Manifestation of the limb prepattern: Limb development in the absence of sonic hedgehog function

The secreted protein encoded by the Sonic hedgehog (Shh) gene is localized to the posterior margin of vertebrate limb buds and is thought to be a key signal in establishing anterior-posterior limb polarity. In the Shh(-/-) mu tant mouse, the development of many embryonic structures, including the lim b, is severely compromised. in this study, we report the analysis of Shh(-/ -) mutant limbs in detail. Each mutant embryo has four limbs with recogniza ble humerus/femur bones that have anterior-posterior polarity. Distal to th e elbow/knee joints, skeletal elements representing the zeugopod form but l ack identifiable anterior-posterior polarity. Therefore, Shh specifically b ecomes necessary for normal limb development at or just distal to the stylo pod/zeugopod junction (elbow/knee joints) during mouse limb development. Th e forelimb autopod is represented by a single distal cartilage element, whi le the hindlimb autopod is invariably composed of a single digit with well- formed interphalangeal joints and a dorsal nail bed at the terminal phalanx . Analysis of GDF5 and Hoxd11-13 expression in the hindlimb autopod suggest s that the forming digit has a digit-one identity. This finding is corrobor ated by the formation of only two phalangeal elements which are unique to d igit one on the foot. The apical ectodermal ridge (AER) is induced in the S hh(-/-) mutant buds with relatively normal morphology. We report that the a rchitecture of the Shh(-/-) AER is gradually disrupted over developmental t ime in parallel with a reduction of Fgf8 expression in the ridge. Concomita ntly, abnormal cell death in the Shh(-/-) limb bud occurs in the anterior m esenchyme of both fore- and hindlimb. It is notable that the AER changes an d mesodermal cell death occur earlier in the Shh(-/-) forelimb than the hin dlimb bud. This provides an explanation for the hindlimb-specific competenc e to form autopodial structures in the mutant. Finally, unlike the wild-typ e mouse limb bud, the Shh(-/-) mutant posterior limb bud mesoderm does not cause digit duplications when grafted to the anterior border of chick limb buds, and therefore lacks polarizing activity. We propose that a prepattern exists in the limb field for the three axes of the emerging limb bud as we ll as specific limb skeletal elements. According to this model, the limb bu d signaling centers, including the zone of polarizing activity (ZPA) acting through Shh, are required to elaborate upon the axial information provided by the native limb field prepattern. (C) 2001 Academic Press.