Figure 5. Rreb1 maintains epithelial organization in the early mouse embryo.

(A) Sagittal confocal optical section of the anterior of E7.5 Rreb1 wild-type and homozygous mutant embryos. Arrowheads highlight cells abnormally protruding from the VE overlying the epiblast. Sb, 25 μm. (B) Confocal maximum intensity projections (MIP) of immunostained E7.5 embryos showing ruffling of the extraembryonic VE (n = 7/8 E7.5 Rreb1^-/- embryos exhibit endoderm ruffling). Sb, 100 μm. (C) Confocal optical sections showing transverse cryosections of E7.5 Afp-GFP Rreb1 wild-type and homozygous mutant embryos. Boxes indicate regions shown in higher magnification in adjacent panels. Arrowhead indicates abnormal accumulation of Afp + VE cells and underlying Afp- DE cells at the anterior embryonic-extraembryonic boundary in Rreb1^-/- (n = 7/52 E7.5 Rreb1^-/- embryos exhibit anterior endoderm accumulations). Sb, 50 μm. (D, E) Maximum intensity projections (MIPs) of wholemount E7.5 embryos and confocal optical sections of transverse cryosections. (D) Dashed lines mark approximate plane of section. Sb, 50 μm. (E) Dashed yellow line outlines the epiblast. Sb, 50 μm. Asterisks mark abnormal gaps between tissue layers, which was the most common defect observed (n = 38/52 E7.5 Rreb1^-/-). (F) Representative images of Rreb1^+/+ and Rreb1^-/- embryos highlighting the epithelial defects observed: (i) abnormal accumulations of cells in the epiblast, (ii) epiblast folding (n = 8/52 52 E7.5 Rreb1^-/- embryos exhibit abnormal epiblast folding), in this case the epiblast is folded such that the putative anterior (aEpi) and posterior (pEpi) regions are adjacent to one another, (iii) formation of multilayered regions (highlighted with brackets) in the, typically monolayer, endoderm and epiblast. Sb 25 μm, high mag sb, 10 μm. (G–I) Confocal MIPs (G,H) and confocal optical sections showing transverse cryosections of Afp-GFP; Rreb1^+/+ and Rreb1^-/- embryos (I). Boxes indicate region shown in higher magnification in H. Sb, 50 μm. Pr, proximal; Ds, distal; A, anterior; P, posterior; R, right; L, left; Epi, epiblast; aEpi, anterior epiblast; pEpi, posterior epiblast; PS, primitive streak; Endo, endoderm; ACD, allantois core domain; AOM, allantois outer mesenchyme; Ch, chorion; Meso, mesoderm; ExVE, extraembryonic visceral endoderm; EmVE, embryonic visceral endoderm; DE, definitive endoderm; NE, neurectoderm; Al, allantois.

Figure 5—figure supplement 1. Rreb1 mutant embryos have perturbed epithelial architecture.

(A) Confocal optical sections showing transverse cryosections in the extraembryonic region of E6.5 embryos. Arrowheads highlight regions where cell layers are abnormally separated from one another. Sb, 25 μm. (B) Brightfield images of Rreb1^+/+ and Rreb1^-/- littermates at embryonic day 7.5. Arrowheads highlight the abnormal accumulation of cells at the anterior embryonic-extraembryonic boundary. (C) Arrows highlight SOX17-expressing definitive endoderm cells within the wings of mesoderm. Sb, 50 μm (A,B) and 25 μm (C). (D) Confocal sagittal optical sections of immunostained embryos. The Rreb1^-/- embryo displays abnormal epithelial folding. Sb, 50 μm. (E) Schematic depicting methodology for angle measurements. We measured the angle of the elongated nuclear axis of epiblast cells relative to the underlying Laminin basement membrane (BM). Sb, 10 μm. (F) Quantification of the angle between the elongated nuclear axis and the BM of E6.5 epiblast cells. Bars represent median and IQR. Each point represents a single cell. (G) Quantification of the coefficient of variation (COV) for the nucleus-BM embryo angle in each embryo (individual points). Bars represent mean and IQR. *** p=≤0.0005, unpaired t-test. (H) Confocal optical sections of transverse cryosections in lateral (i) and anterior (ii) regions of E7.5 embryos. Arrowheads highlight regions where cell layers are abnormally separated from one another. Sb, 25 μm. (I) Confocal optical sections of transverse cryosection of immunostained E7.5 Rreb1^-/- embryo. Arrowheads highlight a break in apical F-ACTIN through which epiblast cells are protruding. Box indicates region shown at higher magnification. Sb, 25 μm. (J) Confocal optical sections of transverse cryosections of immunostained E7.5 embryos. In wild-type embryos, epiblast cells divide adjacent to the cavity (arrowheads), maintain apical F-ACTIN and remain within the epithelium. In Rreb1^-/- embryos, we also observed dividing cells outside of the epithelium (arrowheads), within the amniotic cavity. Sb, 25 μm. (K) Confocal maximum intensity projections (left) and optical sections of transverse cryosections of immunostained embryos stained for phosphorylated Histone H3 (pHH3), which marks mitotic cells. Sb, 50 μm. (L) Quantification of proliferation in Rreb1^+/+ and Rreb1^-/- littermates. We quantified the absolute number of pHH3-positive cells per 10 μm cryosection (left panel) and the % of pHH3 mitotic cells in each germ layer per 10 μm cryosection (right panel) for three entire embryos. There was no significant difference (unpaired t-test) in proliferation rate between genotypes, other than in the ExE, which is likely a reflection of the low sample number in that region. Each point represents a single dividing cell. Bars represent mean and IQR. (M) Transverse cryosection of a lateral region of E7.5 epiblasts immunostained for the basal marker, Laminin, and apical marker, ZO-1. Rreb1^-/- embryos maintain appropriate expression of polarity markers. To note, we observed strong anti-N-CADHERIN and ZO-1 VE fluorescence, which correlates with an apparent difference in the structure of the outer VE layer compared to wild-type embryos. This signal is also observed with other antibodies and likely represents non-specific binding. Sb, 25 μm. (N) Histogram showing fluorescence levels, in arbitrary units (a.u.), of Laminin and ZO-1 immunostaining measured along the apical-basal axis of a representative region of the epiblast epithelium from image in panel. Pr, proximal; Ds, distal; A, anterior; P, posterior; L, left; R, right; PS, primitive streak; Endo, endoderm; Epi, epiblast; ExVE, extraembryonic visceral endoderm; Meso, mesoderm; ExE, extraembryonic ectoderm.

Figure 5—figure supplement 2. Severity of Rreb1 mutant phenotypes is dependent on genetic background.

(A) Sagittal confocal optical sections of Rreb1^+/+ and Rreb1^-/- embryos at E7.5. Sb, 50 μm. (B) Confocal optical sections of transverse cryosections through the proximal primitive streak of E7.5 embryos. Sb, 50 μm. (C) Confocal maximum intensity projection (MIP) of the primitive streak of an E7.5 Rreb1^+/+ embryo. We used these data to measure the distance between LAMININ basement membrane breaks at the primitive streak. (D) Graph showing the distance between LAMININ basement membrane breaks as measured in panel C. n = 3 Rreb1^+/+ and 4 Rreb1^-/- embryos (measurements were taken from the proximal-most five sections through the primitive streak per embryo).