The functional characterization of vertebrate homologs of Drosophila genes participating in a variety of developmental processes, suggests that some of the principles controlling cellular differentiation and determination in flies are also applicable to mammals. Accordingly, we hypothesized that genes involved in Drosophila cell fate determination during CNS and PNS development are likely conserved in vertebrates. Prospero mutant flies exhibit transcriptional misregulation of several genes, including the neuronal subset-specific genes, which leads to severe defects in axonal outgrowth and glial cell differentiation. We have identified a murine gene (Prox1) with sequence similarity to the prospero gene. The analysis of the Prox1 expression pattern in mice revealed interesting similarities to the fly prospero gene. In the developing CNS, Prox1 mRNA is found to be restricted to the subventricular region (SVR) that contains mostly postmitotic neurons. Prox1 expression was also detected in the developing lens, pancreas, liver and heart. In the lens, expression is seen in the region where fibers elongate prior to cellular differentiation, whereas in the pancreas, expression starts to diminish upon endocrine differentiation. Thus, Prox1 may participate not only in the differentiation of neuronal cells, but also of other cell types during early vertebrate development. We recently created mice nullizygous for Prox1 using homologous recombination in ES cells. Prox1 -/- embryos die in utero at E14.5 from a variety of phenotypic alterations in the spinal cord, neural crest, liver and lens. These alterations suggest that Prox1 plays a general role during murine cellular differentiation. We want to identify the mechanisms by which Prox1 controls this complex process; and in particular whether the observed phenotype is a consequence of alterations in cell migration, cell proliferation, or cell death. We will use a variety of cellular markers in the affected tissues of Prox1 -/- embryos to determine whether Prox1 function is necessary and sufficient for neuronal, neural crest, and lens differentiation, and liver and pancreas development. Target genes regulated by Prox1 during liver and pancreas differentiation will be revealed by subtractive cDNAs cloning using cDNA prepared from wild type an and Prox1 -/- foregut endoderm. Prox1 interacting proteins in the CNS and endoderm will be identified by a yeast-two hybrid approach. These experiments will further define the mechanisms and pathways involved in vertebrate cell differentiation, and should help to understand the role of Prox1 during this process.
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