A mechanistic insight into building the embryonic head

Abstract

Embryonic development is driven by molecular instructions encoded by transcriptional regulators that underpin the formation of the body plan. Following gastrulation, the foundation of the embryonic head emerges, being the first major body part to form. Analysis of gastrulating mouse embryos has revealed that the LIM homeobox 1 (LHX1) transcription factor (TF) is indispensable for head and face development. However, the precise function of LHX1 in the initiation of craniofacial morphogenesis has not been fully elucidated. The aim of this project was to identify the direct transcriptional targets and co-factors of LHX1 during the establishment of the embryonic head. We generated an LHX1 anchored gene regulatory network (GRN), utilising multi-omics analytics including RNA-seq, ATAC-seq and DamID-seq in vivo, using mouse embryos. Our data shows that during mid gastrulation, LHX1 is responsible for the modulation of Wnt signalling in the anterior embryo. In late gastrulation, LHX1 acts with OTX2 and FOXA2 to directly downregulate the pluripotency network in the anterior mesendoderm. LHX1 also regulates genes responsible for anterior identity and establishes key signalling gradients including Wnt, Hedgehog, retinoic acid, and FGF signalling. We identified genes expressed in the anterior region of head fold stage mouse embryos such as Kctd1 and Foxd4 as novel downstream targets of LHX1. To understand the function of these TFs during head development, we used chimeric embryos harbouring CRISPR-Cas9-edited mESCs and stem cell-derived embryo models. We showed that Foxd4 is essential for neurulation in the rostral neural tube and for the specification of the cranial neural crest population. A loss of Kctd1 impacted the canonical Wnt signalling pathway, derailing anterior mesendoderm lineage development. Overall, these findings revealed the role of LHX1 in the early formation of head tissue and defined a GRN anchored by LHX1 during embryonic development.