Protocadherin-1 is expressed in the notochord of mouse embryo but is dispensable for its formation

Abstract

Notochord is an embryonic midline structure that serves as mechanical support for axis elongation and the signaling center for the surrounding tissues. Precursors of notochord are initially induced in the dorsal most mesoderm region in gastrulating embryo and separate from the surrounding mesoderm/endoderm tissue to form an elongated rod-like structure, suggesting that cell adhesion molecules may play an important role in this step. In Xenopus embryo, axial protocadherin (AXPC), an orthologue of mammalian Protocadherin-1 (PCDH1), is indispensable for the assembly and separation from the surrounding tissue of the notochord cells. However, the role of PCDH1 in mammalian notochord remains unknown. We herein report that PCDH1 is expressed in the notochord of mouse embryo and that PCDH1-deficient mice form notochord normally. First, we examined the temporal expression pattern of pcdh1 and found that pcdh1 mRNA was expressed from embryonic day (E) 7.5, prior to the stage when notochord cells detach from the surrounding endoderm tissue. Second, we found that PCDH1 protein is expressed in the notochord of mouse embryos in addition to the previously reported expression in endothelial cells. To further investigate the role of PCDH1 in embryonic development, we generated PCDH1-deficient mice using the CRISPR-Cas9 system. In PCDH1-deficient embryos, notochord formation and separation from the surrounding tissue were normal. Structure and marker gene expression of notochord were also unaffected by loss of PCDH1. Major vascular patterns in PCDH1-deficient embryo were essentially normal. These results suggest that PCDH1 is dispensable for notochord formation, including the tissue separation process, in mammalian embryos. We successfully identified the evolutionary conserved expression of PCDH1 in notochord, but its function may differ among species.

Digoxigenin‐labeled RNA probes for untranslated regions enable the isoform‐specific gene expression analysis of myosin heavy chains in whole‐mount in situ hybridization

“…Whole‐mount in situ hybridization (WISH) was performed as previously described (Fukunaga et al, 2021; Yokoyama et al, 2009). Briefly, the embryos and dissected adult hindlimb muscles were fixed with 4% paraformaldehyde (PFA) at 4°C overnight, and dehydrated with methanol.…”

“…Whole-mount in situ hybridization (WISH) was performed as previously described (Fukunaga et al, 2021;Yokoyama et al, 2009).…”