Vertebrate Limb Development

Question 1

Explain the positioning of the three axis in the context of vertebrate limbs

Answer 1

1. Proximal-Distal Axis:
   - Proximal closest to body, distal furthest away
   - Humerus at proximal end and digits and carpals at distal end
2. A-P axis
   - Top to bottom
   - Digit 1 at A and Digit 5 at P
3. D-V axis
   - Back of the hand to palm

Question 2

Why are hox genes important in limb formation?

Answer 2

Provide a ‘code’ for where arms and legs should be via limb bud in lateral mesoderm

• Hoxc6 provides the forelimb field

Question 3

What transcription factors have differential expression between the forelimb and the hindlimb?

Answer 3

Hox proteins –> RA –> Tbx TFs

• Tbx5 and Tbx4 have precise differential expression between forelimb and hindlimb
• Tbx5 = forelimb
• Tbx4=hindlimb
• Loss of each = loss of limb expression

Question 4

What experiments have been conducted with Tbx4 and Tbx5 to show its role in limb formation?

Answer 4

• Tbx4 transforms wing identity into leg in ectopic position also loss of it means loss of hindlimb
• In mice, losing tbx4 only causes partial loss of hindlimb - implying there are other working factors

Question 5

What happens if you KO an enzyme for retinoic acid synthesis?

Answer 5

• lose limb identity entirely
• Retinoic acid provides molecular link between hox proteins and Tbx TFs
• RA produced in mesenchymal cells in lateral mesoderm

Question 6

What is responsible for the outgrowth of limbs and how has this been experimentally shown?

Answer 6

• FGF solely responsible for outgrowth of limb bud
• Specifically FGF10 found in forelimb and hindlimb
• Implanted FGF beads in flanks of chick embryo between Fl and Hl , chicks developed entirely new limbs

Question 7

Explain the process of limb outgrowth and the positive feedback loops which occur within it

Answer 7

• Tbx5 (e.g.) promotes the expression of FGF10 and Wnt2
• FGF10 is in the mesenchyme of the developing limb bud and promotes mitosis
• Positive feedback loop where they all promote eachothers expression
• Fgf4/8 and Wnt3 are in the overlying ectoderm
• FGF10 has a positive feedback loop with both of these molecules, enhancing their expression as coordination of the mesoderm and ectoderm is essential form limb outgrowth

Question 8

What is the morphology of the early limb bud?

Answer 8

• AER forms at tip of limb bud (Apical Ectodermal Ridge), expresses Wnt3 and Fgf8
• Behind AER is the ‘progress zone’ made of mesenchymal cells of lateral plate mesoderm
• Progress zone contains HIGHLY PROLIFERATIVE CELLS
• As limb bud grows out the progress zone associates with AER the whole time and new cells grow behind it

Question 9

What is the role of the ZPA?

Answer 9

‘Zone of polarising activity’
- defines A-P axis long limb so it specifies digit identity
- also induces formation of the AER
- located at the posterior margin of the limb bud and secretes Shh
- lower conc of Shh = cells are more proximal
- higher conc. of Shh = cells are more distal

Question 10

What happens if you remove the AER?

Answer 10

DEPENDS ON WHEN YOU REMOVE IT
- The earlier you remove it, the less distal features are present
- Shows the AER is necessary for the progress zone and limb outgrowth
- Removing AER early means progress zone stops and cells cannot differentiate as much

Question 11

What is the mechanism behind proximo-distal patterning of the vertebrate limb?

Answer 11

• Fate of the cells depends on the AMOUNT OF TIME THEY SPEND IN THE PROGRESS ZONE
• limited proliferation = stylopod as they differentiate early and closer to body
• increased proliferation = zylopod
• intermediate levels of proliferation = zeugopod

Question 12

What are the signalling elements in proximo-distal patterning of the vertebrate limb?

Answer 12

• RA coming from the proximal end
• FGF8 coming from the distal end (ectodermal ridge)
  High RA = stylopod (proximal)
  High FGF8= autopod (distal)
  Intermediate of both = zeugopod

Question 13

How do the models of the proximo-distal signalling and the proliferating progress zone model come together to form a hybrid model?

Answer 13

• It is believed true that e.g. RA influences the proximal fate of cells in the progress zone to become stylopod
• However in order for the more distal areas to develop, the progress zone has to proliferate and extend further from the source of RA to escape it
• The time spent in the progress zone depends on how ‘far’ they escape from RA and therefore their differentiating impact

Question 14

What are the genes expressed in the Stylopod, Zeugopod and Autopod?

Answer 14

Stylopod = Meis
Zeugopod = Hox11
Autopod = Hox13

Question 15

What happens when you add an anterior graft of ZPA?

Answer 15

• Creates a mirror image duplication
• Autopod with two posterior ends as in digits go 4,3,2,2,3,4
• Over expressing Shh does the exact same thing, showing Shh is responsible for the posteriorizing effect

Question 16

What leads to digit 1 identity?

Answer 16

• No exposure to Shh at all

Question 17

How are all 3 axis coordinated?

Answer 17

Shh expression from ZPA are maintaining expression of FGFs in the AER and likewise so outgrowth is coordinated with patterning

Question 18

What is the process of D-V patterning of the vertebrate limb?

Answer 18

• Wnt7a expressed in dorsal ectoderm
• Engrailed1 expressed in ventral ectoderm
• Engrailed1 comes about by BMPs and engrailed1 inhibits Wnt7a
• Wnt7a drives expression of Lmx1b throughout the ectoderm and mesoderm of dorsal limb but (forms boundary)
• Lmx1b is just 1 TF controlling many other genes, so many more are expressed across a D-V axis
• Genes can be involved in repelling muscles ligaments to stop tissues invading the wrong places

Question 19

What happens in KO experiments with Wnt7a and Lmx1b?

Answer 19

• knock out Wnt7a or Lmx1b you lose dorsal identity of the limb (it turns ventral - paw pads)

Question 20

What is the homolog for Lmx1b in drosophila?

Answer 20

apterous