Spindle orientation along the developmental axes in echinoderm embryos

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DESCRIPTION (provided by applicant): A common feature of animal development is that following fertilization, embryos undergo a species-specific, stereotypical pattern of cleavages that sequester developmental determinants into specific daughter cells. And while lineage tracing and cell ablation studies have firmly established the significance of these developmentally asymmetric cell divisions, study of the mechanisms that align the mitotic spindle relative to the developmental axes has been largely limited to a few model organisms. Sea urchins undergo radial cleavage, with each cleavage plane bisecting the plane of the previous division. By the end of the fourth division, factors associated with the vegetal cortex ar sequestered by asymmetric cell division into a population of cells that will in turn direct the formation of the endomesoderm. And despite the intense scrutiny placed on the developmental role of micromeres in sea urchins and asymmetric cell division in other systems, we understand little of the mechanisms that facilitate spindle positioning relative to the animal-vegetal axis during echinoderm development. In almost all cell types studied to date, the mitotic spindle obeys Hertwig's rule and aligns itself along the long axis of the cell, facilitated by cortical puling forces generated by the microtubule motor, dynein. This proposal seeks to understand how the mitotic spindle is positioned relative to developmental axes during the initial cleavages of the sea urchin embryo. We hypothesize that factors that define cell polarity in early blastomeres align the nucleus and its duplicated centrosomes prior to the onset of mitosis to influence spindle orientation during both symmetric and asymmetric cell division. To test this hypothesis, we will perform a series of live cell analyses in sea urchin and starfish embryos, whose complementary features will aid in defining what we believe is a highly conserved mechanism for spindle alignment in early embryos. The lines of experimentation that form the Specific Aims of this proposal will: 1) Define the role of the PAR complex in defining blastomere polarity and spindle orientation during early cleavage; and 2) Assess the role of polarity factors and Disheveled during micromere formation. Together, is anticipated that the these studies will lead to a mechanistic understanding of how polarity factors act to effect a highly stereotypical and precise pattern of cell divisions that will ultimately lead to axis determination and the specification of the germ layers on echinoderm embryos.