anti pdx1  (R&D Systems)

Journal: Developmental Cell

Article Title: Pancreas agenesis mutations disrupt a lead enhancer controlling a developmental enhancer cluster

doi: 10.1016/j.devcel.2022.07.014

Figure Lengend Snippet: Selective Plane Illumination Microscopy was used for 3D reconstructions of in toto -stained embryonic pancreas from E11.5 Ptf1a +/+ (top) and Ptf1a enhΔ/enhΔ (bottom) embryos. Pancreatic buds were stained for PDX1 (red), PTF1A (green) and glucagon (blue), related to Figure 2 C.

Article Snippet: Mouse Anti-Rat monoclonal Pdx1 IF , DSHB , Cat# F6A11, RRID: AB_1157904.

Techniques:

Journal: Developmental Cell

Article Title: Pancreas agenesis mutations disrupt a lead enhancer controlling a developmental enhancer cluster

doi: 10.1016/j.devcel.2022.07.014

Figure Lengend Snippet: Ptf1a enhP controls Ptf1a expression in mouse multipotent pancreatic progenitors (A) HE staining of pancreas from adult control and Ptf1a enhΔ/enhΔ mice. (B) PTF1A immunofluorescence was preserved in acinar cells from adult Ptf1a enhΔ/enhΔ pancreas. (C) 3D reconstructions of E11.5 pancreatic buds from in toto immunofluorescence stainings for PTF1A (green), PDX1 (red), and glucagon (GCG, blue). See also . (D) PTF1A (green) was depleted in dorsal pancreas from E11.5 Ptf1a enhΔ/enhΔ embryos. PDX1 (red) and NKX6.1 (blue) were co-stained to label MPCs. (E) PTF1A expression in sagittal sections from control and mutant E12.5 neural tube, hypothalamus, cerebellum, and retinal cells. See also Figure S1 .

Article Snippet: Mouse Anti-Rat monoclonal Pdx1 IF , DSHB , Cat# F6A11, RRID: AB_1157904.

Techniques: Expressing, Staining, Control, Immunofluorescence, Mutagenesis

Journal: Developmental Cell

Article Title: Pancreas agenesis mutations disrupt a lead enhancer controlling a developmental enhancer cluster

doi: 10.1016/j.devcel.2022.07.014

Figure Lengend Snippet: Modeling PTF1A enhancer mutations in human MPCs (A) qRT-PCR of human MPCs for pancreatic progenitor markers (n = 7–13 independent differentiation experiments per genotype, using 6 PTF1A enhΔ/enhΔ clones—3 lines with 127 bp and 3 lines with 321-bp deletions, see Figure S2 B—and 4 PTF1A +/+ control lines. Graphs show means ± SEM. Mann-Whitney ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001). (B) Quantification of FACS data for PDX1+ NKX6-1+ stage-4 in vitro derived MPCs (n = 8–10 independent differentiation experiments per genotype; ns, not significant). (C) Immunofluorescence of human MPCs (stage 4) shows absence of PTF1A in PTF1A enhΔ/enhΔ lines, without changes in NKX6-1. See also Figure S2 .

Article Snippet: Mouse Anti-Rat monoclonal Pdx1 IF , DSHB , Cat# F6A11, RRID: AB_1157904.

Techniques: Quantitative RT-PCR, Clone Assay, Control, MANN-WHITNEY, In Vitro, Derivative Assay, Immunofluorescence

Journal: Developmental Cell

Article Title: Pancreas agenesis mutations disrupt a lead enhancer controlling a developmental enhancer cluster

doi: 10.1016/j.devcel.2022.07.014

Figure Lengend Snippet: PTF1A enhP creates an active enhancer cluster in mouse and human MPCs (A) Regulatory landscape of the human PTF1A locus in PTF1A +/+ and PTF1A enhΔ/enhΔ MPCs. Six H3K27ac-enriched putative enhancers and the PTF1A promoter, most of which show strong mediator (MED1) binding, are shaded in gray. All show absent activity in PTF1A enhΔ/enhΔ MPCs (q < 0.05). ChIP-seq tracks show a MACS2 −log 10 p values. (B) scATAC-seq profiles for MPCs from Ptf1a +/+ and Ptf1a enhΔ/enhΔ E10.5 pancreatic buds showed chromatin accessibility at Ptf1a and E1-E6 regions orthologous to human enhancers, highlighted in gray. All showed loss in Ptf1a enhΔ/enhΔ cells (q < 0.1, log 2 FC < −0.5). Conservation tracks show multiple alignments between 100 vertebrate species. (C and D) H3K27ac at the PTF1A locus in 2 hPSC-derived pancreatic progenitor datasets ( Alvarez-Dominguez et al., 2020 ; Geusz et al., 2021 ). Both used a protocol that generates two stages of early pancreatic progenitors: PP1 PDX1+ cells that do not express MPC markers such as NKX6-1, PP2 PDX1+, and NKX6.1+ cells that are comparable with stage 4 MPCs from the current study. In both datasets, H3K27ac enrichment at PTF1A enhP preceded that of all other enhancers. (E) Summary model illustrating how PTF1A enhP precedes and activates the enhancer cluster in the PTF1A locus. See also Figure S7 .

Article Snippet: Mouse Anti-Rat monoclonal Pdx1 IF , DSHB , Cat# F6A11, RRID: AB_1157904.

Techniques: Binding Assay, Activity Assay, ChIP-sequencing, Derivative Assay

Journal: Developmental Cell

Article Title: Pancreas agenesis mutations disrupt a lead enhancer controlling a developmental enhancer cluster

doi: 10.1016/j.devcel.2022.07.014

Figure Lengend Snippet:

Article Snippet: Mouse Anti-Rat monoclonal Pdx1 IF , DSHB , Cat# F6A11, RRID: AB_1157904.

Techniques: Flow Cytometry, Control, Recombinant, Enzyme-linked Immunosorbent Assay, Lysis, Amplification, Fluorescence, cDNA Synthesis, SYBR Green Assay, Sequencing, Software, Microscopy

Journal: Nature Communications

Article Title: Identification of dynamic undifferentiated cell states within the male germline

doi: 10.1038/s41467-018-04827-z

Figure Lengend Snippet: Characterization of PDX1+ spermatogonia. a Representative whole-mount IF of WT (top 3 rows, n = 3 mice), Oct4-GFP and Pdx1 GFP adult tubules ( n = 2 mice per genotype). Antibodies to PDX1 and GFRα1 are goat polyclonals and distinguished by nuclear vs . cell surface staining respectively. Scale bars, 50 μm. b Mean percentage of GFRα1+ cells/chains PDX1+ ± s.e.m. from WT analysis in a ( n = 4 mice, >250 cells/chains per sample). c Representative whole-mount IF of adult Plzf-mC/CreER; Z/EG tubules D3 post-TAM ( n = 2 mice). Arrowheads: PDX1+ A s . Inset shows detail of indicated area. Scale bar, 50 μm. d Representative IF of adult WT testis sections ( n = 4 mice). Insets: details of selected areas. EOMES+ GFRα1+ (arrowheads) and EOMES− GFRα1+ cells (bracket) are shown. Tubule stage is indicated. Scale bar, 50 μm. e Representative IF of adult Oct4-GFP testis section ( n = 2 mice). Insets: immunostaining within indicated area. EOMES+ GFP− (arrowheads) and EOMES− GFP+ (bracket) spermatogonia are indicated. Asterisks: autofluorescent interstitium. Scale bar, 50 μm. f Representative flow cytometry of fixed and permeabilized testis cells from Oct4-GFP adults ( n = 2). PLZF+ cells shown. g Representative IF of adult WT testis sections ( n = 4 mice). Insets: immunostaining within indicated area. PDX1+ EOMES+ spermatogonia are indicated (arrowheads). Tubule stage is shown. Asterisk: autofluorescent interstitium. Scale bar, 50 μm. h Representative flow cytometry of fixed and permeabilized WT adult testis cells ( n = 4). Mean numbers of EOMES+ and PDX1+ cells in PDX1+ and EOMES+ gates respectively are shown ± s.e.m. i Representative flow cytometry for KI67 in indicated PLZF+ populations of fixed and permeabilized adult WT testis cells ( n = 4 mice). j Quantification of flow cytometry from i . Percentage of indicated PLZF+ fractions KI67+ . Horizontal bars: mean values ( n = 4 mice). k Representative IF of adult WT testis sections demonstrating PDX1+ A undiff localisation (arrowheads) within tubules ( n = 4 mice). Asterisks: autofluorescent interstitium. Tubule stages are shown. Scale bar, 50 μm. l Quantification of spermatogonial localisation from k . Mean values ± s.e.m. are shown ( n = 4 mice, 56–95 tubule sections per mouse). Significance vs. tubule-tubule localisation is indicated. m Representative IF of Id4 IRES-GFP adult testis sections ( n = 4 mice). Insets show immunostaining within indicated area. Arrowheads: PDX1+ EOMES+ GFP+ spermatogonia. Asterisk: PDX1 low EOMES+ GFP+ cell. Tubule stage is shown. Scale bar, 50 μm. n Representative flow cytometry of indicated PLZF+ populations of fixed and permeabilized adult Id4 IRES-GFP testis cells ( n = 4 mice). Mean numbers of PDX1+ and EOMES+ A undiff expressing GFP ± s.e.m. o Flow cytometry of Id4 IRES-GFP testis cells as in n . PLZF+ fractions are shown. Mean numbers of PLZF+ GFP+ cells positive for EOMES and PDX1 are indicated ± s.e.m. ( n = 4 mice). Significance was calculated by two-tailed Student’s t -test (*** P < 0.001, **** P < 0.0001)

Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R&D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R&D Systems, 1:1000).

Techniques: Staining, Immunostaining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of dynamic undifferentiated cell states within the male germline

doi: 10.1038/s41467-018-04827-z

Figure Lengend Snippet: Stem cell potential and molecular characteristics of PDX1+ A undiff . a Isolation of PDX1+ and PDX1− A undiff from Plzf-mC/CreER; Pdx1 GFP/+ adults by flow cytometry. A undiff are mCherry+ CD9+ c-KIT−. Gates for GFP+ and GFP− cells were set according to Plzf-mC/CreER control (left profile). Percentage of cells in GFP+ gate from representative sample is shown ( n = 7). b Pdx1-GFP+ and GFP− adult A undiff fractions were transplanted into recipient testis and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative donor colonies. Panels on right show higher magnification details of indicated areas and grayscale panels show individual immunostaining. Scale bar, 100 μm. c Colony-forming efficiency of Pdx1-GFP+ and GFP− A undiff fractions in transplantation assays from b . Data is presented as mean number of colonies per 10 5 donor cells ± s.e.m. ( n = 15 recipient testes for Pdx1-GFP− cells and n = 13 for Pdx1-GFP+ cells). Donor cells were pooled from a total of 7 Plzf-mC/CreER; Pdx1 GFP/+ adults. d Mean length ± s.e.m. of donor colonies was measured from tiled microscope images from experiment of c . e Mean number of GFP+ cells/donor colony ± s.e.m. were calculated for a set of whole-mount samples from transplant assay of c ( n = 58 colonies from Pdx1-GFP− cells and n = 63 from Pdx1-GFP+). f Heatmap illustrates expression of indicated genes from RNA-Seq analysis of Pdx1-GFP+ and GFP− A undiff fractions isolated as in a ( n = 4 mice). Differentially expressed genes (DEG) are in bold. Cut-off for DEG is false discovery rate (FDR) < 0.05 and absolute fold change ≥1.5. g Heatmap showing differentially expressed MAPK pathway genes from KEGG analysis of RNA-Seq data from f . h Quantitative RT-PCR analysis of indicated genes in mCherry+ CD9+ c-KIT− A undiff isolated from Plzf-mC/CreER; Pdx1 GFP/+ and Plzf-mC/CreER; Pdx1 +/+ control adults. Mean values ± s.e.m. are shown ( n = 6 mice per genotype). i Representative IF of testis sections from aged (8 months) mice of the indicated genotypes ( n = 3 mice). VASA and PLZF staining identifies germ cell and spermatogonial populations respectively. Scale bar, 50 μm. Significance was calculated by two-tailed Student’s t -test (** P < 0.01, not significant (ns) P > 0.05)

Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R&D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R&D Systems, 1:1000).

Techniques: Isolation, Flow Cytometry, Control, Expressing, Immunostaining, Transplantation Assay, Microscopy, RNA Sequencing, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of dynamic undifferentiated cell states within the male germline

doi: 10.1038/s41467-018-04827-z

Figure Lengend Snippet: PDX1+ and EOMES+ spermatogonia during development and regeneration. a Representative whole-mount IF of tubules from WT mice of indicated ages (PND; postnatal day) ( n = 2 mice per age). Scale bars, 50 μm. b Representative IF of testis sections from WT mice of indicated ages ( n = 3 mice per timepoint). Insets show details of indicated areas. Arrowheads: EOMES+ PDX1+ cells. Asterisks: EOMES+ PDX1− cells. Scale bars, 50 μm. c , d Flow cytometry of fixed and permeabilized testis cells from WT mice of indicated ages. Mean percentages of PLZF+ c-KIT− A undiff expressing PDX1 and EOMES are shown ± s.e.m. ( n = 3–5 mice/time point). e WT adults were treated with busulfan (10 mg/kg) and tubules analysed by whole-mount IF 14 days later ( n = 2 mice). Top panels: regenerative areas with GFRα1+ A al . Bottom: non-regenerative areas lacking GFRα1+ A al . Insets show details of indicated regions. Scale bar, 50 μm. f Regeneration assay of e 4 weeks post busulfan (2 areas shown). Arrowheads: PDX1+ A undiff . Scale bar, 50 μm. g Representative flow cytometry of fixed and permeabilized testis cells from WT adults untreated or busulfan treated as in e ( n = 4 busulfan-treated mice and n = 5 untreated). PLZF+ c-KIT− A undiff are shown. Percentage of A undiff KI67+ and EOMES+ are indicated. h Mean percentages of PLZF+ c-KIT− A undiff expressing PDX1, EOMES and KI67 from g are shown ± s.e.m. i Representative flow cytometry of PDX1 in indicated populations from g ( n = 4 mice per condition). Percentages of cells PDX1+ are indicated. j PDX1 levels (median fluorescent intensity) in EOMES+ PLZF+ cells from i . Mean values ± s.e.m. shown. k Quantitative RT-PCR of A undiff from Plzf-mC/CreER mice of indicated ages or 10 days post busulfan as in e . Expression levels are corrected to β-actin and normalized to an adult sample. Mean values ± s.e.m. are indicated ( n = 4 PND10 and busulfan-treated, n = 5 PND20, n = 6 adults). Selected significance values are shown. l Model of A undiff functional states. Self-renewing (curved arrows) GFRα1+ A undiff adopt different states identified by variable expression of Pdx1 , Eomes and Lhx1 . A fraction of GFRα1+ A undiff lacks PDX1, EOMES and LHX1 (grey in panel) and may represent a transitional state destined to become differentiation-primed. Niche signals differentially support self-renewing states. Given dynamic niche properties, different states predominate in development and homeostatic plus regenerative testis. The state marked by PDX1, EOMES and LHX1 is specific to homeostatic testis and potentially optimized for life-long germline maintenance. Pdx1 is downregulated and Eomes plus Lhx1 upregulated in states suited for short-term expansion during development and regeneration. Oct4-GFP+ differentiation-primed A undiff convert back to a self-renewing state under optimised culture conditions or by transplantation into recipient testis with vacant niches. Significance was calculated by two-tailed Student’s t -test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R&D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R&D Systems, 1:1000).

Techniques: Flow Cytometry, Expressing, Quantitative RT-PCR, Functional Assay, Transplantation Assay, Two Tailed Test