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1126 Cards in this Set

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. 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.
The animal cap will be converted to mesoderm. The endoderm will give rise to endodermal derivatives only.
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?
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).
List two of the general mesodermal inducing factors?
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.
What is the primary purpose of the Nieuwkoop center?
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.
Describe the role of Dishevelled in the Wnt pathway. Can it be used to determine the difference between canonical and non-canonical signaling?
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.
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.
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).
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?
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.
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?
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).
What is the archenteron?
The embryonic gut; it is formed during gastrulation
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?
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.
What is an inhibitory signal in the somite that helps guide trunk neural cell migration? Where is it expressed?
EphrinB1 which is expressed in the posterior somite.
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.
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.
What happens when you electroporate chick embryos with Sox9 in the neural tube?
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.
Where do neural crest cells form in relation to the neural plate of the epidermis?
The form at the boundary of the neural plate and epidermis.
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?
a) anencephaly b) spinal bifida
Describe the 3 primary vesicles of the brain. What will each of them become?
prosencephalon = forebrain
mescencephalon = midbrain
rhombencephalon = hindbrain
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?
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).
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?
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.
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?
FoxD3 needs to be downregulated.
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?
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.
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.
-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).
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.
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).
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?
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?
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.
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.
What would you predict would be the phenotype of a netrin mutant mouse?
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.
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?
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.
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?
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.
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?
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.
What do you think would happen if you forced expression of ephrinA2 throughout the entire limb bud?
-This experiment hasn’t been done. But one prediction would be the complete absence of dorsally projecting motor axons, which are repelled by ephrins.
Based on material covered in lecture, what do you think would happen to neuronal migration in a Robo1 mutant embryo? Why?
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.
What is the predicted phenotype of Robo3 null embryos? Why?
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.
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?
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.
How is the gray crescent formed?
Cortical rotation
What are the domains of the blastula?
Animal cap, marginal zone, and vegetal base
What is the animal cap made of?
Ectoderm
What is the marginal zone made of?
Mesoderm
What is the vegetal base made of?
Endoderm
What happens when the sperm enters the frog egg?
There's a cortical rotation of 30* which makes the gray crescent.
What do microtubules do following sperm entry in a frog egg?
They're negative at the centriole and grow outward with positive ends. These positive ends move up along the sides.
What does beta-catenin do?
It's the dorsalizing signal.
What happens to embryos when they're severely depleted of beta catenin?
They become severely ventralized = belly pieces.
What's the experiment you use to find out if beta-catenin plays a role in DV patterning?
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.
What happens if you inject Wnt into ooctyes before fertilization?
They become super dorsalized.
Why should you inject Wnt before fertilization to see dorsalization?
Wnt is a maternal factor.
What does Wnt do when the sperm gets inside the egg?
It rotates 30* and activates the canoncial Wnt pathway.
Where does the wnt come from in the DV axis formation?
From the vegetal cells.
How many cells in the blastula?
20K
Name 3 mesodermal markers?
Xbra
chordin
FGF8
What happens when you stick a wt cap on a wt base of a blastula?
You get an increase of FGF8 and a decrease of chordin.
Mesoderm induction requires what two signals?
Vg1 and VegT
What happens in Vg1 and VegT mutants?
You get belly pieces.
Where is Vg1 and VegT located in the blastula?
In the bottom vegetal portion.
Another name for Vg1
Nodal
What does Vg1 do?
It activates Smad 2,3 trxn factors.
What happens if you stick a wt animal cap on a vg1- base?
You get a severe loss of mesoderm markers. No chordin and reduced Xbra and fgf8.
What does chordin do?
It's a BMP antagonist
What's the nieuwkoop center?
The dorsal vegetal region of the blastula
What factors are high in the Nieuwkoop center?
beta-catenin and VegT/Vg1. There's also a higher level of Xnr and a gradient of BMP4.
What specific structure is in the Nieuwkoop center?
The organizer.
What does beta-catenin in the Nieuwkoop center lead to? Show the pathway?
beta-catenin w/ VegT/Vg1 --> high nodal --> organizer
What's the pathway for VegT/Vg1 alone?
VegT/Vg1 --> low Nodal --> Ventral mesoderm
What happens when you transfer a doral blastopore lip onto a blastula?
You make a two-headed monster
What does the blastopore lip do?
It is the organizer and induces host tissue to form the body axis.
4 properties of the organizer:
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
What does Noggin antagonize?
BMP
What does BMP do?
It's involved in ventral patterning
Where is noggin expressed?
In the organizer and notochord
What happens if you inject BMP4 into a blastula?
You get a belly piece.
What is the default of the organizer?
Neural ectoderm
What is a great neural marker?
Sox2
What's the initiating step of gastrulation?
The marginal zone cells become bottle shaped.
How does the blastopore form?
Via invagination at the marginal zone.
What is the archenteron?
The beginning of the gut of the embryo.
Name the example of epiboly in gastrulation.
The ectoderm engulfs the endoderm via epiboly.
What is the convergent extension?
It's the stretching and organization of the organizer.
Explain what a Keller explant is:
You take off the organizer and you put it under a microscrope and you will see that it will undergo convergent extension.
What molecules are involved in convergent extension?
Non-canonical Wnt signaling is used to induce cell polarity by activating Dsh (Disheveled).
What can convergent extension live without?
Canonical Wnt signaling
Where does the head mesoderm come from?
The mesoderm that is pushed up from the blastopore.
How can you make multiple heads from head mesoderm?
You inject Cerberus which inhibits Wnt8, BMPs, Nodal and causes more head mesoderm.
Where is Cerberus expressed?
In the anterior endomesoderm.
What happens if you mess with the anterior endomesoderm?
You get headless embryos.
What happens to Lim1 -/- mutants?
They don't have heads.
How common are neural tube defects?
1/1000 live births
What happens in primary neurulation?
Neural plate folds to generate hollow neural tube.
What happens in secondary neurulation?
A solid cylinder of cells cavitates to form hollow neural tube.
What are the 3 structures of the neural plate?
1. The neural plate
2. Neural crest
3. Epidermis
What is the neural plate made out of?
Ectoderm
What drives the folding of the neural tube?
Microtubes and actin.
How does the cephalic region of the neural tube close?
It zippers toward the anterior neuropore.
What happens if your neural tube fails to close?
You get spinal bifida or anencephaly.
How does the neural tube close at the midbrain?
A process called buttoning created by cellular bridges.
What factor is needed for neural tube closure?
B9 (folic acid).
Where is the folate receptor expressed?
In the dorsal neural tube.
How can you decrease neural tube defects?
By taking folic acid at the beginning of the pregnancy. It decreases it by as much as 70%.
what is Mthfd11?
An enzyme in the pathway that makes folic acid available to the cell.
What happens in Mthfd11 mutants?
They have an open hindbrain. Their neural tube doesn't close.
What is the result from the neural tube closing?
Occlusion of the neural tube allows pressure to swell in the lumen of the developing brain.
In zebrafish, what causes the ventricles to swell?
The Na/K ATPase which causes an osmotic gradient.
Name the 3 developmental structures of the brain:
1. prosencephalon
2. mesenchephalon
3. rhombencephalon
forebrain comes from:
prosencephalon
midbrain comes from:
mesencephalon
hindbrain comes from:
rhombencephalon
What happens to Otx2 mutants?
They are missing their forebrain and midbrain.
What happens to Gbx2 mutants?
They're missing their metencephalon (rostral hindbrain).
What does Gbx2 do?
It restricts Otx2 expression
What does the Otx/Gbx border do?
It sets up the mid/hindbrain boundary.
What does Gbx2 establish?
The metencephalon
What does Otx2 establish?
The mesencephalon
What is the isthumus?
The major molecular organizing center in brain development.
Another name for rostral?
Anterior
Another name for caudal?
Posterior
What is Hoxb8 a marker for?
caudal (posterior) neural identity
Where is RA expressed?
In the caudal paraxial mesoderm
What does RA induce?
RA from the paraxial mesoderm induces caudal neurons.
What is Shh a morphogen for?
It's a ventral morphogen
Where is the highest level of Shh?
In the notochord.
What is the Shh pathway?
Shh binds --> Patched --I Smo --> Upregulation trxn
How can you visualize the Hh gradient?
You make a mouse with Shh::GFP
What happens to Ptch1-/- mutants?
They way overexpress Shh.
What happens to Shh mutant embryos?
They lack ventral neurons
Name 2 factors induced by high Shh.
Nkx6.1 and Nkx2.2
Name two factors induced by low Shh.
Pax7 and Dbx1
What factor is repressed in high Shh?
Pax6
How do you electroporate something?
You shock the cells and DNA will move to one side.
What does ectopic Nkx6.1 do?
It causes the loss of Dbx2
What does ectopic Dbx2 do?
Leads to loss of Nkx6.1
What is the fourth germ layer?
Neural crest
Where are neural crest cells born?
In the dorsal neural tube.
What is EMT?
The change that neural crest cells undergo called the epithelial to mesenchymal transition and then they migrate to the periphery.
What is RhoB?
It is the factor expressed during neural crest initiating migration
When do crest cells express RhoB?
When they undergo EMT.
What do neural crest cells express just prior to migration?
Sox9.
What do Sox9 and RhoB do?
They induce Neural Crest differentiation and EMT.
What do neural crest cells end up generating?
Sensory and sympathetic neurons.
What is the sclerotome?
They'll make sensory and sympathetic neurons.
Trunk crest cells only migrate through...
that anterior half of somites.
What is expressed in the posterior half-somite?
Ephrin B1
What do crest cells express?
EphB3.
What repells crest cells from going into the posterior portion of the somite?
Ephrin B1.
What is the stripe assay?
The way to show that neural crest cells are repelled by ephrin B1.
What do path 2 crest cells generate?
They make melanocytes.
Where do path 2 crest cells migrate?
Between the dermamyotome and the surface ectoderm.
What does FoxD3 do?
It's essential for generating NCC and says on Pathway 1.
What does FoxD3 repress?
A gene called Mitf, which is the master regulator of melanocyte fate.
What does Mitf do?
It's the regulator of melanocyte fate.
Mitf1 is essential for...
melanocyte stem cells.
Why do we turn gray?
the cells expressing mitf1 are lost.
Where do the pharyngeal arches come from?
The migration of specific streams of cranial neural crest cells.
Pharyngeal arches are populated by what kind of cell?
Neural crest cells.
What happens to pdgfra mutants?
They're defective for neural crest migration
What is Pdgf?
It's a cranial neural crest chemoattractant.
What is the anatomy of an arch?
Muscle in the middle, wrapped in crest cells with endoderm on top and ectoderm on the bottom.
What does the first pharyngeal arch contribute to?
Malleus
Incus
Mechel's cartilage
What does the second pharyngeal arch contribute to?
The stapes and styloid process
What does the 3rd pharyngeal arch make?
Hyoid bone
What do pharyngeal arch 4 and 5 make?
Thyroid cartilage
When genetically fate mapping a mouse, where do the facial structures come from?
Neural crest cells and mesodermally derived cells.
Where do facial prominences come from?
The first pharyngeal arch.
3 facial structures from the 1st pharyngeal arch.
1. midline facial skeleton
2. upper jaw and palatal skeleton
3. lower jaw
What occurs at the border of Shh and Fgf8?
It's the distal tip of the upper break
What happens if you make extra Fgf8-Shh boundaries?
You get multiple ectopic beaks in a bird.
What makes up an axon growth cone?
Filopodia and lamellipodia.
What are filopodia?
The microspike fingers of the growth cone
What are lamellipodia?
The webbing of the growth cone.
What does the axon growth cone do?
It senses the environment and connects to the appropriate targets.
What is pathway selection?
Axon chooses route through the environment to its target.
What is target selection?
Axon recognizes target and initiates synaptogenesis.
Addess selection:
Synapses are strengthed and lost
Name the two structures of the neural tube that contribute to motor axon projections?
Lateral motor column
Medial motor column
Where does the medial motor column project to?
The dermamyotome
What are the subcategories of the lateral motor column?
Lateral subdivision --> dorsal
Medial subdivision --> ventral
Where do LMCl project to?
Dorsal musculature
Where do LMCm project to?
Ventral musculature
Ephrins are concentrated in what part of the limb bud?
The ventral part
EphA4 expressing axons project...
dorsally
EphA4 is sufficient to drive axons...
dorsally.
What does Lim1 do?
It activates Eph4A.
What does Isl1 do?
It represses Eph4A.
What is the pathway for LMCl axons?
Lim1 --> Eph4A --> dorsal projection
What must axons to at the ventral midline?
They must project to the ventral midline and only cross once.
What are Robo and Slit?
Slit is a ligand in the midline of drosophila.
Robo is a receptor on axons.
What happens to slit mutants?
Axons never leave the midline.
How do Slit and Robo interact?
Robo1 is a receptor and when Slit binds to it, it inhibits DCC.
Slit binding to Robo1 results in what?
Chemorepulsion
What happens when Robo3 is expressed?
It inhibits Robo1 so now DCC can work and the axon can cross the midline.
What does Netrin bind to?
DCC.
What happens after the axon crosses the midline?
Robo1 turns back on and blocks DCC chemoattraction
What does the brainbow help you do?
You can see the neuronal connections based on the random turning on of GFPs.
How does brainbow work?
It's a Cre-base recombination resulting in expression of 3 different fluorescent proteins.
What is the axon chemoattractant?
Netrin binding to DCC receptor.
What is the axon chemorepellant?
Slit binding to Robo1.
Where does somitic formation occur?
In the presomitic mesoderm
What is the somite clock?
The time period of somite formation.
What is the Notch pathway?
Delta (membrane-bound ligand) --> Notch (receptor) --> Lfng --I Hairy --I trxn of Hes and Lfng
What is the Clock and Wavefront model?
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.
What does the clock part of the Clock and Wavefront model do?
It regulates the periodic generation of somites.
What does the Wavefront part of the model do?
It defines the region that will become a somite.
What is the central oscillator controlled by?
Notch/Delta signaling
Another name for Hairy...
Hes
What is the oscillating expression of Hairy?
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.
How does Notch affect the Wavefront model?
It provides signaling for the border information. Notch signaling is sufficient to cause segmentation.
What happens to Delta-like 3 mutants?
They have defects in somite formation, leading to rib fusions.
What signaling is necessary for somite formation?
Notch signaling
What happens to MESP2 mutants?
They have defects in ribs and vertebrae.
What happens to Lfng mutant mice?
They lose Notch cycling
How does Lnfg aid in oscillating expression?
Lnfg is a negative regulator of Notch, but is very unstable. So it leads to an oscillating signal.
How does Wnt signaling affect somite segmentation?
It helps position determination front.
What does Axin2 do?
Inhibits Wnt activity and is short-lived.
If Notch can't oscillate, how does Wnt react?
They still show axin2 expression oscillation so Wnt pathways target genes still oscillate.
What happens if beta-catenin can't be degraded? This is relating to somite formation.
There's no Wnt activity. There are no visible somites.
What happens to somite formation when there's no beta-catenin?
Oscillation still occurs, but it's abnormal. Wnts do not appear to be a pacemaker for oscillations.
What regulates the size of somites?
Fgf
What happens if you stick an FGF-soaked bead near the undetermined zone?
It makes a smaller somite.
How is FGF related to the wavefront?
It regulates segmentation size by changing the "size" of the wavefront.
What happens at somite IV?
Somite segmentation is determined. The receding Fgf8 marks a boundary.
What is the determination front in a molecular definition?
It is the posterior boundary of Mesp2 gene expression.
What happens when the wavefront breaks?
It's the region at which presomitic mesoderm first acquires segmental identity.
What happens to cells anterior to the determination front?
They are more epithelialized due to a downregulation of fgf8. They undero a mesenchyal - epithelial transformation.
How is somitogenesis in fgf8 nulls?
It's normal.
Why do fgf8 nulls still have normal somitogenesis?
fgf8 is redundant for fgf4.
How can you knock out fgf8/4?
You have do conditionally KO.
What happens when you conditionally KO fgf4/8?
They have major somite defects. They make a bunch of somites.
What happens to the the presomitic mesoderm in fgf4/8 cKOs?
It all undergoes somitogenesis.
What does the wavefront prevent?
Early differentiation.
How is the determination front in fgf4/8 cKOs?
It's expanded in the PSM.
In the Clock and Wavefront model, what is keeping the somites from differentiating prematurely?
the fgf/Wnt gradient.
How are snakes somitogenesis different from others?
They have slower wavefront
A slower wavefront leads to...
lots of somites.
What pinches off somites?
Eph/ephrin interactions.
What does Eph4A do in somite formation?
It precedes somite formation and is restricted to the anterior half of the somite.
Where are ephrins expressed during somitogenesis?
In the posterior somite.
How does Eph signaling affect somitogenesis?
It positions somite boundaries.
How does epithelialization of a somite work?
Integrins and N-cadherin polarize outer cells into an epithelium. It occurs immediately after somite fission.
Which process provies the major force to extend the anterior-posterior axis of frog embryos during gastrulation?
Convergent extension
Name a general mesodermal inducing factor.
Vg1
Name 4 neural crest derivatives:
1. The stapes
2. The peripheral neuvous system
3. The hyoid bone
4. The mandible
What branchial arch gives rise to most of the face?
First
Which molecular interaction is most important for helping scult the motor neuron in the neural tube?
Nkx2.2 -- Pax6
Name the pathway that mediates convergent extension.
Non-canonical Wnt signaling.
You take the dorsal life of a gastrulating frog embryo and transplant it to the ventral side of the host embryo. What happens?
The embryo forms another body axis.
You electroporate a chick neural tube with Sox9. What happens to the electroporated cells?
They become neural crest cells.
3 things you need to make a targeting vector that can modify the genome of mouse ES cells.
1. left homology arm
2. right homology arm
3. Positive selection cassette eg NeoR
You express Eph4A in all motor neurons. What happens?
All the projections will go in the dorsal part of the limb bud.
What general route do non-melanocyte trunk neural cells take when they migrate? How could you block or inhibit this route?
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.
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?
A. Will not express ventral neural tube markers
B. Will not express ventral neural tube markers
C. Will express ventral neural tube markers.
How could you use genetic fate mapping to determine the fate of somites?
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).
You treat recently fertilized frog eggs with Nocodazole. What will this do? What will the embryo look like as a result?
This will block microtubule polymerization, preventing cortical rotation. The embryo will be a belly piece.
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.
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.
Why are pregnant women encouraged to take folic acid?
Because folic acid supplements reduce the prevalence of neural tube defects.
What do you think would happen to somite development if you incubated embryos in an inhibitor that reduces but does not eliminate fgf signaling.
You would have a reduced gradient of the wavefront. This would likely give rise to fewer and bigger somites.
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?
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.
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?
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.
You take vegetal tissue from a frog embryo and culture it together with an animal cap tissue. What happens to both tissues?
The vegetal tissue will form endoderm. The animal cap tissue will form mesoderm.
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.
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.
When are the kidneys formed?
Kidney formation is a later developmental event, occurring after the formation of the primary A-P axis.
What are the kidneys made up of?
The kidneys come from intermediate mesoderm. Development relies on multiple interactions between the epithelial ureteric bud and the adjacent mesenchyme.
What two structures interact to make the kidney?
The epithelial ureteric bud and the adjacent mesenchyme.
What is the functional unit of the kidney?
The nephron.
How many nephrons are in a kidney?
A human kidney has 1 million nephrons.
What are the kidneys specified from in relation to the neural tube?
The kidneys come from the intermediate mesoderm which is inbetween the paraxial and lateral plate mesoderm on either side of the neural tube.
How does the kidney form from the intermediate mesoderm?
The intermediate mesoderm travels down caudally and forms the pronephic duct. On the inside of the duct are structures forming called the pronephric tubules.
What is the pronephros?
The pronephic duct + the pronephric tubules
What is the 2nd stage of kidney development?
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.
What happens as the mesonephric duct is forming?
The pronephros begins to disappear.
What is the function of the pronephros?
It's strictly developmental. The pronephros does not have any of the function of the kidneys.
What does the mesonephric duct do?
It begins performing the functions of the kidney and receiving blood from capillaries.
What is another name for the nephric duct?
The wolffian duct
How are the collecting tubules of the developing kidney generated?
The metanephrogenic mesenchyme induces the ureteric duct to undergo branching morphogenesis which turns into tubules.
What is the reciprocal induction in kidney development?
The mesenchyme induces branching morphogenesis of the ureteric bud.
What happens to the ureteric bud without mesenchyme?
The bud does not branch.
What does the ureteric bud do to the metanephrogenic mesenchyme?
The ureteric bud makes the metanephrogenic mesenchyme condense upon the bud at the tips of the branches.
What happens to the mesenchyme when it is cultured without the ureteric bud?
The mesenchyme dies.
What is mesenchyme?
A loosely organized, mainly mesodermal embryonic tissue that develops into connective and skeletal tissues, including blood and lymph.
What is the ureteric bud?
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.
What happens after the mesenchyme condenses onto the tips of the branches?
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.
What does GDNF do?
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.
What secretes GDNF?
The metanephrogenic mesenchyme.
What has Ret receptors?
The ureteric bud.
What happens to the ureteric bud without GDNF?
The ureteric bud doesn't form.
What does Wnt9b do?
It's essential for kidney formation. Without Wnt9b, you don't make kidneys.
What happens if you have a Wnt9b-/- early in development?
Nothing seems to happen. The ureteric bud forms and initial branching is normal. So Wnt9b is not important for early development of the kidneys.
How is Wnt9b important for kidney formation?
Wnt9b expression induces Wnt4, which is critical for renal vesicle formation.
What happens to Wnt4 expression in Wnt9b mutants?
There is no Wnt4 expression. Wnt4 is essential for epithelialization of nephrons (renal vesicle formation).
What does Wnt4 do?
Wnt4 is essential for renal vesicle formation.
How does Wnt9b specify renal vesicle formation?
By inducing Wnt4.
What happens if you put a Wnt4 KO in the presence of Wnt9b?
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.
What are renal vesicles?
They're the nephron progenitor.
What is the mechanism of Wnt9b for making renal vesicles?
Wnt9b in the ureteric bud goes into the MM. The MM secretes Wnt4 which makes renal vesicles.
What is the structure of the metanephric mesenchyme?
The metanephri mesenchyme is spatially distinct. Different parts of the mesenchyme express Pax2, FoxD1, and cytokeratin.
Where is Pax2 expressed in the metanephric mesenchyme?
Pax2 is expressed in the medial region, kind of like a heart.
Where is FoxD1 expressed in the metanephric mesenchyme?
Distally on the edges.
Where is cytokeratin expressed in the metanephric mesenchyme?
Squished in the middle.
What does Six2 do?
Six2 is a key regulator of nephron progenitor populations.
Where is Six2 expressed?
Only in the cap metanephric mesenchyme which will later give rise to nephrons.
What does Six2 define?
Six2 defines a multipotent nephron progenitor population. It is required to maintain a pool of nephron progenitors.
How can you use genetic fate mapping to figure out what the hell Six2 does?
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.
How could you possibly generate new nephrons as a treatment for a degenerative kidney disease?
You could give someone Six2.
How are Six2 and Wnt9b different?
Six2 expands the nephrons and Wnt9b differentiates the nephrons.
What happens if you have a KO for Wnt9b and Six2?
Nephrons are neither expressed nor differentiated.
What forms muscle?
The dermamyotome which comes from the somites.
What differentiates muscle?
MyoD, Myf5, and Myogenin.
What does the somitocoel break up into?
As a mature somite, it breaks up into the epithelial dermamyotome and the mesenchymal sclerotome.
The somite turns into what kinds of muscles relative the the neural tube?
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.
What factors induce abaxial myotome?
Bmp and Fgf from the lateral plate mesoderm. Abaxial myotome is the farthest from the neural tube.
What factors induce primaxial myotome?
Wnt1/Wnt3a and low Shh. This part of the somite is closest to the neural tube.
What is the basic unit of a muscle?
The muscle fiber. It is a multinucleated cell. Myosin heavy chain provides its contractile capabilities and is a marker of differentiated muscle.
What are the 3 steps that convert myoblasts into muscles?
Determination of myotome cells --> multiplication of dividing myoblasts --> multiplication stops and cells align
What are myoblasts?
Muscle progenitors
What factors do myoblasts express?
MyoD and Myf5.
Describe the conversion of myoblasts to muscles
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.
Which factor tells myoblasts to divide and multiply?
Fgf's
Which factors help the muscle cells to align?
Fibronectin, integrin, cadherin/CAM, myogenin.
What does MyoD do?
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.
What happens if you force expression of MyoD in melanocytes and neurons?
They express myosin heavy chain.
What happens to mouse KO's for MyoD?
They still make skeletal muscle. So MyoD must not be the only thing you need to make muscle.
What do MyoD and Myf5 do together?
MyoD and Myf5 cooperate in muscle differentiation.
What is Myf5?
Myf5 is a related bHLH trxn factor to MyoD.
What happens to Myf5 mutants?
They make skeletal muscle too like MyoD KO's.
What happens to MyoD;Myf5 double null mutants?
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.
What do Fgfs do to myoblasts?
Fgfs maintain myoblasts in an undifferentiated state.
What do myoblasts do without Fgfs?
Myoblasts will exit the cell cycle and differentiate before fusing.
What do aligned myoblasts do?
Aligned myoblasts exit the cell cycle.
What happens if you culture isolated and aligned myoblasts with radioactive thymidine?
Isolated myoblasts will take up the label, but aligned myoblasts will not.
What happens if you're a myogenin mutant?
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.
What does myostatin do?
Myostatin regulates muscle size.
What happens if you're a KO for myostatin?
You have super huge muscles and it looks like you're taking steroids.
What are some myostatin mutants?
The Belgian Blue cow, the whippet dog.
What does the TGFbeta family member do?
It suppresses proliferation and differentiation of muscle.
Where do muscle fibers come from?
Most muscle fibers come from cells in the central dermamyotome that express Pax3 and Pax7. Only a subset of myotome makes muscle fibers.
What factors in the dermamyotome make muscle fibers?
Pax3 and Pax7.
What do Pax3/Pax7 do?
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.
How do Pax3 and Pax7 interact?
Pax3-expressing cells give rise to all Pax7 expressing cells and embryonic myoblasts.
How are muscles regenerated?
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.
Pax3/Pax7 have important early roles in development. How can they be deleted in postnatal embryos?
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.
How can you show that mice need Pax3/7 cells for muscle regeneration at birth?
You damage the muscle. With Pax7+/CE, you see regeneration. With Pax7CE/f, you see no regeneration.
What are the 3 types of Pax alleles that you can have for experimenting?
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
Do adult mice still require Pax3/7?
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.
Muscles form from...
dermamyotome.
What regulates muscle development?
A set of trxn factors.
Most muscles come from...
a reservoir of stem cells.
Muscle stem cells are present...
both in embryos and adults, but have different forms of regulation.
Modern Cre-based strategies can be used for...
ablation, inducible deletion, and fate mapping.
What is the first organ to form?
The heart.
How is the vertebrate cardiogenic mesoderm expressed?
It's a combination of BMP and no Wnt.
What are the key factors in early heart development?
Transcription factors GATA4, Nkx2.5, and Tbx5
Name this homeodomain trxn factor related to drosophilia tinman expression in vertebrate hearts:
Nkx2.5
What happens to Nkx2.5 KO mice?
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.
What happens if you're heterozygous Nkx 2.5 +/- and human?
You have a range of heart defects.
How does Nkx2.5 work?
It synergizes with GATA factors and activates trxn of cardiac genes.
What happens if you overexpress Nkx2.5?
You get an enlarged heart.
How can you make a xenopus embryo have a giant heart?
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.
How can you make a frog without a heart?
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.
How is the loss of Nkx2.5 different between frogs and mice?
Nkx2.5 KO in frogs means you get no heart, but you still get a heart with mice.
What happens if you lose Nkx2.5 in a mouse?
The heart forms, but is arrested after the looping stage. They have reduced or absent expression of differentiation markers like ANF expression.
What is drosophila tinman?
No heart!
Why do you have to use a dom neg Nkx2.5 in frogs?
You can't knock out Nkx2.5. You could use RNAi but the heart develops later in development so that makes it really hard.
Why is the Nkx2.5 null phenotype in mouse different from the dominant neg Nkx2.5 phenotype in frogs?
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.
Which trxn factor is needed for heart differentiation?
ANF
How can you mutate ANF so you can analyze it?
You attach GFP to the promoter.
How do ANF and Nkx2.5 interact?
It's thought that GATA, SRF, and Nkx2.5 all work in a complex to promote the trxn of ANF.
What happens if you muck up the NKE sequence of the ANF promoter site?
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.
If deletion of NKE site has little effection on ANF expression, what is happening?
GATA4, SRF, and Nkx2.5 are probably forming a promoting complex.
What's the fruit fly name for Nkx2.5?
Tinman
What's the fruit fly name for GATA4/5?
Pannier
What's the fruit fly name for Tbx5?
Dorsocross
What's the fruit fly name for BMP?
Dpp
How is fruit fly and vertebrate heart signaling different?
Fruit flies require Wnt signaling and vertebrates don't.
How does the fruit fly heart form?
The heart is specified by tinman, dorsocross, and pannier. They forms these stripes in the developing organism.
What causes the expressive patterning of heart trxn factors in fruit flies?
Wnt and BMP
What is ChIP?
Chromatin immunoprecipitation. This technique shows the specific regions of DNA bound by the trxn factor being assayed.
How did they use ChIP in fruit flies?
They looked to find the trxn factors for the heart.
How do many of the trxn factors interact in heart development?
They tend to clump together to bind 600-1200 genes.
What kind of tissue makes the heart?
The endocardial primordia makes a common heart tube. The tube then has an outer layer of myocardium and an inner layer of endocardium.
What's wrong with the miles apart mutant?
The mutant zebrafish gives rise to cardia bifida, two hearts.
Where is miles apart expressed?
It's not in the heart cells, but in the underlying endoderm on either side of the midline.
What does miles apart do?
It's involved in directing migration of presumptive heart cells.
How can you end up getting cardia bifida?
The heart cells migrate to eachother normally so if they're not directed towards each other, they will make two hearts.
What does the right atrium of the heart do?
It takes in deoxygenated blood.
What happens in the heart of a smoothened mutant?
It has linear heart tube, but the mice never form looped heart tubes.
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?
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.
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?
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.
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?
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.
What do you think would happen if you inserted a bead soaked in VEGF into embyronic mesoderm?
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.
What is the difference between vasculogenesis and angiogenesis?
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).
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?
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.
Sketch a linear heart tube. Where is the future atrium, future ventricle? Where will they be when it loops?
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.
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)
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.
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?
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.
You use chromatin immunoprecipitation to identify myogenin binding regions in developing skeletal muscle. What do you think you will identify?
A) You fdentify myogenin bound regions of chromatin. These will include among them enhancers by which myogenin regulates the activation of muscle differentiation genes.
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?
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?
A) Propose a strategy for genetically labeling blood cells in zebrafish with a specific marker.
A) Generate CymbCre fish and cross to a Bactin::LoP::STOP::LoxP::RFP line. Descendants of cymb+ blood cells will be labeled red.
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?
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.
What do you think happens in the following co-cultures:
wild-type MM + Wnt9B -/- UB
MM should survive (since UBs still contain BMP7 and FGF2) but they will not form renal vesicles (no inductive Wnt signal from UB).
What do you think happens in the following co-cultures: wild-type MM + Wnt4 -/- UB
Should form normal renal vesicles. Wnt4 is only required in MM.
What do you think happens in the following co-cultures: Wnt4 -/- MM + wild-type UB
Will not form renal vesicles.
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?
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).
Do renal vesicles form dorsal or ventral to the ureteric bud? What structures do renal vesicles become?
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).
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.
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
Python embryos completely lack the expression of forelimb buds. Why?
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.
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)?
FGF10
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?
FGF8 (or FGF4); either is sufficient (although neither is individually necessary)
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?
FGF4 and FGF8 must both be knocked out.
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?
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)
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.
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.
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?
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.
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).
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.
If you treated a chick embryo prior to limb outgrowth with a retinoic acid inhibitor, what do you think would happen?
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.
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?
FGF4
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?
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.
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'?
The liver bud, which delaminates and undergoes an epithelial to mesenchymal transformation into the mesenchyme
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?
All mesenchyme will be vascularized and thus have this important signal for inducing bud migration.
Why are premature babies so prone to lung defects?
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.
Why is a lungfish considered to have a fin that is somewhat similar to a tetrapod when compared with currently living fish?
Lungish have a single bone that connects the fin to the body. This is an ancestral stylopod.
What is the name of a cartilage cell? A bone cell?
Chondrocyte, Osteoblast
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?
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.
What is the major difference in bone phenotypes between Ihh mutants and PTHrP mutants?
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.
What is the major difference between Tiktaalik and Eusthenopteron limbs?
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.
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?
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
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?
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.
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?
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.
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?
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).
What is the key transcription factor that separates exocrine and endocrine pancreatic lineages?
Neurogenin 3 (Ngn3) is required for the formation of endocrine lineages.
After specification, what is needed for limb outgrowth?
The apical ectodermal ridge.
What is the stylopod?
The proximal arm bone
What is the zeugpod?
the distal arm bone
What is the autopod?
The digits (fingers)
What is the dorsal part of the autopod?
knuckles
The ventral part of the autopod:
palms
The anterior part of the autopod:
thumb
The posterior part of the autopod:
pinkie
What causes initial outgrowth of the limb?
Accumulation of somitic mesoderm (making a limb muscle) and lateral plate mesoderm (making other limb mesodermal components like skeleton).
When do human limb buds form?
Like wk 4/5.
What are thalidomide babies?
Kids with phocomelia (flipper like arms) because their moms took thalidomide for morning sickness.
What is thalidomide?
It's a teratogen that was given for morning sickness in the 50s. Somehow it affects growth of the forelimbs in humans.
What is the mechanism of thalidomide?
Not clear, but probably inhibits growth of blood vessels (angiogenesis) in the developing limbs.
What didn't they know thalidomide was a teratogen?
It's not a teratogen in mice.
Why doesn't a python have limbs?
Pythons are missing the Hox C6/8 boundary that correlates to the forelimb.
What do Hox codes do?
They're like postal codes. The boundaries of Hox expression correlate to the changes in identity of the somites and spinal cord.
Do pythons my limb buds at all?
They have hindlimb buds, but not forelimb buds. The hindlimb buds don't elongate.
In a limb bud, how do you know to get a wing or a leg?
Tbx 5 trxn factor makes a forelimb and Tbx4 makes a hindlimb.
What happens to Tbx5 mutant mice?
They lose their forelimbs, but not their hindlimbs.
What do Tbx5/4 mutations do to humans?
Tbx5 leads to upper extremity defects. Tbx4 leads to small patella syndrome and leg defects
Although necessary, Tbx4 and Tbx5 in mice are not sufficient...
to direct forelimb and hindlimb identity.
What positions limb outgrowth?
Fgf10
Where does Fgf10 become isolated?
Fgf10 becomes restricted in the lateral plate mesoderm that will make a limb.
How can you make an animal with a bunch of extra limbs?
Soak some beads in Fgf10 and shoot it in an embryo. They'll get ectopic limbs.
What happens to Fgf10 mutant mice?
They completel lack limbs. Fgf10 is necessary for limb development. The babies slither like snakes and then the mom eats them.
What's the pathway involving Tbx5/4 and Fgf10?
Wnt2b/8c --> Tbx5/4 --> Fgf10
What factor do you need for limb outgrowth?
Wnt2b
Fgf10 induces expression of...
Fgf8
What is the pathway involving Fgf10 and Fgf8?
Fgf10 in mesoderm --> Wnt3a in AER --> Fgf8 --> Fgf10 maintained
Where is Fgf8 expressed?
In the AER.
What is tetra-amelia in humans and what is it caused by?
You don't have any limbs and it's caused by a mutation of Wnt3.
What happens if you get rid of the AER at different points in development?
The limb stops at more and more distal points the later you get rid of it.
What's the AER?
Apical Ectodermal Ridge.
What does the AER do?
It mediates limb bud extension.
How could thalidomide affect the AER?
It could reduce angiogenesis in the AER.
What happens in an Fgf8 KO?
There's no phenotype. There are actually several Fgfs in the AER that are redundant with each other.
What's in charge of the proximal portion of the limb?
Retinoic acid (RA)
What's in charge of the distal portion of the limb bud?
Fgfs in the AER.
Name two factors that go up as Fgfs go up:
Hox11 and Hoxa13
Name a factor that goes down as Fgfs go up:
Meis1
How do you make a recombinant limb?
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.
Where is Meis1 expressed?
Proximally.
Where is Hoxa11 expressed?
Midway in the limb bud.
Where is Hoxa13 expressed?
Distally in the limb bud.
Where is Shh expressed in the limb bud?
In the ZPA at the bottom.
What is the ZPA?
The zone of polarizing activity.
What does Shh define in the limb bud?
The posterior portion of the limb bud.
What happens to Shh mutants' limbs?
They only make a thumb.
How can you get an autopod mirror image duplication?
You shoot a Shh bead into the anterior portion of the limb bud.
What does mirror image autopod duplication tell you about the ZPA?
Shh is sufficient for ZPA activity.
What controls Shh in the ZPA?
A cis-regulatory domain 850kb away from the Shh gene.
What is the Shh-Fgf signaling loop?
Shh --> gremline --> Fgfs --> Shh
What do low Fgf and high Fgf do to the Shh signaling loop?
Low Fgf maintain Shh and leave Gremlin alone. High doses accumulate and repress Gremlin.
What happens when Gremlin is repressed?
The AER breaks down.
What is the predicted Shh-dependent enhancer of Gremlin?
It's a Gli-bound site way downstream of Gremlin that might be how Shh activates Gremlin.
How does the Gli-binding region affect Gremlin?
Binding to the Gli-binding region drives gli-dependent limb extension.
What happens after limb bud patterning?
Condensation of mesenchyme into cartilage elements.
What's the order of digit condensation?
4-2-5-3-1
What is digit condensation?
The formation of cartilage masses (rays) that will subsequently develop the hand.
What is the foil experiment?
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.
What's the current model for phalange formation?
The AER which is part of the avascular mesenchyme gives cells to the vascular noncondensed mesenchyme which grows into a digit.
What are chondrocytes?
Cartilage cells.
How does endocondral bone form?
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.
What are hypertrophic chondrocytes?
Cartilage cells that are big and stop proliferating.
What are osteoblasts?
Developing bone cells.
What's the progenitor of osteoblasts?
perichondrocytes.
Which trxn factor is the master regulator for cartilage?
Sox9.
What happens if you only have one copy of Sox9?
You have campomelic dysplasia (bowing and bending of long bones).
What happens to your digits if you're without Sox9?
You don't have digits. You don't show any cartilage.
What's the second step of bone formation?
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.
What is the growth plate?
The proliferating region of the growing bone.
What does Ihh do?
It's in charge of the development of long bones.
What's wrong with an Ihh KO?
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.
What is the endocondral ossification pathway?
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.
What does PTHrP do?
It encourages chondrocyte proliferation and inhibits Ihh.
What does Ihh do?
It encourages chrondrocyte differentiation and promotes PTHrP in the perichondrial cells.
What happens to PTHrP mutants?
It causes dwarfism and premature ossification.
What does the Ihh-PTHrP feedback loop do?
It regulates the timing of hypertrophy.
How are the bones vascularized?
VegF and Matrix Metalloproteinases secreted by hypertrophic chondrocytes are responsible for vascularization of bone.
How is bone homeostasis maintained?
It's by the activity of the osteoblasts and the osteoclasts (destroy bone).
What are osteoclasts and where do they come from?
Osteoclasts are bone destroyers and they come from the blood cell lineage.
How do Fgfs relate to bone?
They also regulate chondrocyte proliferation. There are like 22 of them.
How are Fgfs related to dwarfism?
Activating Fgf signaling prematurely causes chondrocytes to differentiate prematurely. The mutations cause achondroplasia which is the most common form of dwarfism. It's dominant.
Why don't humans have webbing?
The webbing cells undergo apoptosis.
How do BMPs affect webbing?
BMPs lead to apoptosis of the interwebbing cells.
How can you make your own webbed creature?
You shoot it with Gremlin-soaked beads which inhibit BMP and then the interwebbed cells don't undergo apoptosis.
What happens to BMP mutants?
They have webbed feet.
How are bat and mice limbs different?
Bat embryos still have webbing.
What gene is upregulated in bats?
Prx expression is upregulated in the cartilage and distal limb elements compared to mice.
How did they make a bat mouse and what was the result?
They gave the mouse a bat Prx enhancer and it gave the mouse longer limbs. Prx KO's had bendy small limbs.
Why do horses have a penis-looking foot?
Horses progressively lost digits.
What is the jerboa?
It's a future model system because it appears to be losing digits.
What's so cool about lungfish?
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.
What is acanthostega?
An early tetrapod.
Why is eusthenopteron important?
He seemed to have a stylopod and zugopod.
Why is tiktaalik important?
It's a fossil intermediate containing digit-like elements (autopod).
What happens if you overexpress late-phase HoxD13 in zebrafish limbs?
They have more of a limb.
3 structures formed from endoderm development?
Liver, lungs and pancreas.
What does the endoderm specifically make?
The foregut, midgut, and hindgut.
What specifies endoderm?
Nodal. It turns on downstream differentiation markers that segregate endoderm from mesoderm.
How do you make the foregut and hindgut?
The tube folds to make the foregut and then folds again to make the hindgut.
What closes up in the gut tube?
The midgut and the hindgut.
How does the liver form?
It gets signals off of the heart. That's why with cardio bifida you get two livers. It comes from the foregut.
How do you know that the liver gets signals from the heart?
If you stick cardiac mesoderm with the foregut, you get a proliferation of cells. Foregut alone and nothing happens.
What's the signal the heart secretes to the foregut?
Fgf4.
What does Hhex do?
It's essential for budding of the liver.
What happens to embryos het for Hhex?
They're fine.
What promotes liver bud outgrowth?
Endothelial cells even without circulating blood. You can put endothelial cells in situ.
What is the pancreas composed of?
alpha, beta, sigma, and PP cells. It's composed of acinar cells, ductal epithelium and endocrine cells.
What do acinar cells do?
They produce digestive enzymes.
How is the pancreas specified?
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.
What does Pdx1 do?
It marks the dorsal and ventral pancreatic buds.
What happens if you're a Pdx mutant?
You fail to differentiate your pancreas.
What induces pancreatic differentiation?
Blood vessels.
What's the marker for pancreatic beta cells?
Insulin.
What induces insulin expression?
Endothelium.
What's needed for the formation of the pancreas?
Endothelium.
What does Ngn3 do?
It makes endocrine cells.
What happens to Ngn3 mutants?
They fail to make endocrine cells although they make exocrine fine. You don't end up getting insulin and glucagon.
What does Nkx2.2 do?
It's critical for generating beta cells.
Nkx2.2 mutants...
lose expression of alpha and beta cells and have lots of ghrelin. Nkx2.2 is a trxn activator and repressor.
What is ghrelin?
They signal that tells you you're hungry.
Is it better to have repressive or activator Nkx2.2?
The repressor forms rescues production of essentially all alpha cells and some beta cells.
How do you find out what makes beta cells in adults?
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.
What were the results of the stem cell population for beta cells experiment?
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.
What happens if you injure the beta cells? Where do the new beta cells come from?
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.
Steps to make beta cells from ES cells:
1. Convert ES cells to endoderm.
2. Make endoderm into posterior foregut.
3. Cells express Insulin, Glucagon, somatostatin.
How can you tell if beta cells from ES cells are functional?
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.
Can you make endocrine from exocrine?
Yes if you have access to Ngn3, Pdx1, and Mafa (M3).
What does VegF do with vascular stuff?
It's essential for vascular patterning.
What's the VegF receptor?
Flk1
Flk1 mutants...
have no blood vessels. Hets are fine though.
Angiogenesis:
The growth of blood vessels from pre-existing vascular tubes.
Vasculogenesis:
Initial formation of blood vessels which come from a single cells.
Macular degeneration:
caused by abnormal blood vessels that grow under the retina.
What does vascular tube formation need?
It needs endoderm during vasculogenesis.
How do you make the BBB?
You need Wnts
What happens if you're a double KO for Wnt7a/7b?
You have no BBB. You get blood pooling in your prefrontal cortex and spine.
What does Wnt7a/7b do?
It controls expression of BBB-specific markers.
What's a marker of BBB?
Glut1
You blood populations comes from...
endothelial cells.
What is the most primitive bilaterally symmetric animal?
Planaria.
What's so special about planaria?
They're really good at regenerating.
How are planaria able to regenerate?
They have a population of cells called neoblasts that are involved in regeneration.
What family are planaria a part of?
Platyhelminthes.
What are 3 general parts of a planarian?
1. Eyes with photoreceptors
2. Branched gut system
3. Pharynx on the ventral side where they eat and poop through the same tube. Ew.
What happens if you cut a planarian in half?
The top part will make a posterior blastema and the bottom part will make an anterior blastema and you will generate two planarians.
blastema:
regeneration bud.
The anterior portion of the planarian is...
the head region.
The posterior portion of the planarian is...
the bottom end region.
The only proliferating cells in planarians are...
neoblasts.
How do you experimentally show that neoblasts give rise to regenerating parts?
You label neoblasts with BrDU before you amputate them. The blastema contains many BrDU labeled cells.
What's wrong with irradiated planarians?
They can't regenerate.
What happens if you inject purified neoblasts into an irradiated planarian?
You can restore its ability to regenerate.
What three factors does a planarian lose when it's irradiated?
Neoblasts, smedwi-1, smedwi-2.
Which smedwi seems to be the most important?
smedwi-2 seems to be more important for regeneration.
What does smedwi-2 do?
It's essential for regeneration and homeostasis.
What happens if you use smedwi-2 RNAi in planarians?
They fail to form blastemas - the phenotype is similar to if the planarians were irradiated.
What does a radiated planarian head look like?
It gets all shrunken in; it's kinda gross.
What does a smedwi-2 RNAi planarian head look like?
It looks similar to the irradiated planarian head. It head starts to get shrunken in which is creepy.
What does smedwi-2 RNAi do to the neoblasts?
The neoblasts can't function normally. You can see in the experiment that with smedwi-2 RNAi, there is no tissue surrounding the neoblasts.
What happens if you put a wild type planarian chunk into an irradiated planarian?
You can save the whole planarian and they look all happy.
What happens if you put an irradiated chunk into an irradiated planarian?
It just dies and degrades after around 29 days.
What's another name for smedwi-2?
Smed-piwi. Thanks for making me remember yet another name.
You irradiate a planarian and then you take a wild type chunk and stain for smed-piwi. What happens?
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.
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?
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.
How do you experiment with directional migration in planarians?
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.
What kind of cells do you see when regenerating a decapitated planarian?
agat-1
What's the pathway for cell differentiation in planarians?
neoblasts --> prog-1 --> agat-1
What are the stem cells of the planarian?
Neoblasts
How do you make the Janus phenotype in planarians?
You have to knock down beta catenin in the planarian and you'll end up getting a head at the place you chopped off.
What does beta catenin do in the planarian?
It is required for blastema AP polarity.
How do you get rid of beta catenin in the planarian?
RNAi.
How does beta catenin relate to the axial levels of the planarian?
If you deplete beta-catenin with RNAi, you can cut a multiple different places and you'll still get a head that forms.
What's the six headed monster?
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.
What happens if you deplete beta-catenin and then chop off just the most anterior portion of the planarian?
They still regenerate heads right by their heads.
Based on RNAi studies, how is beta-catenin functioning in the planarian?
It probably doesn't simply inhibit anterior formation. It probably promotes posterior fates instead on a gradient level.
What's the insitu Wnt signaling for planarian?
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.
What's another way you mess with beta-catenin in the planarian?
You could inhibit beta-catenin half way in the planarian and look at the fate of the planarian.
What is a planarian stem cell marker?
Smedwi-2 (aka Smed-piwi)
How do amputated planarian pieces deterime AP identity?
Through beta-catenin dependent mechanisms.
How do you perform RNAi on a planarian?
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.
Why can't you use RNAi on humans through feeding them dsRNA?
Humans see dsRNA as a virus and will attack the dsRNA.
Urodeles can regenerate...
limbs and hearts.
Zebrafish can regenerate...
hearts.
How do you prove experimentally that zebrafish can regenerate their hearts?
Cut part of their heart off and do insitu hybridization for cardiac myosin heave chain and a dye indicating fibrin. It'll grow back.
How can you see if cardiomyocytes come from cardiomyocytes?
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.
What happens to mature cardiomyocytes after injury?
They dedifferentiate into early embryonic heart cells again and express embryonic cardiac differentiation markers like Nkx 2.5, tbx5.
What three factors are found in the zebrafish regenerating heart?
Mef2 and Myosin Heavy Chain (MHC) are in the cardiomyocytes and DAPI is in the nuclei.
2 factors in the regenerating cardiomyocytes:
Mef2 and MHC
Factor in the regerating nuclei of the zebrafish heart:
DAPI
What factor shows activation during zebrafish heart injury?
GATA4. That's what is labeled with GFP.
During zebrafish cardiac injury, what factor do you see in the injury site?
GATA-4
How can you determine the contribution of GATA4 descendants in the heart?
Use GATA4 CRE ER. When you injure the zebrafish heart, the GFP-expressing cells migrate down to the injury site and de-differentiate.
How much does the human heart increase from birth to adulthood?
30-50 fold
How are most human heart cells reacting as the heart grows?
Via hypertrophy instead of cell division.
How do you test the origin of heart cells in humans?
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.
Turnover rate of cardiomyocytes at age 20:
1%
Turnover rate of cardiomyocytes at age 75:
0.4%
What's the difference between a proximal amputation and a distal amputation on a salamander?
The proximal amputation goes more slowly, but you ultimately end up with the same regenerated limb.
In urodeles, the blastema has a cap made of...
apical epidermal cells.
What are the 5 stages of urodele limb regeneration?
1. Amputation
2. Healing
3. Dedifferentiation
4. Cone stage
5. Palette stage
6. Notch stage
7. Digit stage
What happens in the dedifferentiation stage of regeneration?
Proliferation of blastema cells and the presence of the apical epidermal cap.
What happens in the palette stage of regeneration?
The dedifferentiated cells redifferentiate.
What are the 3 assays you can do to look at PD identity of blastemal cells?
1. Confrontation assay
2. Grafted blastema assay
3. Level of Blastema assay
What's the confrontation assay?
If you stick a proximal blastema by a distal blastema, the proximal blastema will engulf the distal blastema.
What's the grafted blastema assay?
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.
What is the level of the blastema assay?
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.
Why would a proximal blastema attached to a proximal stump become super proximal?
Structures from the shoulder to the wrist arise by growth from the proximal partner. The blastema itself can't proximalize.
How can you make a 3-armed salamander monster?
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!
Why does making an "I'm a little teapot" salamander monster work?
Distal regeneration occurs even when the cut surface is proximal facing. For some reason...
What does retinoic acid do to a stub?
It reprograms the blastema. In the presence of retinoic acid, the salamander forms a completely new limb because retinoic acid is a proximal signal.
What happens if you treat a wrist blastema with retinoic acid?
It makes a completely new limb. So you get an super long limb.
What happens if progenitors for regeneration are lineage-restricted?
You find out that there are no stem cells. Skeletal cells make more skeletal cells.
What can the blastema regenerate?
It has a "memory" of where the amputation occurred. It can regenerate all tissues distal, but not proximal from the point of amputation.
What do you see from the regenerated limbs of axolotls?
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.
What factor resets the memory of a blastema?
Retinoic acid.
What are the two main stages of sex determination?
1. Germ cell development
2. Sex determination (dose compensation)
Early in development, how do germ cells differentiate themselves form somatic cells (2 ways)?
1. They're sequestered very early in development.
2. They are in some aspects immortal.
Where are germ cells specified?
In the periphery of embryos.
How do germ cells get to the gonads?
They have to migrate.
3 stages of germ cell development:
1. Specification
2. Migration
3. Differentiation
What are P-granules?
Germ line-specific cells from the earliest stages in c elegans that act as instructors of germ line development.
What are the 2 roles of p-granules?
1. Instructors of germ line development
2. bind RNA and promote germ-cell specific trxn.
What is Pie-1?
A trxn factor that prevents the phosphorylation of RNA pol II and blocks trxn in germ cells during development.
What's wrong with Pie-1 mutants?
They have embryonic genes in the P-cell.
What happens if you put Pie-1 in somatic cells?
It causes repression of somatic transcripts.
Where are primordial germ cells induced?
In the posterior region of the embryo immediately next to the extraembryonic ectoderm.
How many primordial germ cells are made from induction?
20
When does specification of the PGC occur?
E 7.5
What are PGCs?
primordial germ cells
What does Blimp1 do?
It's the earliest marker of PGCs and is a trxnal repressor.
When does migration of PGCs occur?
E 9.5
When does reprogramming of PGCs occur?
E 11.5
What day does mammalian sex differentiation occur?
E 12.5
What happens to Blimp1 mutants? 2 things.
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).
How do you perform single cell cDNA analysis of PGC cells?
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.
What are pole cells?
Drosophila germ cell progenitors. They start at the tip and migrate to the midgut where they attach and align with the gonadal mesoderm.
What are the 4 steps of pole cell migration?
1. Attachment to endoderm and migration through the midgut
2. Attachment to mesoderm
3. Alignment with gonadal mesoderm
4. Two streams of migrating cells.
Which direction do the drosophila germs cells migrate in the midgut?
Dorsally before splitting into two streams.
What are Wunen genes?
Wunen and Wunen-2 genes are redundant lipid phosphate phosphatases that act as chemorepellants expressed in the ventral midgut.
What do lipid phosphate phosphatases do?
They remove extracellular phospholipid substrates.
What do Wunen phosphatases do?
They guide PGCs by repelling them.
Why do PGCs only migrate through the endoderm dorsally?
They are repelled by ventral Wunen.
Why do PGCs split into two streams?
They are repelled by additional Wunen expressed in the CNS.
Wunen is necessary for...
proper lateral migration.
What happens if you knock out wunen but not wunen-2?
Nothing because the phosphatases are redundant for each other.
What happens to Wunen; Wunen-2 double mutants?
Their PGCs are unable to migrate laterally when they are in the middle of the embryo. They start going every which way.
How can you visualize PGC migration?
You can use a PGC-specific antibody.
How can you rescue a Wunen;wunen-2 double mutant?
CNS-specific expression in embryos can partially rescue gonad expression.
How do mice PGCs migrate?
They move into the endoderm (hindgut) and then migrate into the body wall.
What happens with mice PGCs at E 9?
The cells are motile, but do not leave the hindgut epithelium.
What are the 3 locations that mice PGCs migrate through?
hindgut --> body wall --> genital ridges
What are the two forms of X-inactivation in mammals?
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.
How are tortoishell cats the way they are?
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.
What is x-chromosome inactivation in mice?
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.
What is Xist RNA?
It's a 17kb non-coding RNA which is produced only by the inactive X.
Where is Xist RNA seen?
In mice and only in females. You have to have more than 1 X chromosome.
How is Xist trxn regulated?
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.
Which chromosome produces Xist RNA?
Only the inactive X (Xi). It negatively regulates itself.
How does an active X chromosome keep from getting shut off by Xist?
The active chromosome produces Tsix which creates dsRNA with Xist and then gets degraded.
What exactly does Xist do to the inactive chromosome?
It stabilizes and recruits repressive histone markers.
Where are germ cells usually specified?
In the periphery of the embryos (pole cells, P-granules)
How do germ cells differ from somatic cells during development?
They keep a low profile and transcriptionally repress somatic cell fates.
How are gonads formed?
The migration of the germ cells and their combining to somatic cells.
What is Sxl?
It is an RNA binding protein and is the master sex regulator in drosophila.
What does early Sxl trxn stimulate?
Female drosophila sex development
Which sex is default in drosophila?
Male
What does Sxl control?
Dosage compensation.
What does Sxl control?
Dosage compensation
Sxl is only functional in...
females.
How do drosophila compensate for XX in females?
They overexpress X proteins in males. This is opposite of mammals which use bar bodies.
Sxl and Tra don't work in males because...
it has an extra exon which tells the protein to terminate so that it becomes nonfunctional.
What is the pathway for Sxl?
Sxl --> Tra --> Dsx
What does DSX-M do?
It is the protein that makes male drosophila.
Promoters of Sxl:
Pe and Pm.
Where do Pe and Pm occur?
The Sxl promoters both occur in both sexes. Pe occurs only in females.
What is the difference between the Pe and Pm transcript?
The Pe transcript is functional, but the Pm requires pre-existing Sxl to be spliced properly to make functional Sxl.
XCE =
X chromosome counting elements
What does active transformer (tra) do?
Makes female-specific doublesex (Dsx)
What does Dsx do?
Processes Fruitless
What is fruitless in charge of?
Sexual behavior
Which drosophila trxn factor is the most important for sex determination?
Dsx.
What are 4 downstream effects of female Dsx?
1. female-specific growth genital disc
2. yolk proteins
3. spermathecal ducts
4. female pigmentation
4 downstream effects of male Dsx?
1. Male-specific growth genital disc
2. Paragonia
3. Clasper
4. Male pigmentation
Sxl inhibits...
Msl2
Msl2 is not inhibited in...
males
What happens if Sxl isn't activated early?
You get male junk.
Pe is only active for...
30-40 minutes
What does Msl2 do?
It opens the chromatin so that trxn is increased in the male x chromosome.
What are the two different pathways in drosophila?
1. Plumbing
2. Sexual preference
Sexual behavior of wt female drosophila:
No courting behavior
Sexual behavior of wt male drosophila:
1. Chase the female
2. Play a song
3. Lick her
What is essential for male sex behavior?
Correct splicing for Fruitless
What happens to males lacking Fru sex-specific transcripts?
They have normal sexual differentiation, but are completely sterile because they don't persue females.
What happens to Fru(male) females?
She persues wt females.
What is the primary sex determination of mammals?
Creation of gonads
The gonad is initially...
bipotential; it can become either ovary or testis
What is mammalian secondary sex determination?
The phenotype outside the gonads (duct systems, external genitalia)
at 4 weeks in human gonad development, you have...
indifferent gonads. You have a wolffian duct and mesonephric ridge. You also have a genital ridge.
At 6 weeks human gonad development, you have...
You have Mullerian duct development and germ cells in the genital ridge.
8 weeks testis development:
1. Wolffian duct --> vans deferens
2. Mullerian duct
3. Testis develops from genital ridge
8 weeks ovarian development:
1. Wolffian duct degrades
2. Mullerian duct
3. Surface epithelium on genital ridge
16 weeks testis development:
1. Mullerian duct degrading
2. Testis cords have formed.
20 weeks ovarian development:
1. Ovarian cortex and follicles form
2. Wolffian duct degrading
What is SRY?
A trxn factor and is the mammalian sex determining gene
What happens if a female has sry?
It becomes an XX male - male genitalia, but no functional sperm.
2 hormones made from testis:
1. AMH (anti-mullerian hormone)
2. Terosterone
What does AMH do?
Causes Mullerian duct regression
What does testosterone do in development?
Causes differentiation of Wolffian duct into internal male genitalia (epididymis and vas deferens).
Internal male genitalia:
1. epididymis
2. vas deferens
Two kinds of cells in the testis:
1. Sertoli cells
2. Leydig cells
Sertoli cells make...
AMH
Leydig cells make...
Testosterone
2 cells in ovary:
1. Granulosa cells
2. Thecal cells
The mullerian duct creates...
female internal genitalia (uterus, oviducts, cervix, upper vagina)
Female external genitalia comes from...
genital tubercle, urogenital sinus
Testosterone turns on...
DHT
DHT controls...
Penis, prostate, scrotum
Wolffian duct controls...
Male internal genitalia.
What happens to XX mice lacking FoxL2?
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.
Evidence against female default status:
FoxL2 is needed to generate and maintain female gonads.
How can you prove that FoxL2 is needed to maintain female gonads?
Use CRE ER and wait to add tamoxifen. The females will start producing testosterone when FoxL2 gets turned off.
When FoxL2 turns off...
Sox9 gets turned on.
Two mammalian factors that turn the undifferentiated gonad into testis:
1. Sry
2. Sox9
What happens to guevedoces in Dominican Republic?
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.
2 factors needed for external genitalia:
1. testosterone
2. DHT
Androgen Insensitivity Syndrome:
Individuals lack testosterone receptor. The individual can be XY, but has female secondary sex characteristics. They have internal male sex characteristics though.
Sex determination in turtles:
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.
What does the Hh inibiting drug do?
It can reduce your tumors for a bit, but they come back worse later.
Inappropriate expression of signaling molecules after birth causes...
cancer.
Inappropriate Hedgehog signaling in the brain causes...
medulloblastoma
Basal cell carcinoma can be caused by...
inappropriate Hh signaling.
How do neurons in the cerebellum differentiate?
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.
Where to the granule neurons go?
To the external granule layer (EGL).
6 layers of the cerebellum:
1. Ventricular zone
2. Intermediate zone
3. Granule cell layer
4. Purkinje cell layer
5. Marginal zone
6. External granule cell layer
Where do neuroblasts go from the ventricular zone?
They migrate to form Purkinje cell layer.
Why are Purkinje cells so cool?
They're huge and are the sole motor output of the cerebellum.
How do cells in the cerebellum proliferate?
Hh signaling
Hh signaling pathway:
Shh binds to --> Patched receptor --> inhibits Smo
Most common pediatric brain tumor:
medulloblastomas
If you have a Ptch mutation...
Smoothened never gets turned off so Hh is always on.
Where does Shh signaling come from in the cerebellum?
It comes from the PCL and EGL cells respond to it. When you stain for it, Shh seems to be in the EGL layer.
What does Shh signaling do in the cerebellum?
It causes proliferation of GCPs in the cerebellar slice cultures.
Mitogen:
growth factor
Mitogen in the cerebellum:
Shh
What happens if you don't have Anti-Shh in the cerebellum?
GCP don't proliferate. They have a much smaller layer of EGL. Purkinje cells are fine though because they secrete Shh.
Medulloblastoma is rare in...
adults
What is the drug they used for that patient with medulloblastoma?
It's a small molecule inhibitor of smoothened called GDC-0449.
What is the challenge of working with animal models for cancer?
When you take cells out of an animal, they start to behave differently.
What happens to Ptch +/- mice?
50% develop medulloblastomas.
What happens if you're Ptch +/-;p53+/-?
You get an insane amount of cancer.
How many genes are directly activated by Shh signaling?
500 target genes!
How does ChIP work to find Gli-activated proteins?
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.
How do you find the Gli binding sites in medulloblastomas?
You have to breed a Patch1+/- mouse with a tagged Gli mouse line (RosaGli1Flag).
What kind of cancer can you lessen with Smoothened inhibitors?
You can help treat basal cell carcinoma and it works ok on medulloblastomas.
Wnt signaling plays a role in what cancers?
Breast, colorectal, etc.
Pluripotent:
(of an immature or stem cell) capable of giving rise to several different cell types.
What kind of stem cells make muscel?
Satellite cells
Ovarian stem cells are...
Presophiles
Embryonic stem cells are...
pluripotent.
What happens to TCF4-/- mice have gut defects?
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.
What is TCF4 essential for?
Maintaing gut homeostasis.
The absorptive epithelium of the small intestine is ordered into...
vili and crypts.
What do vili do?
They allow for the increase in surface area for absorbing nutrients.
What's the life cycle of individual epithelial cells?
Less than a week.
Stem cells in crypts make how many cells a day?
200/crypt/day in a mouse.
What does Lgr5 do?
It marks small intestinal stem cells.
Where is Lgr5 expression?
Only in a subset of cells at the base of the crypt.
What's the Wnt target gene in small intestine?
Lgr5
Paneth cells:
One of the principle cell types in the small intestine
2 roles of paneth cells:
1. Emit microbial agents descended from stem cells.
2. Are always proliferating
What's the difference between paneth and quiescent muscle cells?
Paneth are always proliferating and quiescent cells only proliferate in damage.
How can you visualize the lgr5 cells in a mouse?
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.
What do paneth cells secrete?
Wnt proteins
What does Lgr5 amplify?
Wnt signaling response in stem cells.
How do you grow mini-gut organoids if you're weird like that?
You grow in vitro from a single Lgf5-expressing cell and it will get 5 cryps around the main lumen and will become functional!
What's wrong with TCF4-/- in relation to Wnt signaling?
They can't activate beta-catenin.
What is FAP?
An APC protein discovered via FAP disease in which patients get colon cancer.
When does FAP become cancerous?
FAP leads to pollups, but when the cells lose p53 and K-ras, that's when they become cancerous.
How do you give a mouse colon cancer?
You mess up its APC alleles and it will proliferate rapidly and give the mouse colon cancer.
What is nuclear reprogramming:
The ability to change the fate of a cell by making a switch in nuclear gene expression.
3 reasons nuclear reprogramming is of interest:
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.
How did they make Dolly?
They transplated the nuclei of cultured mammary gland cells from a sheep into enucleated sheep eggs.
What is nuclear reprogramming and how does it relate to cloning?
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.
What are the steps of the early cloning experiment?
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!)
What happens when you take nuclei later in development?
The percent of embryos that develop normally decreases exponentially.
What did Sir John Gurdon do?
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.
How did they make Dolly according to the slides?
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.
What's the controversy with Dolly?
Some people did think it was really cloning and it was a really rare event - 1/434 injections.
What is OMP?
Marker for differentiated olfactory sensory neurons.
How do you find out the origin of olfactory cells?
You use a CRE that only expressed in terminal olfactory neurons by sticking it to OMP.
Describe the experiment that makes a mouse out of olfactory sensory neurons.
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.
Why can you reprogram an oocyte?
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.
What is the first embryonic cell cycle?
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.
MG-132: