Question

Limb Development.

Different species display characteristic digit patterning along the anterior-posterior axis. The forelimbs of bats are modified such that the posterior digits are elongated and maintain their webbing (to make the functional bat wing). Researchers observed that in addition to the normally timed expression of Sonic hedgehog (Shh) in the bat ZPA, there is a second phase of both Fgf8 and Shh expression in the developing forelimb in the interdigital tissue.

How does the second phase of Fgf8 and Shh contribute to both the elongation of the forelimb digits and to the maintenance of the webbing between the digits?

Answer 1

Sonic hedgehog pathway is involved in unique skeletal development of the limbs in bats. The processes involved in anterior-posterior patterning and development of limb buds require changes in Shh pathway. Anterior-posterior (A-P) patterning of limbs and the size of digits are different in bats as compared to mouse. In bats, the forelimbs have elongated posterior digits that are joined by tissue (webbing). along with short thumbs. On the other hand, the digits of hindlimb do not vary in size.

Shh is widely expressed in the zone of polarizing (ZPA) activity. This early Shh expression occurs with fibroblast growth factor 8 (Fgf8) expansions in the apical ectodermal ridge (AER). Later in development of the limbs, the interdigital tissues start expressing Shh and patched 1 (Ptch1) in forelimbs. Forelimbs thus show gradient expression along the A-P axis. Fgf8 and Shh provide proliferation signals.

In the day 16 (C16) bat embryo, Shh, Ptch1, Fgf8 and gremlin are expressed in the interdigital tissues of bat limbs. Fgf8 is expressed in the interdigital tissues of the C15 embryo forelimb.

In day 14 or C14 embryo, the Shh expression in zone of polarizing activity will ensure a positive feedback loop with Fgf8 expression in the AER of forelimb. This allows outgrowth of the forelimb. This expression occurs in a gradient manner from posterior to anterior direction. The expression of Fgf8 in AER precedes the formation of ZPA. This FgF8 will activate a second wave of Shh expression in the interdigital tissues in C16 forelimbs. This second wave of Shh leads to activation of bone morphogenetic protein 2 expressions at the same site as Shh. However, Bmp2 will then activate Gremlin expression in a gradient but opposite direction- high in anterior pole and less in posterior pole. There is highest Gremlin expression in digits 2 and 3. Gremlin has an inhibitory effect of Bmp2 in interdigital tissues, suppressing its actions in the interdigital tissue. Thus, the Fgf8 expression is now maintained in the interdigital tissue. This will allow the expression of Shh in interdigital tissue.and complete the Shh-Fgf8 feedback loop. This Shh and fgf8 expression is however, not restricted now to ZPA and AER.

Shh and Fgf8 expression is now also observed in the interdigital tissues of the posterior pole and not restricted only to AER and ZPA. Thus, in this second wave, Shh and Fgf8 expression is not confined only to AER and ZPA and can occur in same domain, which does not occur in the first wave. Thus, the Shh-Fgf signaling loop is required to ensure cell proliferation and cell survival. As the FgF8 expression triggers Shh expression in posterior digits of forelimb due to action of gremlin, there will be proliferation and growth of the posterior digits of the forelimbs. As there is expression of Fgf8 in AER and Fgf8 in the ZPA, there is also survival of the interdigital tissues. In case of hind limbs, Fgf8 and shh are also expressed in interdigital tissues, but at lower level. Further, Shh and Ptch are not expressed in gradient manner but are uniform throughout. This leads to differences in forelimb and hindlimb development.