Evolution Evolutionary analyses of hedgehog and Hoxd-10 genes in fish species closely related to the zebrafish

The study of development has relied primarily on the isolation of mutations in genes with specific functions in development and on the comparison of their expression patterns in normal and mutant phenotypes. Comparative evolutionary analyses can complement these approaches. Phylogenetic analyses of Sonic hedgehog (Shh) and Hoxd-10 genes from 18 cyprinid fish species closely related to the zebrafish provide novel insights into the functional constraints acting on Shh. Our results confirm and extend those gained from expression and crystalline structure analyses of this gene. Unexpectedly, exon 1 of Shh is found to be almost invariant even in third codon positions among these morphologically divergent species suggesting that this exon encodes for a functionally important domain of the hedgehog protein. This is surprising because the main functional domain of Shh had been thought to be that encoded by exon 2. Comparisons of Shh and Hoxd-10 gene sequences and of resulting gene trees document higher evolutionary constraints on the former than on the latter. This might be indicative of more general evolutionary patterns in networks of developmental regulatory genes interacting in a hierarchical fashion. The presence of four members of the hedgehog gene family in cyprinid fishes was documented and their homologies to known hedgehog genes in other vertebrates were established. Many vertebrate homologues of Drosophila developmental regulatory genes have recently been identified (1–4). These discoveries provide intriguing evidence that comparable ontogenetic processes, even in species from different phyla, can be regulated by homologous and evolutionarily conserved signaling factors. Despite their high degree of sequence conservation and their similarity of interactions in developmental networks, the expression of these homologous developmental control genes results in drastically a divergent Baupläne like those of insects and vertebrates. The homology and often extensive degree of sequence similarity illustrates the somewhat paradoxical contradiction between biological diversification among different animal phyla and the phylogenetic conservation of some developmental genes and their interactions. Originally identified by Nüsslein-Volhard andWieschaus (5) in Drosophila, the hedgehog (hh) gene has emerged as one of the most interesting and important developmental genes characterized so far. In Drosophila, hh has been reported to regulate embryonic segmentation and patterning (6), whereas in vertebrates one member of the hh gene family, the protein encoded by the Sonic hedgehog (Shh) gene, controls several developmental processes including dorsal–ventral patterning of the neural tube (7), left–right distinction (8, 9), and limb bud morphogenesis (10). One of the most interesting long-range signaling activities of the Shh protein is the regulation of limb bud patterning in vertebrate embryos (1, 3, 11–13). Recent evidence indicates that the effect of Shh signal on mesenchymal limb bud proliferation is indirect and is mediated through a wide variety of secondary transductional factors (10). Notably, the expression of posterior members of the HoxD gene cluster (i.e., Hoxd-10 to Hoxd-13) is associated with Shh gene expression, in the early developmental stages of the mesoderm primordium of fish and tetrapods (14, 15). Furthermore, differential late distal expression patterns of Shh and Hoxd-10 to Hoxd-13 genes in fish and tetrapod limb buds is responsible for the development of fins and limbs (15). Both hh and Shh proteins are expressed as precursors that undergo self-cleaving and secretion events (16, 17). After autoprocessing, Nterminal and C-terminal fragments are generated and locally released. The N-terminal processed form corresponds to exons 1 and 2 and seems to be implicated in shortas well as long-range signaling activities, whereas the C-terminal portion of the protein is encoded by exon 3 and has the autoproteolytic activity (18). The recently determined crystal structure of the N-terminal signaling domain of Shh revealed the presence of an unsuspected zinc-coordinated catalytic site (19). The discovery of this catalytic site, which had not been predicted based on the amino acid sequences, emphasizes that Shh might have features and functions that still await discovery. Herein, we use an alternative approach to improve our understanding of both the evolution of ontogenetic processes and the developmental genes that regulate them (20). This method involves the study of the evolutionary history of developmental genes to characterize the evolutionary and presumably functionally permissible variation within them and to discover conserved functional domains. MATERIALS AND METHODS Samples and DNA Extraction. Total DNA was extracted as described (21) from muscle of individuals of 18 cyprinid species related to the zebrafish (Danio rerio): Danio frankei, Danio kerri, Danio pulcher, Danio sp. aff. albolineatus, Danio sp. aff. tweediei, Devario devario, Devario cf. aequipinnatus, Devario pathirana, Devario malabaricus, Rasbora heteromorpha, Rasbora elegans, Rasbora paviei, Amblypharyngodon chulabhornae, Pseudorasbora cf. parva, Tanichthys albonubes, Puntius tetrazona, Puntius conchonius, and Carassius auratus. PCR Amplification, Cloning, and DNA Sequencing. Four sets of PCR primers were designed to consistently amplify Shh exon 1 (Shh1-F, 59-CTGGCCTGTGGTCCCTGGCAGAGG39; Shh1-R, 59-CTGAGTCATGAGCCGGTCC GCTCC-39), Shh exon2 (Shh2-F, 59-GACAAGCTGAACGCACTGGCCATCTC-39; Shh2-R, 59-CTTTGGACTCGTAATAGACCCThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: MP, maximum parsimony; NJ, neighbor joining; ML, maximum likelihood. Data deposition: The sequences reported in this paper have been deposited in the GenBank data base (accession nos. U51339–U51423 and U68236–U68241). *To whom reprint requests should be addressed. e-mail: ameyer@life.bio.sunysb.edu.