UC Irvine

Germ layer specification is one of the earliest developmental events in metazoan organisms, and relies upon the combinatorial interactions of signaling pathways and transcription factors (TFs), and the epigenetic landscape. A complete understanding of the gene regulatory program that contribute to the specification of the mesoderm and endoderm (‘mesendoderm’) in vivo is a crucial unanswered question. The Nodal signaling pathway, which is zygotically activated in the vegetal hemisphere of the early Xenopus embryo, is transcriptionally mediated by the TF Foxh1 and plays a key role in mesendoderm development. It has been suggested that Foxh1 plays dual regulatory roles in transcriptional mediation, and also functions independently of Nodal signaling. However, little is known about the contribution of Foxh1 to the activation of the mesendoderm program via TF binding to cis-regulatory modules (CRMs).

Here, I have investigated the early functions of Xenopus Foxh1. I have utilized chromatin immunoprecipitation (ChIP) coupled with deep sequencing to investigate the genome-wide in vivo binding patterns of Foxh1 over the time course of early mesendoderm development, and compared Foxh1 occupancy to the recruitment of RNA polymerase II, epigenetic marks, and zygotic factors. I have found that Foxh1 binding is dynamic and bookmarks the early embryonic genome before zygotic genome activation. Furthermore, this early binding recruits the co-repressor Groucho/Tle. I identified a population of Foxh1 persistent peaks that are occupied throughout early mesendoderm development. These persistent peaks are marked as active enhancers, and recruit Smad2/3 and the zygotic pioneer factor Foxa. Therefore, they are likely critical mesendoderm CRMs, likely pioneered by Foxh1.

Further, through mRNA-seq coupled with loss-of-function, I’ve expanded the list of bona-fide direct Foxh1 and Nodal signaling targets throughout mesendoderm development. Foxh1 direct targets appear dynamically regulated across the time course, and I confirm that Foxh1 functions dually as an activator and repressor. Temporal analysis of the expression of Foxh1 repressed targets suggests that Foxh1 plays a role in the regulation of maternal genes.

My findings indicate a complex role for maternal Foxh1 at the top of a temporal hierarchy in the regulation of the early mesendoderm gene regulatory program. I propose a model whereby Foxh1 interacts with transcriptional co-repressors and activators in a context-specific fashion to regulate enhancer activation through both time and space. As the roles of Nodal signaling and Foxh1 in mesoderm and endoderm development are conserved among vertebrates, the findings presented here will have broad impacts in the fields of developmental, evolutionary, and stem cell biology.