Zebrafish smoothened

Intercellular signaling is crucial for embryonic patterning, cell specification and tissue induction. Hedgehog (Hh) encodes a secreted signal originally identified in Drosophila (Nüsslein-Volhard and Wieschaus, 1980). Vertebrate family members have been subsequently isolated (Echelard et al., 1993; Ekker et al., 1995; Krauss et al., 1993; Riddle et al., 1993; Roelink et al., 1994). In vivo and in vitro studies indicate that Hh signaling is required for dorsoventral patterning, specification of cell fates and proliferation in vertebrate neural tissue (Hammerschmidt et al., 1997). Two transmembrane proteins, Patched and Smoothened, mediate Hh signaling intracellularly (Ingham, 1998; Denef et al., 2000). Patched is the Hh receptor, but silences signaling in the absence of Hh. Smoothened interacts with Patched, but not Hh, and is required for signal transduction. Hh binding to Patched may relieve Patched repression of Smoothened (Kalderon, 2000). In vertebrate development, however, the role of Smoothened remains unclear, because loss-offunction mutations in smoothened have not been reported. Constitutively active forms of Smoothened mimic Sonic hedgehog (Shh) function cell-autonomously in patterning neural tube, inducing Hh target genes and specifying ventral cells, supporting evidence that Smoothened mediates Hh signaling (Hynes et al., 2000). Moreover, gain-of-function smoothened mutations are implicated in human basal cell carcinoma (Xie et al., 1998), consistent with loss-of-function mutations of patched (Hahn et al., 1996; Johnson et al., 1996a; Johnson et al., 1996b). Hh signaling is required for development of the vertebrate ventral neural tube. Hh conveys ventral characteristics to anterior diencephalic cells (Dale et al., 1997). Teratogens and mutations that affect Hh signaling in human embryos can cause severe ventral CNS developmental anomalies, including holoprosencephaly (Muenke and Beachy, 2000). Mice with Shh mutations are cyclopic and lack motoneurons, floor plate and ventral forebrain (Chiang et al., 1996). By contrast, zebrafish sonic-you (syu, an ortholog of mammalian Shh) mutants develop normal medial floor plate and motoneurons and have relatively normal ventral forebrain patterning (Schauerte et al., 1998), although they lack lateral floor plate. However, shh overexpression is sufficient to alter forebrain gene expression and morphology (Barth and Wilson, 1995; Macdonald et al., 1995). One explanation for this discrepancy is that development of zebrafish hypothalamus requires both Nodal and Hh signaling (Rohr et al., 2001; Varga et al., 1999). Thus, mutations in syu (shh) alone may have a relatively mild effect. Another possible explanation is that in zebrafish, additional Hh family members, Tiggy-winkle hedgehog (Twhh; Ekker et al., 1995) and Echidna hedgehog (Ehh; Currie and Ingham, 1996), may act redundantly with Shh to pattern ventral neural tube (Nasevicius and Ekker, 2000; Zardoya et al., 1996). Several other zebrafish mutations affect Hh signaling and produce similar phenotypes. Some of these have been termed 3497 Development 128, 3497-3509 (2001) Printed in Great Britain © The Company of Biologists Limited 2001 DEV1705