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178 THE ROLE OF SONIC HEDGEHOG IN THE INDUCTION OF CHICK LIMB REGENERATION/REDEVELOPMENT.
  1. B. E. Halverson1,
  2. N. Mishima1,
  3. J. J. Liu1,
  4. C. U. Pira1,
  5. K. C. Oberg1
  1. 1Loma Linda University Loma Linda, CA.

Abstract

Limb outgrowth is initiated and maintained by the secretion of FGFs in the apical ectodermal ridge (AER), tightly linked to progressive limb pattern formation regulated by SHH emanating from the posteriorly positioned zone of polarizing activity (ZPA). The reciprocal induction of SHH and FGF during limb development has been termed the SHH-FGF loop. Amputation of a chick wing bud during early development results in removal of the AER and the ZPA with truncation of the distal limb. Addition of FGF to the posterior, but not the anterior, stump immediately after amputation induces tissue regeneration and reactivates development of the distal limb structures. SHH expression is up-regulated adjacent to posteriorly applied FGF-soaked beads but not anterior applied FGF and correlates with regenereative capacity. We wondered whether SHH was sufficient to induce regenerative capacity. To determine the role of SHH in limb regenerative capacity, we amputated developing wings (HH stage 23) and ectopically expressed SHH by confined microeclectroporation (CMEP) in the anterior and posterior stump margins in the presence or absence of FGF-soaked beads. Embryos were harvested at day 10 (6 days after amputation) and the limb morphology analyzed by alcian green staining of the skeletal elements. In other experiments, chicks were harvested 30 to 40 hours after electroporation, and whole-mount in situ hybridization (WMISH) to FGF4 was used to identify any ectopic ectodermal FGF expression. Ectopic expression of SHH in the posterior stump alone could not reestablish segmented limb development. In contrast, expression of ectopic SHH in the anterior stump induced segmented regeneration/redevelopment in the absence of FGF (9/20 ≈ 40%). However, WMISH for FGF4 identified ectopic FGF4 expression in the ectoderm overlying ectopic anterior SHH expression, indicating the local presence of both factors. Thus, it appears that both SHH and FGF are required to regenerate lost tissue and initiate redevelopment. Our data suggest that SHH induces both positive and negative regulators of FGF expression. Initial SHH expression in naive limb mesoderm (anterior stump cells) up-regulates FGFs in the overlying ectoderm, while cells with prior exposure to SHH (posterior stump cells) no longer have the ability to up-regulate FGF in response to SHH. This shutdown of the SHH-FGF-loop, overcome in amphibian regeneration, may be a critical step in promoting mammalian limb regeneration.

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