The mouse Hedgehog gene family consists of three members, Sonic, Desert, and Indian hedgehog (Shh, Dhh, and Ihh, respectively), relatives of the Drosophila segment polarity gene, hedgehog (hh). All encode secreted proteins implicated in cell-cell interactions. One of these, Shh, is expressed in and mediates the signaling activities of several key organizing centers which regulate central nervous system, limb, and somite polarity. However, nothing is known of the roles of Dhh or Ihh, nor of the possible function of Shh during later embryogenesis. We have used serial-section in situ hybridization to obtain a detailed profile of mouse Hh gene expression from 11.5 to 16.5 days post coitum. Apart from the gut, which expresses both Shh and Ihh, there is no overlap in the various Hh expression domains. Shh is predominantly expressed in epithelia at numerous sites of epithelial-mesenchymal interactions, including the tooth, hair, whisker, rugae, gut, bladder, urethra, vas deferens, and lung, Dhh in Schwann and Sertoli cell precursors, and Ihh in gut and cartilage. Thus, it is likely that Hh signaling plays a central role in a diverse array of morphogenetic processes. Furthermore, we have compared Hh expression with that of a second family of signaling molecules, the Bone morphogenetic proteins (Bmps), vertebrate relatives of decapentaplegic, a target of the Drosophila Hh signaling pathway. The frequent expression of Bmp-2, -4, and -6 in similar or adjacent cell populations suggests a conserved role for Hh/Bmp interactions in vertebrate development.

Dhh expression in pre-Sertoli cells is one of the earliest indications of male sexual differentiation. Analysis of a null mutant demonstrates that Dhh signaling plays an essential role in the regulation of mammalian spermatogenesis. Loss of Patched expression in Dhh mutants suggests a conservation in the Hedgehog signaling pathway between flies and mice, and indicates that Leydig cells may be the direct target of Dhh signaling.

Midline signaling by Hedgehog (Hh) family members has been implicated in patterning the vertebrate embryo. We have explored the potential regulatory role of cAMP-dependent protein kinase A (PKA) in these events. Zebrafish embryos injected with RNAs encoding Sonic hedgehog (Shh), Indian hedgehog (Ihh), or a dominant-negative regulatory subunit of PKA, PKI, have equivalent phenotypes. These include the expansion of proximal fates in the eye, ventral fates in the brain, and adaxial fates in somites and head mesenchyme. Moreover, ectopic expression of PKI partially rescues somite and optic stalk defects in no tail and cyclops mutants that lack midline structures that normally synthesize Shh. Conversely, all cell types promoted by ectopic expression of hhs and PKI are suppressed in embryos injected with RNA encoding a constitutively active catalytic subunit of PKA (PKA*). These results, together with epistasis studies on the block of ectopic Hh signaling by PKA*, indicate that PKA acts in target cells as a common negative regulator of Hedgehog signaling.[Key Words: Sonic hedgehog; Indian hedgehog; protein kinase A; midline signaling; zebrafish] Received December 20, 1995; revised version accepted February 2, 1996.Members of the hedgehog (hh) gene family encode signaling proteins involved in induction and patterning processes in vertebrate and invertebrate embryos. In vertebrates, hhs constitute a multigene family. Sonic hedgehog (Shh), which has received most of the experimental attention, has been isolated from mouse (Echelard et al. 1993; Chang et al. 1994}, chick (Riddle et al. 1993, rat (Roelink et al. 1994), Xenopus (Ekker et al. 1995), and zebrafish (Krauss et al. 1993). The early expression is limited to the notochord, the floor plate and its anterior extension in the brain, and the zone of polarizing activity (ZPA) in the posterior mesenchyme of the limb bud. All of these sites have been shown to act as organizing centers patterning neighboring tissues by the secretion of morphogenetic signals (e.g., Saunders and Gasseling 1983;Brand-Saberi et al. 1993; Pourquie et al. 1993;Yamada et al. 1993). Recent evidence indicates that Shh is involved in these interactions.For example, ectopically expressed Shh leads to a ventralization of large regions of the mid-and hindbrain in mouse and zebrafish, as revealed by the dorsally expanded expression pattern of HNF-3~ and its zebrafish homolog axial (Echelard et al. 1993;Krauss et al. 1993).In explants of intermediate neuroectoderm at spinal cord levels, Shh protein induces, in a dose-dependent fashion, floor plate, and motor neuron development (Marti et al. 1Corresponding author.1995a; Roelink et al. 1995;Tanabe et al. 1995). In explants taken at midbrain and forebrain levels, Shh also induces the appropriate ventrolateral neuronal cell types, dopaminergic (Heynes et al. 1995;Wang et al. 1995} and cholinergic (Ericson et al. 1995) precursors, respectively. In chick somites (Johnson et al. 1994) and mouse presomitic explants (Fan et al. 1995), Shh promotes the expression...

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The dorsal nasal flap technique is more versatile than has been traditionally appreciated and can allow single-stage reconstruction of many sizes of defects affecting various areas of the nose.