3/ vertebrate limb

Question 1

anterior/posterior, ventral/dorsal axis in human hand

Answer 1

• thumb anterior, pinkie posterior
• back of hand dorsal, palm ventral

Question 2

chick wing as a model

Answer 2

• 3 digits (2, 3 and 4)
• chick wing has an ulna, radius and humerus
• chick limbs are closely related to ours

Question 3

what do all tetrapod limbs share? what does this suggest?

Answer 3

• homologous skeletal features along the proximal distal axis
• evolved from a common ancestor
• (alternative: limb developed twice, convergence, weaker theory)

Question 4

stages of developement

Answer 4

• early cleavage, gastrulation, somitogenesis/neurulation, limb development

Question 5

what is the relationship between limb growth and limb development?

Answer 5

• occur together
• limb grows
• wave of differentiation follows the growth (eg skeletal elements)

Question 6

3 distinct regions in the limb bud

Answer 6

• 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.

Question 7

what happens if you remove the AER? what does this suggest? variations of this experiment?

Answer 7

• 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

Question 8

how many FGFs are in mice

Answer 8

22

Question 9

what does FGF stand for

Answer 9

fibroblast growth factors

Question 10

what FGFs in mice did early researchers think were responsible for limb development? why? how are they expressed?

Answer 10

• FGF8 and FGF4
• expressed strongly at the right time
• expressed in overlapping expression domains

Question 11

FGF experiment 1: gene knock outs. method + conclusion

Answer 11

• 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

Question 12

redundant

Answer 12

inclusion of extra components which are not strictly necessary to functioning

Question 13

what does double mutation of fgf4 and fgf8 phenocopy (look like)?

Answer 13

• AER ablation (removal)

Question 14

Fgf8 beads experiment 1 - removal of AER replacement with fgf8

Answer 14

• 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

Question 15

fgf8 beads experiment 2 - ectopic limb

Answer 15

• 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)

Question 16

what is the ZPA?

Answer 16

• Zone of polarising activity
• pattering the identities - telling cells their different fates

Question 17

ZPA experiment 1: what experiment uncovered ZPA?

Answer 17

• 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

Question 18

ZPA experiment 2:

Answer 18

• 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

Question 19

Turning’s secreted morphogen model: normal limb

Answer 19

• 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

Question 20

Turning’s secreted morphogen model: additional ZPA on anterior of limb bud

Answer 20

• get duplication of foot digits
• distance concentration graph u shaped

Question 21

Turning’s secreted morphogen model: small ZPA on anterior of limb bud

Answer 21

• get extra digit 2

Question 22

what is sonic hedgehog (Shh)

Answer 22

• 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

Question 23

what is Shh required for? effect of -/- phenocopy? Shh soaked beads?

Answer 23

• 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