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Biochem Biophys Rep
2021 Jun 15;27:101047. doi: 10.1016/j.bbrep.2021.101047.
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Protocadherin-1 is expressed in the notochord of mouse embryo but is dispensable for its formation.
Fukunaga K
,
Tanji M
,
Hanzawa N
,
Kuroda H
,
Inui M
.
???displayArticle.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/endodermtissue 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 endodermtissue. 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.
Fig. 1. Thepcdh1mRNA expression in mouse embryos. (A) An RT-PCR analysis showing the expression of pcdh1 mRNA in mouse embryo. β-actin served as a loading control. RT, reverse transcription. (B, C) Whole-mount in situ hybridization using sense and antisense probes of pcdh1 with E9.5 embryos. (D–O) Whole-mount in situ hybridization using antisense probes of pcdh1, shh, and pecam-1 with E9.5, 10.5 and 11.5 embryos. Black arrowhead: intersomitic vessel. White arrowhead: notochord. Scale bar: 0.5 mm. The experiments were performed at least twice, and representative results are shown.
Fig. 2. The PCDH1 protein expression in mouse embryos. (A) HE staining of transverse sections of E10.5 embryo. (B) Immunofluorescence staining for PCDH1 with transverse sections of E10.5 embryo. (C) Immunofluorescence staining for PECAM-1 with transverse sections of E10.5 embryo. DAPI staining (blue) shows nuclear positions. Note that the sections of A-C are consecutively taken from the same sample. White arrowhead: PCDH1-positive and PECAM-1-negative structure. Diagrams illustrating the positions of notochord and vasculature are shown in Figure S3. (D) HE staining of transverse sections of E8.5 embryos. (E) Immunofluorescence staining for PCDH1 with transverse sections of E8.5 embryos. DAPI staining (blue) shows nuclear positions. Note that the sections of D and E are consecutively taken from the same sample. no: notochord. nt: neural tube. pnvp: peri-neural vessel plexus. En: endodermal layer. Scale bar: 20 μm. Three embryos were used for each experiment. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. Generation of pcdh1 knockout mice using the CRISPR-Cas9 system. (A) A schematic drawing showing the position and sequences of gRNAs targeting exon2 of pcdh1 gene. Exons (black boxes) and introns (black lines) indicate the structure of one of seven transcription variants of pcdh1 (NM_029357.3). Targeting sequences and PAM sequences of gRNAs are indicated with underline and a gray background, respectively. (B) Genomic sequences around the targeting site of the wild-type (WT) and Δ11 alleles. Targeting sequences of gRNAs are indicated with underline. Dashes: deleted bases. (C) Gross appearance of pcdh1+/+ and pcdh1Δ11/Δ11 female mice at 8 weeks old. (D) Western blotting to detect PCDH1 protein in livertissue of pcdh1+/+ and pcdh1Δ11/Δ11 adult mice. GAPDH serves as a loading control. (E) Body weight of pcdh1+/+ and pcdh1Δ11/Δ11 mice at 3–8 weeks old. At least three mice were measured for each genotype. Data represent the mean ± SE. A t-test was used for the statistical analyses (*p < 0.05, **p < 0.01, and ***p < 0.001). (F) Body length of pcdh1+/+ and pcdh1Δ11/Δ11 adult male and female mice at 8 weeks old. At least three mice were measured for each genotype. Box plots show the median and 25-75th percentiles (middle line and box, respectively). Whiskers extend to the maximum and minimum data points. A t-test was used for the statistical analyses (*p < 0.05). N.S., not significant
Fig. 4. Notochord of pcdh1 knockout mice. (A, B) Gross appearance of E9.5 pcdh1+/+ and pcdh1Δ11/Δ11 embryos. Scale bars: 0.5 mm. (C, D) HE staining of the cross-sections of E9.5 pcdh1+/+ and pcdh1Δ11/Δ11 embryos. Scale bars: 20 μm. no: notochord. nt: neural tube. (E, F) In situ hybridization of E9.5 pcdh1+/+ and pcdh1Δ11/Δ11 embryos with shh probe. (G–J) Immunofluorescence of cross-sections of E10.5 pcdh1+/+ and pcdh1Δ11/Δ11 embryos. (K) qPCR analysis of E11.5 pcdh1+/+ and pcdh1Δ11/Δ11 embryos. A t-test was used for the statistical analyses (*p < 0.05). Four embryos were used for each genotype in each experiment. N.S., not significant
Fig. 5. Tissues surrounding notochord of pcdh1 knockout mice. (A–H) HE staining of the cross-sections of pcdh1+/+ and pcdh1Δ11/Δ11 embryos. (A–D) E10.5 embryos. Scale bars: 50 μm. (E–H) E13.5 embryos. Scale bars: 200 μm. cv: cardinal vein. da: dorsal aorta. es: esophagus. fg; foregut. lu: lung. no: notochord. nt: neural tube. sc: spinal code. vb: vertebra. Three embryos were used for each genotype in each experiment.
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