Analyses of fugu hoxa2 genes provide evidence for subfunctionalization of neural crest cell and rhombomere cis-regulatory modules during vertebrate evolution.

Hoxa2 gene is a primary player in regulation of craniofacial programs of head development in vertebrates. Here we investigate the evolution of a Hoxa2 neural crest enhancer identified originally in mouse by comparing and contrasting the fugu hoxa2a and hoxa2b genes with their orthologous teleost and mammalian sequences. Using sequence analyses in combination with transgenic regulatory assays in zebrafish and mouse embryos we demonstrate subfunctionalization of regulatory activity for expression in hindbrain segments and neural crest cells between these two fugu co-orthologs. hoxa2a regulatory sequences have retained the ability to mediate expression in neural crest cells while those of hoxa2b include cis-elements that direct expression in rhombomeres. Functional dissection of the neural crest regulatory potential of the fugu hoxa2a and hoxa2b genes identify the previously unknown cis-element NC5, which is implicated in generating the differential activity of the enhancers from these genes. The NC5 region plays a similar role in the ability of this enhancer to mediate reporter expression in mice, suggesting it is a conserved component involved in control of neural crest expression of Hoxa2 in vertebrate craniofacial development.

Novel gene ashwin functions in Xenopus cell survival and anteroposterior patterning.

The novel gene ashwin was isolated in a differential display screen for genes activated or up-regulated early in neural specification. ashwin is expressed maternally and zygotically, and it is up-regulated in the neural ectoderm after the midgastrula stage. It is expressed in the neural plate and later in the embryonic brain, eyes, and spinal cord. Overexpression of ashwin in whole embryos leads to anterior truncations and other defects. However, a second Organizer does not form, and the secondary axial structures may result from splitting of the Organizer, rather than axis duplication. Morpholino oligonucleotide-mediated reduction in ashwin expression leads to lethality or abnormalities in gastrulation, as well as significant apoptosis in midgastrula embryos. Apoptosis is also observed in midgastrula embryos overexpressing ashwin. Coexpression of ashwin with the bone morphogenetic protein-4 antagonist noggin has a synergistic effect on neural-specific gene expression in isolated animal cap ectoderm. Ashwin has no previously characterized domains, although two nuclear localization signals can be identified. Orthologues have been identified in the human, mouse, chicken, and pufferfish genomes. Our results suggest that ashwin regulates cell survival and anteroposterior patterning.

Evidence for antagonism of BMP-4 signals by MAP kinase during Xenopus axis determination and neural specification.

We have previously shown that mitogen-activated protein (MAP) kinase activity is required for neural specification in Xenopus. In mammalian cells, the BMP-4 effector Smad1 is inhibited by phosphorylation at MAP kinase sites (Kretzschmar et al., 1997). To test the hypothesis that MAP kinase inhibits the BMP-4/Smad1 pathway during early Xenopus development, we have generated a Smad1 mutant lacking the MAP kinase phosphorylation sites (M4A-Smad1) and compared the effects of wild-type (WT)- and M4A-Smad1 on axial pattern and neural specification in Xenopus embryos. Although overexpression of either WT- or M4A-Smad1 produced ventralized embryos, at each mRNA concentration, M4A-Smad1 had a greater ventralizing effect than WT-Smad1. Interestingly, overexpression of either form of Smad1 in ventral blastomeres disrupted posterior pattern and morphogenesis; again, more severe defects were produced by expression of M4A-Smad1 than by equal amounts of WT-Smad1. Ectodermal expression of M4A-Smad1 disrupted expression of the anterior neural gene otx2 in vivo and inhibited neural specification in response to endogenous signals in mesoderm-ectoderm recombinates. In contrast, overexpression of WT-Smad1 at identical levels had little effect on either neural specification or otx2 expression. Comparisons of protein levels following overexpression of either WT- or M4A-Smad1 indicate that WT-Smad1 may be slightly more stable than M4A-Smad1; thus, differences in stability cannot account for the increased effectiveness of M4A-Smad1. Our results demonstrate that mutations disrupting the MAPK phosphorylation sites act collectively as a gain-of-function mutation in Smad1 and that inhibitory phosphorylation of Smad1 may be a significant mechanism for the regulation of BMP-4/Smad1 signals during Xenopus development.