Limb outgrowth is initiated and maintained by the secretion of FGFs in the apical ectodermal ridge (AER), whereas progressive limb pattern formation is regulated by SHH emanating from the zone of polarizing activity (ZPA). SHH also up-regulates factors that stimulate posteriorly accentuated FGFs in the AER. In turn, these FGFs maintain SHH expression in the adjacent distal mesoderm to complete the SHH-FGF feedback loop. Interestingly, application of FGF to the anterior mesoderm fails to up-regulate SHH, indicating a requirement for additional factors. We hypothesized that SHH induces this FGF responsiveness in limb mesoderm. To test this hypothesis, we ectopically expressed a SHH-GFP construct in the distal-anterior mesoderm by confined microelectroporation (CMEP) at HH 18-19. We demonstrated subsequent expression of SHH and its downstream target, the patched receptor (PTCH), by whole mount in situ hybridization (WISH). After 18 hours (HH 23-24) of exposure to SHH, we applied an FGF-soaked bead to the anterior margin. After an additional 24 hours, chicks were harvested and the SHH expression was determined by WISH. SHH and PTCH expression were up-regulated 24 hours after CMEP. The initial ectopic expression of SHH correlates with the site of transfection indicated by GFP fluorescence. Interestingly, by 42 hours the expression of SHH has shifted to lie immediately underneath the distal anterior AER and does not correlate with GFP fluorescence. These data signify that endogenous expression and FGF-regulated maintenance of SHH has been established. Furthermore, anteriorly placed FGF-soaked beads locally up-regulate SHH expression. Collectively, these data indicate that exposure of limb mesoderm to SHH activates a unique mechanism to produce SHH in response to FGF and thus link SHH expression tightly to the AER during limb outgrowth.
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