Genetics of mouse head formation: the morphogenetic activity of the Otx2-Lhx1-Dkk1 cascade in the axial mesendoderm

Abstract

In the mouse, the formation of the embryonic head is critically dependent on the function of homeobox transcription factors OTX2 and LHX1, the strict regulation of WNT signaling activity and the inductive signals that are emanating from the axial mesendoderm (AME) at gastrulation stage. Inactivation of Otx2 or Lhx1 results in head truncation, similar to the mutant embryos that are associated with the loss of Dkk1 gene which codes for an antagonist of the WNT signaling cascade. All three genes, Otx2, Lhx1 and Dkk1, are co-expressed in the AME, a tissue that is derived from the epiblast. The focus of this project is to investigate the molecular mechanism underpinning the formation, and the function of the AME for head formation. AME-specific ablation of Otx2 gene (Otx2ameCKO), mediated by the recombinase activity controlled by Foxa2 promoter (Foxa2-merCremer), resulted in a head truncation phenotype. In the AME of the Otx2ameCKO mutant embryos, the expression of Dkk1 was markedly reduced, which is consistent with the finding of Dkk1 being a downstream target of OTX2. In addition, Lhx1 expression was also absent in the AME of the Otx2ameCKO mutant embryos. This suggests that the head phenotype observed in the Otx2 mutants may be associated with the loss of both Lhx1 and Dkk1 activity. To test if Otx2, Lhx1 and Dkk1 may functionally interact for head morphogenesis, AME-specific compound Lhx1;Otx2, Lhx1;Dkk1 and Otx2;Dkk1 heterozygous embryos were generated. All the compound mutants displayed head abnormality, suggesting that these three genes act in synergy for head formation, with Otx2 at the top of the signaling hierarchy. While OTX2 can regulate Dkk1 expression through binding to the H1 conserved regulatory region, it was not known if OTX2 could directly control Lhx1. Bioinformatics search has identified three inter-species conserved regions of the Lhx1 locus (located +30kb, +1.5kb and -6.5kb relative to the ATG start codon) that contain OTX2 binding motifs. Experimental manipulation by RT-qPCR, ChIP-qPCR and luciferase assay have demonstrated that OTX2 activates gene expression when binding to the +1.5kb and the -6.5kb but not the +30kb conserved regions. In summary, these results have highlighted the tissue-specific requirement of Otx2 in the AME for head morphogenesis, and that the OTX2 controls both Dkk1 and Lhx1 expression for head formation. Further analysis of the tissue-specific requirement of LHX1 function in the whole epiblast, Lhx1 gene was ablated specifically in the epiblast by Meox2-Cre (to generate Lhx1epiCKO mutants). At E9.5, the mutant embryos resulted in the loss of head structure. In the Lhx1epiCKO mutants, AME was absent, which raised the possibility that the head phenotype is the consequence of the loss of the signaling tissue. Fluorescent-labelling of the cells in the endoderm layer subjacent to the anterior primitive streak revealed that the movement of the Lhx1-deficient cells was impaired. In the Xenopus, morpholino knockdown of Xlim1 (Lhx1 orthologue) also impaired morphogenetic movement of the chordamesoderm (a structure that is equivalent to the mouse AME), and results in head truncation. One of the protocadherin molecules, Pcdh8, has been demonstrated to be required for chordamesoderm development (the equivalent structure of AME) and is a downstream target of LHX1 in the Xenopus. In mouse, the paralogue of Pcdh8, Pcdh7 was found to co-expressed with Lhx1 in the posterior primitive streak of the mid-streak stage embryos. Bioinformatics search has identified three multi-species conserved regions around the Pcdh7 locus. ChIP-qPCR and luciferase assay confirmed that LHX1 binds directly to these regions and can activate Pcdh7 gene expression in P19 cells. In the Lhx1epiCKO mutants, the expression of Pcdh7 was markedly reduced in the anterior primitive streak and nascent AME, suggesting that the lack of Pcdh7 activity may account for the defective morphogenetic movement of the nascent AME. This data suggest a potential role of Pcdh7in contributing to the morphogenesis of AME, however, at this stage of the study, the Pcdh7-null embryos have not been made, further analysis of the AME structure in these mutant embryos are required. Furthermore, the requirement of Lhx1 in the mesoderm was assessed by generating Lhx1mesCKO. The mutant embryos displayed no head phenotype, suggesting that the function of Lhx1 is not required in the mesoderm for head formation. To address if Lhx1 function is specifically required in the AME for head formation, Lhx1ameCKO mutants were generated using the Foxa2-merCremer recombinase system. The conditional mutant embryo displayed a fully penetrant head truncation phenotype, similar to that associated with the elevated level of WNT signaling activity caused by the loss of Dkk1 function. The expression of Dkk1 and other WNT-related genes expressing in the AME (Hesx1, Gsc and Cer1) were all down-regulated in the Lhx1ameCKO mutants. LHX1 could bind to and activate Dkk1, Hesx1, Gsc and Cer1 regulatory regions. These results suggest that LHX1 acts in the AME by activating genes that antagonize WNT signaling. Therefore, LHX1 regulates an array of downstream genes that are required for the morphogenetic activity for the formation of the AME (Pcdh7) and the WNT signaling cascade suppressing function (Dkk1, Hesx1, Cer1 and Gsc) at two different phases of the formation of the embryonic head respectively. To identify the other putative downstream targets, transcriptome analysis and ChIP-sequencing experiments were performed on embryoid bodies (EBs) derived from embryonic stem (ES) cells that express Lhx1 upon doxycycline induction. The list of genes was cross-referenced with the transcriptome of E7.5 mouse embryos to identify LHX1-dependent genes (identified in EBs) that are expressed during gastrulation. By gene ontology analysis, genes that are associated with neural development by negatively modulate WNT signaling (Zic2, Zic3 and Hesx1) and the integrin-α6β4 signaling pathway for morphogenetic cell movement (Fyn, Lama1, Lamb1 and Lamc1) was identified. Consistent with their putative downstream status, these genes were co-expressed with Lhx1 during gastrulation. In summary, the findings presented here have uncovered a hierarchy of Otx2-Lhx1-Dkk1 activity for head formation, and in particular, different downstream activity of Lhx1 has been identified for the genesis and the signaling function of the axial mesendoderm during head morphogenesis.