Abstract
Gene regulatory networks controlling functional activities of spatially and temporally distinct endodermal cell populations in the early mouse embryo remain ill defined. The T-box transcription factor Eomes, acting downstream from Nodal/Smad signals, directly activates the LIM domain homeobox transcription factor Lhx1 in the visceral endoderm. Here we demonstrate Smad4/Eomes-dependent Lhx1 expression in the epiblast marks the entire definitive endoderm lineage, the anterior mesendoderm, and midline progenitors. Conditional inactivation of Lhx1 disrupts anterior definitive endoderm development and impedes node and midline morphogenesis in part due to severe disturbances in visceral endoderm displacement. Transcriptional profiling and ChIP-seq (chromatin immunoprecipitation [ChIP] followed by high-throughput sequencing) experiments identified Lhx1 target genes, including numerous anterior definitive endoderm markers and components of the Wnt signaling pathway. Interestingly, Lhx1-binding sites were enriched at enhancers, including the Nodal-proximal epiblast enhancer element and enhancer regions controlling Otx2 and Foxa2 expression. Moreover, in proteomic experiments, we characterized a complex comprised of Lhx1, Otx2, and Foxa2 as well as the chromatin-looping protein Ldb1. These partnerships cooperatively regulate development of the anterior mesendoderm, node, and midline cell populations responsible for establishment of the left-right body axis and head formation.
Highlights
After implantation, Nodal and Wnt signaling pathways coordinately instruct the symmetrical cup-shaped epiblast, the founder tissue of the embryo proper, to become appropriately patterned and give rise to the three primary germ layers: the ectoderm, endoderm, and mesoderm (Tam and Loebel 2007; Arnold and Robertson 2009)
At the early headfold (EHF) stage, robust Lhx1 expression becomes confined to the node and midline, with lower levels detectable in the cranial and cardiac mesoderm (Fig. 1D)
As for Lhx1, both Ldb1 and Ssbp3 mutant embryos display anterior patterning defects (Mukhopadhyay et al 2003; Nishioka et al 2005; Enkhmandakh et al 2006). These findings strongly suggest that Lhx1, Ldb1, and Ssbp3 cooperatively govern anterior DE (ADE) specification and midline morphogenesis
Summary
Nodal and Wnt signaling pathways coordinately instruct the symmetrical cup-shaped epiblast, the founder tissue of the embryo proper, to become appropriately patterned and give rise to the three primary germ layers: the ectoderm, endoderm, and mesoderm (Tam and Loebel 2007; Arnold and Robertson 2009). By a process of convergent extension, the node gives rise to the notochord, which, together with the AME, forms a continuous midline cell population (Yamanaka et al 2007), the source of key growth factor signals necessary to promote growth and patterning of the overlying neuroectoderm. As for the Eomes+ cell population (Costello et al 2011), Lhx1+ progenitors exclusively colonize the head and cardiac mesoderm and the entire gut endoderm as well as APS derivatives, including the AME, node, and notochord. Lhx binds to enhancer regions at both Otx and Foxa2 These findings strongly argue that Lhx functions together with its transcriptional partners, Otx and Foxa, to coordinately regulate AME, node, and midline development
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