Dissection of the Mixl1 gene regulation network in endoderm development

Abstract

Gastrulation, a key process in early embryonic development, involves the formation of the three germ layers that will eventually become all tissues of the adult mouse. The Mix paired-like homeobox (Mixl1) transcription factor, expressed in both mouse and human gastrula-stage embryo, is instrumental for the formation of the definitive endoderm and, in mouse and human stem cell models, is correlated with endoderm propensity. However, whether MIXL1 function is coupled to the specification and differentiation of the endoderm has not been established. The aims of this project are to determine the role of Mixl1 in endoderm propensity and to identify a gene regulatory network of Mixl1 during endoderm formation.

In a timed Mixl1-induction model, early expression of Mixl1 in mouse epiblast stem cells produced the highest proportion of endoderm. An integrated analysis of MIXL1 ChIP-, ATAC- and RNA-seq studies has revealed that early induction resulted in reduced accessibility at ectoderm specific enhancers and a shift in inferred trajectory towards an endodermal fate. Mixl1 bound regions harbouring a dual-ATTA motif and resulted in expression changes of downstream genes independent of chromatin accessibility changes. Analysis by single cell gene expression after CRISPR perturbation (Perturb-seq) of direct targets of Mixl1 showed that Mixl1 target genes are engaged with both the activation and inhibition of gene modules downstream of Mixl1. Further, endoderm specification after Mixl1 induction relies on the activation and deactivation of a subset of regulons activated and deactivated by Mixl1 induction.

In summary, this study established a causative link between Mixl1 timing and endoderm formation, identified the chromatin accessibility changes associated with Mixl1 induction and elucidated the downstream gene network after Mixl1 induction.