Journal: bioRxiv

Article Title: Coordination Between Embryo Growth and Trophoblast Migration Upon Implantation Delineates Mouse Embryogenesis

doi: 10.1101/2022.06.13.495767

Figure Lengend Snippet: (A) Left, representative immunofluorescence image of the E4.75 uterus cross-section, simultaneously stained for Fibronectin (FN1, white), GATA4 (green), and nuclei (DNA, blue). Right, 4x zoom into the interface between trophectoderm (TE), uterine epithelium (E), and stroma (S) (outlined). Red arrowheads mark uterine ECM. (B) Representative immunofluorescence image of the E5.25 uterus cross-section, simultaneously stained for pan-Laminin (panLAM, white), GATA4 (green), and nuclei (DNA, blue). Measurements of E5.25 in utero embryo dimension across n = 12. (C) Schematic illustration of peri-implantation embryo culture. Embryos are recovered at E3.5, treated by Tyrode’s solution to remove Zona pellucida (ZP), and embedded into the crypts on the day of recovery (D0). ICM, red; TE, blue; IVC1 and ICV2 stand for “In Vitro Culture” medium 1 and 2, respectively. Inset, schematic illustration of the hydrogel composition. 8-arm Poly(ethylene glycol) (PEG, gray) molecules, crosslinked via metalloprotease-cleavable peptides (Peptide, green), and functionalized with RGDSPG peptide (Arg-Gly-Asp-Ser-Pro-Gly, ‘RGD’, red). (D) Representative immunofluorescence images of the embryos developed in utero until E4.5 and E5.25, simultaneously stained for OCT3/4 (magenta), GATA4 (green), and nuclei (DAPI, blue). (E) Representative immunofluorescence images of the 3E-uterus embryos from day 2 and 3, simultaneously stained for OCT3/4 (magenta), GATA4 (green), and nuclei (DAPI, blue). (F) Scatterplot showing numbers of epiblast (EPI) cells (x-axis) vs numbers of visceral endoderm (VE) cells (y-axis) that cover epiblast in the embryos developed in utero until E5.5 (E3.5 – E5.5) and the embryos developed by 3E-uterus until day 3 (D1 – 3). n = 5 (E3.5), n = 21 (E4.5), n = 28 (E4.75), n = 20 (E5.0), n = 20 (E5.25), n = 21 (E5.5), data from ; n = 20, two replicates pooled (D1), n = 13 of 28, three replicates pooled (D2), n = 12 of 26, three replicates pooled (D3). X/Y scale, log 10. Bottom right, the scheme of the quantified embryo region, EPI (magenta), VE (green). (G) From left to right, boxplots showing egg cylinder’s length, diameter, and the length-to-diameter ratio between embryos developed in utero until E5.25 and 3E-uterus embryos from day 3 (D3). n = 14 and 12, respectively. Data points, shown as black dots, correspond to individual embryos, midline marks the median, boxes indicate interquartile range. P-values were calculated using Student’s t-test and the Mann-Whitney U test. (H) Cell number-based correspondence between in utero and 3E-uterus embryo development. (I) Left, representative immunofluorescence image of the E4.75 uterus cross-section from the pregnant F1 female mated with the H2B-GFP male. Simultaneous immunostaining for TFAP2C (yellow), GFP (green), and nuclei (DNA, blue). Right, immunostaining of the 3E-uterus embryo from day 2 (D2). Yellow arrowheads mark differentiated trophoblast cells. (J) Left, representative immunofluorescence image of the E5.25 uterus cross-section from the F1 female mated with the H2B-GFP male. Simultaneous immunostaining for TFAP2C (yellow), GFP (green), and nuclei (DNA, blue). Right, immunostaining of the 3E-uterus embryo from day 3 (D3). Scale bars, 50 μm, 12.5 μm (4x zoom). White asterisks mark epiblast of the implanted embryos. See also Materials and Methods, .

Article Snippet: Primary antibodies against GATA4 biotinylated (R&D systems, AF2606), SOX2 (Cell Signaling, 23064), TFAP2C (Cell Signaling, #2320), CDX2 (Biogenex Laboratories, MU392AUC), PARD6B (Santa Cruz Biotechnology, sc-67393), pan-Laminin (Novus Biologicals, NB300-144SS), Collagen IV (Millipore, AB756P), Fibronectin (Proteintech, 15613-1-AP), ITGB1 (Millipore, MAB1997), GFP (chromotek, gb2AF488) were diluted at 1:200.

Techniques: Immunofluorescence, Staining, In Utero, Embryo Culture, In Vitro, MANN-WHITNEY, Immunostaining

Journal: Cell Reports Medicine

Article Title: In vitro germ cell induction from fertile and infertile monozygotic twin research participants

doi: 10.1016/j.xcrm.2022.100782

Figure Lengend Snippet: Induction of hPGCLCs in an embryo model of the amniotic sac (A) Diagram of the embryo model. BMP4 was added 18 h after loading hiPSCs into the device. Thirty h after adding BMP4, amniotic sac-like embryo models develop, each containing an amniotic cavity, an amniotic ectoderm-like cell layer, pre-primitive streak epiblast (EPI)-like cells, and hPGCLCs (right). (B) The amniotic ectoderm-like cell layer is TFAP2A+ (n = 12 modeled embryos per participant were evaluated). Scale bar: 30 μm. (C) Representative images of hPGCLCs (triple positive for TFAP2C, NANOG, and SOX17) in the amniotic ectoderm-like cell layer (shown is MZT05-D). Arrows indicate hPGCLCs. (D) The number of specified hPGCLCs in the amniotic ectoderm-like cell layer was quantified from n = 8 embryo models from each participant’s hiPSCs (MZT01 versus MZT02, p = 0.58; MZT04 versus MZT06, p = 0.30). Scale bar: 10 μm. Data are represented as mean ± SEM. Figure created with BioRender.com . Statistical significance was calculated using t test.

Article Snippet: The primary antibodies anti-TFAP2C (sc8977; 1:200, RRID: AB_2286995 ), anti-TFAP2C (sc12762; 1:200, RRID: AB_667770 ), anti-BLIMP1 (Cell Signaling C14A4; 1:100, RRID: AB_2169699 ), anti-SOX17 (GT15094; 1:100, RRID: AB_2195648 ), anti-BRACHYURY/T (Fisher Scientific AF2085; 1:200, RRID: AB_2200235 ), anti-NANOG (ab109250, 1:40, RRID: AB_10863442 ), anti-TFAP2A (sc-12726, 1:200, RRID: AB_667767 ) were incubated overnight at 4°C.

Techniques:

Journal: Cell Reports Medicine

Article Title: In vitro germ cell induction from fertile and infertile monozygotic twin research participants

doi: 10.1016/j.xcrm.2022.100782

Figure Lengend Snippet:

Article Snippet: The primary antibodies anti-TFAP2C (sc8977; 1:200, RRID: AB_2286995 ), anti-TFAP2C (sc12762; 1:200, RRID: AB_667770 ), anti-BLIMP1 (Cell Signaling C14A4; 1:100, RRID: AB_2169699 ), anti-SOX17 (GT15094; 1:100, RRID: AB_2195648 ), anti-BRACHYURY/T (Fisher Scientific AF2085; 1:200, RRID: AB_2200235 ), anti-NANOG (ab109250, 1:40, RRID: AB_10863442 ), anti-TFAP2A (sc-12726, 1:200, RRID: AB_667767 ) were incubated overnight at 4°C.

Techniques:

Journal: Cell Reports Medicine

Article Title: In vitro germ cell induction from fertile and infertile monozygotic twin research participants

doi: 10.1016/j.xcrm.2022.100782

Figure Lengend Snippet: Induction of hPGCLCs in an embryo model of the amniotic sac (A) Diagram of the embryo model. BMP4 was added 18 h after loading hiPSCs into the device. Thirty h after adding BMP4, amniotic sac-like embryo models develop, each containing an amniotic cavity, an amniotic ectoderm-like cell layer, pre-primitive streak epiblast (EPI)-like cells, and hPGCLCs (right). (B) The amniotic ectoderm-like cell layer is TFAP2A+ (n = 12 modeled embryos per participant were evaluated). Scale bar: 30 μm. (C) Representative images of hPGCLCs (triple positive for TFAP2C, NANOG, and SOX17) in the amniotic ectoderm-like cell layer (shown is MZT05-D). Arrows indicate hPGCLCs. (D) The number of specified hPGCLCs in the amniotic ectoderm-like cell layer was quantified from n = 8 embryo models from each participant’s hiPSCs (MZT01 versus MZT02, p = 0.58; MZT04 versus MZT06, p = 0.30). Scale bar: 10 μm. Data are represented as mean ± SEM. Figure created with BioRender.com . Statistical significance was calculated using t test.

Article Snippet: The primary antibodies anti-TFAP2C (sc8977; 1:200, RRID: AB_2286995 ), anti-TFAP2C (sc12762; 1:200, RRID: AB_667770 ), anti-BLIMP1 (Cell Signaling C14A4; 1:100, RRID: AB_2169699 ), anti-SOX17 (GT15094; 1:100, RRID: AB_2195648 ), anti-BRACHYURY/T (Fisher Scientific AF2085; 1:200, RRID: AB_2200235 ), anti-NANOG (ab109250, 1:40, RRID: AB_10863442 ), anti-TFAP2A (sc-12726, 1:200, RRID: AB_667767 ) were incubated overnight at 4°C.

Techniques:

Journal: Cell Reports Medicine

Article Title: In vitro germ cell induction from fertile and infertile monozygotic twin research participants

doi: 10.1016/j.xcrm.2022.100782

Figure Lengend Snippet:

Article Snippet: The primary antibodies anti-TFAP2C (sc8977; 1:200, RRID: AB_2286995 ), anti-TFAP2C (sc12762; 1:200, RRID: AB_667770 ), anti-BLIMP1 (Cell Signaling C14A4; 1:100, RRID: AB_2169699 ), anti-SOX17 (GT15094; 1:100, RRID: AB_2195648 ), anti-BRACHYURY/T (Fisher Scientific AF2085; 1:200, RRID: AB_2200235 ), anti-NANOG (ab109250, 1:40, RRID: AB_10863442 ), anti-TFAP2A (sc-12726, 1:200, RRID: AB_667767 ) were incubated overnight at 4°C.

Techniques: