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what tissues form from the animal cap? ventral? Dorsal?
V epidermis and D the nervous system
where does mesoderm form?
how does it form?
what does mesoderm form? Dorsal to ventral...
- marginal zone.
- Forms from the vegetal portion the marginal zone on the ventral dorsal axis.
- D notochord, somites & heart, blood & kidney V
what do ventral explants from the marginal zone form?
derived from what germ layers?
- V blood, epidermis, mesenchyme
- D notochord, muscle, neural tube
- + both ecto & meso derived
- Ecto - epidermis, neural tube
what/where is a blastocoel and what does it tell us?
it is a fluid filled cavity in animal portion size determines what stage of blastula ( larger later)
Meso patterning dorsalizing signals (5)? Where?
What do they do?
- Noggin, chordin, Frizbee, follistatin, cereberus
- Along dorsal-ventral axis
- repress ventralizers
Meso patterning ventralizing signals? Where?
- BMP4, XWnt8
- along dorsal-ventral axis
4 ways to test for secreted mesoderm inducers?
- add candidate molecule in animal cap culture
- inject mRNA into animal cap at early blastula
- inject mRNA into VegT knockdown to rescue meso
- inject asRNA into vegetal pole - inhibition meso?
when is this zygotic genome activated?
what transcription factor?
after the midblastula transition at stage 8 yielding marginal zone in stage 9. VegT induces mesoderm
what is the midblastula transition?
- late blastula, stage 8
- Zygotic genome active
- cell cycles become asynchronous cells become motile
what and where does the Nieuwkoop center form?
Forms Spemann organizer (in above animal region) from the dorsal vegetal side
what are the 4 types of mesoderm signals? What do they do?
- Ventral vegetal - general meso
- Dorsal vegetal - Nieuwkoop to organizer (in above animal cells)
- Ventral meso - ventralizes
- Dorsal meso - Spemann organizer to axial meso to notochord (by VM inhibition)
What/where increases Xnr signalling? Why?
No expression? Why?
What does Xnr form?
What also acts like Xnr?
- Beta catenin +VegT - dorsal side allows Xnr transcription b/c beta catenin removes Tcf repressor
- Only VegT - ventral vegetal very low Xnr
- W/o either beta catenin or VegT - no expressiom due to repression by Tcf
- Xnr forms head, trunk & tail mesodermal tissue
- Derriere forms trunk & tail meso
What is VegT? Where is it expressed? What does it do? How?
- VegT is a zygotically expressed transcription factor expressed in the vegetal equitorial region that specifies endoderm and induces mesoderm in concentration dependent manner
- low = ventral, high = dorsal
- secreted by Wnt, expressed in ventral marginal zone leading to ventral mesoderm
- inhibited by Frizbee
How does the Wnt pathway influence mesodermal induction?
Wnt bind to the Frizzle receptor which release Disheveled to remove Axin from associating with GSK-3. The Axin-GSK-3 complex causes the degradation of β-catenin. So in the presence of Wnt β-catenin is disinhibited and can accumulate and enter the nucleus to allow transcription of dorsal genes like Xnr which induces head, trunk & tail mesoderm
- secreted dorsalizer expressed in Spemann organizer
- inhibits BMP4
- secreted in organizer region
- inhibits BMP4 & activin
- maternal & zygotic dorsalizing factor expressed in Spemann organizer
- soluble form of Frizzled therefore inhibits XWnt8
secreted inhibitor of Wnts, BMPs & Nodals
What is a morphogen?
- a chemical or molecule that is present in differing concentrations that result in different cellular responses
- gsc is a zygotically expressed homeobox-containing gene that encodes a transcription factor
- expressed in dorsal marginal zone
- marker for the organizer
- high doses of activin can induce its expression
what does it do?
- Xbra is a T-box gene that encodes a transcription factor
- expressed in primitive streak (meso source) & node/notochord (axial meso)
- general meso inducer
- muscle/meso marker
- early response gene beginning during convergence-extension of meso along the midline (activin induce its expression)
injection of gsc mRNA into ventral region of the blastula?
mimics Spemann organizer transplantation with nearly doubled dorsal embryo
injection of Xbra in animal caps?
ventral mesoderm induced
Xbra & gsc response to activin...
in animal caps?
- intermediate doses of activin in animal cap assays activates Xbra expression
- high doses activates gsc
- in vivo: activin gradient induces cells to express Xbra further from activin source (lower conc.)
- cells closest to activin source (higher conc.) express gsc
where & when?
- BMP4 is a TGFβ ligand which leads to the expression of genes that induce ventral mesoderm (a.w.a. bone cartilage & apoptosis)
- therefore a ventralizer expressed thru late blastula, yet dorsal vegetal (organizer region) reduces expression of BMP4 in gastrulation
- inhibited by: noggin/chordin, follistatin, cerberus (dorsalizers exp. in organizer)
TGFβ signalling pathway
- dimeric ligand of TGFβ family (V! BMP or D! TGFβ/nodal/activin) binds heterodimeric receptor
- receptor subunits are phosphorylated inducing its Ser/Thr kinase activity
- SMADs are phosphorylated and recruit coSMADs
- SMAD complex translocates into the nucleus to initiate transcription of target genes
TGFβ dominant-negative mutation?
- mRNA encoding mutated TGFβ receptor is injected into 2 cell embryo leading to truncation and NO signal transduction resulting in NO mesoderm or axial structures.
- (done with activin, yet just points to TGFβs as mesoderm inducers)
what is the mesenchymal-to-epithelial transition?
when does it occur?
- as the primitive streak regresses it will laydown mesoderm tissue
- the paraxial mesoderm (mesenchymal cells) will eventually form somites (epithelial blocks of cells) in the anterior to posterior direction
what are somites?
where do they form and from what?
- somites are blocks of epithelial cells that form from the paraxial mesoderm which forms from the primitive streak
- somites are laid down on either side of the neural tube as the primitive streak regresses to the posterior end (during gastrulation)
what do somites form?
- dermamyotome -> dermatome -> dermis
- -> myotome -> skeletal muscle
- sclerotome -> vertebrae & ribs
what is the sclerotome?
what does it form?
what does it do?
sclerotome forms from somites and will become mesenchymal to surround the neural tube to eventually protect the spinal cord
How are Hox genes expressed?
- they are expressed from the Anterior 3' end (Hox#1) to the Posterior 5' end (Hox#13)
- RA is expressed in the trunk region and acts as a TF to induce Hox gene expression in a conc. dependent manner A (low) -> P (high)
why is retinoic acid important?
how is it synthesized?
what/how is it regulated?
- RA regulates Hox gene expression and therefore coordinated segmentation and somite formation in a conc. dependent manner
- RA binds its receptor (RAR) & acts as a transcription factor
- Vitamin A -> rentinol -> rentinal -- RALDH-2 --> RA
- CYP26 graded expression causes RA degradation
CYP26 vs RALDH-2?
expression controls what?
- CYP26 - expressed in tailbud and rostral hindbrain (degrades RA)
- RALDH-2 - expressed in trunk region (synthesizes RA)
- complementary expression helps pattern Hox gene expression b/c they are regulated by RA
RALDH-2 vs. FGF-8?
effect of RALDH-2 KO?
- RALDH-2 (RA)/FGF-8 counter gradient along AP axis controls somite formation
- RALDH-2 KO mimics FGF8 -> mini somites
- RALDH-2 overexp. mimics FGF8 KO -> large somites
why is FGF8 important?
where is it expressed?
overexpression or KO & molecular tracking?
- FGF8 is growth factor that keeps the PSM in unsegmented form at the posterior end in a conc. dependent manner
- FGF8 KO -> large somites
- FGF8 overexp -> mini somites
- track expt. manipulation by unc4.1 transcription factor (posterior half of somite marker gene)
what factors influence somite patterning?
- Shh (sonic hedgehog) induces Pax1 transcription factor in notochord & floorplate -> ventralizing -> sclerotome
- Wnt in roofplate & ectoderm -> dorsalizing
- BMP4 in lateral plate meso. -> lateralizing
BMP4 vs Noggin in somite formation?
- BMP4 is expressed laterally from the lateral plate mesoderm and is repressed by Noggin which is expressed medially
- Noggin allows somite formation
how are somites segmented? markers?
why is somite resegmentation important?
how does it happen?
- somites are segmented into an anterior and posterior zone
- A = Δ1 & Tbx18 expressed -> dermamyotome
- P = Δ3 & unc4.1 expressed -> sclerotome
- resegmentation involves the seperation of the posterior half of the somite binding to the anterior half of the adjacent somite
- this is important to allow motion (contractile force) b/c now the muscle section (A) is connected to 2 vertebrae
How/why is Notch signalling important for somite segmentation?
- Δ1/Δ3 are Notch receptor ligands
- when they bind Notch its ICD will be release to remove the target gene's repressor (RBP-Jk -> Lnfg, Notch, Hairy, HES) awa activate Lnfg that post-translationally adds sugar chains to the Notch receptor to attract other ligands
- Hairy & HES will inhibit Lnfg directly awa gene exp. by negative feedback to the RBP-Jk receptor = oscillating gene expression & somite segmentation
What type of proteins do the homeobox genes encode?
- homeobox genes encode the homeodomain that can bind to DNA to regulate transcription.
- Hox genes
What are the two primary techniques used in this paper to study homeobox gene function?
- skeletal phenotyping - morphology (of complete loss of function of paralogous Hox genes)
- in situ hybridization - track differential gene expression (confirm there is none with complete knockdown)
Briefly describe what the research team wanted to examine in the paper?
characterize how Hox genes pattern the vertebral axial skeleton and ribs of the thoracic region
What is the origin of axial vertebrae versus the ribs?
- axial vertebrae (& proximal ribs) are somitic mesoderm derived
- the sternum & the sternum ribs are lateral plate mesoderm derived
what is colinearity?
spatial and temporal ordering of gene expression corresponding to their genomic order
What did the researchers in the paper find?
- Hox gene mutants exhibited colinearity in the somitic mesoderm derivatives (the axial vertebrae & proximal ribs)
- Lateral plate mesoderm derivatives (sternum & sternum ribs) are patterned by Hox genes mutants exhibited defects along the entire anterior-posterior sternum.
What does complete loss of function of a Hox gene do?
- It induces anterior homeotic transformations in the most anterior region of its expression zone.
- Hox5 LoF may allow thoracic vertebrae to form at Hox6's most anterior position
What is a homeotic transformation?
the replacement of one structure for another
What are the experimental methods we can use to reveal how Hox genes specify positional info?
- KO - LoF (targeted mutagenesis)
- misexpression - GoF ectopic or overexpression of gene of interest (DNA construct under the control of heterologous promoter)
- microinjection into fertilized egg, incubate in foster mouse & DNA will randomly insert
Transient transgenics vs transgenic line?
- transient - dissect during development
- transgenic line - let embryo develop & reproduce