View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector ABSTRACTS / Developmental Biology 295 (2006) 393 – 402 The dorsal midline of the vertebrate neural tube has been appreciated as a signaling center directing cell fate specification, studied extensively in the spinal cord. Using genetic fate mapping of the Wnt3a lineage, we demonstrate that the dorsal midline is also an important source of migratory neuronal precursors, which participate in a variety of brain structures. Dorsal midline-derived cells in the midbrain form the mesencephalic nucleus of the trigeminal sensory system and participate in the periaqueductal gray and the inferior and superior colliculi. In addition, many nuclei in the pons and the medulla form as a consequence of tangential migrations from the dorsal midline. Wnt3a-expressing precursors in the rhombic lip, a transient dorsal midline structure, give rise to all granule cells, deep cerebellar nuclei, mossy fiber precerebellar nuclei and also generate some Purkinje cells and cerebellar interneurons. Finally, Wnt3a lineage derivatives, arising from early expression domains along the anterior/ posterior axis of the dorsal neural tube, participate in the formation of the central auditory system. Previous reports have indicated that diverse migratory streams originating at the rhombic lip give rise to cells in the isthmus, mesopontine tegmental system and cerebellum; our observations, however, demonstrate that this is not a unique feature of the rhombic lip and implicate the dorsal midline as a site of widespread neuronal migration. Our results further suggest that structures eventually becoming integrated into a functional pathway share a molecular identity early in their developmental history. doi:10.1016/j.ydbio.2006.04.216 194 Effects of Shh on Pax6 and proliferation in transdifferentiation Christian Gutierrez, Jason R. Spence, Mayur Madhavan, Katia Del Rio-Tsonis Miami University, Oxford, OH, USA Only a handful of organisms are able to regenerate the retina after injury. The embryonic chick has the ability to regenerate the retina during a limited window in embryonic development via stem/progenitor cells located in the ciliary margin of the eye or through the process of transdifferentiation, where the retina pigmented epithelium (RPE) dedifferentiates, proliferates and re-differentiates into laminated retina complete with all of the cell types. Transdifferentiation only occurs until E4.5 and when stimulated by a source of fibroblast growth factor (FGF). We have previously shown that overexpression of Sonic Hedgehog (Shh) is able to inhibit FGF stimulated transdifferentiation. Here we examine that Pax6 is regulated by Shh during transdifferentiation. At E3 – E3.5, Pax6 is expressed in the RPE, but by E4 it is present at low levels, and is absent in the RPE by E5. It has previously been shown that Pax6 can induce transdifferentiation of the RPE. We demonstrate that FGF is able to induce Pax6 expression in the RPE after the retina has been removed. Furthermore, we show that Shh is able to dramatically decrease the number of Pax6 positive RPE cells, indicating that Shh plays a role in maintaining RPE identity by inhibiting Pax6 397 expression. We have also shown that when FGF is added to the eye after retinectomy, the increase in Pax6 expression is correlated with an increase in proliferation as assayed by BrdU incorporation. Since it has been shown that Shh inhibits Pax6 expression and transdifferentiation, it is expected that Shh will also limit the amount of proliferation in the RPE. doi:10.1016/j.ydbio.2006.04.217 195 Adherens junctions in chick optic development Ricardo M. Borges, C.Y. Irene Yan* Department of Cell and Developmental Biology, University of Sao Paulo, Sao Paulo, Brazil Early optic development begins when the neuroepithelium of neural tube outpockets bilaterally, forming the optic vesicles. The optic vesicles grow as a continuous epithelial sheet, contact the surface ectoderm and invaginate, forming bilayered optic cups. The inner portion of the optic cup forms the neuroretina (NR), and the outer portion forms the retinal pigmented epithelium (RPE). At the time of its contact with the neuroepithelium, the surface ectoderm thickens into the lens placode and invaginates to form the lens vesicle. Both optic vesicle and lens placode invagination requires maintenance of tissue cohesion. Here, we investigate the dynamics of adherent junctions localization in the eye during optic invagination in chick embryos. Imunohistochemistry for components of Adherens Junctions (AJ) showed that, at optic vesicle stages, neuroepithelial AJ were concentrated in cell membranes lining the ventricular surface, while the surface ectoderm expressed AJ homogeneously between cell boundaries. In invaginating optic vesicles, AJ remained polarized at neuroepithelium ventricular surface, while surface ectoderm labeling became concentrated at the distal portion of the lens placode. In optic cups, labeling was concentrated at the cell membranes that constitute the NR/RPE border, and around the lumen of the lens vesicle. We also detected the presence of the GTPases Rac1 and Cdc42 in optic tissues during these stages, suggesting that localization of AJ during eye morphogenesis is coordinated with differentiation of optic tissues and could be regulated by GTPases. Supported by FAPESP. doi:10.1016/j.ydbio.2006.04.218 196 Wnt5A, a marker for dorsal retinal pigmented epithelium C.Y. Irene Yan*, Eliane Rossi, Fernanda Siwiec Biomedical Sciences Inst., Univ. de São Paulo, São Paulo, SP, Brazil The optic vesicle forms an optic cup with distinct threedimensional axial identity after intense morphogenesis and patterning. These axes can be identified through both anatomical and molecular landmarks. The proximal –distal axis in the 398 ABSTRACTS / Developmental Biology 295 (2006) 393 – 402 bilayered optic cup is defined anatomically by the presence of the prospective neural retina (NR) in the distal layer and the retinal pigmented epithelium (RPE) proximally. Accordingly, molecular markers accompany morphogenesis by restricting their expression to definite compartments. For instance, in the optic cup, the prospective neural retina expresses Chx10, and the RPE, Mitf. However, to facilitate identification of definite events during oculogenesis, there remains a need to identify additional markers of optical development. Thus, we performed here a screen for Wnt ligands that are expressed during eye development. Specifically, we examined the expression of Wnt1, Wnt3, Wnt4-1 and Wnt5A during chick optic vesicles stages up to optic cup formation. Of these four genes, only Wnt5A was consistently expressed in the dorsal optic cup. Although Wnt1, Wnt3 and Wnt4-1 were present in the developing nervous system, neither was found in the optic vesicle or cup. Wnt5A was first detected at the dorsal region of the RPE at stage HH14. In stages HH15 and HH16, its expression domain increased ventrally in the both nasal and temporal domains. Throughout all these stages, the labeling was restricted to the RPE only, and was completely absent in the neural retina. Based on these data, we would like to propose Wnt5A as a marker for early dorsal retinal pigmented epithelium. Corresponding author: reneyan@icb.usp.br. doi:10.1016/j.ydbio.2006.04.219 197 Transdifferentiation in Xenopus laevis eye Alexander S. Jerome, Maria N. Vergara, Katia Del Rio-Tsonis Miami University, Oxford, OH, USA The process of transdifferentiation involves the transformation of a differentiated cell type into another. During this process, a cell must lose its phenotype, the characteristics that make it a unique cell type that performs specific functions, and become ‘‘stem-like’’, being able to proliferate and give rise to different kinds of cells. This outstanding phenomenon occurs under very specific circumstances in vertebrates, and its understanding could have profound implications in the field of regenerative and developmental biology. The eye of the Xenopus laevis tadpole provides a good model to analyze this process. In the present study, we have characterized the process of transdifferentiation of pigmented eye tissues in Xenopus tadpoles, at a stage in which the eye is already fully developed. Our aim is to establish a model that will allow for the study of the molecular mechanisms that drive the process of transdifferentiation. We have been able to induce the transdifferentiation of pigmented epithelium explants into lens when transplanted into host Xenopus eyes. In addition, we have induced retina regeneration from pigmented tissues after complete removal of the retina in vivo. We have also assessed the influence of several growth factors and morphogens on this process. doi:10.1016/j.ydbio.2006.04.220 198 Characterization of silica spicule formation during the resuscitation and in vitro cell culture of Hymeniacidon perleve Wei Zhang 1, Xupeng Cao 2, Xingju Yu 1 1 Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China 2 Graduate School of the Chinese Academy of Sciences, Beijing, China The biogenic silica mineralization in an intertidal marine sponge Hymeniacidon perleve (Porifera: Demospongiae) has been investigated during the developmental process over one year period and in an in vitro sponge cell culture. Tissue samples of different developmental stages from dormancy to bloom and decline were collected. The structural dimensions and development characteristics of silica spicules were measured. It was found that the dimensional characteristics of spicules were restricted by their material properties. The spicule development that was closely linked with the sponge development can be classified as four distinct stages: newly born, growing, maturing and over-matured. In in vitro cell culture of archaeocyte-dominant cell populations (ADCP), a time-lapse microscopic observation was set up for studying the spicule formation and cell – spicule interaction over 1 month period. The first spicule appeared on day 10 during the ADCP culture, and the dynamics of spicule formation mimics the spicule development in the field. To understand the molecular regulation of spicule developments, the silicatein gene, which is responsible for the silicification of sponge spicules has been cloned. In both tissues development and cell cultures, the expressions of silicatein are correlated well with the onset and growth of spicules; however, the changes in the number of spicules formed lag behind the silicatein gene expression. doi:10.1016/j.ydbio.2006.04.221 199 ram-6 is required for Caenorhabditis elegans male sensory rays morphogenesis H.Y. Lee, Y.M. Lam, S.W. Tsang, K.L. Chow Hong Kong University of Science and Technology, Hong Kong Caenorhabditis elegans males develop nine pairs of bilaterally symmetrical peripheral sensory organs known as rays. Although they are all morphologically and positionally distinguishable, each of them develop smooth boundary against the cuticular fan structure. We are interested in a class of ‘‘ram’’ genes essential for the morphogenesis of these sensory rays. A new component, designated as ram-6, was identified in an EMS mutagenesis screen. ram-6 mutants display severe swollen rays phenotype in all of the rays and was shown to complement all other known ram genes. Temperature shift experiments suggested that ram-6 participates not just in ray development but also in early embryonic development. Mutant