Vertebrate Limb Development Flashcards

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1
Q

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

A
  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
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2
Q

Why are hox genes important in limb formation?

A

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

  • Hoxc6 provides the forelimb field
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3
Q

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

A

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
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4
Q

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

A
  • 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
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5
Q

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

A
  • lose limb identity entirely
  • Retinoic acid provides molecular link between hox proteins and Tbx TFs
  • RA produced in mesenchymal cells in lateral mesoderm
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6
Q

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

A
  • 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
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7
Q

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

A
  • 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
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8
Q

What is the morphology of the early limb bud?

A
  • 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
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9
Q

What is the role of the ZPA?

A

‘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

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10
Q

What happens if you remove the AER?

A

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

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11
Q

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

A
  • 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
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12
Q

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

A
  • 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
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13
Q

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

A
  • 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
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14
Q

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

A

Stylopod = Meis
Zeugopod = Hox11
Autopod = Hox13

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15
Q

What happens when you add an anterior graft of ZPA?

A
  • 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
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16
Q

What leads to digit 1 identity?

A
  • No exposure to Shh at all
17
Q

How are all 3 axis coordinated?

A

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

18
Q

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

A
  • 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
19
Q

What happens in KO experiments with Wnt7a and Lmx1b?

A
  • knock out Wnt7a or Lmx1b you lose dorsal identity of the limb (it turns ventral - paw pads)
20
Q

What is the homolog for Lmx1b in drosophila?

A

apterous