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309 Cards in this Set

  • Front
  • Back
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.