Revealing how the mitotic spindle controls asymmetric cell division in vivo

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Summary The goal of this proposal is to discover how new forms of microtubule organization drive the first asymmetric cell divisions of a mammalian organism in vivo. One of the most salient features of multicellular organisms is their vast number of cell types. Many of these are produced by asymmetric cell division, in which a parental cell asymmetrically distributes cell fate determinants, or generates daughter cells with different shape, volume or cell polarity features. In most cell types, the mitotic spindle apparatus plays a fundamental role in asymmetric cell division. It comprises two key parts, the central spindle that interacts with chromosomes and promotes cytokinetic furrow formation, and an array of astral microtubules. These astral microtubules originate from centrosomes at the spindle poles and allow polarized cortical elements and force regulators to differentially control the spindle to drive asymmetric cell division. The mechanisms of asymmetric cell division have been studied in detail in cultured cells and non- mammalian organisms. Yet, the mouse embryo does not inherit centrioles form its parental cells and has been proposed to lack functional centrosomes and astral microtubule arrays. Therefore, it remains unclear how mitotic spindles are organized during the earliest stages of development, and how they may contribute to asymmetric cell division to drive the differentiation of the first mammalian cell types. Here, we combine live-imaging approaches with molecular and biophysical methods to test the function of a new form of mitotic spindle organization, which we found to drive asymmetric cell division in vivo. Unlike previous assumptions of lack of centrosomes and astral microtubule arrays, we found that some cells establish a highly asymmetric spindle with only one astral-like microtubule array. Manipulations of this asymmetric spindle disrupt the segregation of the first cell types. Thus, the central hypothesis of this proposal is that the establishment of this new form of asymmetric spindle organization drives the first asymmetric cell divisions in vivo. To test this, our aims will investigate the mechanisms by which the asymmetric spindle is assembled (Aim 1) and can drive asymmetric cell division (Aim 2). Aim 1 will specifically focus on dissecting the source and function of the molecular regulators required to assemble the asymmetric spindle, the role of cell polarity components, and the physical forces required to bring together the astral array and the central spindle to assemble a complete mitotic spindle apparatus. Aim 2 will investigate the mechanisms by which the asymmetric spindle drives asymmetric cell division. We will specifically test three fundamental mechanisms based on the differential regulation of cell cleavage orientation, daughter cell volume and cell cortex tension.