AUTHOR=McMahon Riley , Sibbritt Tennille , Aryamanesh Nadar , Masamsetti V. Pragathi , Tam Patrick P. L. 

TITLE=Loss of Foxd4 Impacts Neurulation and Cranial Neural Crest Specification During Early Head Development

JOURNAL=Frontiers in Cell and Developmental Biology

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2021.777652

DOI=10.3389/fcell.2021.777652

ISSN=2296-634X

ABSTRACT=<p>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, <italic>Foxd4,</italic> as one downstream target of LHX1 in late-gastrulation E7.75 embryos. Our analysis of single-cell RNA-seq data show <italic>Foxd4</italic> is co-expressed with <italic>Lhx1</italic> and <italic>Foxa2</italic> in the anterior midline tissue of E7.75 mouse embryos, and in the anterior neuroectoderm (ANE) at E8.25 alongside head organizer genes <italic>Otx2</italic> and <italic>Hesx1</italic>. To study the role of <italic>Foxd4</italic> 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 <italic>Foxd4</italic>. In an <italic>in vitro</italic> model of the anterior neural tissues derived from <italic>Foxd4</italic>-loss of function (LOF) mESCs and extraembryonic endoderm cells, expression of head organizer genes as well as <italic>Zic1</italic> and <italic>Zic2</italic> was reduced, pointing to a need for FOXD4 in regulating early neuroectoderm development. Mid-gestation mouse chimeras harbouring <italic>Foxd4</italic>-LOF mESCs displayed craniofacial malformations and neural tube closure defects. Furthermore, our <italic>in vitro</italic> data showed a loss of FOXD4 impacts the expression of cranial neural crest markers <italic>Twist1</italic> and <italic>Sox9</italic>. 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.</p>