In-depth study of mouse and bovine HOXB9 protein distribution during the first step of cell differentiation.

<p><b>A-C:</b> Mouse HOXB9 protein distribution. <b>D-E:</b> Bovine HOXB9 protein distribution. <b>(A-B, D)</b> 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). <b>(C, E)</b> 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).</p

Conditions of whole-mount immunofluorescence on bovine and mouse embryos.

<p>Conditions of whole-mount immunofluorescence on bovine and mouse embryos.</p

Mouse and bovine HOXB9 protein distribution during primitive endoderm formation.

<p><b>A-C:</b> Whole-mount immunofluorescence. <b>(A)</b> Mouse <i>in vivo</i> blastocyst at E4.5. a—b: Zoom on epiblast. <b>(B)</b> Bovine <i>in vitro</i> blastocysts at day 7, 8, 8.5 and 9 post-insemination (D7; D8; D8.5 and D9) and <i>in vivo</i> 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 <b>(a)</b>. 1. epiblast; 2. primitive endoderm; 3. trophectoderm. <b>(C)</b> Bovine blastocyst produced <i>in vivo</i> 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).</p

Mouse and bovine HOXB9 protein distribution in oocytes and from zygotes to blastocysts.

<p><b>A-C:</b> Whole-mount immunofluorescence of <i>in vitro</i> cultured mouse <b>(A)</b> and <i>in vitro</i> produced bovine <b>(B)</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-). <b>(C)</b> 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).</p

Mouse HOXB9 protein distribution in <i>in vivo</i> peri-gastrulating embryos: E7.5 and E7.8.

<p><b>A-C:</b> Whole-mount immunofluorescence. <b>(A)</b> E7.5 late allantoic bud stage (LB). <b>(B)</b> E7.5 early headfold—late headfold stage (EHF—LHF). <b>(C)</b> E7.8 first somites stage. Zoom on allantois <b>(C2)</b>. 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.</p

Secondary antibodies used for proteins detection.

<p>Secondary antibodies used for proteins detection.</p

Analysis of specificity of the anti-HOXB9 antibodies.

<p><b>A-E:</b> Overexpression of mouse <b>(A-B)</b> or bovine <b>(C-E)</b> HOXA9 (A9), HOXB9 (B9), HOXC9 (C9) or HOXD9 (D9) proteins in HEK293T cells. Each protein was fused to a FLAG tag. <b>(A, C)</b> Western-blot using the anti-FLAG antibody (N = 3). <b>(B, D)</b> Western-blot using the anti-HOXB9 antibody 1 <b>(D)</b> or 2 <b>(B)</b> (N = 3). C-: cells transfected with a control vector. The ß-ACTIN was used to control protein loading. <b>(E)</b> 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. <b>F:</b> Immunofluorescence with anti-HOXB9 antibody 2 on E12.5 <i>Hoxb9</i>^-/- knock-out mouse embryos (N = 2). E12.5 wild-type embryos (<i>Hoxb9</i>^+/+) were used as positive control. <i>Hoxb9</i>^+/+ 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.</p

Distribution of bovine HOXB9 protein in <i>in vivo</i> peri-gastrulating embryos.

<p><b>A, B:</b> Immunohistochemistry on D14 <b>(A)</b> and D17 <b>(B)</b> embryos. Merge C-: Negative control without primary antibody. Representative confocal Z-section. Scale bar = 20 μm. <b>C, D</b>: Whole-mount immunofluorescence on D18 embryos <b>(C)</b> followed by immunohistochemistry <b>(D)</b>. The localization of the sectioning is indicated by the white line in <b>C</b>. Epifluorescence images <b>(C)</b> and confocal Z-section <b>(D)</b>. Scale bar = 500 μm. <b>E:</b> 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.</p

Primary antibodies used for proteins detection.

<p>Primary antibodies used for proteins detection.</p

Protein expression of pluripotency genes in the early bovine embryos.

<p>Immunofluorescent detection of OCT4, SOX2 and NANOG protein was done in the pre-implantation bovine embryos/oocytes: GV (Germinal Vesicle), MII (Metaphase 2 oocyte), 4-cell stage, 8–16-cell stage and Morula (25–30 cells) using immunofluorescence (A) and Western Blot (B-D). Protein compartmentalization in the ICM/TE was also studied at E7 (Day 7 blastocyst), E8 (Day 8 blastocyst) and E9 (Day 9 blastocyst) as well. Encircled areas demarcate the ICM.</p