Gastrulation is the fundamental event during embryogenesis that generates the three primary germ layers from the pluripotent epiblast. To understand this intricate process, it is essential to use systems-based approaches to elucidate the genetic regulatory network that controls the tightly coordinated events of patterning, differentiation, and morphogenesis that take place during gastrulation. In this application, we will use sophisticated computational tools for the de novo reconstruction of regulatory networks to investigate mouse gastrulation. In our preliminary studies, we have generated a genetic regulatory network (interactome) for mouse epiblast stem cells (EpiSC) using unbiased reverse-engineering approaches. Using gene expression signatures generated from peri-gastrulation mouse embryos, we have interrogated the EpiSC interactome to identify candidate master regulators of gastrulation, as well as cis-regulatory sequence motifs and cognate transcription factors that are likely to be active during gastrulation. Therefore, based on these preliminary findings, we hypothesize that our systems approach can identify novel master regulators of key biological processes during mouse gastrulation. We will now pursue a comprehensive analysis of the genetic regulatory network that governs mouse gastrulation through three linked specific aims: (1) Identification of master regulators of mouse epiblast during gastrulation by unbiased systems analyses using signatures from peri-gastrulation mouse embryos to interrogate the EpiSC interactome for the identification of candidate master regulators, followed by functional validation studies; (2) Analysis of the transcriptional network that regulates mouse gastrulation by computational identification and experimental validation of cis-regulatory motifs and cognate transcription factors that are likely to function in gastrulation; and (3) Functional analyses of the regulatory network for gastrulation by testing the inferred regulatory network and biological functions of master regulators using signatures from mouse mutants that are defective in gastrulation, and by performing loss-of-function analyses of candidate master regulators in mouse embryos in vivo. Taken together, these studies will provide novel mechanistic insights into a central biological process that is essential for proper embryonic development, and whose perturbation can lead to major congenital defects.
Our proposed studies should have significant implications for understanding the basis of several major categories of birth defects, such as holoprosencephaly and left-right laterality defects, which frequently originate from minor errors in the biological processes that take place during gastrulation. Understanding the genetic regulatory network that controls and coordinates the intricate process of gastrulation should provide greater insight into the origin of these congenital defects and possibly lead to new approaches for their prevention.
