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A study of the genomes of calcisponges shows that they contain at least one ParaHox gene, adding weight to the 'ghost locus' hypothesis that Hox and ParaHox genes were present in the earliest animal ancestor, but subsequently lost in some sponge lineages. Evolution of Antennapedia-class genes in sponges (Adamska HG) The simple body plan of sponges belies the complexity of their genomes, which contain a variety of transcription factors involved in body patterning in more complex creatures, such as Antennapedia class genes that in bilaterians (animals with distinct anterior and posterior ends) include Hox, ParaHox and NK. The genome of the demosponge Amphimedon queenslandica contains a tight cluster of NK genes, but no Hox or ParaHox genes. This led to the hypothesis that Hox and ParaHox genes evolved after sponges diverged from the lineage leading to cnidaria (jellyfishes, sea anemones and others) and bilateria. Study of the overall structure of the sponge genome suggested, however, that Hox and ParaHox might once have been present and later lost -- the 'ghost locus' hypothesis. Maja Adamska and colleagues have studied the genome of two calcisponges, a distinct lineage from demosponges, and show that whereas the NK genes are not clustered, there is at least one ParaHox gene (Cdx). This adds weight to the ghost locus hypothesis and highlights the need to examine the genomes of many different sponge lineages in order to obtain a more complete picture of the ancestral animal complement of Antennapedia genes. Sponges are simple animals with few cell types, but their genomes paradoxically contain a wide variety of developmental transcription factors.sup.1,2,3,4, including homeobox genes belonging to the Antennapedia (ANTP) class.sup.5,6, which in bilaterians encompass Hox, ParaHox and NK genes. In the genome of the demosponge Amphimedon queenslandica, no Hox or ParaHox genes are present, but NK genes are linked in a tight cluster similar to the NK clusters of bilaterians.sup.5. It has been proposed that Hox and ParaHox genes originated from NK cluster genes after divergence of sponges from the lineage leading to cnidarians and bilaterians.sup.5,7. On the other hand, synteny analysis lends support to the notion that the absence of Hox and ParaHox genes in Amphimedon is a result of secondary loss (the ghost locus hypothesis).sup.8. Here we analysed complete suites of ANTP-class homeoboxes in two calcareous sponges, Sycon ciliatum and Leucosolenia complicata. Our phylogenetic analyses demonstrate that these calcisponges possess orthologues of bilaterian NK genes (Hex, Hmx and Msx), a varying number of additional NK genes and one ParaHox gene, Cdx. Despite the generation of scaffolds spanning multiple genes, we find no evidence of clustering of Sycon NK genes. All Sycon ANTP-class genes are developmentally expressed, with patterns suggesting their involvement in cell type specification in embryos and adults, metamorphosis and body plan patterning. These results demonstrate that ParaHox genes predate the origin of sponges, thus confirming the ghost locus hypothesis.sup.8, and highlight the need to analyse the genomes of multiple sponge lineages to obtain a complete picture of the ancestral composition of the first animal genome., Author(s): Sofia A. V. Fortunato [sup.1] [sup.2] , Marcin Adamski [sup.1] , Olivia Mendivil Ramos [sup.3] [sup.4] , Sven Leininger [sup.1] [sup.4] , Jing Liu [sup.1] , David E. K. [...]

Wnt signaling plays an essential role in induction and development of the limb. Missing digits are one consequence of the reduced Wnt signaling in Wnt7a null mice, while extra digits result from excess Wnt signaling in mice null for the Wnt antagonist Dkk1. The extra digits and expanded apical ectodermal ridge (AER) of Dkk1-deficient mice closely resemble En1 null mice. To evaluate the in vivo interaction between En1 and the canonical Wnt signaling pathway, we generated double and triple mutants combining the hypomorphic doubleridge allele of Dkk1 with null alleles of En1 and Wnt7a. Reducing Dkk1 expression in [Dkk1.sup.d/+] [Wnt7a.sup.-/-] double mutants prevented digit loss, indicating that Wnt7a acts through the canonical pathway during limb development. Reducing Dkk1 levels in [Dkk1.sup.d/d][En1.sup.-/-] double mutants resulted in severe phenotypes not seen in either single mutant, including fused bones in the autopod, extensive defects of the zeugopod, and loss of the ischial bone. The subsequent elimination of Wnt7a in [Dkk1.sup.d/d][En1.sup.-/-][Wnt7a.sup.-/-] triple mutants resulted in correction of most, but not all, of these defects. The failure of Wnt7a inactivation to completely correct the limb defects of [Dkk1.sup.d/d][En1.sup.-/-] double mutants indicates that Wnt7a is not the only gene regulated by En1 during development of the mouse limb. Keywords: Limb development; Wnt signaling; AER; Dkk1; En1; Wnt7a

Doubleridge is a transgene-induced mutation characterized by polydactyly and syndactyly of the forelimbs. The transgene insertion maps to the proximal region of chromosome 19. During embryonic development of the mutant forelimb, delayed elevation and compaction of the apical ectodermal ridge (AER) produces a ridge that is abnormally broad and flat. Fgf8 expression persists in the ventral forelimb ectoderm of the mutant until E10.5. Strong expression of Fgf8 and other markers at the borders of the AER at E11.5 gives the appearance of a double ridge. At E11.5, apoptotic cells are distributed across the broadened ridge, but at E13.5, there is reduced apoptosis in the interdigital regions. The Shh expression domain is widely spaced at the posterior margin of the AER. The doubleridge AER is morphologically similar to that of En1 null mice, but the expression of En1 and Wnt7a is properly restricted in doubleridge, and the dorsal and ventral structures are correctly determined, doubleridge thus exhibits an unusual limb phenotype combining abnormal compaction of the AER with normal dorsal/ventral patterning. Keywords: Limb development; AER; Dorsal-ventral patterning; Fgf8; En1; Polydactyly; Syndactyly; Mouse

In the mouse embryo, Dlx5 is expressed in the otic placode and vesicle, and later in the semicircular canals of the inner ear. In mice homozygous for a null Dlx5/LacZ allele, a severe dysmorphogenesis of the vestibular region is observed, characterized by the absence of semicircular canals and the shortening of the endolymphatic duct. Minor defects are observed in the cochlea, although Dlx5 is not expressed in this region. Cristae formation is severely impaired; however, sensory epithelial cells, recognized by calretinin immunostaining, are present in the vestibular epithelium of [Dlx5.sup.-/-] mice. The maculae of utricle and saccule are present but cells appear sparse and misplaced. The abnormal morphogenesis of the semicircular canals is accompanied by an altered distribution of proliferating and apoptotic cells. In the [Dlx5.sup.-/-] embryos, no changes in expression of Nkx5.1(Hmx3), Pax2, and Lfng have been seen, while expression of bone morphogenetic protein-4 (Bmp4) was drastically reduced. Notably, BMP4 has been shown to play a fundamental role in vestibular morphogenesis of the chick embryo. We propose that development of the semicircular canals and the vestibular inner ear requires the independent control of several homeobox genes, which appear to exert their function via tight regulation of BPM4 expression and the regional, organization of cell differentiation, proliferation, and apoptosis. Key Words: Dlx; homeobox gene; transcription factor; bone morphogenetic protein-4; inner ear; apoptosis.

Abstract Background Sox genes are HMG-domain containing transcription factors with important roles in developmental processes in animals; many of them appear to have conserved functions among eumetazoans. Demosponges have fewer Sox genes than eumetazoans, but their roles remain unclear. The aim of this study is to gain insight into the early evolutionary history of the Sox gene family by identification and expression analysis of Sox genes in the calcareous sponge Sycon ciliatum. Methods Calcaronean Sox related sequences were retrieved by searching recently generated genomic and transcriptome sequence resources and analyzed using variety of phylogenetic methods and identification of conserved motifs. Expression was studied by whole mount in situ hybridization. Results We have identified seven Sox genes and four Sox-related genes in the complete genome of Sycon ciliatum. Phylogenetic and conserved motif analyses showed that five of Sycon Sox genes represent groups B, C, E, and F present in cnidarians and bilaterians. Two additional genes are classified as Sox genes but cannot be assigned to specific subfamilies, and four genes are more similar to Sox genes than to other HMG-containing genes. Thus, the repertoire of Sox genes is larger in this representative of calcareous sponges than in the demosponge Amphimedon queenslandica. It remains unclear whether this is due to the expansion of the gene family in Sycon or a secondary reduction in the Amphimedon genome. In situ hybridization of Sycon Sox genes revealed a variety of expression patterns during embryogenesis and in specific cell types of adult sponges. Conclusions In this study, we describe a large family of Sox genes in Sycon ciliatum with dynamic expression patterns, indicating that Sox genes are regulators in development and cell type determination in sponges, as observed in higher animals. The revealed differences between demosponge and calcisponge Sox genes repertoire highlight the need to utilize models representing different sponge lineages to describe sponge development, a prerequisite for deciphering evolution of metazoan developmental mechanisms.