Gastrulation occurs early in mouse development and specifies the three germ layers. The epiblast cells, present before gastrulation, lose potency and transition from pluripotency to multipotent endoderm, mesoderm or ectoderm cells. The gene Mix1 homeobox-like 1 (Mixl1) is vital for this process of gastrulation. Mixl1 is typically expressed in the primitive streak region, a region in the posterior of the mouse embryo from which the mesoderm and endoderm lineages arise. Mixl1-null mice models fail to gastrulate or form endoderm. In vitro experiments involving the in vitro counterpart to the epiblast, epiblast stem cells (EpiSC), suggest that the timing of Mixl1 expression biases the outcome of differentiation. However, it is unknown what role Mixl1 carries out during gastrulation.
To build a gene regulatory network (GRN) of endoderm differentiation, focusing on Mixl1, we differentiated EpiSC lines to model gastrulation and analysed the transcriptome (RNA-seq), chromatin availability (ATAC-seq) and Mixl1 binding activity (ChIP-seq). In addition to utilising established methods to determine the direct effector targets of Mixl1, we also developed a novel ATAC-seq analysis pipeline to predict the GRN of endoderm differentiation.A combination of differential chromatin availability and transcription factor binding prediction using ATAC-seq footprinting was used to expand the GRN past the direct targets of Mixl1. This provides a method to form testable hypotheses on the mechanistics of Mixl1 function, based on the second level of the Mixl1 GRN.
This study provides an understanding of the molecular mechanisms involved in the specification of the endoderm lineage and sheds light on the role of Mixl1 in the early mouse embryo.