6. Limb patterning Flashcards
Segment polarity and homeotic genes
What could be the genetic manipulations in limb development to understand patterning?
Modifications to observe gene expression:
- gene KI
- gene KO
- cDNA overexpression
- genomic BAC constructs
Explain conditional gene targeting
Introduction of Cre loxP system - when Cre added - gene turned off/on
Explain the structure of a limb bud
What are the limb axes that need to be patterned
What are the initial signals in limb bud induction?
Limb induction in lateral plate mesoderm (LPM):
- Fgf10 expressed
- Wnt8c stabilized in hindlimb
- Wnt2b stabilized in forelimb
- in the set limb bud regions Fgf10 -> Fgf8 expression => apical ectodermal ridge (AER) formation
What is the function of Fgf10 in limb development?
Fgf10 necessary for for limb bud induction => Fgf10 KO - no limbs - some initial outgrowth but no limb bud outgrowth
What if exogenous Fgf is introduced in Fgf10-/-?
Ectopic Fgf beads induce limb formation - type of limb depends on Tbx4/5
Tbx5 - forelimb
Tbx4 - hindlimb
-> but later found that not these are the determinants of fore vs hind limb diffferentiation - but actually retinoic acid -> Hox genes control fore vs hind
How was Tbx role in fore vs hind limb development disproved?
Thought that Tbx5 / Tbx4 - but in Tbx5-/- + ectopic Tbx4 -> still developed as forelimb => Tbx4 replaced Tbx5
Both Tbx play similar role - what actually controls is site restricted => retinoic acid -> Hox genes
What controls fore vs hind limb development in limb buds?
Retinoic acid (RA) signaling regionalizes lateral plate mesoderm (LPM) from which limb buds outgrow - RA regionalizes LPM by regulating Hox expression =>
- rostral Hox (fore)
- caudal Hox (hind)
What is the sequence of signalling in fore vs hind limb induction?
What is the main determining region for P-D axis development in the limb?
Apical ectodermal ridge (AER) - necessary for outgrowth of limb bud - AER responsible for P-D patterning - cells under AER = progress zone (PZ)
Limb outgrowth develops sequentially - progress established as time develops - the later AER removed - the more distal development established
Explain limb bud anatomy
Limb bud anatomy:
- apical ectodermal ridge (AER): P-D pattern
- progress zone (PZ): limb outgrowth
- zone of polarising activity (ZPA): A-P pattern
Explain progress zone in the limb bud
Progress zone (PZ) can only divide and contribute to outgrowth while in contact with AER - the more time spent - the more distal structures developed
When lose contact with AER - differentiates into cartilage
Experiment showed that removal of AER -> cell death + decreased proliferation => progenitor cells outside AER can induce outgrowth but not sufficiently enough
What is the most important signalling molecule in P-D axis development in the limb?
In P-D development FGFs important:
(most proximal) Fgf8 > Fgf4 > Fgf9 > Fgf17 (most distal) in terms of importance
Fgf8 -> activates RA - RA diffuses P-D -> activates Meis/Meis2 proximal gene + Hoxa13, Hoxa11 distal genes
What is the role of Meis in P-D axis in limb development?
Meis is a proximal gene:
1. Meis expressed homogenously through P-D axis
2. FGF expressed in AER -> inhibits Meis expression in distal => Meis gradient in P-D axis
3. Meis inhibited -> HoxA cluster - Hoxa11, Hoxa13
Explain how limb A-P axis is patterned?
A-P axis of the limb patterned from the posterior - zone of polarising activity (ZPA) - signals - morphogen conc gradient - Shh
Ecotpic RA beads also induce axis duplication as ZPA grafting
Explain hte importance of Shh in all of development
Shh is a major regulator of development - acts as a morphogen - acts many times in development for different functions - Shh-/- mutants very defected - no distal limb, no head, no normal eye development,
Explain hedgehog -> sonic hedgehog gene relation
Hedgehog gene - Drosophila gene - called hedgehog because -/- mutants have shortened trunk + all covered in dentacles - instead of forming segments
Sonic hedgehog gene - homolg in vertebrates
How Shh expression was mapped?
Shh was fate mapped - Shh expressing cells adopt more posterior fate - not contributing to digits 1 and 2
Explain biphasic model
What is the role of Gli gene and how does it relate to limb patterning?
Gli acts in A-P patterning of the limb - effector of Shh - secreted in posterior - diffuses into anterior more than Shh - Shh and Gli3 are dispensable for limb skeleton formation - regulate digit number and identity
Gli3 downstream of Shh, mjust oth be ative for effect - if Shh-/- -> limbs can still develop some with only Gli3 - but if double KO Gli3-/-, Shh-/- -> same phenotype as Gli3-/-
Explain ZRS related enhancer in Shh expression
ZRS (ZPA regulatory sequence) enhancer required for Shh expression in the limb - mutations result in ZRS associated syndromes
Why is it hard to change Hox gene expression experimentally?
Hox genes difficult to investigate because arranged in a cluster - in a specific architecture - **if smth changed **- disrupts whole expression regulation - which comes externally -> Hox cluster architecture needs to be intact to be appropriatly regulated
Explain the feedback loop in gene expression of Hox and Shh in limb development
What is the first gene expressed in cartilage formation?
Sox9
Explain what is a turing type reaction diffusion model
Turing type reaction diffusion model - two molecules acts as an activator and inhibitor - both diffuse in an area - activator activates production of **inhibitor + itself **- inhibitor inhibits activator => over time oscillations of expressoion
Where is the Turing Network used in limb patterning
Turing network model used by Hox genes to generate periodicity in digit formation
Explain how Shh, Fgf, Gli3R, Wnt, Sox9, BMP and Hox genes are related in limb development
What are the important factors in DV patterning of the limb?
D-V patterning determined by ectodermal component: BMP signalling via BMPR1a -> En1 in ventral ectoderm -> restricts Wnt7 in dorsal ectoderm -> Wnt7 induces Lmx1b
Lmx1b responsible for dorsal limb identity - Lmx1b -/- nail patella syndrome
Explain evolutionary tweaking in limb development
Between species genes the same but their expression is altered - specific to the species
Give an example how different species express same genes but differently to develop limbs
Evolutionary conserved genes but expressed differently between species - ex:
Shh expression in ZPA in limb development of bats is delayed and expanded early - later expression reinitiated for inter-digital tissue
How mice were engineered to express snake limb developmental programme
Proved that evolutionary conserved mechanisms of limb development between different species
How does Shh expression differ in animals with fewer digits?
In fewer digit animals Shh turned off earlier - however - not in cows:
Shh expression early like in mouse - also expansion of Shh protein and signalling but not Otch1
Explain Shh signalling outcome in limb development
How do limb development genes act in regeneration
Salamanders regenerate limbs:
- RA proximalizes regeneration
- muscle does not respect - no P-D boundries
- connective tissue responds to P-D boundries - remember their expression levels of Meis
=> in regeneration blastema cells have P-D determination system from connective tissues
smth blastema patterning in regeneration
What is the similarity between vertebrate limb patterning and Drosophila?
Vertebrate limb bud <=> Drosophila wing imaginal disc
Relationship between ZPA, Shh, AER, FGF, polarization and proliferation in limb development
check paper discussion notes for more info
