rom the identification of genes to the characterization of their functions and interactions. Developmental biologists have long used whole mount in situ hybridization (WISH) to determine gene expression patterns, as a vital tool for formulating and testing hypotheses about function. This paper describes the application of WISH to the study of gene expression in larval and adult schistosomes. Fixed worms were permeablized by proteinase K treatment for hybridization with digoxygenin-labelled RNA probes, with binding being detected by alkaline phosphatase-coupled anti-digoxygenin antibodies, and BM Purple substrate. Discrete staining patterns for the transcripts of the molecules Sm29, cathepsin L, antigen 10.3 and chorion were observed in the tegument cell bodies, gut epithelium, oesophageal gland and vitelline lobules, respectively, of adult worms. Transcripts of the molecules SGTP4, GP18-22 and cathepsin L were localized to tegument cell bodies and embryonic gut, respectively, of lung schistosomula. We also showed that Fast Red TR fluorescent substrate can refine the pattern of localization permitting use of confocal microscopy. We believe that method of WISH will find broad application, in synergy with other emerging post-genomic techniques, such as RNA interference, to studies focused at increasing our molecular understanding of schistosomes

Gsx class proteins are members of the ParaHox homeodomain transcription factor family with conserved roles in specification and patterning of the nervous system. We report the cloning of two Gsx genes, Gsh1 and Gsh2, from the frog Xenopus tropicalis. We demonstrate the existence of a single, intact Xenopus ParaHox cluster, containing Gsh1, Pdx, and Cdx2, plus three degenerate clusters containing Gsh2, Cdx1, and Cdx4. Anterior expression boundaries of genes from the intact ParaHox cluster are co-linear with respect to their genomic organization. We show that Gsh1 and Gsh2 exhibit complex, overlapping patterns of expression within the anterior nervous system from open neural plate stages. We also find that expression of Gsh2, Nkx6, and Msx1 across the medio-lateral axis of the amphibian neural plate is strikingly similar to that of related genes in the Drosophila neuroectoderm. These findings provide further evidence for a conserved pathway regulating dorso-ventral patterning in the Bilateria. Developmental Dynamics 238: 194 -203, 2009.

SUMMARYThe Gsx genes encode members of the ParaHox family of homeodomain transcription factors, which are expressed in the developing central nervous system in members of all major groups of bilaterians. The Gsx genes in Xenopus show similar patterns of expression to their mammalian homologues during late development. However, they are also expressed from early neurula stages in an intermediate region of the open neural plate where primary interneurons form. The Gsx homologue in the protostome Drosophila is expressed in a corresponding intermediate region of the embryonic neuroectoderm, and is essential for the correct specification of the neuroblasts that arise from it, suggesting that Gsx genes may have played a role in intermediate neural specification in the last common bilaterian ancestor. Here, we show that manipulation of Gsx function disrupts the differentiation of primary interneurons. We demonstrate that, despite their similar expression patterns, the uni-directional system of interactions between homeodomain transcription factors from the Msx, Nkx and Gsx families in the Drosophila neuroectoderm is not conserved between their homologues in the Xenopus open neural plate. Finally, we report the identification of Dbx1 as a direct target of Gsh2-mediated transcriptional repression, and show that a series of cross-repressive interactions, reminiscent of those that exist in the amniote neural tube, act between Gsx, Dbx and Nkx transcription factors to pattern the medial aspect of the central nervous system at open neural plate stages in Xenopus.