Table 1.
Primary antibodies used for proteins detection.
Table 2.
Secondary antibodies used for proteins detection.
Table 3.
Conditions of whole-mount immunofluorescence on bovine and mouse embryos.
Fig 1.
Analysis of specificity of the anti-HOXB9 antibodies.
A-E: Overexpression of mouse (A-B) or bovine (C-E) HOXA9 (A9), HOXB9 (B9), HOXC9 (C9) or HOXD9 (D9) proteins in HEK293T cells. Each protein was fused to a FLAG tag. (A, C) Western-blot using the anti-FLAG antibody (N = 3). (B, D) Western-blot using the anti-HOXB9 antibody 1 (D) or 2 (B) (N = 3). C-: cells transfected with a control vector. The ß-ACTIN was used to control protein loading. (E) HEK293T cells overexpressing the bovine HOX9 proteins or transfected with a control vector (C-) were stained simultaneously with anti-HOXB9 (Red) and anti-FLAG (Green) antibodies and DAPI (N = 3). Representative confocal Z-section. Scale bar = 10 μm. F: Immunofluorescence with anti-HOXB9 antibody 2 on E12.5 Hoxb9-/- knock-out mouse embryos (N = 2). E12.5 wild-type embryos (Hoxb9+/+) were used as positive control. Hoxb9+/+ C-: negative control without primary antibody. The boxes represent zooms on neural tube epithelium. HOXB9: Red, DAPI: Blue. Epifluorescence images. Scale bar = 100 μm. N = number of replicates.
Fig 2.
Mouse and bovine HOXB9 protein distribution in oocytes and from zygotes to blastocysts.
A-C: Whole-mount immunofluorescence of in vitro cultured mouse (A) and in vitro produced bovine (B) embryos. Immature oocyte (IO); mature oocyte (MO); 1-cell embryo (1c); 2-cell embryo (2 c); 5- to 8-cell embryo (5–8 c); 9- to 16-cell embryo (9–16 c); mouse pre-compaction stage (E2.5); bovine compact morula (D5); mouse blastocyst (E3.6) and bovine blastocyst (D7.5). Negative control without primary antibody are shown for E2.5 mouse embryos (E2.5 C-) and bovine IO (IO C-). (C) Zoom on bovine HOXB9 distribution at the metaphasic plate of mature oocyte. Nuclei: Blue; HOXB9: Red. The asterisk depicts polar body. White arrow shows metaphasic plate. Representative confocal Z-section. Scale bar = 20 μm (mouse) or 50 μm (bovine).
Fig 3.
In-depth study of mouse and bovine HOXB9 protein distribution during the first step of cell differentiation.
A-C: Mouse HOXB9 protein distribution. D-E: Bovine HOXB9 protein distribution. (A-B, D) Whole-mount immunofluorescence. Mouse compact morula at embryonic day E2.8 (E2.8); mouse blastocyst at E3.3 and E3.6 and bovine blastocyst at day 6, 7.5 and 8 post-insemination (D6, D7.5 and D8). Nuclei: Blue; HOXB9: Red; CDX2: Green. Representative confocal Z-section. Scale bar = 20 μm (mouse) or 50 μm (bovine). (C, E) Quantification of relative fluorescence corresponding to nuclear HOXB9 proteins. The boxplot depicts the distribution of the ratios. The ends of the whiskers represent the lowest or the highest datum still within 1.5 x interquartile range. Dots correspond to outliers. N = number of analyzed embryos. * Significant difference (Linear mixed model, * = 0.01 < p < 0.05, **** = p < 0.0001).
Fig 4.
Mouse and bovine HOXB9 protein distribution during primitive endoderm formation.
A-C: Whole-mount immunofluorescence. (A) Mouse in vivo blastocyst at E4.5. a—b: Zoom on epiblast. (B) Bovine in vitro blastocysts at day 7, 8, 8.5 and 9 post-insemination (D7; D8; D8.5 and D9) and in vivo embryos at day 11 post-insemination (D11). Arrows point to GATA6 positive cells that could correspond to primitive endoderm cells. Zoom on D11 bovine embryo (a). 1. epiblast; 2. primitive endoderm; 3. trophectoderm. (C) Bovine blastocyst produced in vivo at D7. Merge C-: negative control without primary antibody. Nuclei: Blue; HOXB9: Red; GATA4/GATA6: Green; CDX2: Pink. Representative confocal Z-section. Scale bar = 20 μm (mouse) or 50 μm (bovine).
Fig 5.
Mouse HOXB9 protein distribution in in vivo peri-gastrulating embryos: E5.5 and E6.5.
A-B: Whole-mount immunofluorescence. (A) E5.5: HOXB9 proteins were co-stained with OCT4 (A1), GATA4 (A2) or IgG (A3). Arrows indicate HOXB9 that is localized in apical vacuoles of visceral endoderm cells. Orange arrows indicate the reduction of HOXB9 staining intensity observed in trophoblast cells in the center of the ectoplacental cone. Arrowheads indicate variation in HOXB9 nuclear staining between visceral endoderm cells. (B) E6.5: HOXB9 proteins were co-stained with OCT4 (B1), BRACHYURY (T—B2), GATA4 (B3) and CER-1 (B4). Zoom on the ectoplacental cone (B5). Yellow arrows, arrowheads and the asterisk show the flattened, cuboidal and columnar cells of the visceral endoderm, respectively. Blue arrows indicate the strong HOXB9 staining observed in anterior visceral endoderm cells. 1. epiblast; 2. extra-embryonic ectoderm; 3. primary trophoblastic giant cells; 4. embryonic visceral endoderm; 5. extra-embryonic visceral endoderm; 6. anterior visceral endoderm; 7. ectoplacental cone; 8. parietal endoderm; 9. primitive streak. Nuclei: Blue; HOXB9: Red; OCT4/GATA4/IgG/T/CER-1: Green. Representative confocal Z-section. Scale bar = 20 μm.
Fig 6.
Mouse HOXB9 protein distribution in in vivo peri-gastrulating embryos: E7.5 and E7.8.
A-C: Whole-mount immunofluorescence. (A) E7.5 late allantoic bud stage (LB). (B) E7.5 early headfold—late headfold stage (EHF—LHF). (C) E7.8 first somites stage. Zoom on allantois (C2). 1. node; 2. notochord; 3. allantois; 4. mesothelium of the allantois; 5. primitive streak; 6. embryonic visceral endoderm. Nuclei: Blue; HOXB9: Red; T: Green. Representative confocal Z-section. Scale bar = 20 μm.
Fig 7.
Distribution of bovine HOXB9 protein in in vivo peri-gastrulating embryos.
A, B: Immunohistochemistry on D14 (A) and D17 (B) embryos. Merge C-: Negative control without primary antibody. Representative confocal Z-section. Scale bar = 20 μm. C, D: Whole-mount immunofluorescence on D18 embryos (C) followed by immunohistochemistry (D). The localization of the sectioning is indicated by the white line in C. Epifluorescence images (C) and confocal Z-section (D). Scale bar = 500 μm. E: Immunohistochemistry on allantois from D25 embryos. Representative confocal Z-section. Scale bar = 20 μm. Nuclei: Blue; HOXB9: Red; CDX2/VIMENTIN: Green; DLX3: Yellow. 1. epiblast; 2. primitive endoderm; 3. mural trophectoderm; 4. amniotic cavity; 5. amniotic wall; 6. embryonic mesoderm; 7. extra-embryonic mesoderm; 7 + 3 = chorion; 8. coelom; 9 yolk sac cavity; 7 + 2 = yolk sac wall; 10. primitive streak.
Fig 8.
Schematic summary of the HOXB9 protein distribution in mouse (A) and bovine (B) early embryonic development.
Immature oocyte (IO); Mature oocyte (MO); 1-cell embryo (1c); 2-cell embryo (2 c); 5- to 8-cell embryo (5–8 c); 9- to 16-cell embryo (9–16 c); Inner Cell Mass (ICM); Visceral endoderm (VE); Trophoblastic Giant Cells (TGCs); Anterior Visceral Endoderm (AVE). Tissues represented without nucleus in E7.5 embryos were not examined in details.