Manifestation of the Limb Prepattern: Limb Development in the Absence of Sonic Hedgehog Function

Abstract

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(-/-) mutant mouse, the development of many embryonic structures, including the limb, is severely compromised. In this study, we report the analysis of Shh(-/-) mutant limbs in detail. Each mutant embryo has four limbs with recognizable humerus/femur bones that have anterior-posterior polarity. Distal to the elbow/knee joints, skeletal elements representing the zeugopod form but lack identifiable anterior-posterior polarity. Therefore, Shh specifically becomes necessary for normal limb development at or just distal to the stylopod/zeugopod junction (elbow/knee joints) during mouse limb development. The forelimb autopod is represented by a single distal cartilage element, while 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 suggests that the forming digit has a digit-one identity. This finding is corroborated by the formation of only two phalangeal elements which are unique to digit one on the foot. The apical ectodermal ridge (AER) is induced in the Shh(-/-) mutant buds with relatively normal morphology. We report that the architecture of the Shh(-/-) AER is gradually disrupted over developmental time in parallel with a reduction of Fgf8 expression in the ridge. Concomitantly, abnormal cell death in the Shh(-/-) limb bud occurs in the anterior mesenchyme of both fore- and hindlimb. It is notable that the AER changes and mesodermal cell death occur earlier in the Shh(-/-) forelimb than the hindlimb bud. This provides an explanation for the hindlimb-specific competence to form autopodial structures in the mutant. Finally, unlike the wild-type 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 well as specific limb skeletal elements. According to this model, the limb bud 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.

Anteroposterior patterning in the limb and digit specification: Contribution of mouse genetics

“…1C). Together, these results confirmed that Shh is required for patterning the limb bud over a very large area (Chiang et al, 2001;Kraus et al, 2001). Conversely, several mouse mutations leading to polydactyly are characterized by an anterior ectopic Shh expression domain in the limb (Masuya et al, 1995(Masuya et al, , 1997.…”

“…C: In the Shh null mutant, no digit is detectable in the forelimb and a single digit remains in the hindlimb. This digit has a digit1 (hallux) identity according to molecular markers (Chiang et al, 2001). The zeugopod contains a single bone, likely a radius (R?…”

Evolution of vertebrate appendicular structures: Insight from genetic and palaeontological data

“…In autopod patterning, this Shh-mediated anterior restriction of Gli3R is crucial for correct digit identity and number as evident from skeletal morphology of Shh À/À , Gli3 À/À , and Shh À/À : Gli3 À/À null mutants (Fig. 6) (Chiang et al, 2001;Litingtung et al, 2002;te Welscher et al, 2002).…”

“…In the Shh À/À mutants, there is a dramatic loss of intermediate and distal limb elements, with the zeugopod containing a single bone (radius/tibia) and a reduced digit 1 (Chiang et al, 2001) (Fig. 6).…”

“…Schematic representation of limb elements of wild type and genetically manipulated mice. The knockout data suggest that genetic interactions between Shh and Gli3 define digit number and identity (Chiang et al, 2001;Litingtung et al, 2002;te Welscher et al, 2002). Top: The developing limb bud is shown with dotted ovals representing the presumptive digit-forming regions (5-4-3-2 from posterior to anterior direction) under the influence of Shh, whereas the patterning of digit-1 is under the combined regulation of Gli3 and Alx4 transcriptional factors (Harfe et al, 2004;Panman et al, 2005).…”