Limb Bud - Relevant Experiments

Relevant Experiments

  • FGF10 Can Induce Limb Formation, but Tbx, Pitx, and Hox Determine Identity:

By mimicking the initial FGF10 secretions of the lateral plate mesoderm cells, limb development can be initiated. Other signaling molecules are implicated in determining the limb's identity.

  1. Placement of FGF10-containing beads beneath chick ectodermal cells results in the formation a limb bud, AER, ZPA and, subsequently, an entire limb. When the beads created limb buds towards the anterior region, forelimb formation coincided with Tbx5 expression, while hindlimb formation coincided with Tbx4 expression. When beads were placed in the middle of the flank tissue, the anterior portion expressed Tbx5 and forelimb features, while the posterior portion of the limb expressed Tbx4 and hindlimb features.
  2. When chick embryos were engineered to constitutively express Tbx4 (via viral-transfection) throughout their flank tissue, every limb they grew was a leg, even those that formed in the anterior region, which would normally become wings. This confirms the role of Tbx proteins in the type of limb that develops.
  3. Knocking out Tbx4 or Tbx5 knockout prevents FGF10 expression in the lateral plate mesoderm in mice.The Hox pathway may affect Tbx expression, which in turn affects FGF10 expression.
  4. When Pitx1 was incorrectly expressed in mouse forelimbs, several hindlimb-associated genes (Tbx4, HOXC10) were turned on and drastic alterations of the muscles, bones, and tendons shifted the phenotype towards that of a hindlimb. This indicates that Pitx1 – through Tbx4 - plays a role in the emergence of hindlimb properties.
  • HOXD11 Expression Correlates with Shh Secretion:

HOXD11 is expressed most posteriorly, near the ZPA, where the highest levels of Shh expression occur.

  1. When retinoic acid is applied to induce Shh expression, a ZPA is transplanted, or ectopic expression of Shh is stimulated, HOXD11 expression follows.
  • Mesenchyme Cells Determine Limb Identity, but the AER Maintains Limb Outgrowth through FGF secretion:

These experiments reveal that the limb mesenchyme contains the necessary information concerning limb identity, but the AER is needed to stimulate the mesenchyme to live up to its destiny (of becoming an arm, leg, etc.)

  1. When the AER is removed, limb development halts. If an FGF bead is added in the AER’s place, normal limb development proceeds.
  2. When an extra AER is added, two limbs form.
  3. When forelimb mesenchyme is replaced with hindlimb mesenchyme, a hindlimb grows.
  4. When forelimb mesenchyme is replaced with non-limb mesenchyme, the AER regresses, and limb development halts
  • ZPA’s Role in Establishing Polarity and Further Limb Development:

The ZPA first specifies anterior-posterior polarity (and dictates digit identity), and then, by sustaining AER activity, it ensures that the necessary cell proliferation occurs for normal formation of a five-digit limb.

  1. When Shh normally secreted from the ZPA is inhibited (either through use of tamoxifen or Shh-null mutants) the AER morphology, particularly its anterior extent, is perturbed and its FGF8 signaling decreased. As a result of Shh downregulation during limb bud expansion, the number of digits was decreased, but the identities of the formed digits was not altered.

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