Bio 349: Exam 2

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.