development?

Back 264

The pronephros continues down and becomes just the nephric duct, which attaches itself to the cloaca. The nephric duct also has tubules called the mesonephric tubules (mesonephros). The entire combo is called the mesonephric duct.

Front 265

What happens as the mesonephric duct is forming?

Back 265

The pronephros begins to disappear.

Front 266

What is the function of the pronephros?

Back 266

It's strictly developmental. The pronephros does not have any of the function of the kidneys.

Front 267

What does the mesonephric duct do?

Back 267

It begins performing the functions of the kidney and receiving blood from capillaries.

Front 268

What is another name for the nephric duct?

Back 268

The wolffian duct

Front 269

How are the collecting tubules of the developing kidney generated?

Back 269

The metanephrogenic mesenchyme induces the ureteric duct to undergo branching morphogenesis which turns into tubules.

Front 270

What is the reciprocal induction in kidney development?

Back 270

The mesenchyme induces branching morphogenesis of the ureteric bud.

Front 271

What happens to the ureteric bud without mesenchyme?

Back 271

The bud does not branch.

Front 272

What does the ureteric bud do to the metanephrogenic mesenchyme?

Back 272

The ureteric bud makes the metanephrogenic mesenchyme condense upon the bud at the tips of the branches.

Front 273

What happens to the mesenchyme when it is cultured without the ureteric bud?

Back 273

The mesenchyme dies.

Front 274

What is mesenchyme?

Back 274

A loosely organized, mainly mesodermal embryonic tissue that develops into connective and skeletal tissues, including blood and lymph.

Front 275

What is the ureteric bud?

Back 275

The ureteric bud, also known as the metanephrogenic diverticulum, is a protrusion from the mesonephric duct during the development of the urinary and reproductive organs. It later develops into a conduit for urine drainage from the kidneys, which, in contrast, originate from the metanephric blastema.

Front 276

What happens after the mesenchyme condenses onto the tips of the branches?

Back 276

The branch tips form aggregates ventral to the bud which will epithelialize and become renal vesicles, then S-shaped bodies that fuse to the UB (forms the collecting duct), and finally mature nephrons.

Front 277

What does GDNF do?

Back 277

It directs ureteric bud growth. GDNF (glial derived neurotrophic factor) is a ligand for the Ret receptor. GDNF is secreted by the metanephrogenic mesenchyme and it promotes sprouting of the ureteric bud which expresses Ret.

Front 278

What secretes GDNF?

Back 278

The metanephrogenic mesenchyme.

Front 279

What has Ret receptors?

Back 279

The ureteric bud.

Front 280

What happens to the ureteric bud without GDNF?

Back 280

The ureteric bud doesn't form.

Front 281

What does Wnt9b do?

Back 281

It's essential for kidney formation. Without Wnt9b, you don't make kidneys.

Front 282

What happens if you have a Wnt9b-/- early in development?

Back 282

Nothing seems to happen. The ureteric bud forms and initial branching is normal. So Wnt9b is not important for early development of the kidneys.

Front 283

How is Wnt9b important for kidney formation?

Back 283

Wnt9b expression induces Wnt4, which is critical for renal vesicle formation.

Front 284

What happens to Wnt4 expression in Wnt9b mutants?

Back 284

There is no Wnt4 expression. Wnt4 is essential for epithelialization of nephrons (renal vesicle formation).

Front 285

What does Wnt4 do?

Back 285

Wnt4 is essential for renal vesicle formation.

Front 286

How does Wnt9b specify renal vesicle formation?

Back 286

By inducing Wnt4.

Front 287

What happens if you put a Wnt4 KO in the presence of Wnt9b?

Back 287

The mutant metanephric mesenchyme (MM) can't make renal vesicles even in the presence of Wnt9b. If you do a Pax8 in situ hybridization, you'll see that Wnt4 in the MM is required for renal vesicle formation.

Front 288

What are renal vesicles?

Back 288

They're the nephron progenitor.

Front 289

What is the mechanism of Wnt9b for making renal vesicles?

Back 289

Wnt9b in the ureteric bud goes into the MM. The MM secretes Wnt4 which makes renal vesicles.

Front 290

What is the structure of the metanephric mesenchyme?

Back 290

The metanephri mesenchyme is spatially distinct. Different parts of the mesenchyme express Pax2, FoxD1, and cytokeratin.

Front 291

Where is Pax2 expressed in the metanephric mesenchyme?

Back 291

Pax2 is expressed in the medial region, kind of like a heart.

Front 292

Where is FoxD1 expressed in the metanephric mesenchyme?

Back 292

Distally on the edges.

Front 293

Where is cytokeratin expressed in the metanephric mesenchyme?

Back 293

Squished in the middle.

Front 294

What does Six2 do?

Back 294

Six2 is a key regulator of nephron progenitor populations.

Front 295

Where is Six2 expressed?

Back 295

Only in the cap metanephric mesenchyme which will later give rise to nephrons.

Front 296

What does Six2 define?

Back 296

Six2 defines a multipotent nephron progenitor population. It is required to maintain a pool of nephron progenitors.

Front 297

How can you use genetic fate mapping to figure out what the hell Six2 does?

Back 297

You mess with the Six2 gene and make this monster:
Six2::GFPCre x RosaLacZ. This experiment will show you Six2-expressing cells all give rise to the nephron tubule.

Front 298

How could you possibly generate new nephrons as a treatment for a degenerative kidney disease?

Back 298

You could give someone Six2.

Front 299

How are Six2 and Wnt9b different?

Back 299

Six2 expands the nephrons and Wnt9b differentiates the nephrons.

Front 300

What happens if you have a KO for Wnt9b and Six2?

Back 300

Nephrons are neither expressed nor differentiated.

Front 301

What forms muscle?

Back 301

The dermamyotome which comes from the somites.

Front 302

What differentiates muscle?

Back 302

MyoD, Myf5, and Myogenin.

Front 303

What does the somitocoel break up into?

Back 303

As a mature somite, it breaks up into the epithelial dermamyotome and the mesenchymal sclerotome.

Front 304

The somite turns into what kinds of muscles relative the the neural tube?

Back 304

The position of parts of the somite relative to signaling centers determines the fate of the somites. Bmp and Fgf from the lateral plate mesoderm turn into abaxial myotome. Wnt1/Wnt3a and low Shh levels make primaxial myotome.

Front 305

What factors induce abaxial myotome?

Back 305

Bmp and Fgf from the lateral plate mesoderm. Abaxial myotome is the farthest from the neural tube.

Front 306

What factors induce primaxial myotome?

Back 306

Wnt1/Wnt3a and low Shh. This part of the somite is closest to the neural tube.

Front 307

What is the basic unit of a muscle?

Back 307

The muscle fiber. It is a multinucleated cell. Myosin heavy chain provides its contractile capabilities and is a marker of differentiated muscle.

Front 308

What are the 3 steps that convert myoblasts into muscles?

Back 308

Determination of myotome cells --> multiplication of dividing myoblasts --> multiplication stops and cells align

Front 309

What are myoblasts?

Back 309

Muscle progenitors

Front 310

What factors do myoblasts express?

Back 310

MyoD and Myf5.

Front 311

Describe the conversion of myoblasts to muscles

Back 311

The neural tube secretes paracrine factors Wnt and Shh. Myoblasts express MyoD and Myf5. The myoblasts divide and multiply from the Fgf signal. When Fgf runs out, multiplication stops and the cells align with help from fibronectin, integrin, cadherin/CAM, and myogenin.

Front 312

Which factor tells myoblasts to divide and multiply?

Back 312

Fgf's

Front 313

Which factors help the muscle cells to align?

Back 313

Fibronectin, integrin, cadherin/CAM, myogenin.

Front 314

What does MyoD do?

Back 314

MyoD is a muscle determinant. It's a muscle-specific basic helix-loop-helix trxn factor that actives expression of other muscle genes as well as its own expression. It's can turn other cells into muscles, even neurons.

Front 315

What happens if you force expression of MyoD in melanocytes and neurons?

Back 315

They express myosin heavy chain.

Front 316

What happens to mouse KO's for MyoD?

Back 316

They still make skeletal muscle. So MyoD must not be the only thing you need to make muscle.

Front 317

What do MyoD and Myf5 do together?

Back 317

MyoD and Myf5 cooperate in muscle differentiation.

Front 318

What is Myf5?

Back 318

Myf5 is a related bHLH trxn factor to MyoD.

Front 319

What happens to Myf5 mutants?

Back 319

They make skeletal muscle too like MyoD KO's.

Front 320

What happens to MyoD;Myf5 double null mutants?

Back 320

They lack skeletal muscle. Pups are born completely immobile and die within a few minutes. They still make circular vascular smooth muscle, but not skeletal muscle.

Front 321

What do Fgfs do to myoblasts?

Back 321

Fgfs maintain myoblasts in an undifferentiated state.

Front 322

What do myoblasts do without Fgfs?

Back 322

Myoblasts will exit the cell cycle and differentiate before fusing.

Front 323

What do aligned myoblasts do?

Back 323

Aligned myoblasts exit the cell cycle.

Front 324

What happens if you culture isolated and aligned myoblasts with radioactive thymidine?

Back 324

Isolated myoblasts will take up the label, but aligned myoblasts will not.

Front 325

What happens if you're a myogenin mutant?

Back 325

You don't have skeletal muscle. An appropriate number of cells are formed in myogenin mutants, but they don't form multinucleated myotubes or express myosin.

Front 326

What does myostatin do?

Back 326

Myostatin regulates muscle size.

Front 327

What happens if you're a KO for myostatin?

Back 327

You have super huge muscles and it looks like you're taking steroids.

Front 328

What are some myostatin mutants?

Back 328

The Belgian Blue cow, the whippet dog.

Front 329

What does the TGFbeta family member do?

Back 329

It suppresses proliferation and differentiation of muscle.

Front 330

Where do muscle fibers come from?

Back 330

Most muscle fibers come from cells in the central dermamyotome that express Pax3 and Pax7. Only a subset of myotome makes muscle fibers.

Front 331

What factors in the dermamyotome make muscle fibers?

Back 331

Pax3 and Pax7.

Front 332

What do Pax3/Pax7 do?

Back 332

They inhibit MyoD expression. This inhibition can be reversed, allowing these cells to express MyoD and make muscles. Apparently, Pax3+;Pax7+ population gives rise to basically all muscle.

Front 333

How do Pax3 and Pax7 interact?

Back 333

Pax3-expressing cells give rise to all Pax7 expressing cells and embryonic myoblasts.

Front 334

How are muscles regenerated?

Back 334

Muscles can enlarge by cell growth, but done divide. They can be replaced by muscle stem cells. The stem cells have Pax7 in them and lie between the basal lamina and the muscle fiber.

Front 335

Pax3/Pax7 have important early roles in development. How can they be deleted in postnatal embryos?

Back 335

You can do this will a tamoxifen-inducible CRE. CRE is expressed everywhere in the cell until you add tamoxifen which turns on the expression as the CRE goes into the nucleus.

Front 336

How can you show that mice need Pax3/7 cells for muscle regeneration at birth?

Back 336

You damage the muscle. With Pax7+/CE, you see regeneration. With Pax7CE/f, you see no regeneration.

Front 337

What are the 3 types of Pax alleles that you can have for experimenting?

Back 337

1. Pax7 which has CRE ER instead of exon 1. This is called CE.
2. f allele (or c allele): This is the conditional loss of function allele in which exon 1 is between Lox P sites.
3. Normal Pax7

Front 338

Do adult mice still require Pax3/7?

Back 338

No - if you damage the muscle of a Pax7CE/f, they'll grow back muscle just fine. Pax3/7 are markers for adult stem cells, but they don't seem to be important for stem cell activity in adults.

Front 339

Muscles form from...

Back 339

dermamyotome.

Front 340

What regulates muscle development?

Back 340

A set of trxn factors.

Front 341

Most muscles come from...

Back 341

a reservoir of stem cells.

Front 342

Muscle stem cells are present...

Back 342

both in embryos and adults, but have different forms of regulation.

Front 343

Modern Cre-based strategies can be used for...

Back 343

ablation, inducible deletion, and fate mapping.

Front 344

What is the first organ to form?

Back 344

The heart.

Front 345

How is the vertebrate cardiogenic mesoderm expressed?

Back 345

It's a combination of BMP and no Wnt.

Front 346

What are the key factors in early heart development?

Back 346

Transcription factors GATA4, Nkx2.5, and Tbx5

Front 347

Name this homeodomain trxn factor related to drosophilia tinman expression in vertebrate hearts:

Back 347

Nkx2.5

Front 348

What happens to Nkx2.5 KO mice?

Back 348

They die at about 9.5 days due to heart defects. Most of the cardiac muscle genes are expressed and the cardiac muscle does beat.

Front 349

What happens if you're heterozygous Nkx 2.5 +/- and human?

Back 349

You have a range of heart defects.

Front 350

How does Nkx2.5 work?

Back 350

It synergizes with GATA factors and activates trxn of cardiac genes.

Front 351

What happens if you overexpress Nkx2.5?

Back 351

You get an enlarged heart.

Front 352

How can you make a xenopus embryo have a giant heart?

Back 352

Inject Nkx2.5 mRNAs into the cardiac forming region of early xenopus embryos. It will make a giant heart. Also you can assay after heart presence for the formation of heart specific differentiation genes to see the heart.

Front 353

How can you make a frog without a heart?

Back 353

You make a dominant negative Nkx2.5 that doesn't work into the cardiac forming region of early xenopus embryos. They won't have hearts. Assay for heart specific differentiation genes.

Front 354

How is the loss of Nkx2.5 different between frogs and mice?

Back 354

Nkx2.5 KO in frogs means you get no heart, but you still get a heart with mice.

Front 355

What happens if you lose Nkx2.5 in a mouse?

Back 355

The heart forms, but is arrested after the looping stage. They have reduced or absent expression of differentiation markers like ANF expression.

Front 356

What is drosophila tinman?

Back 356

No heart!

Front 357

Why do you have to use a dom neg Nkx2.5 in frogs?

Back 357

You can't knock out Nkx2.5. You could use RNAi but the heart develops later in development so that makes it really hard.

Front 358

Why is the Nkx2.5 null phenotype in mouse different from the dominant neg Nkx2.5 phenotype in frogs?

Back 358

Mice hearts have like 3 more Nkx's. Frogs have a paired atria but 3 chambered heart. Frogs don't have a bunch of Nkx's so you can't completely knock out Nkx2.5 in a frog.

Front 359

Which trxn factor is needed for heart differentiation?

Back 359

ANF

Front 360

How can you mutate ANF so you can analyze it?

Back 360

You attach GFP to the promoter.

Front 361

How do ANF and Nkx2.5 interact?

Back 361

It's thought that GATA, SRF, and Nkx2.5 all work in a complex to promote the trxn of ANF.

Front 362

What happens if you muck up the NKE sequence of the ANF promoter site?

Back 362

It only produces a small reduction in ANF expression, which is surprising because you would think ANF expression would stop. This result probably points to the formation of a complex for promoting expression.

Front 363

If deletion of NKE site has little effection on ANF expression, what is happening?

Back 363

GATA4, SRF, and Nkx2.5 are probably forming a promoting complex.

Front 364

What's the fruit fly name for Nkx2.5?

Back 364

Tinman

Front 365

What's the fruit fly name for GATA4/5?

Back 365

Pannier

Front 366

What's the fruit fly name for Tbx5?

Back 366

Dorsocross

Front 367

What's the fruit fly name for BMP?

Back 367

Dpp

Front 368

How is fruit fly and vertebrate heart signaling different?

Back 368

Fruit flies require Wnt signaling and vertebrates don't.

Front 369

How does the fruit fly heart form?

Back 369

The heart is specified by tinman, dorsocross, and pannier. They forms these stripes in the developing organism.

Front 370

What causes the expressive patterning of heart trxn factors in fruit flies?

Back 370

Wnt and BMP

Front 371

What is ChIP?

Back 371

Chromatin immunoprecipitation. This technique shows the specific regions of DNA bound by the trxn factor being assayed.

Front 372

How did they use ChIP in fruit flies?

Back 372

They looked to find the trxn factors for the heart.

Front 373

How do many of the trxn factors interact in heart development?

Back 373

They tend to clump together to bind 600-1200 genes.

Front 374

What kind of tissue makes the heart?

Back 374

The endocardial primordia makes a common heart tube. The tube then has an outer layer of myocardium and an inner layer of endocardium.

Front 375

What's wrong with the miles apart mutant?

Back 375

The mutant zebrafish gives rise to cardia bifida, two hearts.

Front 376

Where is miles apart expressed?

Back 376

It's not in the heart cells, but in the underlying endoderm on either side of the midline.

Front 377

What does miles apart do?

Back 377

It's involved in directing migration of presumptive heart cells.

Front 378

How can you end up getting cardia bifida?

Back 378

The heart cells migrate to eachother normally so if they're not directed towards each other, they will make two hearts.

Front 379

What does the right atrium of the heart do?

Back 379

It takes in deoxygenated blood.

Front 380

What happens in the heart of a smoothened mutant?

Back 380

It has linear heart tube, but the mice never form looped heart tubes.

Front 381

You isolate a new zebrafish mutant that has cardia bifida. What is cardia bifida? Based on what you know about heart migration, which germ layer do you think is causing the mutant phenotype?

Back 381

Cardia bifida is a condition in which the two heart progenitor patches fail two fuse, instead forming two small, beating hearts on each side of the body. Most defects underlying cardia bifida are actually in the underlying endoderm rather than in the cardiac progenitors themselves. The mutations in the endoderm impede the ability of the cardiogenic mesoderm to migrate to the midline.

Front 382

You clone the mutation described above, and find that it is a previously uncharacterized member of a large group of closely related proteins, several of which are also expressed in the same tissue type predicted in question #1. In followup experiments, you attempt to replicate the phenotype by injecting wild-type zebrafish with morpholinos specifically against this gene (you perform exhaustive controls to show that the morpholino knockdown is specifically removing all detectable protein). Surprisingly, the embryos all appear relatively normal.
A) What does this data suggest to you about the nature of your mutation?

Back 382

It suggests that the mutation is not a null allele since other experimental methods removing the protein do not cause the same phenotype. Instead, this mutation might be a dominant negative allele that is able to interfere not only with its own function but also with other related family members.

Front 383

You clone the mutation described above, and find that it is a previously uncharacterized member of a large group of closely related proteins, several of which are also expressed in the same tissue type predicted in question #1. In followup experiments, you attempt to replicate the phenotype by injecting wild-type zebrafish with morpholinos specifically against this gene (you perform exhaustive controls to show that the morpholino knockdown is specifically removing all detectable protein). Surprisingly, the embryos all appear relatively normal. How could you then test that this was indeed this type of mutation?

Back 383

Dominant negative mutations inhibit the activity of endogenous proteins. You could inject mRNA encoding the mutated form of the protein into wild-type zebrafish embryos to see if this gave a cardi bifida phenotype.

Front 384

What do you think would happen if you inserted a bead soaked in VEGF into embyronic mesoderm?

Back 384

Almost all mesoderm contains angioblasts. The addition of VEGF would cause a massive proliferation of these angioblasts, resulting in hypervascularization. It could also cause directional migration.

Front 385

What is the difference between vasculogenesis and angiogenesis?

Back 385

Vasculogenesis is the formation of of blood vessels from vascular precursor cells (this forms the first blood vessels). Angiogenesis is the formation of blood vessels from pre-exisiting blood vessels (all later vascular development).

Front 386

You delete a hypothetical protein-protein interaction domain on Nkx2.5 that renders it incapable of binding to SRF and GATA? However, it is still able to bind DNA, and you haven't interfered with a known transcriptional activation domain. You then inject this mRNA transcribed from this construct into the future heart forming region of a frog embryo. What are possible scenarios that might happen and why?

Back 386

If it can still bind DNA, it might have normal function; we know from lecture that overexpressing a WT Nkx2.5 causes enlarged hearts so this would be the first possibility. Alternatively, the protein::protein interactions between SRF-Myocardin and GATA4 may be really important (recall that Nkx2.5 can still activate target genes even in the absence of a DNA binding domain presumably through interactions with GATA/SRF that recruit and tether it to the enhancer). In this case, the altered form of Nkx2.5 might either act as a dominant negative in that it could bind to DNA, preventing WT endogenous Nkx2.5 (and other cardiac Nkx genes) from binding - result is a small or no heart at all. Finally, it could simply require protein-protein interactions for function, but might not act as a dominant negative; in this case, you would see no change in the size of the heart.

Front 387

Sketch a linear heart tube. Where is the future atrium, future ventricle? Where will they be when it loops?

Back 387

The atrium is the caudal portion of the tube with; the ventricle is the rostral portion with future outflow tract on top . Later, the tube will loop, bringing the atria on top of the ventricle.

Front 388

In the mid 1990s, Carmeliet et al generated a null allele of VEGF using tetraploid chimeras with homozygous null ES cells generated by selecting with very high levels of Neomycin. Despite this success, the approach has never caught on (and we did not discuss in class) because conditional knockout approaches soon made it possible to obtain similar experimental results with a different strategy. Propose a Cre mediated strategy for generating conditional VEGF mutant embryos (with VEGF deleted throughout the embryo)

Back 388

Since VEGF is lethal even as a heterozygous embryo, you would need to use an inducible Cre (described in lecture on muscle development). First, generate a floxed allele of VEGF (LoxP sites flanking an essential exon(s)) using standard ES cell targeting. The resulting ES cells are VEGF c/+. Generate chimeras and breed heterozygous mice. Intercross the het mouse with a ubiquitous CreER (UbCreER – not a real name) to generate UbCreER +/-; VEGF c/+. Cross this line to VEGF c/+ mice to generate UbCreER +/-; VEGF c/c embryos (occur at a frequency of 1:8). Then inject pregnant females at an early stage with Tamoxifen (well before vascular formation occurs) to activate Cre expression. This would delete the VEGF alleles, creating a null.

Front 389

You decide to ablate (kill / remove) Pax3+;Pax7+ double positive cells in adult mice (this is distinct from experiments discussed in class where Pax7 and Pax3 were deleted from theses cells – but the cells themselves remained (though no longer expressing Pax3/Pax7). What cell type are Pax3/7 double postivei cells? What do you think would be the immediate consequence on an adult animal, if any? What would be a long-term consequence? How could you experimentally determine this?

Back 389

There would be no immediate consequences. Pax3/7 double positive cells mark a satellite cells that are the stem/progenitor cells for repairing injured muscles. The long-term consequence would be a failure to regenerate damaged muscles. You could test this experimentally by damaging muscle (for example, with cardiotoxin) and then determining if there was muscle regeneration. If you removed all satellite cells, you would expect to see no regeneration.

Front 390

You use chromatin immunoprecipitation to identify myogenin binding regions in developing skeletal muscle. What do you think you will identify?

Back 390

A) You fdentify myogenin bound regions of chromatin. These will include among them enhancers by which myogenin regulates the activation of muscle differentiation genes.

Front 391

You use chromatin immunoprecipitation to identify myogenin binding regions in developing skeletal muscle. How could you test some of these to determine if your approach is working? How could you cherry-pick your list for the best candidates?

Back 391

B) You could look for myogenin-bound regions that are near known muscle differentiation genes. Then clone the DNA into a minimal promoter::reporter and generate transgenic embryos. Does it have expression in forming muscle?

Front 392

A) Propose a strategy for genetically labeling blood cells in zebrafish with a specific marker.

Back 392

A) Generate CymbCre fish and cross to a Bactin::LoP::STOP::LoxP::RFP line. Descendants of cymb+ blood cells will be labeled red.

Front 393

A) Propose a strategy for genetically labeling blood cells in zebrafish with a specific marker. B) If you looked at embryos later on, do you think that the endothelial cells would also be labeled by this strategy?

Back 393

A) Generate CymbCre fish and cross to a Bactin::LoP::STOP::LoxP::RFP line. Descendants of cymb+ blood cells will be labeled red.
B) Endothelial cells will not be labeled red because we discussed data in class where blood cells come from a specific sub-population of endothelial cells at the bottom of the aorta.

Front 394

What do you think happens in the following co-cultures:
wild-type MM + Wnt9B -/- UB

Back 394

MM should survive (since UBs still contain BMP7 and FGF2) but they will not form renal vesicles (no inductive Wnt signal from UB).

Front 395

What do you think happens in the following co-cultures: wild-type MM + Wnt4 -/- UB

Back 395

Should form normal renal vesicles. Wnt4 is only required in MM.

Front 396

What do you think happens in the following co-cultures: Wnt4 -/- MM + wild-type UB

Back 396

Will not form renal vesicles.

Front 397

What do you think should happen if you implanted a bead soaked in GDNF into the rostral intermediate mesoderm of a mouse embryo (where pronephric ducts would normally form) and then cultured it in vivo?

Back 397

It will induce ectopic epithelial buds (ureteric buds) from the Wolffian duct. In theory, it is possible that these could then make ectopic kidneys (if the embryos could be cultured for long enough).

Front 398

Do renal vesicles form dorsal or ventral to the ureteric bud? What structures do renal vesicles become?

Back 398

RV form ventral to the UB. They are epithelial structures that form from aggregates of metanephric mesenchyme. They will become the future nephron and will fuse with the ureteric bud (which becomes the collecting duct).

Front 399

You have just generated a new mutant line of mice that you hope will be a model for understanding kidney development. Mutant embryos die shortly after birth, and when you examine them, they have extremely small kidneys. Describe the strategy you would need to do undertake to determine the molecular cause of this phenotype.

Back 399

The key strategy needs to be to uncover the first time-point at which the kidney (or any organ) starts to show a phenotype (like described for Wnt9b in lecture). The end phenotype above is not terribly informative with regards to the underlying molecular mechanism. Assay mutant embryos starting with the beginning of kidney formation and looking progressively later until a phenotype is apparent. Based on this early phenotype, you can then formulate a hypothesis for the cause of the mutation and then experimentally test the hypothesis

Front 400

Python embryos completely lack the expression of forelimb buds. Why?

Back 400

Pythons have expression of HoxC8 all the way to the anterior boundary of the spinal cord. Forelimbs form at the anterior boundary of HoxC6-B5 in almost all vertebrates. The failure of this boundary creates thoracic vertebrate all the way up to the very first vertebrate (the atlas vertebrate) so there is likely not the molecular boundary necessary to initiate limb bud formation.

Front 401

Limb outgrowth is a complex set of signals between the budding mesoderm and the adjacent ectoderm. A) Which key mesodermal component induces the formation of the apical ectodermal ridge (AER)?

Back 401

FGF10

Front 402

Limb outgrowth is a complex set of signals between the budding mesoderm and the adjacent ectoderm. B) What molecule is sufficient to substitute for the AER in directing limb outgrowth?

Back 402

FGF8 (or FGF4); either is sufficient (although neither is individually necessary)

Front 403

Limb outgrowth is a complex set of signals between the budding mesoderm and the adjacent ectoderm. C) Which molecule(s) are necessary for AER-directed limb outgrowth?

Back 403

FGF4 and FGF8 must both be knocked out.

Front 404

Transgenic mice containing Prx1Cre drive expression of Cre throughout limb mesenchyme, while another line, Msx2Cre, drives expression specifically in the AER. If you were to generate a conditional double knockout of FGF4 and FGF8 in the specifically in the limb mesenchyme, what would be the predicted phenotype? What about in the AER?

Back 404

There would not be a predicted phenotype with PrxCre since FGF4/8 are expressed only in the AER (which is in the overlying ectoderm). Deleting a gene where it is not expressed will not have a phenotype. Msx2Cre embryos will have no hindlimbs and severely malformed forelimbs containing stylopods, small zeugopod elements and some digits (3.30.12 lecture)

Front 405

A) Write the official genotype of a PrxCre FGF4/8 double knockout and a PrxCre FGF4/8 double heterozygous embryo (for the c and wt alleles). Use “c” for the conditional allele (also called the LoxP-flanked allele or floxed allele). B) Diagram what the FGF4 locus would look like in cells in the metanephric mesenchyme and want they would look like in limb bud mesenchyme.

Back 405

A)
-Double knockout: PrxCre+/-; Fgf4c/c; Ffg8c/c
-Double het: PrxCre+/-; Fgf4c/+; Ffg8c/+
B) See Jackies’ discussion section for diagram…but LoxP-flanked region will be recombined (only a single LoxP site remaining) in limb mesenchyme (since as specified in #7 this is where PrxCre is active). There would still be LoxP flanked sites for both genes in the MM.

Front 406

What would happen to an embryo if you specifically knocked out the Shh limb enhancer? How would this embryo differ from a knockout of the Shh gene?

Back 406

The Shh limb expression is completely controlled by the limb enhancer. Mice in which this region is knocked out have severely truncated, malformed limbs with only one digit (a thumb), the exact same mutation as in Shh null embryos (in which the gene itself is knocked out). However, the enhancer knockout mice will only have abnormal limbs. Their neural tubes and all other regions requiring Shh expression will be normal, in contrast to Shh null embryos.

Front 407

A pharmaceutical compound, SU5402, blocks FGF signaling by inhibiting the FGF receptor. What limb phenotype would you predict to occur if you added this compound to a chick embryo shortly before any limb outgrowth was apparent and then incubated it for another 48 hours (easy to do in chick - you just cut a hole in the shell, add your factor, and then close the hole with tape).

Back 407

No limb outgrowth at all. The inhibitor should inhibit the action of FGF10. FGF10 mutants have no limbs. So the prediction would be no limb buds would form at all.

Front 408

If you treated a chick embryo prior to limb outgrowth with a retinoic acid inhibitor, what do you think would happen?

Back 408

It would not form proximal elements. Early, there would be a lack of Meis1 (a retinoic acid-responsive gene that is a marker of the proximal region). Later, the limb element would form the zeugopod and autopod – but no stylopod.

Front 409

The earliest study concerning liver induction was performed by Willer and Rawles (1931), who observed that chick explants with two hears (cardia bifida) often developed two livers, but never developed a second liver in the absence of cardia bifida. A) With the benefit of hindsight, what was the major inductive molecule secreted by the hearts?

Back 409

FGF4

Front 410

The earliest study concerning liver induction was performed by Willer and Rawles (1931), who observed that chick explants with two hears (cardia bifida) often developed two livers, but never developed a second liver in the absence of cardia bifida. B) What embryonic tissue is stimulated by this molecule, and what is the earliest known marker of the liver?

Back 410

FGF4 induces foregut endoderm directly adjacent to the cardiac mesoderm to become hepatic endoderm. The transcription factor, Hhex, is the earliest known marker of the region.

Front 411

In 1968, Nicole Le Dourain showed that the septum transversum mesenchyme can be substituted with a variety of different mesenchymal tissues and still promote migration. A) What tissue was she referring to when she said 'migration'?

Back 411

The liver bud, which delaminates and undergoes an epithelial to mesenchymal transformation into the mesenchyme

Front 412

In 1968, Nicole Le Dourain showed that the septum transversum mesenchyme can be substituted with a variety of different mesenchymal tissues and still promote migration. B) In the context of more modern experiments, what is a possible molecular explanation for her observation?

Back 412

All mesenchyme will be vascularized and thus have this important signal for inducing bud migration.

Front 413

Why are premature babies so prone to lung defects?

Back 413

Because AT2 cells (which secrete surfactants in the alveoli) differentiate between 24-34 weeks in humans. Premature babies are often treated with steroids (to accelerate maturation of AT2 cells) as well as by giving them surfactants.

Front 414

Why is a lungfish considered to have a fin that is somewhat similar to a tetrapod when compared with currently living fish?

Back 414

Lungish have a single bone that connects the fin to the body. This is an ancestral stylopod.

Front 415

What is the name of a cartilage cell? A bone cell?

Back 415

Chondrocyte, Osteoblast

Front 416

Ihh-PTHrP interactions regulate the rate of bone growth. While Ihh has several roles in bone growth, which specific interaction is involved in regulating PTHrP, and why is this considered a negative feedback loop?

Back 416

Ihh is only expressed in hypertrophic chondrocytes that stopped cell proliferation. Ihh secreted from this region is essential for activating and maintaining PTHrP (its not known if this activation is direct or indirect). PTHrP is the most important factor for maintaining chondrocytes in a proliferative state, and it is these proliferating chondrocytes that extend the length of the forming bones. So this is a classic negative feedback loop because hypertrophic cells, through Ihh, maintain a factor (PTHrP) that inhibits their formation by maintaining the cells above them in a proliferative state.

Front 417

What is the major difference in bone phenotypes between Ihh mutants and PTHrP mutants?

Back 417

Ihh mutants will form no real mineralized bone. They will initially form cartilage, but since Ihh induces PTHrP expression, the cartilage element will stop growing. You will get a very small cartilaginous element (the alizarin red present in the Ihh k/o slide is not real mineralized bone).
In contrast, PTHrP mutants lack the critical proliferative agent for proliferating chondrocytes. The cartilage element will be initially formed but won’t proliferate. As a result, the element will prematurely exit cell cycle and you will get much smaller limbs that are prematurely ossified with no growth plates.

Front 418

What is the major difference between Tiktaalik and Eusthenopteron limbs?

Back 418

Tiktaalik is the earliest fossil related to the tetrapod lineage that has autopod appendages (mainly wrist). Eusthenopteron has stylopod and zeugopodal-like bones, but lacks digit-like appendages.

Front 419

You decide to fate map developing digits after the initial appearance of the digit rays but before any detectable phalanges have appeared. You label proximal cells in limb buds directly with di-I (a red dye that binds to membranes that is often used for fate mapping). Using di-O (a related dye that is green), you also label distal cells in limb buds. You then disassociate the limb buds and culture them. What do you see?

Back 419

Red cells will clump together with other red cells while green cells will do the same? Each of these clusters will then form chondrocytes, suggesting that progenitors for different skeletal elements in the limb have differential adhesion properties

Front 420

Several short-limbed dogs, including Dachshunds and Bassett Hounds, have an extra copy of Fgf4 aberrantly expressed in their limbs. Why do you think this makes their limbs shorter?

Back 420

The activation of FGF receptor 3 (FGFr3) inhibits chondrocyte proliferation. In many types of achondroplastic dwarfism, the limbs are shorter because of activating mutations in FGFr3, which cause excessive inhibition of chondrocytes, resulting in shorter limbs. It seems likely that the extra copy of FGF4 is similarly overactivating FGFr3.

Front 421

Dor et al used a pulse-chase approach to label beta cells in adult mice. Describe their experimental approach and conclusions. What would have been the result if a non-beta cell progenitor gave rise to other beta cells?

Back 421

Dor et al crossed a mouse line expressing a beta-cell specific, tamoxifen inducible Cre with a Cre reporter line (irreversibly expresses Alkaline phosphatase upon Cre-mediated activation). Without tamoxifen, the CreER protein is sequestered in the cytoplasm and is therefore unable to activate the reporter gene (which requires nuclear localization of Cre). They treated adult mice with an intermediate dose of tamoxifen that allowed for activation of the Cre reporter in ~25% of beta cells when assayed a few days post-induction. They saw no difference in the percent of labeled cells – even a year later. B/c the half-life of beta cells is only ~3 months, and because there is a tremendous increase in the total number of beta cells over this time, they conclude that beta cells are giving rise to new beta cells. If, instead, they had seen a progressive reduction in the percentage of labeled beta cells, it would have suggested that a non-beta cell progenitor population was present.

Front 422

Zhou et al 2008 described an experimental approach for generating beta cells, showing that forcibly expressing three transcription factors in exocrine cells transformed them from exocrine cells to endocrine fates (including beta cells). The data we showed in class stopped short of proving that the ‘new’ beta cells were derived from non-beta cells. Describe an experiment and result that would directly demonstrate that these new cells were descended from amacrine cells?

Back 422

You would need a tamoxifen inducible Cre line that is only active in the exocrine cells and a Cre reporter. If you injected with tamoxifen at the same time when you infected the mice with the transcription factor cocktail, cells that expressed endocrine markers (such as insulin) and GFP+ (indicating viral infection) should also express b-galactosidase (assuming RosaLacZ is the Cre reporter).

Front 423

What is the key transcription factor that separates exocrine and endocrine pancreatic lineages?

Back 423

Neurogenin 3 (Ngn3) is required for the formation of endocrine lineages.

Front 424

After specification, what is needed for limb outgrowth?

Back 424

The apical ectodermal ridge.

Front 425

What is the stylopod?

Back 425

The proximal arm bone

Front 426

What is the zeugpod?

Back 426

the distal arm bone

Front 427

What is the autopod?

Back 427

The digits (fingers)

Front 428

What is the dorsal part of the autopod?

Back 428

knuckles

Front 429

The ventral part of the autopod:

Back 429

palms

Front 430

The anterior part of the autopod:

Back 430

thumb

Front 431

The posterior part of the autopod:

Back 431

pinkie

Front 432

What causes initial outgrowth of the limb?

Back 432

Accumulation of somitic mesoderm (making a limb muscle) and lateral plate mesoderm (making other limb mesodermal components like skeleton).

Front 433

When do human limb buds form?

Back 433

Like wk 4/5.

Front 434

What are thalidomide babies?

Back 434

Kids with phocomelia (flipper like arms) because their moms took thalidomide for morning sickness.

Front 435

What is thalidomide?

Back 435

It's a teratogen that was given for morning sickness in the 50s. Somehow it affects growth of the forelimbs in humans.

Front 436

What is the mechanism of thalidomide?

Back 436

Not clear, but probably inhibits growth of blood vessels (angiogenesis) in the developing limbs.

Front 437

What didn't they know thalidomide was a teratogen?

Back 437

It's not a teratogen in mice.

Front 438

Why doesn't a python have limbs?

Back 438

Pythons are missing the Hox C6/8 boundary that correlates to the forelimb.

Front 439

What do Hox codes do?

Back 439

They're like postal codes. The boundaries of Hox expression correlate to the changes in identity of the somites and spinal cord.

Front 440

Do pythons my limb buds at all?

Back 440

They have hindlimb buds, but not forelimb buds. The hindlimb buds don't elongate.

Front 441

In a limb bud, how do you know to get a wing or a leg?

Back 441

Tbx 5 trxn factor makes a forelimb and Tbx4 makes a hindlimb.

Front 442

What happens to Tbx5 mutant mice?

Back 442

They lose their forelimbs, but not their hindlimbs.

Front 443

What do Tbx5/4 mutations do to humans?

Back 443

Tbx5 leads to upper extremity defects. Tbx4 leads to small patella syndrome and leg defects

Front 444

Although necessary, Tbx4 and Tbx5 in mice are not sufficient...

Back 444

to direct forelimb and hindlimb identity.

Front 445

What positions limb outgrowth?

Back 445

Fgf10

Front 446

Where does Fgf10 become isolated?

Back 446

Fgf10 becomes restricted in the lateral plate mesoderm that will make a limb.

Front 447

How can you make an animal with a bunch of extra limbs?

Back 447

Soak some beads in Fgf10 and shoot it in an embryo. They'll get ectopic limbs.

Front 448

What happens to Fgf10 mutant mice?

Back 448

They completel lack limbs. Fgf10 is necessary for limb development. The babies slither like snakes and then the mom eats them.

Front 449

What's the pathway involving Tbx5/4 and Fgf10?

Back 449

Wnt2b/8c --> Tbx5/4 --> Fgf10

Front 450

What factor do you need for limb outgrowth?

Back 450

Wnt2b

Front 451

Fgf10 induces expression of...

Back 451

Fgf8

Front 452

What is the pathway involving Fgf10 and Fgf8?

Back 452

Fgf10 in mesoderm --> Wnt3a in AER --> Fgf8 --> Fgf10 maintained

Front 453

Where is Fgf8 expressed?

Back 453

In the AER.

Front 454

What is tetra-amelia in humans and what is it caused by?

Back 454

You don't have any limbs and it's caused by a mutation of Wnt3.

Front 455

What happens if you get rid of the AER at different points in development?

Back 455

The limb stops at more and more distal points the later you get rid of it.

Front 456

What's the AER?

Back 456

Apical Ectodermal Ridge.

Front 457

What does the AER do?

Back 457

It mediates limb bud extension.

Front 458

How could thalidomide affect the AER?

Back 458

It could reduce angiogenesis in the AER.

Front 459

What happens in an Fgf8 KO?

Back 459

There's no phenotype. There are actually several Fgfs in the AER that are redundant with each other.

Front 460

What's in charge of the proximal portion of the limb?

Back 460

Retinoic acid (RA)

Front 461

What's in charge of the distal portion of the limb bud?

Back 461

Fgfs in the AER.

Front 462

Name two factors that go up as Fgfs go up:

Back 462

Hox11 and Hoxa13

Front 463

Name a factor that goes down as Fgfs go up:

Back 463

Meis1

Front 464

How do you make a recombinant limb?

Back 464

You remove the mesodermal cells from the ectodermal jacket and repack with disassociated mesodermal cells. You end up with semi-normal limb bud with the right expression of PD markers.

Front 465

Where is Meis1 expressed?

Back 465

Proximally.

Front 466

Where is Hoxa11 expressed?

Back 466

Midway in the limb bud.

Front 467

Where is Hoxa13 expressed?

Back 467

Distally in the limb bud.

Front 468

Where is Shh expressed in the limb bud?

Back 468

In the ZPA at the bottom.

Front 469

What is the ZPA?

Back 469

The zone of polarizing activity.

Front 470

What does Shh define in the limb bud?

Back 470

The posterior portion of the limb bud.

Front 471

What happens to Shh mutants' limbs?

Back 471

They only make a thumb.

Front 472

How can you get an autopod mirror image duplication?

Back 472

You shoot a Shh bead into the anterior portion of the limb bud.

Front 473

What does mirror image autopod duplication tell you about the ZPA?

Back 473

Shh is sufficient for ZPA activity.

Front 474

What controls Shh in the ZPA?

Back 474

A cis-regulatory domain 850kb away from the Shh gene.

Front 475

What is the Shh-Fgf signaling loop?

Back 475

Shh --> gremline --> Fgfs --> Shh

Front 476

What do low Fgf and high Fgf do to the Shh signaling loop?

Back 476

Low Fgf maintain Shh and leave Gremlin alone. High doses accumulate and repress Gremlin.

Front 477

What happens when Gremlin is repressed?

Back 477

The AER breaks down.

Front 478

What is the predicted Shh-dependent enhancer of Gremlin?

Back 478

It's a Gli-bound site way downstream of Gremlin that might be how Shh activates Gremlin.

Front 479

How does the Gli-binding region affect Gremlin?

Back 479

Binding to the Gli-binding region drives gli-dependent limb extension.

Front 480

What happens after limb bud patterning?

Back 480

Condensation of mesenchyme into cartilage elements.

Front 481

What's the order of digit condensation?

Back 481

4-2-5-3-1

Front 482

What is digit condensation?

Back 482

The formation of cartilage masses (rays) that will subsequently develop the hand.

Front 483

What is the foil experiment?

Back 483

You take a piece of foil and put it in the vascular region above the ray, you'll see the foil incorporated in different places in the digit. If you add it early, it will be more proximal. If you add the foil in the avascular region, it only ends up being distal. You can do the same experiment by injecting the retrovirus with beta gal.

Front 484

What's the current model for phalange formation?

Back 484

The AER which is part of the avascular mesenchyme gives cells to the vascular noncondensed mesenchyme which grows into a digit.

Front 485

What are chondrocytes?

Back 485

Cartilage cells.

Front 486

How does endocondral bone form?

Back 486

Mesenchymal cells condense and then form chondrocytes. The chondrocytes at the center stop proliferating and become hypertrophic. Perichondrial cells on either side of the hypertrophic cells become osteoblasts forming the bone collar.

Front 487

What are hypertrophic chondrocytes?

Back 487

Cartilage cells that are big and stop proliferating.

Front 488

What are osteoblasts?

Back 488

Developing bone cells.

Front 489

What's the progenitor of osteoblasts?

Back 489

perichondrocytes.

Front 490

Which trxn factor is the master regulator for cartilage?

Back 490

Sox9.

Front 491

What happens if you only have one copy of Sox9?

Back 491

You have campomelic dysplasia (bowing and bending of long bones).

Front 492

What happens to your digits if you're without Sox9?

Back 492

You don't have digits. You don't show any cartilage.

Front 493

What's the second step of bone formation?

Back 493

Hypertrophic chondrocytes direct the formation of a mineralized matrix and attract blood vessels. They also undergo apoptosis. Then the osteoblasts will invade along the blood vessels and eventually become bone. During later bone growth, cells continue to proliferate between the two regions.

Front 494

What is the growth plate?

Back 494

The proliferating region of the growing bone.

Front 495

What does Ihh do?

Back 495

It's in charge of the development of long bones.

Front 496

What's wrong with an Ihh KO?

Back 496

1. Massive decrease in the proliferation of chondrocytes.
2. Increased fraction of chondrocytes that are post-mitotic (no longer proliferating). This is because Ihh mutants no longer synthesize PTHrP.

Front 497

What is the endocondral ossification pathway?

Back 497

PTHrP is secreted from the top perichondrial cells and chondrocytes at the ends of the long bones. PTHrP keeps chondrocytes proliferating (inhibits Ihh). When it gets far enough away, Ihh makes the chondrocytes differentiate and stimulates the production of PTHrP at the ends of the bones.

Front 498

What does PTHrP do?

Back 498

It encourages chondrocyte proliferation and inhibits Ihh.

Front 499

What does Ihh do?

Back 499

It encourages chrondrocyte differentiation and promotes PTHrP in the perichondrial cells.

Front 500

What happens to PTHrP mutants?

Back 500

It causes dwarfism and premature ossification.

Front 501

What does the Ihh-PTHrP feedback loop do?

Back 501

It regulates the timing of hypertrophy.

Front 502

How are the bones vascularized?

Back 502

VegF and Matrix Metalloproteinases secreted by hypertrophic chondrocytes are responsible for vascularization of bone.

Front 503

How is bone homeostasis maintained?

Back 503

It's by the activity of the osteoblasts and the osteoclasts (destroy bone).

Front 504

What are osteoclasts and where do they come from?

Back 504

Osteoclasts are bone destroyers and they come from the blood cell lineage.

Front 505

How do Fgfs relate to bone?

Back 505

They also regulate chondrocyte proliferation. There are like 22 of them.

Front 506

How are Fgfs related to dwarfism?

Back 506

Activating Fgf signaling prematurely causes chondrocytes to differentiate prematurely. The mutations cause achondroplasia which is the most common form of dwarfism. It's dominant.

Front 507

Why don't humans have webbing?

Back 507

The webbing cells undergo apoptosis.

Front 508

How do BMPs affect webbing?

Back 508

BMPs lead to apoptosis of the interwebbing cells.

Front 509

How can you make your own webbed creature?

Back 509

You shoot it with Gremlin-soaked beads which inhibit BMP and then the interwebbed cells don't undergo apoptosis.

Front 510

What happens to BMP mutants?

Back 510

They have webbed feet.

Front 511

How are bat and mice limbs different?

Back 511

Bat embryos still have webbing.

Front 512

What gene is upregulated in bats?

Back 512

Prx expression is upregulated in the cartilage and distal limb elements compared to mice.

Front 513

How did they make a bat mouse and what was the result?

Back 513

They gave the mouse a bat Prx enhancer and it gave the mouse longer limbs. Prx KO's had bendy small limbs.

Front 514

Why do horses have a penis-looking foot?

Back 514

Horses progressively lost digits.

Front 515

What is the jerboa?

Back 515

It's a future model system because it appears to be losing digits.

Front 516

What's so cool about lungfish?

Back 516

They have articulating bones in their fins and lungs and they seem like a limb progenitor. They have a single, stylopod-like connection to the body.

Front 517

What is acanthostega?

Back 517

An early tetrapod.

Front 518

Why is eusthenopteron important?

Back 518

He seemed to have a stylopod and zugopod.

Front 519

Why is tiktaalik important?

Back 519

It's a fossil intermediate containing digit-like elements (autopod).

Front 520

What happens if you overexpress late-phase HoxD13 in zebrafish limbs?

Back 520

They have more of a limb.

Front 521

3 structures formed from endoderm development?

Back 521

Liver, lungs and pancreas.

Front 522

What does the endoderm specifically make?

Back 522

The foregut, midgut, and hindgut.

Front 523

What specifies endoderm?

Back 523

Nodal. It turns on downstream differentiation markers that segregate endoderm from mesoderm.

Front 524

How do you make the foregut and hindgut?

Back 524

The tube folds to make the foregut and then folds again to make the hindgut.

Front 525

What closes up in the gut tube?

Back 525

The midgut and the hindgut.

Front 526

How does the liver form?

Back 526

It gets signals off of the heart. That's why with cardio bifida you get two livers. It comes from the foregut.

Front 527

How do you know that the liver gets signals from the heart?

Back 527

If you stick cardiac mesoderm with the foregut, you get a proliferation of cells. Foregut alone and nothing happens.

Front 528

What's the signal the heart secretes to the foregut?

Back 528

Fgf4.

Front 529

What does Hhex do?

Back 529

It's essential for budding of the liver.

Front 530

What happens to embryos het for Hhex?

Back 530

They're fine.

Front 531

What promotes liver bud outgrowth?

Back 531

Endothelial cells even without circulating blood. You can put endothelial cells in situ.

Front 532

What is the pancreas composed of?

Back 532

alpha, beta, sigma, and PP cells. It's composed of acinar cells, ductal epithelium and endocrine cells.

Front 533

What do acinar cells do?

Back 533

They produce digestive enzymes.

Front 534

How is the pancreas specified?

Back 534

It comes from the foregut right by the hepatic endoderm. The dorsal and lateral pancreas come together. The dorsal pancreatic endoderm is by the notochord and suppresses Shh.

Front 535

What does Pdx1 do?

Back 535

It marks the dorsal and ventral pancreatic buds.

Front 536

What happens if you're a Pdx mutant?

Back 536

You fail to differentiate your pancreas.

Front 537

What induces pancreatic differentiation?

Back 537

Blood vessels.

Front 538

What's the marker for pancreatic beta cells?

Back 538

Insulin.

Front 539

What induces insulin expression?

Back 539

Endothelium.

Front 540

What's needed for the formation of the pancreas?

Back 540

Endothelium.

Front 541

What does Ngn3 do?

Back 541

It makes endocrine cells.

Front 542

What happens to Ngn3 mutants?

Back 542

They fail to make endocrine cells although they make exocrine fine. You don't end up getting insulin and glucagon.

Front 543

What does Nkx2.2 do?

Back 543

It's critical for generating beta cells.

Front 544

Nkx2.2 mutants...

Back 544

lose expression of alpha and beta cells and have lots of ghrelin. Nkx2.2 is a trxn activator and repressor.

Front 545

What is ghrelin?

Back 545

They signal that tells you you're hungry.

Front 546

Is it better to have repressive or activator Nkx2.2?

Back 546

The repressor forms rescues production of essentially all alpha cells and some beta cells.

Front 547

How do you find out what makes beta cells in adults?

Back 547

You do a pulse-chase strategy with an insulin promoter CreER and Z/AP LacZ thing to detect stem cells. Use beta cell specific inducible Cre and cross with a Cre reporter (LacZ). You see it in about 30% of all beta cells.

Front 548

What were the results of the stem cell population for beta cells experiment?

Back 548

Beta cells are not derived from progenitor/stem cell populations - at least not in healthy mice. Beta cells probably come from beta cells. They stuck beta cells on a plate for a long time and there was still staining so beta was coming from beta.

Front 549

What happens if you injure the beta cells? Where do the new beta cells come from?

Back 549

Beta cells can come from endogenous progenitors if injured. In injury Ngn3 starts up again and makes beta cells. We don't know where Ngn3 comes from.

Front 550

Steps to make beta cells from ES cells:

Back 550

1. Convert ES cells to endoderm.
2. Make endoderm into posterior foregut.
3. Cells express Insulin, Glucagon, somatostatin.

Front 551

How can you tell if beta cells from ES cells are functional?

Back 551

Yeah, they stick them in a mouse penis and they kill the pancreas with STZ. And they produce insulin from the human ones. Take those out and the mice are ruined.

Front 552

Can you make endocrine from exocrine?

Back 552

Yes if you have access to Ngn3, Pdx1, and Mafa (M3).

Front 553

What does VegF do with vascular stuff?

Back 553

It's essential for vascular patterning.

Front 554

What's the VegF receptor?

Back 554

Flk1

Front 555

Flk1 mutants...

Back 555

have no blood vessels. Hets are fine though.

Front 556

Angiogenesis:

Back 556

The growth of blood vessels from pre-existing vascular tubes.

Front 557

Vasculogenesis:

Back 557

Initial formation of blood vessels which come from a single cells.

Front 558

Macular degeneration:

Back 558

caused by abnormal blood vessels that grow under the retina.

Front 559

What does vascular tube formation need?

Back 559

It needs endoderm during vasculogenesis.

Front 560

How do you make the BBB?

Back 560

You need Wnts

Front 561

What happens if you're a double KO for Wnt7a/7b?

Back 561

You have no BBB. You get blood pooling in your prefrontal cortex and spine.

Front 562

What does Wnt7a/7b do?

Back 562

It controls expression of BBB-specific markers.

Front 563

What's a marker of BBB?

Back 563

Glut1

Front 564

You blood populations comes from...

Back 564

endothelial cells.

Front 565

What is the most primitive bilaterally symmetric animal?

Back 565

Planaria.

Front 566

What's so special about planaria?

Back 566

They're really good at regenerating.

Front 567

How are planaria able to regenerate?

Back 567

They have a population of cells called neoblasts that are involved in regeneration.

Front 568

What family are planaria a part of?

Back 568

Platyhelminthes.

Front 569

What are 3 general parts of a planarian?

Back 569

1. Eyes with photoreceptors
2. Branched gut system
3. Pharynx on the ventral side where they eat and poop through the same tube. Ew.

Front 570

What happens if you cut a planarian in half?

Back 570

The top part will make a posterior blastema and the bottom part will make an anterior blastema and you will generate two planarians.

Front 571

blastema:

Back 571

regeneration bud.

Front 572

The anterior portion of the planarian is...

Back 572

the head region.

Front 573

The posterior portion of the planarian is...

Back 573

the bottom end region.

Front 574

The only proliferating cells in planarians are...

Back 574

neoblasts.

Front 575

How do you experimentally show that neoblasts give rise to regenerating parts?

Back 575

You label neoblasts with BrDU before you amputate them. The blastema contains many BrDU labeled cells.

Front 576

What's wrong with irradiated planarians?

Back 576

They can't regenerate.

Front 577

What happens if you inject purified neoblasts into an irradiated planarian?

Back 577

You can restore its ability to regenerate.

Front 578

What three factors does a planarian lose when it's irradiated?

Back 578

Neoblasts, smedwi-1, smedwi-2.

Front 579

Which smedwi seems to be the most important?

Back 579

smedwi-2 seems to be more important for regeneration.

Front 580

What does smedwi-2 do?

Back 580

It's essential for regeneration and homeostasis.

Front 581

What happens if you use smedwi-2 RNAi in planarians?

Back 581

They fail to form blastemas - the phenotype is similar to if the planarians were irradiated.

Front 582

What does a radiated planarian head look like?

Back 582

It gets all shrunken in; it's kinda gross.

Front 583

What does a smedwi-2 RNAi planarian head look like?

Back 583

It looks similar to the irradiated planarian head. It head starts to get shrunken in which is creepy.

Front 584

What does smedwi-2 RNAi do to the neoblasts?

Back 584

The neoblasts can't function normally. You can see in the experiment that with smedwi-2 RNAi, there is no tissue surrounding the neoblasts.

Front 585

What happens if you put a wild type planarian chunk into an irradiated planarian?

Back 585

You can save the whole planarian and they look all happy.

Front 586

What happens if you put an irradiated chunk into an irradiated planarian?

Back 586

It just dies and degrades after around 29 days.

Front 587

What's another name for smedwi-2?

Back 587

Smed-piwi. Thanks for making me remember yet another name.

Front 588

You irradiate a planarian and then you take a wild type chunk and stain for smed-piwi. What happens?

Back 588

You see the cells from the chunk repopulate the lethally irradiated cells of the host. It makes the planarian look like a murderous demon, but it saves the planarian.

Front 589

If you're really mean, you irradiated the planarian and then decapitate it. Then you attach the irradiated headless thing to a section of wild type and stain for smed-piwi. What do you see?

Back 589

You see the smed-piwi begin to migrate up toward the decapitated portion. The smed-piwi doesn't move up if you haven't cut off the head. This is an example of directional migration.

Front 590

How do you experiment with directional migration in planarians?

Back 590

Irradiate a planarian and cut off its head. Now attach the irradiated section to a wild-type section with a smed-piwi marker. You'll see the cells migrating up to make a new head.

Front 591

What kind of cells do you see when regenerating a decapitated planarian?

Back 591

agat-1

Front 592

What's the pathway for cell differentiation in planarians?

Back 592

neoblasts --> prog-1 --> agat-1

Front 593

What are the stem cells of the planarian?

Back 593

Neoblasts

Front 594

How do you make the Janus phenotype in planarians?

Back 594

You have to knock down beta catenin in the planarian and you'll end up getting a head at the place you chopped off.

Front 595

What does beta catenin do in the planarian?

Back 595

It is required for blastema AP polarity.

Front 596

How do you get rid of beta catenin in the planarian?

Back 596

RNAi.

Front 597

How does beta catenin relate to the axial levels of the planarian?

Back 597

If you deplete beta-catenin with RNAi, you can cut a multiple different places and you'll still get a head that forms.

Front 598

What's the six headed monster?

Back 598

It's this planarian, that mad scientists depleted beta-catenin in using RNAi. Then they chopped all over the planarian and got a new head at ever cut.

Front 599

What happens if you deplete beta-catenin and then chop off just the most anterior portion of the planarian?

Back 599

They still regenerate heads right by their heads.

Front 600

Based on RNAi studies, how is beta-catenin functioning in the planarian?

Back 600

It probably doesn't simply inhibit anterior formation. It probably promotes posterior fates instead on a gradient level.

Front 601

What's the insitu Wnt signaling for planarian?

Back 601

Beta-catenin is involved in Wnt signaling so if you stain for 6 different Wnts, you'll see them in different places throughout the planarian. Now you cut the planarian in three parts (head, trunk, tail) and stain for the Wnts again. You will see the specific Wnts in the same place in each of the sections.

Front 602

What's another way you mess with beta-catenin in the planarian?

Back 602

You could inhibit beta-catenin half way in the planarian and look at the fate of the planarian.

Front 603

What is a planarian stem cell marker?

Back 603

Smedwi-2 (aka Smed-piwi)

Front 604

How do amputated planarian pieces deterime AP identity?

Back 604

Through beta-catenin dependent mechanisms.

Front 605

How do you perform RNAi on a planarian?

Back 605

You take homogenized liver and infect it with e coli which has a plasmid that makes dsRNA. The planarian eats the liver and dsRNA which will break down the specific mRNA you want.

Front 606

Why can't you use RNAi on humans through feeding them dsRNA?

Back 606

Humans see dsRNA as a virus and will attack the dsRNA.

Front 607

Urodeles can regenerate...

Back 607

limbs and hearts.

Front 608

Zebrafish can regenerate...

Back 608

hearts.

Front 609

How do you prove experimentally that zebrafish can regenerate their hearts?

Back 609

Cut part of their heart off and do insitu hybridization for cardiac myosin heave chain and a dye indicating fibrin. It'll grow back.

Front 610

How can you see if cardiomyocytes come from cardiomyocytes?

Back 610

Use a beta-actin2:RSG zebrafish with CRE ER. Under normal conditions, the beta actin will glow red. Then after you injure the heart, when you add tamoxifen, the CRE will induce the gene to make GFP. The new cardiomyocytes glow green so they come from cardiomyocytes.

Front 611

What happens to mature cardiomyocytes after injury?

Back 611

They dedifferentiate into early embryonic heart cells again and express embryonic cardiac differentiation markers like Nkx 2.5, tbx5.

Front 612

What three factors are found in the zebrafish regenerating heart?

Back 612

Mef2 and Myosin Heavy Chain (MHC) are in the cardiomyocytes and DAPI is in the nuclei.

Front 613

2 factors in the regenerating cardiomyocytes:

Back 613

Mef2 and MHC

Front 614

Factor in the regerating nuclei of the zebrafish heart:

Back 614

DAPI

Front 615

What factor shows activation during zebrafish heart injury?

Back 615

GATA4. That's what is labeled with GFP.

Front 616

During zebrafish cardiac injury, what factor do you see in the injury site?

Back 616

GATA-4

Front 617

How can you determine the contribution of GATA4 descendants in the heart?

Back 617

Use GATA4 CRE ER. When you injure the zebrafish heart, the GFP-expressing cells migrate down to the injury site and de-differentiate.

Front 618

How much does the human heart increase from birth to adulthood?

Back 618

30-50 fold

Front 619

How are most human heart cells reacting as the heart grows?

Back 619

Via hypertrophy instead of cell division.

Front 620

How do you test the origin of heart cells in humans?

Back 620

Between WWII and 1963, there was a lot of C14 in the air. C14 was incorporated into dividing cells and can be used to date the age of cells. You find that people born before WWII have higher C14 levels in their cardiomyocytes compared to when they were born. Similarly, people born after WWII have lower levels than they were born with. This tells you that cardiomyocytes do proliferate, but slowly.

Front 621

Turnover rate of cardiomyocytes at age 20:

Back 621

1%

Front 622

Turnover rate of cardiomyocytes at age 75:

Back 622

0.4%

Front 623

What's the difference between a proximal amputation and a distal amputation on a salamander?

Back 623

The proximal amputation goes more slowly, but you ultimately end up with the same regenerated limb.

Front 624

In urodeles, the blastema has a cap made of...

Back 624

apical epidermal cells.

Front 625

What are the 5 stages of urodele limb regeneration?

Back 625

1. Amputation
2. Healing
3. Dedifferentiation
4. Cone stage
5. Palette stage
6. Notch stage
7. Digit stage

Front 626

What happens in the dedifferentiation stage of regeneration?

Back 626

Proliferation of blastema cells and the presence of the apical epidermal cap.

Front 627

What happens in the palette stage of regeneration?

Back 627

The dedifferentiated cells redifferentiate.

Front 628

What are the 3 assays you can do to look at PD identity of blastemal cells?

Back 628

1. Confrontation assay
2. Grafted blastema assay
3. Level of Blastema assay

Front 629

What's the confrontation assay?

Back 629

If you stick a proximal blastema by a distal blastema, the proximal blastema will engulf the distal blastema.

Front 630

What's the grafted blastema assay?

Back 630

If you cut off a salamander arm, it will make a blastema. Now cut off that blastema and stick it somewhere on the arm stub. The more proximal you put it, the longer the two arms will be. The more distal the two blastemas and you'll just have autopods.

Front 631

What is the level of the blastema assay?

Back 631

The tissue of the limb stump will regenerate to the level of the blastema. If you switch the blastemas of a proximal and a distal amputation, you'll get one really short and one really long limb. This is because the proximal blastema gets attached to the proximal and becomes super proximal. The other limb is fine.

Front 632

Why would a proximal blastema attached to a proximal stump become super proximal?

Back 632

Structures from the shoulder to the wrist arise by growth from the proximal partner. The blastema itself can't proximalize.

Front 633

How can you make a 3-armed salamander monster?

Back 633

You sew up its arm into its stomach so it looks like it's doing "I'm a little teapot." Then you cut the teapot arm in half and it grows 2 arms, one out of its stomach!

Front 634

Why does making an "I'm a little teapot" salamander monster work?

Back 634

Distal regeneration occurs even when the cut surface is proximal facing. For some reason...

Front 635

What does retinoic acid do to a stub?

Back 635

It reprograms the blastema. In the presence of retinoic acid, the salamander forms a completely new limb because retinoic acid is a proximal signal.

Front 636

What happens if you treat a wrist blastema with retinoic acid?

Back 636

It makes a completely new limb. So you get an super long limb.

Front 637

What happens if progenitors for regeneration are lineage-restricted?

Back 637

You find out that there are no stem cells. Skeletal cells make more skeletal cells.

Front 638

What can the blastema regenerate?

Back 638

It has a "memory" of where the amputation occurred. It can regenerate all tissues distal, but not proximal from the point of amputation.

Front 639

What do you see from the regenerated limbs of axolotls?

Back 639

They appear to be derived from multiple progenitor cells that represent major tissue lineages rather than a single pluripotent cell, ie there are no stem cells.

Front 640

What factor resets the memory of a blastema?

Back 640

Retinoic acid.

Front 641

What are the two main stages of sex determination?

Back 641

1. Germ cell development
2. Sex determination (dose compensation)

Front 642

Early in development, how do germ cells differentiate themselves form somatic cells (2 ways)?

Back 642

1. They're sequestered very early in development.
2. They are in some aspects immortal.

Front 643

Where are germ cells specified?

Back 643

In the periphery of embryos.

Front 644

How do germ cells get to the gonads?

Back 644

They have to migrate.

Front 645

3 stages of germ cell development:

Back 645

1. Specification
2. Migration
3. Differentiation

Front 646

What are P-granules?

Back 646

Germ line-specific cells from the earliest stages in c elegans that act as instructors of germ line development.

Front 647

What are the 2 roles of p-granules?

Back 647

1. Instructors of germ line development
2. bind RNA and promote germ-cell specific trxn.

Front 648

What is Pie-1?

Back 648

A trxn factor that prevents the phosphorylation of RNA pol II and blocks trxn in germ cells during development.

Front 649

What's wrong with Pie-1 mutants?

Back 649

They have embryonic genes in the P-cell.

Front 650

What happens if you put Pie-1 in somatic cells?

Back 650

It causes repression of somatic transcripts.

Front 651

Where are primordial germ cells induced?

Back 651

In the posterior region of the embryo immediately next to the extraembryonic ectoderm.

Front 652

How many primordial germ cells are made from induction?

Back 652

20

Front 653

When does specification of the PGC occur?

Back 653

E 7.5

Front 654

What are PGCs?

Back 654

primordial germ cells

Front 655

What does Blimp1 do?

Back 655

It's the earliest marker of PGCs and is a trxnal repressor.

Front 656

When does migration of PGCs occur?

Back 656

E 9.5

Front 657

When does reprogramming of PGCs occur?

Back 657

E 11.5

Front 658

What day does mammalian sex differentiation occur?

Back 658

E 12.5

Front 659

What happens to Blimp1 mutants? 2 things.

Back 659

Their PGCs fail to migrate. You can stain the PGCs with alkaline phosphatase and see they don't migrate. They also fail to repress somatic cell markers (Hox genes).

Front 660

How do you perform single cell cDNA analysis of PGC cells?

Back 660

Isolate the RNA, transcribe to cDNA with RT, assay by PCR and run on a gel to look for specific genes, probably via Western blotting.

Front 661

What are pole cells?

Back 661

Drosophila germ cell progenitors. They start at the tip and migrate to the midgut where they attach and align with the gonadal mesoderm.

Front 662

What are the 4 steps of pole cell migration?

Back 662

1. Attachment to endoderm and migration through the midgut
2. Attachment to mesoderm
3. Alignment with gonadal mesoderm
4. Two streams of migrating cells.

Front 663

Which direction do the drosophila germs cells migrate in the midgut?

Back 663

Dorsally before splitting into two streams.

Front 664

What are Wunen genes?

Back 664

Wunen and Wunen-2 genes are redundant lipid phosphate phosphatases that act as chemorepellants expressed in the ventral midgut.

Front 665

What do lipid phosphate phosphatases do?

Back 665

They remove extracellular phospholipid substrates.

Front 666

What do Wunen phosphatases do?

Back 666

They guide PGCs by repelling them.

Front 667

Why do PGCs only migrate through the endoderm dorsally?

Back 667

They are repelled by ventral Wunen.

Front 668

Why do PGCs split into two streams?

Back 668

They are repelled by additional Wunen expressed in the CNS.

Front 669

Wunen is necessary for...

Back 669

proper lateral migration.

Front 670

What happens if you knock out wunen but not wunen-2?

Back 670

Nothing because the phosphatases are redundant for each other.

Front 671

What happens to Wunen; Wunen-2 double mutants?

Back 671

Their PGCs are unable to migrate laterally when they are in the middle of the embryo. They start going every which way.

Front 672

How can you visualize PGC migration?

Back 672

You can use a PGC-specific antibody.

Front 673

How can you rescue a Wunen;wunen-2 double mutant?

Back 673

CNS-specific expression in embryos can partially rescue gonad expression.

Front 674

How do mice PGCs migrate?

Back 674

They move into the endoderm (hindgut) and then migrate into the body wall.

Front 675

What happens with mice PGCs at E 9?

Back 675

The cells are motile, but do not leave the hindgut epithelium.

Front 676

What are the 3 locations that mice PGCs migrate through?

Back 676

hindgut --> body wall --> genital ridges

Front 677

What are the two forms of X-inactivation in mammals?

Back 677

1. During early cleavage stages, the paternal X-chromosome is inactivated in all cells. It stays inactivated in the extraembryonic tissue but is reactivated in the embryo proper.
2. In the embryo, then one of the two X-chromosomes is randomly inactivated in each cell.

Front 678

How are tortoishell cats the way they are?

Back 678

During X-chromosome inactivation and as the embryo grows, one of the two x-chromosomes will randomly inactivate in each cell. For that reason, some cells will have a black fate and some will have an orange fate.

Front 679

What is x-chromosome inactivation in mice?

Back 679

The mouse has Xist RNA which is a 17kb non-coding RNA produced only from the inactive X (Xi). It blankets the entire chromosome and is thought to initiate chromosome-wide silencing.

Front 680

What is Xist RNA?

Back 680

It's a 17kb non-coding RNA which is produced only by the inactive X.

Front 681

Where is Xist RNA seen?

Back 681

In mice and only in females. You have to have more than 1 X chromosome.

Front 682

How is Xist trxn regulated?

Back 682

Xist binds to a specific region on the X-chromosome (the Xic). Xist is regulated by binding to an antisense transcript on the same transcript called Tsix. Tsix is required for an active X.

Front 683

Which chromosome produces Xist RNA?

Back 683

Only the inactive X (Xi). It negatively regulates itself.

Front 684

How does an active X chromosome keep from getting shut off by Xist?

Back 684

The active chromosome produces Tsix which creates dsRNA with Xist and then gets degraded.

Front 685

What exactly does Xist do to the inactive chromosome?

Back 685

It stabilizes and recruits repressive histone markers.

Front 686

Where are germ cells usually specified?

Back 686

In the periphery of the embryos (pole cells, P-granules)

Front 687

How do germ cells differ from somatic cells during development?

Back 687

They keep a low profile and transcriptionally repress somatic cell fates.

Front 688

How are gonads formed?

Back 688

The migration of the germ cells and their combining to somatic cells.

Front 689

What is Sxl?

Back 689

It is an RNA binding protein and is the master sex regulator in drosophila.

Front 690

What does early Sxl trxn stimulate?

Back 690

Female drosophila sex development

Front 691

Which sex is default in drosophila?

Back 691

Male

Front 692

What does Sxl control?

Back 692

Dosage compensation.

Front 693

What does Sxl control?

Back 693

Dosage compensation

Front 694

Sxl is only functional in...

Back 694

females.

Front 695

How do drosophila compensate for XX in females?

Back 695

They overexpress X proteins in males. This is opposite of mammals which use bar bodies.

Front 696

Sxl and Tra don't work in males because...

Back 696

it has an extra exon which tells the protein to terminate so that it becomes nonfunctional.

Front 697

What is the pathway for Sxl?

Back 697

Sxl --> Tra --> Dsx

Front 698

What does DSX-M do?

Back 698

It is the protein that makes male drosophila.

Front 699

Promoters of Sxl:

Back 699

Pe and Pm.

Front 700

Where do Pe and Pm occur?

Back 700

The Sxl promoters both occur in both sexes. Pe occurs only in females.

Front 701

What is the difference between the Pe and Pm transcript?

Back 701

The Pe transcript is functional, but the Pm requires pre-existing Sxl to be spliced properly to make functional Sxl.

Front 702

XCE =

Back 702

X chromosome counting elements

Front 703

What does active transformer (tra) do?

Back 703

Makes female-specific doublesex (Dsx)

Front 704

What does Dsx do?

Back 704

Processes Fruitless

Front 705

What is fruitless in charge of?

Back 705

Sexual behavior

Front 706

Which drosophila trxn factor is the most important for sex determination?

Back 706

Dsx.

Front 707

What are 4 downstream effects of female Dsx?

Back 707

1. female-specific growth genital disc
2. yolk proteins
3. spermathecal ducts
4. female pigmentation

Front 708

4 downstream effects of male Dsx?

Back 708

1. Male-specific growth genital disc
2. Paragonia
3. Clasper
4. Male pigmentation

Front 709

Sxl inhibits...

Back 709

Msl2

Front 710

Msl2 is not inhibited in...

Back 710

males

Front 711

What happens if Sxl isn't activated early?

Back 711

You get male junk.

Front 712

Pe is only active for...

Back 712

30-40 minutes

Front 713

What does Msl2 do?

Back 713

It opens the chromatin so that trxn is increased in the male x chromosome.

Front 714

What are the two different pathways in drosophila?

Back 714

1. Plumbing
2. Sexual preference

Front 715

Sexual behavior of wt female drosophila:

Back 715

No courting behavior

Front 716

Sexual behavior of wt male drosophila:

Back 716

1. Chase the female
2. Play a song
3. Lick her

Front 717

What is essential for male sex behavior?

Back 717

Correct splicing for Fruitless

Front 718

What happens to males lacking Fru sex-specific transcripts?

Back 718

They have normal sexual differentiation, but are completely sterile because they don't persue females.

Front 719

What happens to Fru(male) females?

Back 719

She persues wt females.

Front 720

What is the primary sex determination of mammals?

Back 720

Creation of gonads

Front 721

The gonad is initially...

Back 721

bipotential; it can become either ovary or testis

Front 722

What is mammalian secondary sex determination?

Back 722

The phenotype outside the gonads (duct systems, external genitalia)

Front 723

at 4 weeks in human gonad development, you have...

Back 723

indifferent gonads. You have a wolffian duct and mesonephric ridge. You also have a genital ridge.

Front 724

At 6 weeks human gonad development, you have...

Back 724

You have Mullerian duct development and germ cells in the genital ridge.

Front 725

8 weeks testis development:

Back 725

1. Wolffian duct --> vans deferens
2. Mullerian duct
3. Testis develops from genital ridge

Front 726

8 weeks ovarian development:

Back 726

1. Wolffian duct degrades
2. Mullerian duct
3. Surface epithelium on genital ridge

Front 727

16 weeks testis development:

Back 727

1. Mullerian duct degrading
2. Testis cords have formed.

Front 728

20 weeks ovarian development:

Back 728

1. Ovarian cortex and follicles form
2. Wolffian duct degrading

Front 729

What is SRY?

Back 729

A trxn factor and is the mammalian sex determining gene

Front 730

What happens if a female has sry?

Back 730

It becomes an XX male - male genitalia, but no functional sperm.

Front 731

2 hormones made from testis:

Back 731

1. AMH (anti-mullerian hormone)
2. Terosterone

Front 732

What does AMH do?

Back 732

Causes Mullerian duct regression

Front 733

What does testosterone do in development?

Back 733

Causes differentiation of Wolffian duct into internal male genitalia (epididymis and vas deferens).

Front 734

Internal male genitalia:

Back 734

1. epididymis
2. vas deferens

Front 735

Two kinds of cells in the testis:

Back 735

1. Sertoli cells
2. Leydig cells

Front 736

Sertoli cells make...

Back 736

AMH

Front 737

Leydig cells make...

Back 737

Testosterone

Front 738

2 cells in ovary:

Back 738

1. Granulosa cells
2. Thecal cells

Front 739

The mullerian duct creates...

Back 739

female internal genitalia (uterus, oviducts, cervix, upper vagina)

Front 740

Female external genitalia comes from...

Back 740

genital tubercle, urogenital sinus

Front 741

Testosterone turns on...

Back 741

DHT

Front 742

DHT controls...

Back 742

Penis, prostate, scrotum

Front 743

Wolffian duct controls...

Back 743

Male internal genitalia.

Front 744

What happens to XX mice lacking FoxL2?

Back 744

They develop male gonads. The follicle cells begin to look like Sertoli cells and secrete testosterone (Leydig cells) You can convert eggs into sperm. They eggs apoptose.

Front 745

Evidence against female default status:

Back 745

FoxL2 is needed to generate and maintain female gonads.

Front 746

How can you prove that FoxL2 is needed to maintain female gonads?

Back 746

Use CRE ER and wait to add tamoxifen. The females will start producing testosterone when FoxL2 gets turned off.

Front 747

When FoxL2 turns off...

Back 747

Sox9 gets turned on.

Front 748

Two mammalian factors that turn the undifferentiated gonad into testis:

Back 748

1. Sry
2. Sox9

Front 749

What happens to guevedoces in Dominican Republic?

Back 749

Their bodies dont produce DHT so they start lives out with female genitalia and then produce outer male genitalia during puberty. They can have kids.

Front 750

2 factors needed for external genitalia:

Back 750

1. testosterone
2. DHT

Front 751

Androgen Insensitivity Syndrome:

Back 751

Individuals lack testosterone receptor. The individual can be XY, but has female secondary sex characteristics. They have internal male sex characteristics though.

Front 752

Sex determination in turtles:

Back 752

It depends on the temperature that the eggs incubate. 31* (hotter) become ovaries. 26* (cooler) become testis. Many turtles, some lizards, and all crocodiles are determined by temperature.

Front 753

What does the Hh inibiting drug do?

Back 753

It can reduce your tumors for a bit, but they come back worse later.

Front 754

Inappropriate expression of signaling molecules after birth causes...

Back 754

cancer.

Front 755

Inappropriate Hedgehog signaling in the brain causes...

Back 755

medulloblastoma

Front 756

Basal cell carcinoma can be caused by...

Back 756

inappropriate Hh signaling.

Front 757

How do neurons in the cerebellum differentiate?

Back 757

The first neuroblasts migrate to form the external granular layer. Granule neurons are produced in the External Granule cell layer and migrate back towards the lumen to form the granule cell layer. The EGL is gradually depleted.

Front 758

Where to the granule neurons go?

Back 758

To the external granule layer (EGL).

Front 759

6 layers of the cerebellum:

Back 759

1. Ventricular zone
2. Intermediate zone
3. Granule cell layer
4. Purkinje cell layer
5. Marginal zone
6. External granule cell layer

Front 760

Where do neuroblasts go from the ventricular zone?

Back 760

They migrate to form Purkinje cell layer.

Front 761

Why are Purkinje cells so cool?

Back 761

They're huge and are the sole motor output of the cerebellum.

Front 762

How do cells in the cerebellum proliferate?

Back 762

Hh signaling

Front 763

Hh signaling pathway:

Back 763

Shh binds to --> Patched receptor --> inhibits Smo

Front 764

Most common pediatric brain tumor:

Back 764

medulloblastomas

Front 765

If you have a Ptch mutation...

Back 765

Smoothened never gets turned off so Hh is always on.

Front 766

Where does Shh signaling come from in the cerebellum?

Back 766

It comes from the PCL and EGL cells respond to it. When you stain for it, Shh seems to be in the EGL layer.

Front 767

What does Shh signaling do in the cerebellum?

Back 767

It causes proliferation of GCPs in the cerebellar slice cultures.

Front 768

Mitogen:

Back 768

growth factor

Front 769

Mitogen in the cerebellum:

Back 769

Shh

Front 770

What happens if you don't have Anti-Shh in the cerebellum?

Back 770

GCP don't proliferate. They have a much smaller layer of EGL. Purkinje cells are fine though because they secrete Shh.

Front 771

Medulloblastoma is rare in...

Back 771

adults

Front 772

What is the drug they used for that patient with medulloblastoma?

Back 772

It's a small molecule inhibitor of smoothened called GDC-0449.

Front 773

What is the challenge of working with animal models for cancer?

Back 773

When you take cells out of an animal, they start to behave differently.

Front 774

What happens to Ptch +/- mice?

Back 774

50% develop medulloblastomas.

Front 775

What happens if you're Ptch +/-;p53+/-?

Back 775

You get an insane amount of cancer.

Front 776

How many genes are directly activated by Shh signaling?

Back 776

500 target genes!

Front 777

How does ChIP work to find Gli-activated proteins?

Back 777

You get a Gli generally with a Flag Tag and you have the Gli flag tag bind to unknown sequences and then you use a restriction digest and chop it all up. Next throw in some beads that have an antibody to the flag tag. The antibody will bind to any pieces that have Gli protein attached. Stick in a magnet and wash - the antibody will attach to the magnet and everything else will wash away. Finally, you release the proteins and DNA and sequence the DNA to find out the Gli targets.

Front 778

How do you find the Gli binding sites in medulloblastomas?

Back 778

You have to breed a Patch1+/- mouse with a tagged Gli mouse line (RosaGli1Flag).

Front 779

What kind of cancer can you lessen with Smoothened inhibitors?

Back 779

You can help treat basal cell carcinoma and it works ok on medulloblastomas.

Front 780

Wnt signaling plays a role in what cancers?

Back 780

Breast, colorectal, etc.

Front 781

Pluripotent:

Back 781

(of an immature or stem cell) capable of giving rise to several different cell types.

Front 782

What kind of stem cells make muscel?

Back 782

Satellite cells

Front 783

Ovarian stem cells are...

Back 783

Presophiles

Front 784

Embryonic stem cells are...

Back 784

pluripotent.

Front 785

What happens to TCF4-/- mice have gut defects?

Back 785

They're born, but die in 24hrs. They fail to maintain proliferative compartments in the intestinal epithelium. At birth, the entire epithelium is composed of non-dividing, differentiated cells.

Front 786

What is TCF4 essential for?

Back 786

Maintaing gut homeostasis.

Front 787

The absorptive epithelium of the small intestine is ordered into...

Back 787

vili and crypts.

Front 788

What do vili do?

Back 788

They allow for the increase in surface area for absorbing nutrients.

Front 789

What's the life cycle of individual epithelial cells?

Back 789

Less than a week.

Front 790

Stem cells in crypts make how many cells a day?

Back 790

200/crypt/day in a mouse.

Front 791

What does Lgr5 do?

Back 791

It marks small intestinal stem cells.

Front 792

Where is Lgr5 expression?

Back 792

Only in a subset of cells at the base of the crypt.

Front 793

What's the Wnt target gene in small intestine?

Back 793

Lgr5

Front 794

Paneth cells:

Back 794

One of the principle cell types in the small intestine

Front 795

2 roles of paneth cells:

Back 795

1. Emit microbial agents descended from stem cells.
2. Are always proliferating

Front 796

What's the difference between paneth and quiescent muscle cells?

Back 796

Paneth are always proliferating and quiescent cells only proliferate in damage.

Front 797

How can you visualize the lgr5 cells in a mouse?

Back 797

You make a mouse with an Lgr5-GFP Cre ER allele. Once you activate with tamoxifen, after day 1, you see GFP in the base. Over time though, it will start to cover the whole vili.

Front 798

What do paneth cells secrete?

Back 798

Wnt proteins

Front 799

What does Lgr5 amplify?

Back 799

Wnt signaling response in stem cells.

Front 800

How do you grow mini-gut organoids if you're weird like that?

Back 800

You grow in vitro from a single Lgf5-expressing cell and it will get 5 cryps around the main lumen and will become functional!

Front 801

What's wrong with TCF4-/- in relation to Wnt signaling?

Back 801

They can't activate beta-catenin.

Front 802

What is FAP?

Back 802

An APC protein discovered via FAP disease in which patients get colon cancer.

Front 803

When does FAP become cancerous?

Back 803

FAP leads to pollups, but when the cells lose p53 and K-ras, that's when they become cancerous.

Front 804

How do you give a mouse colon cancer?

Back 804

You mess up its APC alleles and it will proliferate rapidly and give the mouse colon cancer.

Front 805

What is nuclear reprogramming:

Back 805

The ability to change the fate of a cell by making a switch in nuclear gene expression.

Front 806

3 reasons nuclear reprogramming is of interest:

Back 806

1. Identifying how reprogramming takes place can help us understand how cell differentiation and specialized gene expression is usually maintained.
2. Could be the first step in cell-replacement therapy
3. It enables the culture of lines of cells to be analyzed for therapeutic drugs.

Front 807

How did they make Dolly?

Back 807

They transplated the nuclei of cultured mammary gland cells from a sheep into enucleated sheep eggs.

Front 808

What is nuclear reprogramming and how does it relate to cloning?

Back 808

It's the switch in gene expression of one cell type to that of another cell type. For cloning, it means reprogramming the nucleus of a differentiated cell into a pluripotent cell.

Front 809

What are the steps of the early cloning experiment?

Back 809

1. Remove chromosomes and spindle from the cell of an animal pole
2. Extract and lyse the donor cell
3. Stick the donar nucleus into the enucleated cell
4. PUt the somatic cell nucleus in an activated egg
5. You get a frog (yay!)

Front 810

What happens when you take nuclei later in development?

Back 810

The percent of embryos that develop normally decreases exponentially.

Front 811

What did Sir John Gurdon do?

Back 811

He took the nucleus from an albino frog and put it in a brown frog and would keep taking it out putting it in other enucleated cells and eventually was able to reprogram the cell.

Front 812

How did they make Dolly according to the slides?

Back 812

They took an egg from an oocyte donor. They removed other cells from another sheep and grew them and then stuck them in the enucleated egg. The egg and cell fused and an embryo was cultured for 7 days. A blastocyst formed and then they put it in the surrogate mom and voila Dolly.

Front 813

What's the controversy with Dolly?

Back 813

Some people did think it was really cloning and it was a really rare event - 1/434 injections.

Front 814

What is OMP?

Back 814

Marker for differentiated olfactory sensory neurons.

Front 815

How do you find out the origin of olfactory cells?

Back 815

You use a CRE that only expressed in terminal olfactory neurons by sticking it to OMP.

Front 816

Describe the experiment that makes a mouse out of olfactory sensory neurons.

Back 816

Rudolph Jaenisch's group showed that either terminally differentiated olfactory sensory neurons or lymphocytes can be used to generate mice. You put the nuclei in ES cells, culture a blastocyst and then stick it in a surrogate.

Front 817

Why can you reprogram an oocyte?

Back 817

It probably still has some maternal factors. It's under prolongued miotic arrest. The egg can be unstable the egg has no nucleus for a while during the experiment. But it works in an oocyte.

Front 818

What is the first embryonic cell cycle?

Back 818

1. After fertilization, the reprogramming factors are initially in the cytoplasm are in the nucleus.
2. You can treat zygotes with nocodazole which results in mitotic arrest until the drug's removed.
3. During this arrest, NEB has already happened so reprogramming can last longer.
MG-132 inhibits proteasome, prevents degradation of cyclin B.

Front 819

MG-132:

Back 819

Inhibits proteosome and prevents degradation of cyclin B (blocks metaphase to anaphase)

Front 820

Nocodazole:

Back 820

Inhibits spindle formation and results in mitotic arrest.

Front 821

Could zygotes be used instead of oocytes for Reprogramming?

Back 821

They froze the zygote and used it to clone an ES cell, but the zygotes were inviable. Lots of cells from fertilization and polyspermy - it causes messed up zygotes.

Front 822

What development stage is more ethical in in vitro fertilization treatment?

Back 822

The author Egli argued that it's more ethical to use defective zygotes because using a human zygote has the capacity to form an embryo. So you should use one with multiple nuclei.

Front 823

How did they make the human ES cells in the new data?

Back 823

They took skin fibroblasts and human blastocysts.

Front 824

What was the goal when they were making iPS cells?

Back 824

To try to reprogram cells without having to use eggs.

Front 825

Fbx15?

Back 825

It is turned on in ES cells, but off in somatic cells.

Front 826

Fbx15 is not essential...

Back 826

for ES cells.

Front 827

How did Yamanaka make iPS cells?

Back 827

They infected mouse embryonic fibroblast viruses with 24 candidate genes possibly required for generating stem cell fates and ended up with some colonies.

Front 828

How did Yamanaka see which cells were iPS cells?

Back 828

He gave the Fbx15 Neomycin resistance so that as long as the cells were iPS cells, they would produce neo R on a neo plate and survive.

Front 829

What are 4 critical factors for iPS cells?

Back 829

1. c-Myc
2. Klf4
3. Sox2
4. Oct 3/4

Front 830

What happens if you try to make iPS cells without Sox2?

Back 830

You can still make colonies, but they look really weird.

Front 831

What do iPS colonies look like?

Back 831

ES cells

Front 832

What can you use iPS cells for?

Back 832

You can make chimeric embryos with GFP expression and can make various tissue types in adult chimeras.

Front 833

problems with iPS cells:

Back 833

1. Retroviuses permanently integrate into the genome. So that's bad because the viruses could reactivate.
2. iPS cells aren't very efficient (10^-4 of the population)
3. Two of the Yamanka factors (C-Myc and Klf-4) are oncogenes and could make a tumor.

Front 834

Which Yamanaka factors are oncogenes?

Back 834

c-Myc and Klf4.

Front 835

What are the most narrowed down two critical Yamanaka factors?

Back 835

Oct4 and Sox2

Front 836

iPSCs make all three...

Back 836

embryonic germ layers: endoderm, mesoderm, ectoderm

Front 837

Are iPSCs just as good as ES cells?

Back 837

Yeah.

Front 838

What happens if you don't culture ES cells?

Back 838

They start to make cardiomyocytes and neurons?

Front 839

How do you differentiate ESCs and IPSCs into motor neurons?

Back 839

You culture ES cells with different levels of RA or Shh/RA and you get different progenitors.

Front 840

What happens if you culture ES cells with RA?

Back 840

You get dorsal neural progenitors.

Front 841

What happens if you culture ES cells with Shh and RA?

Back 841

You get ventral neural progenitors.

Front 842

How do ESCs and IPSCs compare when trying to give them neural fates?

Back 842

They differentiate into motor neurons at similar rates.

Front 843

How could you make IPSCs for people with Type 1 diabetes?

Back 843

You could take skin fibroblast biopsies, generate human iPS cell lines which could then be differentiated into beta cells.

Front 844

What's the biggest problem with IPSCs?

Back 844

They have troublesome memories. They seem to retain imprinting form what they used to be.

Front 845

What are the worst IPSCs from?

Back 845

Skin fibroblasts neural cells.

Front 846

It's harder to make IPSCs in what kind of cells?

Back 846

Older cells.

Front 847

IPSCs are better at making ____ than ___

Back 847

bone; blood

Front 848

What's causing the memory of these IPSCs?

Back 848

Genes have methylation markers that are maintained throughout life of the cell.

Front 849

How can you rescue IPSC neural cells from remembering?

Back 849

You can treat them with 5-Azac

Front 850

5-Azacytidine:

Back 850

Drug inhibiting DNA methylation

Front 851

Dor et al used a pulse-chase approach to label beta cells in adult mice. Describe their experimental approach and conclusions. What would have been the result if a non-beta cell progenitor gave rise to other beta cells?

Back 851

Dor et al crossed a mouse line expressing a beta-cell specific, tamoxifen inducible Cre with a Cre reporter line (irreversibly expresses Alkaline phosphatase upon Cre-mediated activation). Without tamoxifen, the CreER protein is sequestered in the cytoplasm and is therefore unable to activate the reporter gene (which requires nuclear localization of Cre). They treated adult mice with an intermediate dose of tamoxifen that allowed for activation of the Cre reporter in ~25% of beta cells when assayed a few days post-induction. They saw no difference in the percent of labeled cells – even a year later. B/c the half-life of beta cells is only ~3 months, and because there is a tremendous increase in the total number of beta cells over this time, they conclude that beta cells are giving rise to new beta cells. If, instead, they had seen a progressive reduction in the percentage of labeled beta cells, it would have suggested that a non-beta cell progenitor population was present.

Front 852

Zhou et al 2008 described an experimental approach for generating beta cells, showing that forcibly expressing three transcription factors in exocrine cells transformed them from exocrine cells to endocrine fates (including beta cells). The data we showed in class stopped short of proving that the ‘new’ beta cells were derived from non-beta cells. Describe an experiment and result that would directly demonstrate that these new cells were descended from amacrine cells?

Back 852

You would need a tamoxifen inducible Cre line that is only active in the exocrine cells and a Cre reporter. If you injected with tamoxifen at the same time when you infected the mice with the transcription factor cocktail, cells that expressed endocrine markers (such as insulin) and GFP+ (indicating viral infection) should also express b-galactosidase (assuming RosaLacZ is the Cre reporter).

Front 853

What is the key transcription factor that separates exocrine and endocrine pancreatic lineages?

Back 853

Neurogenin 3 (Ngn3) is required for the formation of endocrine lineages.

Front 854

When you graft a distal (wrist-level) axolotl blastema to another animal that has a proximal limb amputation, replacing the original host blastema, you still form a normal limb. Which portion will be derived from the host, and which portion from the blastema?

Back 854

The region up to the wrist will be derived from the host (intercalary regeneration). The region distal to the wrist level will be derived from the blastema.

Front 855

When you graft a distal (wrist-level) axolotl blastema to another animal that has a proximal limb amputation, replacing the original host blastema, you still form a normal limb. Which portion will be derived from the host, and which portion from the blastema?
-The region up to the wrist will be derived from the host (intercalary regeneration). The region distal to the wrist level will be derived from the blastema.
Repeat the experiment in #1 except that the donor animal is first treated with high doses of retinoic acid for several days. What happens?

Back 855

High levels of retinoic acid will completely proximalize the blastema. When grafted, it will then form a complete limb after the proximal stump. This will result in a limb with extra appendages.

Front 856

Injury to zebrafish ventricles results in the initial activation of genes throughout the entire region of a specific tissue in the heart. Which tissue?

Back 856

A sub-population of GATA4+ cardiomyocytes adjacent to the epicardium.

Front 857

What is the functional effect of mammalian cells lacking Xist?

Back 857

There will be no inactivation of X-chromosomes and there will therefore be a lethal increase in X-chromosome genes.

Front 858

What is Xist? How is it controlled? How does it inactivate X-chromosome transcription?

Back 858

Xist is a long non-coding RNA that binds to the X-chromosome at a specific region (Xic) and randomly initiates inactivation of one of the X-chromosomes. Xist is only transcribed and presence on the inactive X-chromosome. Prior to X-inactivation, Xist is transcribed at low levels on both X-chromosomes, but is inhibited by being bound by an antisense non-coding transcript called Tsix. After initiating inactivation of one X-chromosome, Xist transcription is highly upregulated, eventually binding to sites along the entire X-chromosome. Xist facilitates the recruitment of histone modification enzymes that are markers of silenced regions, and also bind along the inactive X-chromosome.

Front 859

What are the differences in how PIE-1 and Blimp1 repress somatic gene transcription in germline progenitor cells?

Back 859

While both negatively regulate transcription, PIE-1 represses all Pol2 mediated zygotic transcription (by inhibiting transcriptional elongation of Pol2) while Blimp1 is a sequence specific transcriptional repressor that doesn't turn off all somatic genes.

Front 860

How is germline specification different in C. elegans versus mice?

Back 860

Germ cells are autonomously specified in C. elegans (sequestration of P-granules and PIE-1) while in mice they are induced (by BMPS) in the extraembryonic tissue.

Front 861

The mechanism by which primordial germ cells (PGCs) migrate from the hindgut endoderm into the genital ridges to populate the two future gonads is not understood in mice. Propose a testable hypothesis for how PGCs might split into two streams as they migrate out of the hindgut in mice based on your knowledge of the process in Drosophila. Your hypothesis should include specific molecule(s) as well as where you hypothesize they should be expressed, and how you might alter/remove them.

Back 861

Wunen and Wunen-2 mediate midline repulsion of PGCs as they migrate out of the Drosophila hindgut, thereby allowing them to form two discrete populations that migrate laterally to populate the two gonads. Based on this, a reasonable hypothesis is that they might play an analogous role in mice. If so, they might be expressed in a midline structure (CNS or more likely mesodermal). If so, you would need to determine if there are mouse orthologues (by searching the mouse genome for genes with similar structure - or the literature; as described on the discussion board there are functional orthologues of Wunens in mammals that can even substitute for Wunen activity in flies) and where they are expressed (by in situ hybridization). Then, you would need to generate null mice, and look for defects in germ cell migration….

Front 862

Why do you think Sex lethal (Sxl) is in fact lethal when mutated in female flies?

Back 862

Because females will then have twice as much transcription from genes on their X-chromosome.

Front 863

How is Sxl null mutant female similar or different to a null mutant in Transformer (Tra)?

Back 863

Sxl lethal is upstream of Tra; both have the same effect that in their absence, they will generate male-specific isoforms of the sex determination transcription factor Doublesex (Dsx). They are different because Sxl has an additional role in inhibiting Msl genes, thereby preventing amplification of transcription in female cells. Thus, transformer only effects the sex determination pathway and not the doseage compensation pathway. Because of this, Tra mutants are not lethal, and XX females will display male morphology and male courting behavior (since they will have a male isoform of Fruitless).

Front 864

Why don't XX mice expressing an Sry transgene produce functional sperm?

Back 864

Sry is sufficient to initiate the male sexual development pathway. However, additional proteins necessary for making sperm are also present on the Y-chromosome.

Front 865

What happens to the Mullerian duct in males? In females?

Back 865

It degenerates in males, where Sertoli cells (which are present in the fetal testis, secrete anti-mullerian hormone that promotes its apoptosis. In females it forms the oviduct and contributes to the internal genitalia.

Front 866

Which fetal cells secrete testosterone?

Back 866

The Leydig cells (along with the Sertoli cells the two major somatic copmonents of the testis.

Front 867

What is the difference between individuals with mutations in the testosterone receptor vs. mutations in synthesizing DHT?

Back 867

If the receptor is mutated, the testes will form. However, the testosterone secreted by the testes cannot be sensed, leading to the development of female secondary sexual characteristics. Individuals with DHT mutations still form internal genital structures, and in the case of the guevedoces, will form full male secondary structures at puberty (presumably because there is a surge of testosterone).

Front 868

Speculate on the phenotype of a female fly with normal sex determination genes except for a mutated version of Fruitless that can only generate a male isoform.

Back 868

The fly will be a fertile female fly but will exhibit male mating behavior (e.g. will court females).

Front 869

You generate embryonic flies with a deletion in the Pe (early) promoter for Sxl. What is the phenotype for females, and why?

Back 869

Sxl will be transcribed later from the Pm promoter, but this will not encode for a functional protein. In females, this will result in the activation of Msl2, leading to a lethal overdose of transcription from the X chromosome.

Front 870

What is the progenitor population that gives rise to granule cells in the cerebellum? Is this population retained in adults?

Back 870

The external granule layer (EGL). This layer is not retained past a relatively early postnatal period (2 years in humans) – not present in adults.

Front 871

If you used an EGL-specific Cre to remove Shh, what would happen?

Back 871

Nothing! The EGL responds to (does not produce) Shh secreted by the Purkinjie Cells.

Front 872

Elevated levels of Wnt signaling have been implicated in colon cancer. Propose a plausible mouse model system that would allow you to study this WITHOUT perturbing Wnt signaling during development or in other tissues (besides the colon).

Back 872

Need some type of strategy that allows for the elevation of Wnt signaling in a Cre-specific fashion. One strategy would be an inducible Cre that would activate Wnt signaling in the colon (by perturbing or deleting a negative regulator or activating a positve factor. For example inducing a dominant negative in a Cre-dependent fashion…or, conditionally deleting a negative regulator (Axin) in adult colon tissue.

Front 873

How do the experiments outlined in Egli et al differ from classic nuclear transfer?

Back 873

Instead of transferring nuclei into enucleated oocytes, they transferred chromosomes into zygotes that have had their chromosomes removed. Importantly, the zygotes do not have their nuclei removed. Instead, they are frozen in metaphase when the nuclear envelope has broken down (and all nuclear reprogramming components are therefore retained in the zygote).

Front 874

How did Egli et al. freeze their cells in metaphase? Why was this necessary?

Back 874

They froze cells by arresting them in metaphase with the drug nocodazole (this destabilized microtubules and prevents the mitotic spindle from forming). They then removed Nocodazole, allowing the spindles to form completely synchronously in all their cells, and added MG-132, which inhibits the proteasome-based degradation of Cyclin B. This second drug freezes them at a stage when the mitotic spindles have formed. This was necessary because this is the only stage at which chromosomes can be viewed and removed under the microscope without adding toxic dyes and viewing under UV.

Front 875

Why does dysregulated, activated Wnt signaling cause intestinal cancer?

Back 875

Lgr5-expressing stem cells require Wnt signaling for their maintenance (they get this from their neighboring Paneth cells). If there is more Wnt signaling, there will be more stem cells. These will form a poly that is, at this stage, benign. With an additional mutation in another gene (P53, K-ras), these cells will transform into an adenocarcinoma.

Front 876

Instead of nuclear transfer, you decide it will be far easier to generate an induced pluripotent stem cell line (iPS cell) from cardiomyocytes and then use this to clone mice. How would you generate an iPS cell line from a cardiomyocyte?

Back 876

Infect primary cardiomyocytes with the Yamanaka factors (Sox2, Oct4, Klf4, Myc - don't actually have to use Myc in newer approaches). Look for ES cell-like colonies growing on the dish over time (here you would be greatly aided by the fact that differentiated cardiomyocytes undergo little or no proliferation).

Front 877

Describe the positive selection screen used by Takahashi and Yamanaka to isolate the 4 "Yamanaka" factors.

Back 877

They used Fbx15, a gene that is only expressed in ES cells (but not essential) to drive neomycin resistance. They then isolated fibroblasts from mice that were Fbx15::Neo-R. These cells are differentiated and Fbx15 is therefore not expressed; thus these cells are neomycin sensitive. They then infected these cells with 24 viruses expressing 24 candidate genes that they hypothesized might be involved in making pluripotent cells. When all 24 were added, they obtained several neomycin-resistant colonies that closely resembled ES cells. They then sequentially removed 1 factor from the 24, infecting pools of 23 factors. This narrowed down the number of candidates to 10. They then showed that only 4 of these were required to make iPS cells.

Front 878

What are enhancers?

Back 878

Non-coding elements that can be located either close or far away from the genes.

Front 879

What do enhancers do?

Back 879

They can modulate activity regardless of their orientation.

Front 880

What are enhancers bound by?

Back 880

Clusters of transcription factors.

Front 881

What are promoters?

Back 881

Docking sites for RNA pol II.

Front 882

How can you experimentally ID enhancers?

Back 882

Candidate enhancers are inserted into a piece of DNA that is then injected into embryos to generate transgenics. You can then use in situ hybridization to visualize where the enhancer and gene are.

Front 883

Transgenics:

Back 883

The manipulation of a gene to visualize the gene or enhancer.

Front 884

What are 4 regions of a typical developmental gene?

Back 884

1. Enhancer regions
2. Promoter regions
3. Transcribed regions
4. Coding regions

Front 885

What happens to primary RNA transcripts?

Back 885

They are processed (spliced) into mature mRNA and exported to the cytoplasm.

Front 886

Two things that can occur to cytoplasm mRNAs?

Back 886

1. They can be stored.
2. They can be actively translated into proteins.

Front 887

Bauplan:

Back 887

The most basic anatomical structure for the vertebrate embryo.

Front 888

3 things ectoderm turns into:

Back 888

1. Epidermal cells of skin
2. Neurons of the brain
3. Pigment cells

Front 889

5 things mesoderm turns into:

Back 889

1. Notochord
2. Bone tissue
3. Tubule cell of the kidney
4. Red blood cells
5. Facial muscle

Front 890

3 things endoderm turns into:

Back 890

1. Stomach cells
2. Thyroid cells
3. Lung cells

Front 891

What are von Baer's 4 laws?

Back 891

1. The general features of a large group of animals appear earlier in development than do specialized features of a specific group.
2. Less general characters develop from the more general.
3. The embryo of a given species, instead of passing through the adult stages of lower animals, departs more and more from them.
4. The early embryo of a higher animal is never like a lower animal, but only like its early embryo.

Front 892

The phylotypic stage:

Back 892

The stage of development at which all members of a taxon (especially insects and vertebrates) show the maximum morphological similarity.

Front 893

What is the bauplan?

Back 893

The virtual embryo that has the generalized feature of a large group of animals.

Front 894

What is the major goal of evolutionary developmental biology?

Back 894

To uncover previously invisible homologies that contribute to the body plan.

Front 895

What are the 2 different nerves that innerve the middle ear?

Back 895

1. The trigeminal nerve
2. The Facial nerve

Front 896

What does the Trigeminal nerve innervate?

Back 896

The tensor tympani muscle of the Malleus

Front 897

What does the Facial nerve innervate?

Back 897

The Stapedius muscle of the Stapes.

Front 898

3 things derived from the 1st pharyngeal arch:

Back 898

1. upper and lower jaws
2. Malleus and Incus
3. Trigeminal ganglion

Front 899

3 things derived from the 2nd pharyngeal arch:

Back 899

1. Facial muscles
2. Portions of the throat
3. Stapes, facial nerve

Front 900

Why are two tiny muscles in the middle ear innervated by completely different nerves?

Back 900

The trigeminal nerve innervates the Malleus because both are derived from the first arch. The facial nerve innervates the stapes because both are derived from the second pharyngeal arch.

Front 901

Compare mammal, reptile, and fish ear bones.

Back 901

Fish have no middle ear bones
Reptiles have 1 ear bone - the stapes.
Mammales have 3 ear bones.

Front 902

How was it shown that leg length in dogs is controlled by few genes?

Back 902

Cross a tall and short dog in the parent generation and you'll see short and long in the F2 which have unique combinations of the two original heights. It's in a 3:1 ratio.

Front 903

What two features are controlled by relatively few genes in dogs?

Back 903

1. Leg length
2. Upper vs. lower jaw

Front 904

How are marien and freshwater sticklebacks different?

Back 904

Marine stickleback have pelvic armor and freshwater don't.

Front 905

Whats the experiment you can do with sticklebacks?

Back 905

Cross male and female from each species to make F1 hybrids. Then make an F2 generation. Look at the microstellite markers. 40% of variation can be attributed to one locus.

Front 906

What does Pitx1 do?

Back 906

It's a limb specific gene in mice. The mutation in mice is lethal, but embryos have smaller hindlimbs.

Front 907

How is Pitx1 related to sticklebacks?

Back 907

Both marine and freshwater sticklebacks have the Pitx1 wt gene, but it's expressed in the mouth AND the pelvic plate for marine sticklebacks.

Front 908

What happens when Pax6 is misexpressed in drosophila?

Back 908

They get ectopic eyes all over the place - like on their knees.

Front 909

What happens to Pax6+/- humans?

Back 909

They have aniridia which means that their iris is messed up.

Front 910

Are eyes an example of convergent or divergent evolution?

Back 910

Convergent.

Front 911

Whats the genetic basis for diversity?

Back 911

Recombination during meiosis.

Front 912

How does meiosis differ from mitosis?

Back 912

It has 2 rounds of cell division with only one round of DNA replication.

Front 913

Oocytes:

Back 913

Eggs

Front 914

How much bigger are mammalian oocytes from a somatic cell?

Back 914

100-500 fold difference.

Front 915

Why are oocytes so big?

Back 915

They have to store more nutrients, mRNAs, mitrochondria, etc.

Front 916

Vertebrate oocytes are arrested in...

Back 916

meiotic prophase.

Front 917

How much DNA do oocytes have?

Back 917

Twice as much so 4 gene copies.

Front 918

7 things that are amplified in oocytes:

Back 918

1. mitochondria (100K)
2. RNA polymerase (60-100K)
3. DNA polymerase (100K)
4. Ribosomes (200K)
5. tRNA (10K)
6. Histones (15K)
7. dNTPs (2500)

Front 919

What is the cell support in drosophila oocytes?

Back 919

The cell undergoes 4 incomplete divisions to make 15 nurse cells and 1 oocyte. The oocyte is surrounded by follicle cells.

Front 920

How long will oocytes remain in arrest in humans?

Back 920

12-50 years

Front 921

What are the components of the secondary oocyte?

Back 921

Oocyte and PB1

Front 922

What are the components of an ovum?

Back 922

Egg, PB1, and PB2.

Front 923

When do your number of oocytes majorly drop?

Back 923

Like right after birth.

Front 924

What attaches to the polyA tail in mRNA?

Back 924

PABP

Front 925

What is Maskin?

Back 925

A trxnal repressor

Front 926

What happens when you repress maskin with an antibody?

Back 926

There's an increase in expression of Cyclin B1, Map kinase, eIF4E.

Front 927

What complex is made to silence mRNA?

Back 927

It's the eIF4E-maskin-CPEB complex and CPSF is unable to bind.

Front 928

What binds the Guanidine cap in active mRNA?

Back 928

eIF4E, eIF4G, eIF3, 40S.

Front 929

What is the complex on the CPE region of active mRNA?

Back 929

Phosphorylated CPEB, maskin, and CPSF

Front 930

Fertility concerns impact how many people?

Back 930

6.1 million, about 10% of men and women of reproductive age.

Front 931

How many babies in the US are born from IVF?

Back 931

250K.

Front 932

How are dairy cows bred in the US?

Back 932

By artificial insemination using just a few bulls with sorted sperm to make over 80% of female calves.

Front 933

What are blastomeres?

Back 933

The large cells of the cleavage stage embryos.

Front 934

What is MPF

Back 934

maturation promoting factor. It's the complex of Cyclin B and cdc2. MPF must be activated to move from G to M phase.

Front 935

How much bigger is the newly fertilized frog?

Back 935

It has a volume over 1 million times larger than the average differentiated cell.

Front 936

What dictates cleavage of the early embryo?

Back 936

Centrosomes

Front 937

Centriole:

Back 937

barrel-shaped structure of microtubules

Front 938

When do centrosomes (paired centrioles) replicate?

Back 938

During S phase.

Front 939

What is the Sand Dollar Experiment?

Back 939

In a zygote, you put a glass ball which displaces the mitotic spindle. The cleavage furrow ends up being inhibited. It ends up making an extra cleavage furrow...

Front 940

What are the steps of sea urchin cleavage?

Back 940

The zygote has an interphase nucleus and two centrosomes appear on either side of the nucleus. Spindle fibers and metaphase chromosomes appear in between the centrosomes and then the cell divides.

Front 941

What is the importance of axis orientation of a spindle positioned near the animal pole?

Back 941

You end up making the polar bodies by having the centrosomes up near the top.

Front 942

What is MZT?

Back 942

Maternal zygotic transition - the clearing of maternal transcripts

Front 943

What's the first part of early development?

Back 943

1. Maternal zygotic transition: clearing of maternal transcripts
2. Activation of zygotic transcription

Front 944

What's the second part of early development?

Back 944

1. The concept of cell specification and cell fate
2. Sea urchin specification and Wnt signaling

Front 945

What do miRNAs do?

Back 945

They bind to mRNA and inactive them and are a signal for Dicer chop up the mRNA.

Front 946

What is MiR-430?

Back 946

It's a miRNA specifically transcribed by the zygote at the start of MZT. It leads to the degredation of maternal mRNAs.

Front 947

Why is it impossible to knock out MiR-430 and how do you get around that?

Back 947

MiR-430 has multiple family members so it's not possible to knock them all out. However, you can knock out Dicer in zebrafish. Dicer is an enzyme that is required for processing all miRNAs.

Front 948

What happens to miRNAs if you don't have Dicer?

Back 948

There are no active miRNAs.

Front 949

What kind of gene is Dicer in early development?

Back 949

It's a maternal effects gene.

Front 950

Based on research with zebrafish Dicer mutants, what do we know about MiR-430?

Back 950

It clears hundreds (but not all) maternal transcripts.

Front 951

Name 3 of the first trxn factors to be translated from maternal mRNA.

Back 951

1. Nanog
2. Sox19b
3. Pou5f1 (known as Oct4)

Front 952

Name the trxn factors essential for zygotic trxn:

Back 952

1. Nanog
2. Sox19b
3. Pou5f1

Front 953

What are LOF's?

Back 953

Zebrafish lacking the essential factors for zygotic transcription.

Front 954

What is RPKM?

Back 954

The standardized measurement for the amount of RNA expression.

Front 955

4 features of autonomous specification:

Back 955

1. Typical of invertebrates
2. Cytoplasmi determinants
3. Invariant cleavage and lineages
4. Specification precedes cell rearrangements

Front 956

Trochoblasts:

Back 956

Ciliated cells of the mollusc Patella.

Front 957

What happens if you isolate trochoblasts?

Back 957

They just develop into more trochoblasts.

Front 958

4 features of conditional specification:

Back 958

1. Cell rearrangements precede specification
2. Fates not invariant
3. Regulative development
4. Cell-cell interactions

Front 959

Name two kinds of specification in early development:

Back 959

1. Conditional specification
2. Autonomous specification

Front 960

How do you test conditional specification?

Back 960

You can take normal back cells and transplant them to the belly region and they back cells will turn into belly cells in the embryo due to cell-cell interactions.

Front 961

What happens if you remove cells with a glass needle in a blastula?

Back 961

You'll still have normal development of an embryo because they'll just make more cells and fix itself.

Front 962

What is the Driesch experiment on conditional specification?

Back 962

the embryo at the 4 cell stage and separate the four cells. You'll end up getting normal larvae from single cells at the 4-cell embryo stage.

Front 963

What happens if you isolate the animal hemisphere alone of a sea urchin blastomere?

Back 963

It ends up making a dauerblastula and has complete animalization.

Front 964

What happens if you attach the animal hemisphere to the micromere (which is at the bottom) of a sea urchin blastomere?

Back 964

You essentially rescue the larva a bit. You get endoderm from the animal layers.

Front 965

What happens if you isolate the micromeres of a sea urchin blastomere?

Back 965

They generate the skeleton in culture. They're autonomoulsy specified and act as an organizing center.

Front 966

What's the canonical Wnt pathway?

Back 966

Wnt --> Frizzles --> Disheveled --I GSK-3 --I beta-catenin --> trxn

Front 967

What is the micromere specification signal?

Back 967

Beta-catenin

Front 968

How many fusion event in fertilization?

Back 968

4 fusion events

Front 969

2 kinds of polyspermy blocks

Back 969

1. fast blocks
2. slow blocks

Front 970

How does the first cleavage event after fertilization occur?

Back 970

It involves synchronous cycles of M and S phases in the absense of RNA trxn.

Front 971

What does fertilization mean?

Back 971

Fusion of two gametes

Front 972

What is the vitelline envelope?

Back 972

Also known as the zona pellucida, it's the extracellular matrix involved in sperm recognition.

Front 973

What is Resact?

Back 973

It's a chemotactic peptide in sea urchins. The sperm follow a concentration gradient of Resact.

Front 974

What lab study can you do with Resact?

Back 974

You can place Resact in a plate and the sperm will migrate toward it.

Front 975

What's the acrosome reaction?

Back 975

1. Ca2+ mediates the fusion of acrosomal and cell membrare. I
2. t's the first membrane fusion event.
3. Release of the enzymes degrade egg extracellular.
4. Initiated by contact with egg jelly: species specific

Front 976

What is immunostaining?

Back 976

It's a method for detecting protein localization within the embryo.

Front 977

What does bindin do?

Back 977

It's a protein that localizes to the acrosome process.

Front 978

What is the fertilization cone?

Back 978

It's a cone formed in the egg by polymerization of actin. It allows for the entry of sperm. It's the 2nd membrane fusion event.

Front 979

What's an example of fast block to polyspermy?

Back 979

There's a change in the membrane potential of the egg which starts 1-3 seconds after fertilization.

Front 980

How can you reverse fast block in frog eggs?

Back 980

You prevent depolarization of the egg and it leads to increased polyspermy.

Front 981

What's an example of slow block to polyspermy?

Back 981

It's the cortical granule reaction. This is the 3rd membrane fusion event.

Front 982

What is the cortical granule reaction?

Back 982

Serine proteases release the vitelline envelope. Glycoaminoglycans result in water intake in the perivitelline space, forming fertilization envelope. Peroxidase hardens the fertiliztion envelope by cross-linking Tyr residues. Hyalin is a glycoprotein that coasts the outer surface of the cell membrane, provides suppore during cleavage.

Front 983

What are 4 molecules that aid in cortical granule reaction?

Back 983

1. Serine proteases - release the vitelline envelope
2. Glycosaminoglycans - result in water intake forming a fertilization envelope
3. Peroxidase - hardens the fertilization envelope with Tyr cross-linkage
4. Hyalin - glycoprotein that coats the outer surface to provide support during cleavage.

Front 984

What causes the cortical granule reaction in the first place?

Back 984

A wave of Ca2+

Front 985

What releases Ca2+ in the cortical granule reaction?

Back 985

The ER.

Front 986

2 events caused by sperm fusing to the egg membrane:

Back 986

1. Na+ influx --> membrane potential change --> block
2. Kinase stimulation --> Ca release

Front 987

5 downstream results of Ca2+ release from sperm-egg fusion:

Back 987

1. Degradation of cyclin --> restoration of mitotic cell cycle
2. Membrane biosynthesis
3. Slow block of polyspermy
4. formation of hyaline layer
5. Stimulation of protein synthesis

Front 988

4 steps of the union of haploid genomes

Back 988

1. Chromatin decondenses
2. Microtubule aster forms
3. Pronuclei migrate towards each other and separately synthesize more DNA and fuse into zygotic nucleus
4. 4th membrane fusion event

Front 989

What's the length of embryogenesis in C elegans?

Back 989

16 hrs

Front 990

How many somatic cells in c elegans adults?

Back 990

959

Front 991

What are Par proteins?

Back 991

They are a series of maternal effects genes that affect the cleavage planes and assymetry of early cell divisions.

Front 992

What happens to par mutants?

Back 992

They have symmetrical early cleavages and arrest as a blob of cells.

Front 993

What are p-granules in c elegans?

Back 993

They are germ-line specific from the earliest stages and are though to be instructors of germ line development.

Front 994

What do p-granule proteins bind to?

Back 994

They bind to RNA and are thought to promote germ-cell specific transcription.

Front 995

Where is Pie-1 expressed?

Back 995

It's specifically expressed in P-cells and P-granules (maternal transcript)

Front 996

What does Pie-2 do?

Back 996

It represses all RNA pol 2 trxn in the early germ line (P2 and derivatives).

Front 997

What happens to pie-1 mutants?

Back 997

P2 becomes EMS.

Front 998

What is Skn-1 required for?

Back 998

It's required to make endoderm and mesoderm.

Front 999

What represses Skn-1?

Back 999

Pie-1

Front 1000

What happens to skn-1 mutants?

Back 1000

They lack endoderm and mesoderm. They make more of cell type C (body muscle and hypodermis).

Front 1001

What does POP-1 do?

Back 1001

It inhibits the formation of E fate (endoderm).

Front 1002

Skn-1 controls...

Back 1002

an endomesodermal gene regulatory network for EMS fate.

Front 1003

What does Skn-1 activate?

Back 1003

Med-1 and Med-2 which direct all EMS fates.

Front 1004

What can you use the bag of worms screen for?

Back 1004

To ID key regulators in early development.

Front 1005

When are D/V and A/P axis laid out in development?

Back 1005

Before fertilization.

Front 1006

When are the oocyte axes set up?

Back 1006

During oogenesis.

Front 1007

What genes initiate AP and DV patterning?

Back 1007

Gurken and Torpedo

Front 1008

Where can you find Gurken and Torpedo?

Back 1008

In drosophila.

Front 1009

What are torpedo and gurken and where are they located?

Back 1009

Torpedo is a receptor and gurken is the protein that binds to torpedo. You find them in the posterior side in the oocyte of the drosophila.

Front 1010

Where is gurken concentrated?

Back 1010

Dorsally

Front 1011

Where do you find pole cells in drosophila?

Back 1011

They're at the bottom...on the posterior side?

Front 1012

What do pole cells lead to in drosophila?

Back 1012

They lead to germ cells.

Front 1013

How can you make a germline chimera in drosophila?

Back 1013

You have an embryo from a wt mother and an embryo from a mother deficient in torpedo. You switch the pole cells. The torpedo-deficient cells will be in the wt female. The wt germ cells will be in the torpedo-deficient. The torp-deficient germ mom will have a torp-deficient oocyte in wt follicle. It will lead to normal DV patterning. The torp def female wil have wt germ cells with torp-def follicle which will lead to no patterning.

Front 1014

What does torpedo repress?

Back 1014

pipe expression.

Front 1015

Where is pipe expressed?

Back 1015

Ventrally.

Front 1016

What is pipe?

Back 1016

It's a sulfotransferase that activates a serine protease pathway.

Front 1017

What does Easter do?

Back 1017

It cleaves Spatzle.

Front 1018

What does Spatzle bind to?

Back 1018

It binds and activates Toll.

Front 1019

What does Toll do?

Back 1019

It activates Pelle kinase.

Front 1020

What does Pelle kinase do?

Back 1020

It phosphorylates Cactus causing it to be degraded

Front 1021

What happens when Cactus is degraded?

Back 1021

Dorsal is released and travels into the nucleus.

Front 1022

When is dorsal protein activated?

Back 1022

Only have 90 min post-fertilization.

Front 1023

What is the dorsal protein pathway?

Back 1023

Pipe --> Easter --> Spa --> Toll --> Pelle --I Cactus --> Dorsal

Front 1024

Where is dorsal located?

Back 1024

In the ventral nuclei.

Front 1025

What happened to nuclear dorsal in dorsalized embryos?

Back 1025

They lose their nuclear dorsal.

Front 1026

What happens to nuclear dorsal in ventralized embryos?

Back 1026

They have ectopic nuclear dorsal.

Front 1027

How is Spatzle activated?

Back 1027

It's cleaved by Easter

Front 1028

What happens to cells exposed to high nuclear dorsal?

Back 1028

They become mesoderm.

Front 1029

What represses Pipe and where is it repressed?

Back 1029

Gurken represses pipe and it's in the follicle cells.

Front 1030

Which cells express pipe?

Back 1030

Ventral follicle cells.

Front 1031

Where are the microtubules in AP distribution?

Back 1031

In the posterior side.

Front 1032

What happens in maternal bicoid loss?

Back 1032

The embryos lack anterior structures.

Front 1033

What does bicoid do?

Back 1033

It encodes a morphogen responsible for head structures.

Front 1034

What happens if you add bcd (bicoid) to a bcd mutant on the anterior side?

Back 1034

You get normal development.

Front 1035

What happens if you add bcd in the middle of a bcd mutant?

Back 1035

You get a head region in the middle.

Front 1036

What happens if you add bcd to the posterior of a wt embryo?

Back 1036

You get a two headed embryo.

Front 1037

Where is bcd mRNA located?

Back 1037

In the anterior portion of the oocyte.

Front 1038

What does Nanos pattern for?

Back 1038

The posterior

Front 1039

What do bicoid and nanos generate?

Back 1039

The hunchback/caudal gradient.

Front 1040

1 factor bicoid inhibits and 1 factor it activates:

Back 1040

1. inhibits caudal
2. bicoid is a trxn factor activating anterior genes like hunchback

Front 1041

What does bicoid activate?

Back 1041

Anterior genes such as hunchback.

Front 1042

What happens if bicoid mRNA reches the posterior?

Back 1042

There is a Nanos binding element in its 3'UTR that binds bicoid mRNA and degrades it.

Front 1043

How many bicoid-dependent enhancers are there?

Back 1043

66

Front 1044

Changes in Bd concentration are not sufficient to...

Back 1044

change the AP location of Bd-enhancers.

Front 1045

What is Runt?

Back 1045

A txnal repressor.

Front 1046

How are Runt and bicoid related?

Back 1046

They have inverse gradients to each other.

Front 1047

Runt is sufficient to...

Back 1047

antagonize Bd-dependent activation.

Front 1048

How do you affect the precise localization of the gradient of anterior genes?

Back 1048

A bicoid gradient establishes only a rough framework of the downstream genes. The precise localization is shaped by a network of Runt repressors that integrate feedback from a few key Bd target genes.

Front 1049

Name 3 gap genes:

Back 1049

1. Kruppel
2. Hunchback
3. Knirps

Front 1050

What do gap genes?

Back 1050

They lead to the patterning of the embryo and produce inhibitory interactions between each other.

Front 1051

Name 2 pair rule genes:

Back 1051

1. ftz
2. eve

Front 1052

You would like to perform experiments that explore the role of the genes Sall1 and Pitx1 in zebrafish (Danio rerio) development.

1. You decide to assay Sall1 expression by in situ hybridization. Unfortunately, you do not have a construct in your laboratory for the zebrafish Sall1 gene. However, a neighboring laboratory has an probe for the mouse Sall1 gene. Do you have enough information to determine if the probe against the mouse gene would work for in situ hybridization experiments in zebrafish? If so, describe why you think they would work. If not, what additional information do you need?

Back 1052

It is highly unlikely to work. However, you do not have enough information to say conclusively because you do not know how closely the fish and mouse genes resemble each other. If the probes extremely high conservation on the nucleic acid level (virtually identical), they experiment would work. On the other hand if the nucleic acid sequences are substantially different, the probe would not hybridize to the fish gene.

Front 1053

You would like to perform experiments that explore the role of the genes Sall1 and Pitx1 in zebrafish (Danio rerio) development. There are no probes available for Pitx1 so you need to make your own. You generate a plasmid that contains the entire cDNA sequence for the zebrafish Pitx1 gene. The first 10 bases of the "sense" strand are:5'-atggcctcat-3' What does "sense" mean?

Back 1053

The sense strand of DNA is the strand the corresponds to the mRNA strand (of course the mRNA will have U instead of T)

Front 1054

What would be the "antisense" RNA strand for the above sequence? 5'-atggcctcat-3'

Back 1054

5'augaggccau-3'

Front 1055

You prepare an in vitro transcription reaction to synthesize Digoxigenin-UTP labeled RNA for your probe. Which strand from above should be labeled? Why?

Back 1055

You must label the antisense strand so that it will hybridize to the mRNA (sense) within the embryo

Front 1056

You are deeply impressed with the Shapiro et al., 2004 study describing a likely role for Pitx1 in stickleback pelvic fin morphology. In those experiments, they showed that marine sticklebacks express Pitx1 in the pelvic region but freshwater sticklebacks have lost that expression. Is this a mutation in the gene or in a regulatory region? Explain your answer.

Back 1056

It is in a regulatory region. The gene was not mutated in freshwater sticklebacks. However, the gene not expressed in the pelvic fin region in the freshwater fish. Importantly, the gene is still expressed in other regions. This strongly suggests a mutation in a limb-specific regulatory module for the gene.

Front 1057

You are deeply impressed with the Shapiro et al., 2004 study describing a likely role for Pitx1 in stickleback pelvic fin morphology. In those experiments, they showed that marine sticklebacks express Pitx1 in the pelvic region but freshwater sticklebacks have lost that expression. What is the best evidence that this mutation specifically affects gene regulation (and the trait is not, for example caused by salt water)?

Back 1057

Remember that the F1 fish crossed from the marine and freshwater are all reared in captivity under identical conditions. They were then crossed with each other, generating F2 fish (again all under identical lab conditions). They then analyzed which portion of chromosomes are associated with pelvic fin reduction using SNPs specific to freshwater and marine alleles. They found that a region of the chromosome that contained Pitx1 accounted for a siginficant fraction of pelvic fin reduction. Since the fish are otherwise reared under identical condition, this strongly argues that something about the segment of DNA in that region confers pelvic fin reduction (it does not itself establish that it was Pitx1 - they inferred this by comparing with known mouse genes involved in hindlimb formation).

Front 1058

List the three germ layers. For each, give one example of one of the cell types that tissue will ultimately give rise to

Back 1058

Ectoderm (Brain/Skin), Mesoderm (Heart/Bone), Endoderm (Liver/Pancreas)
Many other correct examples for each (page 15 of your book)

Front 1059

Which of the following structure(s) is/are part of a typical vertebrate Bauplan? Notochord, malleus, pelvic spines, heart, pharyngeal arches, stapes

Back 1059

Notochord, Heart, Pharyngeal arches. The other structures are not embryonic. Moreover, the malleus is only found in mammals, the stapes is not found in sharks (rather a hypomandibular arch), pelvic spines are a highly derived structure found in sticklebacks

Front 1060

What is a general derivative of the first pharyngeal arch in all vertebrate animals?

Back 1060

The tongue, the jaw...

Front 1061

Hand2 is a highly conserved gene that plays essential roles in heart and pharyngeal arch development (embryos with a mutation in the Hand2 gene die early in development due to severe heart defects). Consistent with this role, the gene is expressed in both the heart and the pharyngeal arches. There is a well characterized cardiac enhancer and you wish to characterize a pharyngeal arch enhancer. You find a dozen candidate enhancer regions by performing genomic alignments of the Hand2 locus among multiple vertebrate species. How could you experimentally determine which (if any) of these regions actually functioned as pharyngeal arch enhancers?

Back 1061

Clone them into a DNA construct that contains a minimal promoter (ability to bind Pol2) and a reporter gene (LacZ or GFP). Generate transgenic embryos from these constructs. f the DNA construct causes the reporter gene to be expressed in the pharyngeal arch of the embryos, then it is acting as a pharyngeal arch enhancer.

Front 1062

In class, we talked about "deep homology" in Pax6, a transcription factor which is required for eye development in organisms ranging from flies to mammals. This pathway specifically targets photoreceptors. Photoreceptors transmit information to their targets - and, although we did not discuss, in class, this process to occurs with some homologous genes in both Drosophila and humans. One of these genes is another transcription factor, called Math5 in Drosophila and ATOH7 in humans. Recently, it was shown that a mutation in a long-range enhancer for ATOH7 underlies a form of human blindness caused nonsyndromic congenital retinal nonattachment (NCRNA) (Ghiasvand et al., Nat. Neuroscience 2011 vol. 14:578-86). This disease is autosomal recessive, and affects ~1% of the people in a remote village in Iran. The phenotype is nearly identical to that seen in Atoh7 null mouse embryos.

A) In mutated individuals, a large deletion of 6523 base pairs removes an enhancer.

Back 1062

This occurs in humans - you can't make transgenic humans - but since there is a mouse model system, could do in mice or another vertebrate system (the actual study used zebrafish). Generate a minimal promoter::reporter construct (reporter can be GFP or LacZ, which encodes beta-galactosidase) and clone in the 6523bp region. Generate transgenic embryos (this construct is randomly inserted into their genome) and see if it drives expression in the eye during embryonic development.

Front 1063

In class, we talked about "deep homology" in Pax6, a transcription factor which is required for eye development in organisms ranging from flies to mammals. This pathway specifically targets photoreceptors. Photoreceptors transmit information to their targets - and, although we did not discuss, in class, this process to occurs with some homologous genes in both Drosophila and humans. One of these genes is another transcription factor, called Math5 in Drosophila and ATOH7 in humans. Recently, it was shown that a mutation in a long-range enhancer for ATOH7 underlies a form of human blindness caused nonsyndromic congenital retinal nonattachment. Confusingly, there is already a well defined enhancer much closer to ATOH7 in mice (and conserved in humans) that drives enhancer activity in the exact same cell population at the same developmental time. What do you think this second enhancer could be doing?

Back 1063

Since the phenotype (in humans) mirrors a null phenotype (in a mouse model system), it provides strong genetic evidence suggesting that the NCRNA enhancer is critical for driving sufficient levels of ATOH7 expression for proper eye formation. Since the more proximal element also drives expression, perhaps both enhancers are needed to drive sufficient transcription of the gene.

Front 1064

You assay primary oocytes that are still in their primary cell cycle arrest. How many chromosomes are present at this stage?

Back 1064

The are 4 copies of each chromosome.

Front 1065

How many chromosomal copies are extruded in the first polar body? How many in the second polar body?

Back 1065

2, 1

Front 1066

You divide Xenopus oocytes into two groups. You do not treat the control group and you treat the second group with progesterone. What visible change would you expect to see after progesterone treatment?

Back 1066

Nuclear envelope breakdown (also called germinal vesicle).

Front 1067

You divide Xenopus oocytes into two groups. You do not treat the control group and you treat the second group with progesterone. You perform in situ hybridization on both of the above groups with an antisense probe for mos. What is this technique measuring? Which samples do you anticipate detecting a signal?

Back 1067

In situ hybridization measures gene expression. Mos mRNA is present throughout oogenesis so you expect to see staining in cells from both groups.

Front 1068

How does Maskin repress translation? How is this repression relieved? What in your view is the most important mRNA that must be translated upon progesterone translation? Justify your opinion.

Back 1068

Maskin binds to eIF4E, preventing it from being bound by eIF4G, which is the critical protein required to recruit the 40S ribosomal subunit for translational initiation. The repression is relieved when CPEB is phosphorylated, resulting in the extension of a longer poly-A tail, which then stabilized interactions between eIF4G and eIF4E, outcompeting interactions between eIF4E and Maskin. Mos is the most important protein because experiments showed that in the absence of new protein synthesis, the injection of Mos protein is sufficient to cause germinal vesicle breakdown.

Front 1069

The table below indicates the S-phase and M-phase for zygotes immediately upon fertilization and in the next two complete cell cycles are listed. Indicate the presence (+) or absence (-) of each of the component during each stage.

Back 1069

Fertilization (1-2 Cells) 2-4 Cells
S-Phase M-Phase
Cyclin B mRNA + +
Cyclin B Protein -** +
Cdc2 mRNA + +
Cdc2 Protein + +

**Immediately at fertilization, cyclin B protein is destroyed but they are quickly translated and cyclin B protein starts to build up. In this sense, this first S-phase is not so very different than the others.

Front 1070

You treat Xenopus oocytes with A23817, a calcium ionophore. What will be the first major visual change in the eggs?

Back 1070

It will cause the cortical granule reaction (visible fertilization envelope)

Front 1071

You assay primary oocytes that are still in their primary cell cycle arrest. How many chromosomes are present at this stage?

Back 1071

The are 4 copies of each chromosome.

Front 1072

What causes the vitelline envelope to swell after the cortical granule reaction?

Back 1072

The release of glycosaminoglycans

Front 1073

What are the four membrane fusion events during fertilization?

Back 1073

Acrosome fuses with sperm plasma membrane; sperm plasma membrane fuses with egg plasma membrane (fertilization cone); cortical granules fuse with egg plasma membrane; male and female pronuclei fuse (in some species the nuclear membranes surrounding the pronuclei break down before fusion).

Front 1074

You have used molecular biological techniques to generate a fusion protein of Cyclin B and GFP (CyclinB::GFP). You perform extensive tests that show that the fusion protein is indeed as active as regular Cyclin B and that it also glows green under the appropriate fluorescent filters. You inject large (but not toxic) amount of this mRNA into oocytes immediately before fertilization.

A. What do you anticipate seeing as you watch these cells under a fluorescent microscope over the next several hours?

Back 1074

Cyclin B protein is degraded at the end of mitosis. Early cell cleavage is successive cycles of M-S-M-S….The cells should glow green, turn off, glow green, turn off, etc. This would only stop at the onset of zygotic transcription (or earlier if the mRNAs degraded before then).

Front 1075

You have used molecular biological techniques to generate a fusion protein of Cyclin B and GFP (CyclinB::GFP). You perform extensive tests that show that the fusion protein is indeed as active as regular Cyclin B and that it also glows green under the appropriate fluorescent filters. You inject large (but not toxic) amount of this mRNA into oocytes immediately before fertilization. What would you expect to see if you also injected these cells with cycloheximide and why?

Back 1075

The Newport and Kirschner experiment showed that CHX injection causes cells to quickly arrest in S-phase. Cyclin B protein levels are absent (or very low) during S-phase even in a normal cell. However, CHX blocks protein translation so none of the RNA in the cell will be translated and you will not see any green protein.

Front 1076

Sketch a just-fertilized C. elegans egg at the single cell stage. Indicate that following components: sperm binding site, centrosome, anterior, posterior.

Back 1076

Sketch should show sperm at future posterior of eggs (which is dictated by the centrosome from the sperm)

Front 1077

Sketch the same egg as above - but this egg is derived from a Par-2 null mutant mother. Can you predict the future anterior and posterior regions? Why or why not?

Back 1077

Sketch should show sperm binding site and centrosome - but Par-2 mutants are symmetrical with no anterior or posterior axis. So you could not label these regions.

Front 1078

Which of the following crosses in an organism having a recessive mutant allele in a hypothetical maternal effects gene called 'Maternal' would result in an embryonic phenotype? Here, '-' refers to the mutant allele while '+' is the wild-type allele.
a) Mat +/- eggs x Mat +/- sperm
b) Mat +/- eggs x Mat -/- sperm
c) Mat -/- eggs x Mat -/- sperm
d) Mat -/- eggs x Mat +/- sperm
e) Mat +/+ eggs x Mat -/- sperm

Back 1078

C,D. Maternal effects phenotypes depend on the genotype of the mom (eggs) while the genotype of the embryo itself or the paternal chromosome is irrelevant.

Front 1079

For each cross(es) above generating a maternal effects embryonic phenotype, what fraction of embryos should contain a phenotype?

Back 1079

For all crosses, maternal effects mutations should be 100% penetrant.

Front 1080

The Rappaport sand dollar experiment presented in class support a model where cleavage furrows are generated by the centrosomes and do not require chromosomes or a mitotic spindle. If those components had been required, what would have been the final outcome of the experiment?

Back 1080

Only three cells.

Front 1081

Once present, are the micromeres of a sea urchin specified or determined? Justify your answer.

Back 1081

Determined. When transplanted to the animal side of a sea urchin embryo, they still retain their identity (indeed they respecify the animal cells into endoderm and mesoderm, creating a mirror-image embryo).

Front 1082

Sketch a cell indicating where you would expect to find following components of the Wnt pathway in a cell actively receiving a Wnt signal (membrane, cytoplasm, nucleus, outside the cell): Wnt, Frizzled, B-catenin.

Back 1082

Wnt is a secreted ligand (synthesized in cells and secreted outside the cell - so present in the cytoplasm and mostly extracellularly). Frizzled is a receptor (membrane), Beta-catenin is in the cytoplasm in the absence of Wnt. In the presence of Wnt signaling, B-catenin moves to the nucleus.For more info., see pages 92-93 of the book.

Front 1083

You remove the EMS blastomere from a C. elegans embryo and culture in isolation:
A. What will be the identity of the two daughter cells? What cell type(s) would they give rise to?

Back 1083

Both cells will be MS. MS cells give rise to mesoderm

Front 1084

Sandwich the EMS blastomere between two P2 blastomeres (obviously derived from two different embryos). What do the EMS blastomores give rise to? How could you experimentally show these are descendants of EMS and not P2?

Back 1084

Both EMS daughter cells will become E. These give rise to endoderm. You could look for the presence of endodermal markers, but would also have to have some type of lineage tracing strategy (injecting a fluorescent dye, mRNA encoding GFP….) since in an experiment you don’t know ahead of time what will happen.

Front 1085

What happens to microRNAs if there is no Dicer around?

Back 1085

MicroRNAs will not be processed into the short single-stranded 22nt form of an active miRNA. This will have the effect of making all miRNAs inactive.

Front 1086

You have identified a maternal transcript that is very efficiently cleared during MZT in zebrafish embryos. You hypothesize that it is controlled by MiR-430 as we discussed in class. Design an experiment that will allow you to compellingly demonstrate that this transcript is SPECIFICALLY controlled by MiR430. Your experiment should include experimental reporter constructs and controls and you should indicate what portion of the transcript is being tested in your constructs.

Back 1086

miRNAs bind to the 3'UTR so the first thing to do is to look at the sequence of the 3'UTR (available in public databases) and see if there is homology to the MiR430 seed region. If no homology, it suggests this is being regulated in an MiR430-independent mechanism. If, as expected, there is one or more seed regions, you clone the 3'UTR of your gene downstream of a construct containing the coding region for a reporter gene e.g. GFP - you call this construct A. You also clone the same 3'UTR into the reporter but first generate point mutants in the MiR430 binding regions (construct B). If GFP expression in construct A goes away at MZT but persists at MZT in construct B, the experiment supports your hypothesis. A nice control to ensure your RNA construct isn't simply being degraded is to con-inject a second mRNA construct encoding a reporter that does not quickly degrade (has no mIR-430 binding regions and is known to have strong expression for a long itme - could be the dsRed control.

Front 1087

You wish to identify the set of mRNAs that are activated zygotic transcription in Xenopus embryos. Describe two experimental strategies that would allow you to do this.

Back 1087

Divide a pool of early embryos into two pools. One fraction (Fraction A) is untreated. Treat the other fraction (Fraction B) with a transcriptional inhibitor (e.g. alpha-amanatin). Extract total RNA from both fractions and process it for massively high-throughput RNA-sequencing (RNA-seq). After you get the data, which contains tens of millions of independent reads, you look for reads corresponding to intronic sequences. Such reads represent nascent, still unspliced transcripts that are indicative of zygotic transcription (maternal transcripts were spliced before fertilization). An alternative strategy would be to look for transcripts present in Fraction A that are either reduced no longer present in Fraction B.

Front 1088

Do you think MiR430 is processed from a maternal or zygotic transcript? Why

Back 1088

It is zygotically transcribed. If this were a maternal transcript, it would degrade maternal transcripts way too early (before zygotic transcription).

Front 1089

Skn-1 is a master regulator of EMS fate. It activates the transcription factors Med1 and Med2, which in turn activate End1 and End3. Both E and MS have Med1, 2 present but only E activates End1 and End3. Why?

Back 1089

Because these genes are also regulated by the transcriptional repressor Pop-1. If Pop-1 is around it prevents End1,3 transcription even in Med1,2 are also present. MOM (Wnt) activity secreted by P2 only cause a depression of POP1 in the EMS region directly adjacent to P2. As a result when EMS divides, the blastomere closest to P2 becomes E while the other cell (MS) represses Endoderm fate and becomes mesoderm (driven by Tbx35).

Front 1090

Skn-1 mRNAs are maternal transcripts that are present in oocytes and then translated only in a subset of cells after fertilization. SKN-1 protein is present in both EMS and P2.
A. Why doesn't SKN-1 protein cause P2 to become E/MS in wild-type embryos?

Back 1090

Because PIE-1 prevents Pol2 transcription - Skn1 is not active.

Front 1091

You are interested in understanding how Skn-1 mRNA is selectively translated in only two of the four blastomeres.You decide to test two ideas. One is that Skn1 mRNA is localized to the P1 blastomere during the transition from 1-2 cell stage (this could in principle be by active recruitment of the mRNA in P1 or destruction of the mRNA in AB). The second is that the mRNA is present everywhere but is translationally repressed in AB. Design experiments to test the ideas in A and B.

Back 1091

You could do in situ hybridization with an antisense probe against Skn1 to detect mRNA expression in WT 1 and 2 cell stage embryos. If mRNA is localized to the posterior, it would support idea #1. If mRNA is localized throughout the embryo, it indicates idea #1 is wrong (in fact in the literature mRNA is localized throughout the embryo). To test translational represssion, you could take the Skn1 3'UTR and clone it downstream of a GFP reporter. Then use this to make transgenic embryos and see of GFP co-localizes to P1. The actual mechanism by which Skn1 is translationally repressed is unknown.

Front 1092

Cactus is a maternal effects gene that is essential for dorsal-ventral patterning. Describe its function?

Back 1092

Cactus is present in the cytoplasm and binds to the Dorsal transcription factor, preventing it from entering the nucleus by blocking the nuclear localization signal. Upon Toll receptor activation, the Pelle kinase phosphorlyates Cactus, causing it to be degraded. Dorsal then enters the nucleus

Front 1093

. Predict the phenotype of a Cactus null mutation on the embryo.

Back 1093

Dorsal expression in all nuclei -> complete ventralization

Front 1094

Generate germline mosaic adult flies by grafting Cactus mutant pole cells onto wild-type embryos, whose own pole cells have been removed. What would be the phenotype, if any, for the resulting embryos derived from a cross of these mosaic females with wild-type males?

Back 1094

Will make a mosaic fly that has produces Cactus -/- oocyte with wild-type (Cactus +/+) follicle cells. All fertilized eggs from this mosaic fly will produce ventralized embryos because Cactus is required in the egg only.

Front 1095

Generate germline mosaic adult flies by grafting wild-type pole cells onto Cactus mutant embryos, whose own pole cells have been removed. What would be the phenotype, if any, for the resulting embryos derived from a cross of these mosaic females with wild-type males?

Back 1095

Will make a mosaic fly that has a wild-type oocyte (Cactus +/+) surrounded by Cactus -/- follicle cells. All fertilized eggs from this mosaic fly will be normal because Cactus is not required in the follicle cells.

Front 1096

You intercross Gurken +/- male and female flies to produce Gurken null mutant embryos. What is the predicted phenotype? Why?

Back 1096

The resulting Gurken null embryos will have a wild-type phenotype because the mothers were Gurken +/-. Gurken is a maternal effects gene, and so the genotype of an embryos mother gives the phenotype of all of its progeny.

Front 1097

What would be the phenotype of progeny generated by crossing Gurken -/- female flies with wild-type males?

Back 1097

All embryos would be ventralized because a lack of Gurken will cause a lack of Torpedo receptor activation in dorso-anterior follicle cells. Without Torpedo, the follicle cells will not repress Pipe, and they will therefore express Pipe. Expressing Pipe causing the initiation of a protease cascade that is activates the Spatzle ligand, allowing it to activate the Toll receptor, causing Cactus to be degraded and Dorsal to then translocate to the nucleus. N.B. The Dorsal protein is not translated until about 90 minutes after fertilization. So the D-V gradient is already in place before fertilization - but in an mRNA-fashion.

Front 1098

What is the dorsal mutant phenotype? What is dorsal? What is the germ layer composition of the region of cells receiving the highest dose of dorsal?

Back 1098

The dorsal mutant phenotype is a dorsalized embryo. Dorsal is a transcription factor that acts as a morphogen to activate ventral cell populations in a dose-dependent fashion on the future ventral side of the embryo. The cell receiving the highest dose of dorsal become mesoderm.

Front 1099

How is bicoid mRNA localized anteriorly? If mRNA is restricted anteriorly, how would you predict a protein gradient would be established?

Back 1099

The 3'UTR of Bicoid mRNA attached to dyneins (microtubule "motor" proteins that carry things towards the minus end of microtubules). It also requires the “escort’ proteins Expuerantia and Swallow. Recall that Gurken signaling to Torpedo in posterior of the cell causes microtubule orientation to be arranged such that the minus ends are all at the anterior end of the oocyte. Not specifically applying to this answer but related is that non-anterior Bd mRNA is translationally repressed by Nanos.

Front 1100

You identify a previously unknown gene, Drosophillin, that is expressed several vertical stripes along the A-P axis of a syncitial stage Drosophila embryo. Zygotic mutants for this gene exhibit a novel phenotype, and you decide to undertake a research project that examines how the gene is transcriptionally activated. You examine gene expression (using which technique?) and determine that the one of the stripes of gene expression (stripe 1) occurs in the anterior embryo in a domain overlapping Hunchback (Hb) and Biocid (Bd) protein expression domains (which technique measures this?). The gene is not expressed until several hours after the onset of Hb and Bd protein expression, and based on the correlation in expression patterns, you hypothesize that stripe 1 of Drosophillin is a directly activated Hb, Bd (or both). Describe a genetic experiment that would allow you to determine if Drosophillin is genetically downstream of either of these?

Back 1100

You examine gene expression using in situ hybridization. You use immunostaining to detect Hb and Bd protein expression using antibodies against Hb and Bd with fluorescent-conjugated secondaries. Examine the expression of Drosophilin mRNA in Hb and Bd mutant embryos using in situ hybridization. If there was a loss of the imaginary Drosophilin stripe 1 gene expression in 1 or both of the mutants, it indicates that it is genetically downstream of that gene. For example if Stripe 1 is absent in Bd mutants, then it is genetically downstream of Bd.

Front 1101

What is the best technique for determining if an early embryonic cell is in the S-phase at a given timepoint?

Back 1101

Labeling DNA with radioactive nucleotides.

Front 1102

What kind of cells are nurse cells?

Back 1102

Germ cells

Front 1103

What is Par-2 involved in?

Back 1103

Regulating AP polarity in c elegans

Front 1104

The P blastomere in c elegans is ______ specified. The E blastomere in c elegans is ____ specified.

Back 1104

Autonomously; conditionally

Front 1105

The acrosome reaction and the cortical granule reaction are derived forms of...

Back 1105

exocytosis

Front 1106

You fertilize sea urchin eggs then immediately inject them with a calcium chelator (something that strongly binds all free calcium). You do this quickly and the injection is completed about 5 seconds after fertilization. What do you think happens?

Back 1106

The embryos will likely have polyspermy because this disrupts the cortical granule reaction.

Front 1107

A morphogen is...

Back 1107

a substance that can determine cell fates in a concentration-dependent manner.

Front 1108

4 factors that participate in the bicoid gradient:

Back 1108

1. Exuperantia
2. Vasa
3. Nanos
4. Dynein

Front 1109

What protein directly binds to the cap of the mRNAs?

Back 1109

eIF4E

Front 1110

What molecule acts as a direct trxnal activator in reponse to a canonical Wnt ligand?

Back 1110

Beta-catenin

Front 1111

Scientists believe that limbs are evolutionarily derived from fins. What evidence would you need in order to be convinced that this was consistent with Von Baer's Laws?

Back 1111

The embryonic origin of both fins and limbs would need to be the same.

Front 1112

Based on what we discussed in class, do you think MiR-430 is maternally or zygotically transcribed? Why?

Back 1112

It is zygotically transcribed because it clears out maternal transcripts. If expressed maternally, it would likely clear out maternal transcripts even before fertilization.

Front 1113

You identify Drosophila embryos with a distinct phenotype. You determine that they have a mutation resulting in constitutive activation of the Toll receptor (it is always on). What is the phenotype and why?

Back 1113

These embryos will be completely ventralized because activating the Toll receptor (normally in the ventral region by activated spatzle) causes nuclear Dorsal accumulation.

Front 1114

You identify Drosophila embryos with a distinct phenotype. You determine that they have a mutation resulting in constitutive activation of the Toll receptor (it is always on). Do you think this would be a recessive mutation? Why?

Back 1114

No - it will not. If it is turning a receptor on (instead of getting rid of activity), it would be dominant.

Front 1115

You cross pipe +/- females with pipe +/- male flies. What will the resulting embryos look like? Why?

Back 1115

All embryos will look normal. This is because pipe is a maternal effects gene (once the embryos grow up, the progeny of pipe -/- embryos will all be dorsalized.

Front 1116

You wish to ID the set of mRNAs that are activated zygotic trxn in xenopus embryos. Describe a specific experimental strategy that would allow you to specifically ID these mRNAs while excluding maternal RNAs?

Back 1116

Option 1: Treat embryos with an inhibitor of trxn and then perform RNA-seq on these embryos and compare the sequences to control, untreated embryos. Transcripts absent in the inhibitor but present in the control group are likely to be zygotic transcripts.
Option 2: Perform RNA-seq on oocytes and specifically look into intronic sequences (will occur only transiently since they are spliced out of primary transcripts but will only occur in newly transcribed, zygotic mRNAs.

Front 1117

What is the biggest difference in the specification of the AP axis between c elegans and drosophila? Think big picture only.

Back 1117

The posterior axis of c elegans is defined by the sperm (which will go to the nearest oblong end from its entry point). In contrast, the AP axis of flies is specified during oogenesis.

Front 1118

Polar bodies are extremely small compared to the oocyte. Which organelle/structure is directly responsible for defining the size of the polar body?

Back 1118

The centrosome.

Front 1119

You generate oocytes that only contain a mutated form of CPEB that cannot be phosphorylated but is otherwise normal. You then treat them with progesterone. You come back a few hours later and look under the microscope. What do you see and why?

Back 1119

No change - the oocytes will fail to undergo NEB. This is because if CPEB can't be phosphorylated, Mos will remain trxnally repressed. This will prevent the activation of the downstream events that ultimately generate an active MPF that drives NEB.

Front 1120

You are interested in understanding how Dicer is regulated in zebrafish. List the genotype of the crosses and only the informative genotypes that would allow you to determine if embryonic development requries maternal transcripts only zygotic transcripts only or materal and zygotic transcripts. How would you analyze the data to make your conclusion?
Maternal only:
Zygotic only:
Maternal and zygotic:

Back 1120

Maternal only: Female Dicer -/-
(don’t need to specify that she was generated by
transplantation) x Male Dicer +/+;
Zygotic only: Dicer +/-
males x and Dicer +/-
females to generate zygotic nulls (Dicer -/-
)
Maternal and zygotic: Female Dicer -/-
x Male Dicer +/-
to generate Dicer -/-
embryos.
The key would be looking at the severity of the phenotypes. If maternal only, the
maternal and zygotic mutation should be no worse. Likewise, if zygotic only, the
maternal zygotic mutation should be no worse.

Front 1121

You identify a Bicoid-responsive enhancer and confirm that it is expressed in
Drosophila embryos by generating transgenic embryos containing an enhancer::LacZ
reporter.
A) What do you think would happen to reporter gene expression if you crossed the
transgenic into a mutant embryo lacking any Biocid?

Back 1121

Reduced or absent reporter activity

Front 1122

You identify a Bicoid-responsive enhancer and confirm that it is expressed in
Drosophila embryos by generating transgenic embryos containing an enhancer::LacZ
reporter.
What do you think would happen to reporter gene expression if you crossed the
transgenic into a mutant embryo lacking any Runt?

Back 1122

The reporter activity would be expanded

Front 1123

You identify a Bicoid-responsive enhancer and confirm that it is expressed in
Drosophila embryos by generating transgenic embryos containing an enhancer::LacZ
reporter. What do you think would happen to reporter gene expression if you crossed the
transgenic into a mutant embryo expressing high levels of Runt everywhere?

Back 1123

The reporter activity would be reduced or absent

Front 1124

When P2 divides in a Pie1-/-
embryo, the C blastomere adopts an MS fate while P3 becomes another
E cell.
A. This should be slightly surprising to you (or at least you should be able to think of a plausible
alternative fate). If I hadn’t told you this, what fate do you think P2 might have adopted?
Explain what you think might be going on.

Back 1124

In the absence of Pie-1, Skn1 activity will not be inhibited. Therefore, Skn1 will convert P2 into
another EMS-like cell. Without a P2 blastomere, it would seem likely that there might not be MOM1
around to induce an E fate. Therefore, I have predicted the P2 would divide to form two MS cells. The
phenotype suggests that the EMS-like cell retains MOM1 expression and further suggests that MOM1
can self-induce an E fate.

Front 1125

When P2 divides in a Pie1-/-
embryo, the C blastomere adopts an MS fate while P3 becomes another
E cell. Propose a detailed experiment that would allow you to test this

Back 1125

Several credible experiments. One would be to dissect the EMS-like cell and make an explant containing it as well as a wildtype EMA. If the EMS-like cells retains MOM1, it could cause the wild type EMS cell to produce an E cell (MOM1 would self-induce this fate). You would need to use a marker of E cell identity (eg End1) and describe how you would detect it. (eg, in situ hybridization. Lots of variations on this.

Front 1126

When P2 divides in a Pie1-/-
embryo, the C blastomere adopts an MS fate while P3 becomes another
E cell.
You decide that you need a faster way of interpreting data. Describe the features of a
transgenic worm that would allow you to visually identify the E cell in living worms?

Back 1126

You could generate a minimal promoter GFP (or some other fluorescent protein to allow for detection
in living worms) construct that also included an endoderm enhancer from an endodermal gene (such
as End1). You received partial credit if marking it with POP1 even though POP1 is actually not unique
to that cell type and is an inductive signal.