BM formation in Lamc1EQ/EQ zygotes.

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BM formation in Lamc1EQ/EQ zygotes. BM formation in Lamc1EQ/EQ zygotes. (A–H) Whole-mount immunostaining of WT (A, C, E, G) and Lamc1EQ/EQ (B, D, F, H) blastocysts of E4.0 (A–D) and E4.3 (E–H) embryos. The asterisks indicate primitive endoderm cells. (I, J) Whole-mount immunostaining of E3.5 Lamc1EQ/+ (I) and Lamc1EQ/EQ (J) blastocysts cultured ex vivo for 48 h. Magenta, laminin; green, perlecan (A, B, E, F, I, J) or integrin α6 (C, D, G, H); blue, nuclei. Bars, 50 μm. The genotypes of the preimplantation embryos were determined by genomic PCR. (K, L) In situ binding of integrin α7x2β1 (green) to E5.5 embryonic sections. The RM was counterstained with an anti-laminin-α1 mAb (magenta). Blue, nuclei. (M, N) Immunofluorescence staining for laminin-α1 (cyan), Cdx2 (magenta), and Oct3 (green) in WT or Lamc1EQ/+ (M) and Lamc1EQ/EQ (N) E5.5 sections. The dashed lines indicate the region where extraembryonic ectoderm cells are associated with the BM. The open arrowheads indicate extraembryonic ectoderm cells dissociated from the BM. (O) Immunofluorescence staining for laminin (cyan) and laminin-α5 (green) in Lamc1EQ/EQ E5.5 sections. Blue, nuclei. The asterisks and open triangles indicate visceral endoderm cells and parietal endoderm cells, respectively. The arrowheads indicate the laminin-α5–positive epiblast BM. Bars, 50 μm. (P) Summary of the strategy for quantitative image analysis of RM length in the E5.5 egg cylinder. The RM length (double-headed arrow) was measured by image tracing. (Q) RM lengths measured in E5.5 Lamc1EQ/EQ embryos and control WT or Lamc1EQ/+ embryos. Data represent means ± SD. ***P < 0.001, significant difference by Welch's t test. The effect size was 3.13 and the statistical power was 1.0. ExE, extraembryonic ectoderm; Epi, epiblast. Daiji Kiyozumi et al. LSA 2018;1:e201800064 © 2018 Kiyozumi et al.