Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet: EPI and PrE Expression Levels Are Reduced in ATP1-Inhibited Embryos (A) Immunofluorescence images of TE (Cdx2), EPI (Sox2), and PrE (Gata4) fate in pre-treatment control (E3.5 WT), Atp1 inhibited (E4.0 500 μM and E4.0 250 μM), and end-stage control (E4.0 DMSO) embryos. Lumen boundaries outlined by dashed white line and mean lumen volume in white text. Scale bars, 10 μm. (B) Boxplot of lumen volume for E3.5 WT (N = 21), E4.0 DMSO (N = 24), E4.0 250 μM Atp1 inhibited (N = 14) and E4.0 500 μM Atp1 inhibited (N = 31) embryos indicating that the impact on lumen volume is concentration dependent. (C) Boxplot of fluorescence levels of Cdx2 (gray), Sox2 (green), and Gata4 (magenta) in E4.0 500 μM Atp1 inhibited embryos compared to E4.0 DMSO controls. (D) Schematic 2D representation of 3D analysis method for spatial segregation of ICM lineages. P 1,2,3,4 are 3D points. L ↔ is a 3D line ( P 1 P 2 ↔ equivalent) that defines the embryonic-abembryonic axis. d ‾ is the 3D line segment ( P 3 P 4 ‾ equivalent) that measures the perpendicular distance from the center of a cell to L ↔ . See Image Analysis for formal definitions of all geometric entities. (E) Boxplot of spatial overlap between EPI and PrE lineages within E4.0 control (DMSO, N = 15), E4.0 Atp1 inhibited (500 μM, N =13) and simulated data of maximal overlap in E4.0 WT embryos (Simulation, N = 27). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. n.s., not significant. For boxplots: central mark indicates the median; lower edge, 25%; upper edge, 75%; lower whisker, Q1 − (1.5 × IQR), where IQR = Q3 − Q1; upper whisker, Q3 + (1.5 × IQR). See also and ; .

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Expressing, Immunofluorescence, Control, Concentration Assay, Fluorescence, Whisker Assay

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet: PrE Specification and Spatial Segregation of ICM Lineages Is Impaired by Mechanical Inhibition of Lumen Expansion (A) Brightfield images of mechanical deflation. Magenta asterisk marks the needle tip. Dotted magenta line indicates lumen boundary. (B) Immunofluorescence images of EPI (Sox2) and PrE (Gata4) fate in pre-manipulation control (E3.5 WT), E4.0 post-manipulation control (E4.0 WT), and E4.0 mechanically inhibited (E4.0 Mechanical) embryos. Magenta arrowheads indicate the position of cells expressing high levels of Gata4 within the ICM. White dotted line indicates lumen boundaries. Average lumen volume in white text. Scale bars, 10 μm. (C) Boxplot of fluorescence levels of Sox2 (green) and Gata4 (magenta) in mechanically inhibited (Mech., N = 33) and post-manipulation control (WT, N = 28) E4.0 embryos. (D) Boxplot of spatial overlap between EPI and PrE lineages within post-manipulation control (WT, N = 27), mechanically inhibited (Mech., N = 33), E4.0 procedural control (Control, N = 11), and E4.0 simulation of complete overlap in WT conditions (Simulation, N = 27). ∗∗∗∗ p < 0.0001, ∗∗ p < 0.01. n.s., not significant. For boxplots: central mark indicates the median; lower edge, 25%; upper edge, 75%; lower whisker, Q1 − (1.5 × IQR), where IQR = Q3 − Q1; upper whisker, Q3 + (1.5 × IQR). See also Figure S6 and .

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Inhibition, Immunofluorescence, Control, Expressing, Fluorescence, Whisker Assay

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet: Perturbation of FGF4 Signaling in the Lumen Impacts Molecular Specification of EPI and PrE Lineages (A) Immunofluorescence images of EPI (Sox2) and PrE (Gata4) fate in E3.5 post-FGF4 deposition (E3.5 FGF4 Inj.), E3.5 post-PD173074 deposition (E3.5 PD Inj.), and E3.5 post-PBS deposition (E3.5 PBS Inj.). White dotted line indicates lumen boundaries. Average lumen volume in white text. Scale bars, 10 μm. (B) Boxplot of fluorescence levels of Sox2 (green) and Gata4 (magenta) in E3.5 post-FGF4 deposition (FGF4 Inj., N = 24), E3.5 post-PD173074 deposition (PD Inj., N = 21), and E3.5 post-PBS deposition (PBS Inj., N = 13) embryos. (C) Boxplot of luminal volume in E3.5 post-FGF4 deposition (FGF4 Inj., N = 24), E3.5 post-PD173074 deposition (PD Inj., N = 21), and E3.5 post-PBS deposition (PBS Inj., N = 13). ∗∗∗∗ p < 0.0001, ∗∗ p < 0.01. n.s., not significant. For boxplots: central mark indicates the median; lower edge, 25%; upper edge, 75%; lower whisker, Q1 − (1.5 × IQR) where IQR = Q3 − Q1; upper whisker, Q3 + (1.5 × IQR). See also Figure S7 .

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Immunofluorescence, Fluorescence, Whisker Assay

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet: Luminal Deposition of FGF4 Partially Rescues EPI-PrE Specification in ATP1-Inhibited Embryos (A) Immunofluorescence images of EPI (Sox2) and PrE (Gata4) fate in E3.5 post-FGF4 deposition and Atp1 inhibition (E3.5 FGF4 250 μM), E3.5 post-PBS deposition and Atp1 inhibition (E3.5 PBS 250 μM), and E3.5 control embryos (E3.5 DMSO). White dotted line indicates lumen boundaries. Average lumen volume in white text. Magenta arrowhead indicates cell with high Gata4 expression relative to neighboring cells. Scale bars, 10 μm. (B) Boxplot of fluorescence levels of Sox2 (green) and Gata4 (magenta) in E3.5 post-FGF4 deposition and Atp1 inhibition (250 μM FGF4 Inj., N = 42 embryos), E3.5 post-PBS deposition and Atp1 inhibition (250 μM PBS Inj., N = 12), and E3.5 control embryos (DMSO, N = 12). (C) Boxplot of luminal volume in E3.5 post-FGF4 deposition and Atp1 inhibition (250 μM FGF4 Inj., N = 49 embryos), E3.5 post-PBS deposition and Atp1 inhibition (250μM PBS Inj., N = 9), and E3.5 control embryos (DMSO, N = 38). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. n.s., not significant. For boxplots: central mark indicates the median; lower edge, 25%; upper edge, 75%; lower whisker, Q1 − (1.5 × IQR), where IQR = Q3 − Q1; upper whisker, Q3 + (1.5 × IQR).

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Immunofluorescence, Inhibition, Control, Expressing, Fluorescence, Whisker Assay

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Recombinant, Software

Journal: Developmental Cell

Article Title: Lumen Expansion Facilitates Epiblast-Primitive Endoderm Fate Specification during Mouse Blastocyst Formation

doi: 10.1016/j.devcel.2019.10.011

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used in this study: rabbit anti-pERM (Cell Signaling, 3726), mouse anti-Cdx2 (BioGenex, MU392A-UC), goat anti-Sox2 (Santa Cruz Biotechnology, sc-17320), goat anti-Sox2 (R&D Systems, AF2018-SP), rabbit anti-Sox2 (Cell Signaling, 23064), rabbit anti Gata4 (Santa Cruz Biotechnology, sc-9053), goat anti-Gata4 (R&D Systems, AF2606-SP), rabbit anti-GFP (MBL, 598), mouse anti-Rab11 (BD Biosciences; 610656), rat anti-integrin-β1 (Merck Millipore, MAB1997), goat anti-biotinylated Gata4 (R&D Systems, BAF2606) mouse anti-Oct3/4 (Santa Cruz Biotechnology, sc-5279) and mouse anti-Hsp47 (Enzo Life Sciences, M16.10A1).

Techniques: Recombinant, Software

Journal: Biology of Reproduction

Article Title: Sertoli Cells Dictate Spermatogonial Stem Cell Niches in the Mouse Testis 1

doi: 10.1095/biolreprod.110.087320

Figure Lengend Snippet: Evaluation of the number of SSCs in testes of mice with experimentally induced increase of Sertoli cell numbers. A) Experimental strategy using PTU treatment during the neonatal period to increase Sertoli cell numbers at puberty in Rosa donor mice and transplantation analyses to assay for SSC numbers. Each recipient mouse received MACS-isolated THY1+ germ cells from a PTU-treated donor in one testis and MACS-isolated THY1+ germ cells from a control donor in the other testis. The numbers of blue colonies of spermatogenesis are a measure of SSC content in the microinjected donor cell suspension. B) Immunohistochemistry staining for GATA4+ nuclei (arrows) in cross sections of testis from a PTU-treated Rosa mouse. Within seminiferous tubules, GATA4 expression is localized specifically to Sertoli cell nuclei. Bar = 50 μm. C) Numbers of GATA4+ nuclei within cross sections of seminiferous tubules from PTU-treated and control Rosa mice. Data are mean ± SEM for three testes from different mice; *P = 0.01. D) Numbers of SSCs in testes of PTU-treated and control Rosa donor mice. SSC number is derived from quantification of colonies within recipient testes arising from 1 × 105 THY1+ donor germ cells microinjected and normalized to the number of THY1+ cells isolated by MACS. Data are mean ± SEM for three independent experiments using different donors for each treatment and 10–12 recipient testes for each replicate experiment; *P = 0.05.

Article Snippet: Sertoli cell nuclei were then labeled by incubating sections overnight at 4°C with goat anti-mouse GATA4 polyclonal antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transplantation Assay, Isolation, Control, Suspension, Immunohistochemistry, Staining, Expressing, Derivative Assay

Journal: Biology of Reproduction

Article Title: Sertoli Cells Dictate Spermatogonial Stem Cell Niches in the Mouse Testis 1

doi: 10.1095/biolreprod.110.087320

Figure Lengend Snippet: Evaluation of SSC niche numbers in testes of mice with experimentally induced increase of Sertoli cell numbers. A) Experimental strategy using germ cell transplantation to assay for numbers of niches in recipient mice with normal numbers of Sertoli cells and recipient mice treated with PTU during postnatal development to increase Sertoli cell content. MACS-isolated THY1+ germ cells from normal Rosa donors were used as a source of SSCs for microinjection into PTU and non-PTU-treated recipient testes. B) Numbers of GATA4+ nuclei within cross sections of seminiferous tubules from PTU/busulfan-treated and control busulfan-treated 129XC57 mice. Data are mean ± SEM for three different replicate samples of each treatment; *P = 0.05. C) Numbers of donor-derived colonies of spermatogenesis, a reflection of colonized SSC niches, in testes of PTU/busulfan-treated and control busulfan-treated recipient mice 2 mo after transplantation. Data are mean ± SEM for three independent transplantation experiments and 10–12 recipient testes for each treatment; *P = 0.01. D) Number of blood vessels within the interstitial space in cross sections of testes from PTU/busulfan-treated and control busulfan-treated mice. Data are mean ± SEM for three different mice in each treatment. E) Percentage of seminiferous tubules in close association with blood vessels in testes of PTU/busulfan-treated and busulfan-treated control mice. Data are mean ± SEM for three different mice of each treatment. F) Length of seminiferous tubule basement membrane bordered by blood vessels or interstitial tissue in testes of PTU/busulfan-treated and busulfan-treated control mice. Data are mean ± SEM for three different mice in each treatment.

Article Snippet: Sertoli cell nuclei were then labeled by incubating sections overnight at 4°C with goat anti-mouse GATA4 polyclonal antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transplantation Assay, Isolation, Microinjection, Control, Derivative Assay, Membrane

Journal: bioRxiv

Article Title: NEDD4 promotes Sertoli cell proliferation and adult Leydig cell differentiation in the murine testis

doi: 10.1101/2025.02.19.639149

Figure Lengend Snippet: Section immunofluorescence on 14.5 dpc XY Nr5a1- Cre; Nedd4 flox/flox testes alongside XY littermate controls stained for Sertoli cell markers SOX9 (green in A , E ), GATA4 (green in B , G ) and AMH (magenta in D , I ), extracellular matrix protein laminin (magenta in B , G ), Leydig cell marker CYP11A1 (green in C , H ), germ cell marker DDX4 (magenta in A , E , F , J ), pluripotency marker POU5F1 (magenta in C , H ), meiosis marker SYCP3 (green in D , I ) and granulosa cell marker FOXL2 (green in E , J ). The anterior pole of each gonad is positioned at the top of each panel. Scale bars = 100 μm. K ) RT-qPCR analyses of Sox9, Amh, Fgf9, Fgfr2, Inhba, Cyb26b1, Pou5f1, Nanog, Nodal, Nanos2, Dazl, Stra8 and Sycp3 expression at 14.5 dpc on XY Nr5a1- Cre; Nedd4 flox/flox gonads (blue, n=5) and XY littermate controls (grey, n=5). Values are normalized to Sdha and are expressed relative to controls. Mean ± SEM; t-test; n.s. = not significant.

Article Snippet: Primary antibodies used were mouse anti-NEDD4 (BD Transduction Laboratories, 611481; 1:100), goat anti-DDX4 (R&D Systems, RDSAF2030; 1:300), rabbit anti-SOX9 [ ]1:200), rabbit anti-SRY [ ] 1:100), rabbit anti-FOXL2 [ ](1:300), goat anti-GATA4 (Santa Cruz, sc1237; 1:300), rabbit anti-Laminin (Sigma Aldrich, L9393; 1:300), rabbit anti-CYP11A1 [ ]1:300), mouse anti-POU5F1 (Santa Cruz, sc5279; 1:100), mouse anti-SYCP3 (Abcam, ab97672; 1:100), goat anti-AMH (Santa Cruz, sc6886; 1:300), sheep anti-SOX9 [ ] 1:200), rat anti-NR5A1 (Transgenic Inc, KO610; 1:500), rabbit anti-TH (Sigma-Aldrich AB152; 1:100), mouse anti-Ki67 (BD Transduction Lab, 550609; 1:100) and mouse anti-NR2F2 (Perseus Proteomics, PP-H7147-00; 1:200).

Techniques: Immunofluorescence, Staining, Marker, Quantitative RT-PCR, Expressing