Temporal Analysis of Combinatorial Gene Function during Vertebrate Body Elongation

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Project Summary The spinal column is composed of neural and mesodermal tissues, and birth defects, such as scoliosis, that affect the spinal column can originate from abnormal development of either tissue. There are many forms of spinal malformation that can occur in development, including scoliosis, or misshapen bone within the spinal column. Genetic studies, including our zebrafish analyses, show that mutations that perturb somitogenesis lead to scoliosis phenotypes in zebrafish, mice and humans. Many questions remain about the molecular basis for congenital spinal malformations. Embryonic development requires many gene regulatory networks (GRN) that are activated and repressed overtime which underlie the dynamic control of morphogenesis. These GRNs are used repeatedly throughout development but give rise to different developmental outcomes. These differences are critical to achieving the wide array of cellular phenotypes required for development of an entire organism while using a relatively small number of genes. The process of elongation of the vertebrate body axis requires tissue patterning, cellular differentiation and cell migration of multiple tissues simultaneously. In order for all of these complex processes to occur in the embryo they must be tightly regulated by the dynamic control of multiple GRNs over time. This project uses an innovative application of deactivated Cas9 technology to investigate how two transcription factors regulate mesodermal differentiation and cell migration of during early spinal column development. Transgenic lines will be generated to create heat shock controlled bi-partite CRISPRi and CRISPRa systems to down- and up-regulate expression of transcription factors required for spinal column development. Phenotypes will be characterized by morphology and RT-qPCR. RNA sequencing will be used to identify the genes downstream of these transcription factors. In Aim 2, the CRISPRi/CRISPRa systems will be used to perform a targeted genetic screen to interrogate the roles of these target genes in the regulation of cell migration during body elongation. This work will elucidate the roles of these transcription factors, allowing a better understanding of combinatorial gene function in development as well as the genetic and cellular dynamics of vertebrate body elongation.