CRISPR based screen for small GTPase regulators of morphogenesis in Xenopus

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Abstract Many birth defects arise from flaws in the cell behaviors that drive normal tissue organization, including the prevalent cases of neural tube defects, and craniofacial defects. There has been considerable progress in understanding the mechanics of cell behavior; in contrast, the upstream control, at the level of cytoskeletal regulatory proteins, and the transcriptional control of expression of the regulators has been documented in fewer cases. Nonetheless, we can point to several examples where a specific morphogenetic event is ultimately regulated by the expression of genes that encode regulators of small GTPases, particularly Rho. Here we hypothesize that we will gain insights into many embryonic contexts of morphogenesis by screening directly for regulators of small GTPases that are essential for subsets of the early morphogenetic events of gastrulation and neurulation. Rather than conduct open-ended screens, we focus on GTPase Regulators, where we have argued that there is a precedent for their general importance in morphogenesis. There are about 150 Rho GAPs and GEFs listed in the community database, Xenbase, with descriptions of their temporal expression, and sometimes, their transcript localization. We will prioritize genes that are zygotically expressed to avoid regulators of general cellular processes, and then knock out targets using G0 (aka F0) Crispr, which is sufficient to identify phenotypes. Using high-throughput imaging we will screen for phenotypes associated with gastrulation and neurulation defects. Hits will then be further characterized to determine if they are locally or globally expressed. This project will ultimately generate the molecular tools to manipulate and analyze the force generation and intercellular interactions that determine how cells move in the embryo. As with the insights into neural tube defects that have come from understanding neurulation movements in frogs, we will establish principles to examine other morphogenetic events and failures that lead to birth defects.