Project Summary For 25 years now, our laboratory has used live cell imaging with fluorescently marked proteins, and the forward genetics approach of isolating temperature-sensitive (TS) and embryonic-lethal C. elegans mutants, to investigate cytoskeletal function during embryonic cell divisions. More recently, we have (i) incorporated higher throughput positional cloning methods made possible by Illumina DNA sequencing technology to expand our effort to identify TS mutations in essential C. elegans genes as a community resource, and (ii) employed CRISPR/Cas9 genome editing technology to augment our genetics and live cell imaging approaches. Over the past five years, in addition to developing higher throughput positional cloning approaches, we have focused on two fundamentally important cell biological processes: (i) the orientation of cell division axes during development to establish multicellular architectures, and (ii) the mechanisms that nucleate and organize microtubules into a bipolar structure during oocyte meiotic spindle assembly, which occurs in the absence of the centrosomal microtubule organizing centers that mediate mitotic spindle assembly. Our research program over the next five years will focus on expanding our identification of TS mutations in essential C. elegans genes and making them generally available to the research community, extending our analysis of a previously unknown but widely conserved microtubule- independent and cortical actomyosin-dependent mechanism for orienting cell division axes during animal development, and improving our understanding of acentrosomal oocyte meiotic spindle assembly and function.