Somites are the most prominent segmented feature of vertebrate embryos. These metameric blocks of paraxial mesoderm emerge in an anterior-to-posterior progression along the body axis, and subsequently differentiate into axial skeleton, segmental musculature and dermis. The process of somitogenesis involves both patterning and morphogenetic mechanisms that begin during gastrulation. Initially, involution/ingression, as well as convergent extension movements place presomitic mesoderm into a paraxial position underneath ectoderm. Ensuing establishment of segmental pattern in presomitic mesoderm involves activity of a molecular clock that is directly or indirectly is linked to Notch/Delta intercellular signaling. Subsequently, an incompletely understood set of morphogenetic cellular behaviors generates individual somites, which are composed of a mass of internal mesenchymal cells surrounded by epithelial border cells. Currently, it is not understood how gastrulation movements influence segmental patterning of the presomitic mesoderm. Furthermore, the cellular behaviors that execute somite morphogenesis, as well as their genetic regulation, remain incompletely defined. This proposal will address the above problems combining a novel multi-level confocal imaging of living embryos with mutational and gene mis-expression analyses uniquely feasible in zebrafish. Utilizing in vivo multi-level time lapse confocal microscopy we will define the sequence of morphogenetic cellular behaviors that underlie somite formation. Specifically, we will quantify the amount of cellular intercalation, intercellular contacts and cell shape changes of prospective border and internal cells that occur during somitogenesis. Fate mapping experiments will reveal how the convergent extension movements of gastrulation are connected to elaboration of segmental pattern in early presomitic mesoderm and to the initial cell fate choice between epithelial border and internal mesenchymal cells. We will also test whether the convergence and/or extension movements are required for specification of internal cells by analyzing their development in mutants with defined patterning and/or convergence extension defects. We will determine the role of internal cells in somite differentiation by analyzing cellular fates in the somites of knypek/trilobite double mutants that lack internal somitic cells. Finally, we will identify specific cellular behaviors during somite morphogenesis that are dependent on Delta-Notch and Ephrin signaling by analyzing cell behaviors in embryos in which Delta-Notch or Ephrin signaling are specifically activated or inhibited in individual presomitic mesodermal cells.
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