3/ vertebrate limb Flashcards
(23 cards)
anterior/posterior, ventral/dorsal axis in human hand
- thumb anterior, pinkie posterior
- back of hand dorsal, palm ventral
chick wing as a model
- 3 digits (2, 3 and 4)
- chick wing has an ulna, radius and humerus
- chick limbs are closely related to ours
what do all tetrapod limbs share? what does this suggest?
- homologous skeletal features along the proximal distal axis
- evolved from a common ancestor
- (alternative: limb developed twice, convergence, weaker theory)
stages of developement
- early cleavage, gastrulation, somitogenesis/neurulation, limb development
what is the relationship between limb growth and limb development?
- occur together
- limb grows
- wave of differentiation follows the growth (eg skeletal elements)
3 distinct regions in the limb bud
- ectoderm: apical ectodermal ridge AER made from bunched epithelia
- mesoderm: progress zone - mesenchymal cells below the AER. most distal
- mesoderm: Differentiating cells below progress zone. away from distal towards proximal.
what happens if you remove the AER? what does this suggest? variations of this experiment?
- truncates the limb
- older AER can replace AER and work the same. this shows AER is not instructive to tell cells what to become but just permissive for growth (you dont get digits forming on a truncated limb)
- If you remove the AER and incubate, cells continue to differentiate but stop growing
how many FGFs are in mice
22
what does FGF stand for
fibroblast growth factors
what FGFs in mice did early researchers think were responsible for limb development? why? how are they expressed?
- FGF8 and FGF4
- expressed strongly at the right time
- expressed in overlapping expression domains
FGF experiment 1: gene knock outs. method + conclusion
- fgf8 knockout has a mild limb defect (missing femur)
- fgf4 knockout has no limb defect
- redundancy - duplicating overlapping functions. when one is knocked out the other can take over.
- fgf8 and fgf4 are redundant
redundant
inclusion of extra components which are not strictly necessary to functioning
what does double mutation of fgf4 and fgf8 phenocopy (look like)?
- AER ablation (removal)
Fgf8 beads experiment 1 - removal of AER replacement with fgf8
- remove AER and add beads. more beads = more limb development
- fgf8 soaked beads can substitute for AER in a dose dependent manner
- AER only required as a source of FGF since fgf8 soaked beads rescue AER ablation
fgf8 beads experiment 2 - ectopic limb
- ectopic expression of fgf in the flank leads to ectopic limbs
- fgf signalling is sufficient for limb development
- (fgf signalling has other roles outside of limb development)
what is the ZPA?
- Zone of polarising activity
- pattering the identities - telling cells their different fates
ZPA experiment 1: what experiment uncovered ZPA?
- took a region (the ZPA) from the bottom of a donor wing bud and transferred it to bottom of a host wing bud
- host wing bud (ZPA) was at the top of the wing bud, and it was removed
- resulted in a mirror image of a chick’s foot
ZPA experiment 2:
- cut off leg bud, remove mesodermal tissue, leaves just ectodermal jacket of leg bud
- centrifuge mesodermal tissue and pop it back into leg bud ectodermal jacket and put that back into chick
- you get toes developed distally - no A/P axis, lost ability to polarise (what you get when signalling is removed)
- then put bottom ZPA into top mixed up leg bud - normal limb with reversed polarity
Turning’s secreted morphogen model: normal limb
- In model ZPA on posterior side
- concentration of morphogen is highest closest to the polarising region (ZPA) - diffuses out to create conc grad
- highest concentration results in one cell fate - digit 4 in chicks and lowest conc results in digit 2
- digits in distinct conc thresholds
Turning’s secreted morphogen model: additional ZPA on anterior of limb bud
- get duplication of foot digits
- distance concentration graph u shaped
Turning’s secreted morphogen model: small ZPA on anterior of limb bud
- get extra digit 2
what is sonic hedgehog (Shh)
- ZPA moves along to distal end of limb as it grows
- sonic hedgehog correlates to ZPA movement
- when duplicated in flies they got many bristles, thus name
- Shh secreted ligand
what is Shh required for? effect of -/- phenocopy? Shh soaked beads?
- A/P axis. when knocked out, lose A/P axis
- Shh -/- mutant phenocopies removal of ZPA
- Shh soaked beads induce posterior cell fates in a dosage dependent manner
- human disorders like multiple digits, polarity defect and missing digits caused by SHH mutations
