Summary The zebrafish iguana locus encodes Dzip 1 , a novel zinc-finger protein required for proper regulation of Hedgehog signaling

During animal development, cell fates are specified in response to gradients of signaling molecules within the embryo. Members of the Hedgehog (Hh) family of intercellular signaling molecules control a variety of developmental processes, ranging from segment patterning in Drosophila to organogenesis, left-right asymmetry and dorsoventral patterning of the spinal cord and forebrain in vertebrates (reviewed by Ingham and McMahon, 2001; McMahon et al., 2003). Aberrant regulation of Hh signaling in humans causes developmental defects such as holoprosencephaly (HPE) (reviewed by Wallis and Muenke, 2000) and postaxial polydactyly (Radhakrishna et al., 1997), and can also lead to various types of cancers, including basal cell carcinoma and medulloblastoma (reviewed by Goodrich and Scott, 1998; Ruiz i Altaba, 1999b; Villavicencio et al., 2000). Because Hh signaling plays such a central role in development and disease, the Hh signaling pathway has been investigated in considerable detail. Genetic and in vitro studies in Drosophilahave revealed that Hh signals are transduced by binding of Hh ligands to the Patched (Ptc) cell-surface receptor, resulting in the activation of the transmembrane protein Smoothened (Smo). In Drosophila, the intracellular regulation of Hh signaling is mediated by post-translational modifications of Cubitus interruptus (Ci), a zinc-finger-containing transcription factor of the Gli family that can be both an activator and a repressor of Hh target genes (Methot and Basler, 2001). In the absence of Hh signal, proteolytic cleavage converts Ci to a transcriptional repressor (Aza-Blanc et al., 1997; Wang and Holmgren, 1999). In the presence of Hh signals, cleavage of Ci is inhibited and a full-length activator isoform predominates. In vertebrates, at least three Gli genes, Members of the Hedgehog (Hh) family of intercellular signaling molecules play crucial roles in animal development. Aberrant regulation of Hh signaling in humans causes developmental defects, and leads to various genetic disorders and cancers. We have characterized a novel regulator of Hh signaling through the analysis of the zebrafish midline mutant iguana (igu). Mutations in igu lead to reduced expression of Hh target genes in the ventral neural tube, similar to the phenotype seen in zebrafish mutants known to affect Hh signaling. Contradictory at first sight, igu mutations lead to expanded Hh target gene expression in somites. Genetic and pharmacological analyses revealed that the expression of Hh target genes in igu mutants requires Gli activator function but does not depend on Smoothened function. Our results show that the ability of Gli proteins to activate Hh target gene expression in response to Hh signals is generally reduced in igu mutants both in the neural tube and in somites. Although this reduced Hh signaling activity leads to a loss of Hh target gene expression in the neural tube, the same low levels of Hh signaling appear to be sufficient to activate Hh target genes throughout somites because of different threshold responses to Hh signals. We also show that Hh target gene expression in igumutants is resistant to increased protein kinase A activity that normally represses Hh signaling. Together, our data indicate that igu mutations impair both the full activation of Gli proteins in response to Hh signals, and the negative regulation of Hh signaling in tissues more distant from the source of Hh. Positional cloning revealed that the igu locus encodes Dzip1, a novel intracellular protein that contains a single zinc-finger protein-protein interaction domain. Overexpression of Igu/Dzip1 proteins suggested that Igu/Dzip1 functions in a permissive way in the Hh signaling pathway. Taken together, our studies show that Igu/Dzip1 functions as a permissive factor that is required for the proper regulation of Hh target genes in response to Hh signals.