Abstract
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, Foxd4, as one downstream target of LHX1 in late-gastrulation E7.75 embryos. Our analysis of single-cell RNA-seq data show Foxd4 is co-expressed with Lhx1 and Foxa2 in the anterior midline tissue of E7.75 mouse embryos, and in the anterior neuroectoderm (ANE) at E8.25 alongside head organizer genes Otx2 and Hesx1. To study the role of Foxd4 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 Foxd4. In an in vitro model of the anterior neural tissues derived from Foxd4-loss of function (LOF) mESCs and extraembryonic endoderm cells, expression of head organizer genes as well as Zic1 and Zic2 was reduced, pointing to a need for FOXD4 in regulating early neuroectoderm development. Mid-gestation mouse chimeras harbouring Foxd4-LOF mESCs displayed craniofacial malformations and neural tube closure defects. Furthermore, our in vitro data showed a loss of FOXD4 impacts the expression of cranial neural crest markers Twist1 and Sox9. 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.
Highlights
The head is the first major body part to form immediately following gastrulation in vertebrate embryos, arising from the anterior germ layer tissues
We showed that Foxd4 is co-expressed with head organizer genes Lhx1 and Foxa2 in the anterior mesendoderm (AME) and notochord of late-gastrulation embryo, it is co-expressed with Otx2 and Hesx1 in the anterior neuroectoderm (ANE) of the early somite stage embryo
Using in vitro and in vivo models generated using CRISPR-Cas9 gene edited mouse embryonic stem cells (mESCs), we showed that the loss of FOXD4 function resulted in a reduction in head organizer activity and the disruption of cranial neural crest (CNC) development
Summary
The head is the first major body part to form immediately following gastrulation in vertebrate embryos, arising from the anterior germ layer tissues. Knock-out mouse models for key transcription factors Lhx and Foxa, expressed in both the AVE and AME, result in severe truncation of the embryonic head (Ang and Rossant, 1994; Shawlot and Behringer, 1995). Earlier work has identified many of the downstream targets of LHX1 in the AME are involved in the suppression of WNT signaling including Gsc, Dkk and Cer (Fossat et al, 2015; McMahon et al, 2019). To further study the potential target of LHX1 in the E7.75 mouse embryos, a conditional Lhx1-LOF model was used to identify the genes that are down-regulated with Lhx1-LOF (Sibbritt et al, 2018). Genes identified as potential targets of LHX1 include head organizer transcription factors Hesx and Otx, as well as Foxd. Knockout of either genes resulted in a truncated head at earlyorganogenesis stage (Matsuo et al, 1995; Martinez-Barbera et al, 2000)
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