Spindle orientation along the developmental axes in echinoderm embryos

描述(申请人提供)：动物发育的一个共同特征是，在受精后，胚胎经历特定物种的、刻板的卵裂模式，将发育决定因素隔离到特定的子细胞中。虽然谱系追踪和细胞消融研究已经确定了这些发育不对称细胞分裂的重要性，但对有丝分裂纺锤体相对于发育轴对齐的机制的研究在很大程度上局限于少数模式生物。海胆经历径向分裂，每个分裂平面将前一个分裂的平面一分为二。到第四次分裂结束时，与植物皮质相关的因子被不对称的细胞分裂隔离成一组细胞，这些细胞反过来又指导内胚层的形成。尽管微体在海胆中的发育作用和在其他系统中的不对称细胞分裂受到了密切关注，但我们对棘皮动物发育过程中促进纺锤体相对于动植物轴定位的机制知之甚少。到目前为止，在几乎所有研究的细胞类型中，有丝分裂纺锤体遵循赫特威格规则，并在微管马达Dynein产生的皮质拉力的推动下，沿着细胞的长轴排列。这项建议试图了解在海胆胚胎最初的卵裂过程中，有丝分裂纺锤体是如何相对于发育轴定位的。我们假设，在早期分裂球中定义细胞极性的因素在有丝分裂开始之前对齐细胞核及其复制的中心体，以影响对称和不对称细胞分裂期间的纺锤体方向。为了验证这一假设，我们将对海胆和海星胚胎进行一系列活细胞分析，它们的互补特征将有助于确定我们认为是高度保守的早期胚胎纺锤体排列机制。形成这一建议的具体目标的实验路线将：1)确定PAR复合体在确定卵裂球极性和纺锤体早期分裂方向中的作用；以及2)评估极性因素和毛茸茸的因素在微核形成过程中的作用。总之，预计这些研究将导致对极性因素如何作用于高度刻板和精确的细胞分裂模式的机械性理解，这最终将导致轴的确定和棘皮动物胚胎胚层的规范。

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.