Genetic Regulation of Germ Layer Formation in Vertebrate Development

Abstract

Early embryogenesis is a profoundly important time during which morphological rearrangements pattern the growing embryo. Research throughout animals has demonstrated that both the morphological changes and the underlying genetics are highly conserved. Despite the importance of this process our understanding of the genetic pathways regulating it is incomplete, especially concerning the stage of early gastrulation. During early gastrulation, the cells in the embryo undergo coordinated and large scale migrations. The goal of my research has been to overcome historical limitations in identifying genetic regulators of this process. To this end, we used RNA sequencing and candidate approaches to look for previously unidentified genes. Using these methods, we identified a novel, small, highly conserved secreted peptide that we named Toddler. We used TALEN and CRISPR/Cas9 gene editing technology, light sheet microscopy, transplantations, RNA sequencing and other techniques to generate and characterize toddler mutants. toddler mutants are inviable and have defects in many organ systems including their heart, vasculature and digestive system. We found that Toddler acts downstream of Nodal signaling during gastrulation and signals through the GPCRs Apelin Receptor A and B. Toddler signaling directly regulates animal pole directed migration of mesodermal cells and interacts genetically with the GPCR Cxcr4a to indirectly regulate migration of endodermal cells. We also found that Toddler’s receptor may be sensitive to mechanical force and other perturbations. Thus, the Toddler pathway is a good candidate for the genetic cue linking forces generated during morphogenesis to gene induction and cell internalization. These results demonstrate that toddler is a conserved essential regulator of cell migration during early gastrulation and is a proof of principal that other novel signals, including those that interact with toddler, are yet to be found.