Loss of Impacts Neurulation and Cranial Neural Crest Specification During Early Head Development.

The specification of anterior head tissue in the late gastrulation mouse embryo relies on signaling cues from the visceral endoderm and anterior mesendoderm (AME). Genetic loss-of-function studies have pinpointed a critical requirement of LIM homeobox 1 (LHX1) transcription factor in these tissues for the formation of the embryonic head. Transcriptome analysis of embryos with gain-of-function LHX1 activity identified the forkhead box gene, as one downstream target of LHX1 in late-gastrulation E7.75 embryos. Our analysis of single-cell RNA-seq data show is co-expressed with and in the anterior midline tissue of E7.75 mouse embryos, and in the anterior neuroectoderm (ANE) at E8.25 alongside head organizer genes and . To study the role of during early development we used CRISPR-Cas9 gene editing in mouse embryonic stem cells (mESCs) to generate bi-allelic frameshift mutations in the coding sequence of . In an model of the anterior neural tissues derived from -loss of function (LOF) mESCs and extraembryonic endoderm cells, expression of head organizer genes as well as and was reduced, pointing to a need for FOXD4 in regulating early neuroectoderm development. Mid-gestation mouse chimeras harbouring -LOF mESCs displayed craniofacial malformations and neural tube closure defects. Furthermore, our data showed a loss of FOXD4 impacts the expression of cranial neural crest markers and . Our findings have demonstrated that FOXD4 is essential in the AME and later in the ANE for rostral neural tube closure and neural crest specification during head development.