The LHX1 Gene Regulatory Network Represses Pluripotency and Modulates Multiple Signaling Pathways to Specify the Embryonic Head [Embryo_RNA-seq]

The development of the embryonic head in mice relies on the activity of the transcription factor LIM homeobox 1 (LHX1) in the anterior epiblast. In this study, we sought to unravel the molecular role of LHX1 during and immediately following gastrulation. We utilized CRISPR-Cas9 gene editing in embryo models as well as DamID-seq, RNA-seq, and ATAC-seq in mouse embryos. These techniques enabled us to reveal the genome wide targets of LHX1 for the first time in gastrulation stage mouse embryos. Integrated analysis of the omics datasets uncovered that LHX1 is instrumental in decommissioning the pluripotency network and determining the anterior fate of the precursor tissue of the embryonic head. We found that LHX1 directly binds to and regulates genes involved in WNT and FGF signaling pathway inhibition. We also identified a downstream target of LHX1, Kctd1, expressed in the anterior tissue, which acts to modulate canonical WNT activity. These findings identified a component of the gene regulatory network anchored by LHX1 that governs the development of the embryonic head. Overall design: RNA-seq was run on day six embryoid bodies derived from wild type (R1 ESC), Lhx1-knockout or Kctd1-knockout mouse embryonic stem cells. RNA-seq was run on chimeric mouse embryos with high contribution of doxycycline inducible FLAG-Lhx1 or FLAG-Lhx1-? ESCs. Following 24 hours of doxycycline treatment in utero, E7.25 or E7.75 chimeric embryos were dissected and RNA was extracted from individual embryos. DamID-seq peaks were called for LHX1 from ESC and XEN derived neuruloids as well as, E7.75 chimeric mouse embryos. ATAC-seq was run on E7.25 and E7.75 embryos from wild-type (control) or Lhx1-epiCKO embryos.