Journal: Nature

Article Title: A maternal brain hormone that builds bone

doi: 10.1038/s41586-024-07634-3

Figure Lengend Snippet: a , Trabecular and cortical fractional BV, mechanical strength (three-point bend) and BMAT levels in long bones of Esr1 fl/fl and Esr1 Nkx2.1-cre females fed standard diet (SD) or HFD for 17 weeks ( N = 4–6 per group). b , Representative images of tibia from female mice (aged 27 weeks) fed SD or HFD for 17 weeks labelled for calcein and Alizarin Red (top, white arrows and magnified from Extended Data Fig. ) and osmium stained with lipid droplets (bottom, yellow arrows). c , Heatmap of top DEGs changed in the ARC of Esr1 Nkx2.1-cre females at 12 weeks of age (adapted from ref. ) and at 27 weeks of age fed SD or HFD. Scale is log fold change. d , Transcript levels of Ccn3 and Penk in the ARC of 3.5-week-old mutant females, measured by quantitative PCR (qPCR). N = 2–3 per group. e , Ccn3 and Penk expression by RNAscope of the ARC in mutant female Esr1Nkx 2.1cre mice fed either SD or HFD. Scale bar, 100 µm. ME, median eminence. f , Staining for ERα (pink) and CCN3 (green) in brain sections from posterior ARC and SCN regions of Esr1 fl/fl female (10-week-old) and Esr1 Nkx2.1-cre female and male (12-week-old) mice. Scale bar, 200 µm. oc, optic chiasm. g , CCN3 and KISS1 overlapping expression in Esr1 Nkx2.1-cre female medial basal posterior ARC (yellow arrowheads). Scale bar, 100 µm. h , Ccn3 (green) Esr1 (cyan) and Kiss1 (red) transcripts from posterior ARC brain sections in control and mutant Esr1 Nkx2.1-cre females ( Kiss1 only, yellow arrowheads; Ccn3 only, white arrowheads). Scale bar, 50 µm. One-way ANOVA in a (Tukey’s and Šidák’s multiple-comparisons test). Unpaired Student’s t -test, two-tailed for d . ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars ± s.e.m.

Article Snippet: Blots were then probed overnight at 4 °C with anti-CCN3 antibody (R&D Systems, AF1976; 1:3,000) in TBS-T with 5% serum.

Techniques: Staining, Mutagenesis, Real-time Polymerase Chain Reaction, Expressing, RNAscope, Control, Two Tailed Test

Journal: Nature

Article Title: A maternal brain hormone that builds bone

doi: 10.1038/s41586-024-07634-3

Figure Lengend Snippet: a , Heatmaps of top 50 DEGs listed to the right following analyses of bulk RNA-Seq datasets of Esr1 fl/fl and Esr1 Nkx2.1-Cre age-matched female littermates maintained on standard breeder chow (SD) or high-fat diet (HFD) for 17 weeks starting at 10 weeks of age; samples include microdissected ARC (left panel), whole pituitary glands (middle panel) or liver tissue (right panel). The cluster of secreted proteins/peptides for the ARC attenuated by HFD are highlighted in red text. Legend for each heatmap shows relative Z-Scores. b , Normalized reads for candidate genes from the ARC; Penk in pituitary with either SD or HFD (N = 2–4). c , Relative expression of transcripts as listed in the female hypothalamus at 2.5 weeks of age shown in bar graphs with individual points (N = 4–8). d , Relative expression of transcripts in microdissected ARC harvested from control Esr1 fl/fl and mutant Esr1 Nkx2.1-Cre age-matched females (red) or males (blue), (N = 2–5). e , CCN3 (green) expression in the posterior ARC of mutant female, control virgin female and control intact male. Scale bars = 100 µm. One-Way ANOVA for panel b, Unpaired Student’s T-test 2-tailed for panels c and d, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. ns = not significant. Error Bars ± SEM. Abbreviations: ARC arcuate nucleus. ME median eminence.

Article Snippet: Blots were then probed overnight at 4 °C with anti-CCN3 antibody (R&D Systems, AF1976; 1:3,000) in TBS-T with 5% serum.

Techniques: RNA Sequencing Assay, Expressing, Control, Mutagenesis

Journal: Nature

Article Title: A maternal brain hormone that builds bone

doi: 10.1038/s41586-024-07634-3

Figure Lengend Snippet: a – c , Osteogenic differentiation assays (14 days). a , Differentiation of mouse ocSSCs (from 2-week-old male and female mice) treated with mCCN3, met-ENK and Bam22P. Inset, cells stained with Alizarin Red (Al Red) ( n = 3). Veh, vehicle. b , c , Human ocSSCs treated with human CCN3 (hCCN3) during in vitro osteogenesis. b , Human ocSSCs from a 14-year-old male treated with hCCN3, met-ENK, Bam22P, gastric-related peptide (GRP) and follistatin (FST). c , Additional ocSCCs from 15-year-old (left), 72-year-old (middle) and 61-year-old (right) patients treated with hCCN3 (red bars) ( n = 3), with representative images of wells stained with Alizarin Red to the right of the graph. F, female; M, male. d – f , Whole femur bone cultures treated with plasma or mCCN3 daily for 5 days. d , Left, %BV/TV for control Esr1 fl/fl female (red) and male (blue) 6–8-week-old femurs treated with plasma from Esr1 Nkx2.1-cre females; contralateral femurs treated with plasma from Esr1 fl/fl mice ( N = 19, 10). Right, per cent change in %BV/TV of contralateral female (red bar) or male (blue bar) femurs. e , Representative µCT images from treated femurs. f , Left, %BV/TV control female ( N = 11) and male ( N = 8) 10–11-week-old femurs treated daily for 5 days with mCCN3 (3 nM) compared with untreated baseline contralateral femur control. Right, per cent change in female (red bar) or male (blue bar) %BV/TV treated with CCN3 or saline normalized to baseline ( N = 5–11). g , Per cent change in %BV/TV in Esr1 fl/fl mice following daily CCN3 injections (i.p. 7.5 µg kg –1 ) or saline for 21 days, normalized to mean %BV/TV of saline controls ( N = 6, 8 females and 7, 6 males). h , Representative µCT images from treated femurs. i , %BV/TV (left) and mechanical strength of callus (right) 21 days after fracture in aged male mice after slow-release mCCN3 (1 or 2 µg) treatment (phosphate-buffered saline (PBS) N = 4; 1 µg N = 5, 6; and 2 µg N = 4). j , Representative µCT images and cross-sections of callus from 24-month-old C57BL/6 male femurs. One-way ANOVA in a – c and i (Dunnett’s ( a , b , i ) and Tukey’s ( c ) multiple-comparisons test). Paired Student’s t -test, one-tailed for left panels in d , f , and unpaired Student’s t -test, two-tailed for right panels in d , f and g . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars ± s.e.m.

Article Snippet: Blots were then probed overnight at 4 °C with anti-CCN3 antibody (R&D Systems, AF1976; 1:3,000) in TBS-T with 5% serum.

Techniques: Staining, In Vitro, Control, Saline, One-tailed Test, Two Tailed Test

Journal: Nature

Article Title: A maternal brain hormone that builds bone

doi: 10.1038/s41586-024-07634-3

Figure Lengend Snippet: a , Effects of chronic infusion of Naloxone over 28 days with fractional bone volume plotted for control Esr1 fl/fl and mutant Esr1 Nkx2.1-Cre age-matched females. The ages of female mice at the beginning of treatment were 10–12 weeks of age, which was delivered via an implanted mini-osmotic pump (0.5 mg/24 hrs) over 28 days. Legend in bar graph (N = 4 per group). b , Representative images of duplicate wells of Alizarin staining in Control media, osteogenic media minus or plus different doses of human CCN3 with magnified images of one well in far-right images of each panel. Representative images of duplicate wells of Alizarin staining in culture wells with osteogenic defined media minus or plus human CCN3. Some images from panels c and d are duplicated from Main Fig. . c , Bar plots of change in fractional bone volume from whole femurs harvested from control females and then cultured with isolated plasma from Esr1 fl/fl and Esr1 Nkx2.1-Cre age-matched female littermates. Plasma (15 µl) was added daily for 1–7 days of culture as described in the Methods Section. d , Plots of fractional bone volume were determined after culturing the right femur (females) or right femur (males) in media treated with 0.9 % NS (Saline). Baseline values were obtained for freshly isolated left femur from the same mouse and immediately fixed in 4% PFA for analysis without culturing (Baseline). e . Plots of fractional bone volume were determined after culturing the right femur from 18-month-old C57BL/6 female mice in media treated with 0.9 % NS (Saline) or 3 nM mCCN3 compared to baseline. f , Representative images of H&E stained sections of the contralateral left and right femurs from the same female and male mouse at Baseline, Saline, or after treatment with mCCN3. g , Box and whiskers plots of bone parameters after saline (black) or mCCN3 (red) daily treatments of control females. h . Stiffness of callus 21 days post-fracture with images from Modified Periodic Acid‐Schiff (PAS) staining shown for callus region. One Way ANOVA for panels a and h (Tukey’s multiple-comparisons test). Paired Student’s T-test 1-tailed for panels d and e. Unpaired Student’s T-test 2-tailed for panel g. *p < 0.05, **p < 0.01, ns = not significant. Error Bars ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001, ns = not significant. Error Bars ± SEM.

Article Snippet: Blots were then probed overnight at 4 °C with anti-CCN3 antibody (R&D Systems, AF1976; 1:3,000) in TBS-T with 5% serum.

Techniques: Control, Mutagenesis, Staining, Cell Culture, Isolation, Saline, Modification

Journal: Nature

Article Title: A maternal brain hormone that builds bone

doi: 10.1038/s41586-024-07634-3

Figure Lengend Snippet: a , Left, schematic of experiment to induce loss-of-function of CCN3 ( Ccn3 knockdown) in the ARC in mice. Right, Ccn3 -positive neurons in female Esr1 Nkx2.1-cre ARC versus %BV/TV after Ccn3 siRNA injections. b , Ccn3 expression in control, unilateral and bilateral hit with corresponding µCT scans of distal femurs. Scale bar, 500 µm ( N = 6, 4). c , Left, schematic of experiment to induce gain-of-function CCN3 in the liver. Right, ectopic mCCN3 expression in Esr1 fl/fl female hepatocytes following retro-orbital injection of AAVdj-CAG-CCN3 (AAVdj-CCN3) or control (AAVdj-Ctrl) vectors. Inset shows double nuclei. Scale bar, 100 µm. d , mCCN3 immunoblot of heparin–agarose-purified plasma (left) and liver extracts (right, 10 µg total protein) from mice 5 weeks after injection with AAVdj-Ctrl (–) or AAVdj-mCCN3 (+). Recombinant mCCN3 (rCCN3) shown in far left lane. e , %BV/TV (left) of femurs and L5 and mechanical strength (right) of femurs from 3-4-month-old Esr1 fl/fl female mice 5 weeks after injection ( N = 7, 8 femurs and N = 4, 3 L5). f , %BV/TV (left) of femurs and L5 and mechanical strength (right) of femurs from 3–4-month-old Esr1 fl/fl males 5-weeks after injection ( N = 6, 7 femurs and N = 5, 7 L5). g , %BV/TV of femurs and L5 in 5-month-old OVX Esr1 fl/fl females 9 weeks after injection ( N = 8, 8 femurs and N = 8, 8 L5). h , %BV/TV of 20–23-month-old Esr1 fl/fl female femurs 9 weeks after injection ( N = 5, 5). i , Bone formation rate and bone surface (BFR/BS), and number of osteoblasts per bone surface (No. Ob/BS) determined by histomorphometry ( N = 7, 8). Scale bar, 50 µm. j , Number of osteoclasts per bone surface (No. Oc/BS) and lacunar density per bone area as determined by static histomorphometry ( N = 4, 4). Representative images of TRAP (top) and silver nitrate (AgNO3, bottom) staining for femoral osteoclasts and lacunae, respectively. Scale bar, 50 µm. Simple linear regression for a . Unpaired Student’s t -test, one-tailed or two-tailed for e – g , i and j as indicated in Supplementary Table . Mann–Whitney test, one-tailed for h . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars ± s.e.m. Graphic in c was adapted from BioRender ( https://www.biorender.com ).

Article Snippet: Blots were then probed overnight at 4 °C with anti-CCN3 antibody (R&D Systems, AF1976; 1:3,000) in TBS-T with 5% serum.

Techniques: Knockdown, Expressing, Control, Injection, Western Blot, Purification, Recombinant, Staining, One-tailed Test, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: A maternal brain hormone that builds bone

doi: 10.1038/s41586-024-07634-3

Figure Lengend Snippet: a , Expression of mCCN3 protein in female liver transduced with a low dose of AAVdj-CAG- Ccn3 (0.5 * 10 10 GC/mouse) 2 weeks post-injection; panels to the right represent digitally magnified images of individual positive cells. Scale bar = 100 µm. b , Relative levels of Ccn3 transcripts in liver tissue 5 weeks post-injection after transduction of 0.5 * 10 10 , 3 * 10 10 and 15 * 10 10 GC/mouse of AAVdj-CAG- Ccn3 viral vector and control AAV-empty vector (dj) into Esr1 fl/fl male or female littermates, (N = 4–9 per group). c , Western blots of plasma and liver uncropped with Ponceau staining below used in Main Fig. . The relative expression for Ccn3 by qPCR is listed for each sample in lanes. d , Bone volume and dynamic histomorphometry measurements after Calcein and Alizarin red double labeling (5 days apart) of female mice transduced with the lowest dose of AAVdj-CAG- Ccn3 viral vector compared to control vector (black) obtained in femurs from Esr1 fl/fl control females. e , Bone formation rate and mineral apposition rate (MAR) from dynamic histomorphometry measurements with representative images of femur sections described above from aged Esr1 fl/fl females (20–23 months of age) injected with AAVdj-CAG-CCN3. f , Bone volume of femur and L5 of female mice transduced with highest dose of AAVdj-CAG- Ccn3 viral vector. g , TRAP+ osteocytes (%) in femurs from young females (left). In vitro differentiation of osteoclasts from bone marrow isolated from young intact females treated with vehicle or recombinant CCN3 (right). h , Osteoblast surface (Ob.S), osteoclast surface (Oc.S) per bone surface (BS), and silver nitrate staining for lacunae. i . TRAP+ osteocytes (%) in femurs from aged females (left). In vitro differentiation of isolated osteoclasts from aged intact females treated with vehicle or recombinant CCN3 (right). Unpaired Student’s T test 2-tailed for low and high dose groups in panels e, d, g, h. *p < 0.05. ns = not significant. Error Bars ± SEM. Legend is provided above graphs. Abbreviations: MNCs mononucleated cells.

Article Snippet: Blots were then probed overnight at 4 °C with anti-CCN3 antibody (R&D Systems, AF1976; 1:3,000) in TBS-T with 5% serum.

Techniques: Expressing, Transduction, Injection, Plasmid Preparation, Control, Western Blot, Staining, Labeling, In Vitro, Isolation, Recombinant

Journal: Nature

Article Title: A maternal brain hormone that builds bone

doi: 10.1038/s41586-024-07634-3

Figure Lengend Snippet: a , Representative images of coronal brain sections from Esr1 fl/fl females stained for ERα (magenta) and CCN3 (green) in the posterior medial basal ARC during pregnancy and postpartum stages (lactation and post weaning) ( N ≥ 2 for each time point). Scale bar, 50 µm. b , Colocalization of Ccn3 (green), Kiss1 (red) and Esr1 (magenta and cyan) transcripts in the ARC of a lactating control ( Esr1 fl/fl ) female at 7 days. Scale bar, 50 µm. c , Relative Ccn3 quantified from microdissected ARC tissue obtained from Esr1 fl/fl virgin, Esr1 Nkx2.1-cre mutant virgin and Esr1 fl/fl lactating (7 DPP) female mice ( N = 5, 3, 3). d , CCN3 (green) and vimentin (red, VIM) in the posterior ARC of virgin Esr1 Nkx2.1-cre mutant and lactating or OVX Esr1 fl/fl control mice (1 week after surgery). e – i , Ccn3 knockdown (KD) in the ARC of lactating female mice. e , Schematic of injection of shRNA Ccn3 (sh Ccn3 ) or shRNA control (shCtrl) vectors into the ARC of control Esr1 fl/fl females with experimental timeline (left) and representative images of CCN3 at 12 DPP (right). Scale bar, 500 µm. LCD, low-calcium diet. f , g , Litter sizes ( f ) and BV of femurs ( g ) from mothers injected with shCtrl or sh Ccn3 ( N = 5, 9) fed SD (0.8%Ca 2+ ). h , Average pup weight (5–6 pups per litter) nursed by dams injected with shCtrl or sh Ccn3 and fed SD ( N = 5,9) or LCD (0.01% Ca 2+ , N = 4,2). i , Litters at 8 DPP nursed by dams injected with shCtrl or sh Ccn3 and fed a LCD, with survival values in parentheses. Scale bar, 1 cm. j , Brain-derived MBH (that is, CCN3) replaces E2 as an osteoanabolic hormone during lactation and counteracts the catabolic actions of mammary-gland PTHrP to promote healthy bone formation, thereby ensuring adequate calcium supplies for milk and maternal skeleton integrity during lactation. One-way ANOVA for c (Šidák’s multiple-comparisons test). Unpaired Student’s t -test, two-tailed for f , and one-tailed for g . Three-way ANOVA for h (Tukey’s multiple-comparisons test). * P < 0.05, ** P < 0.01, **** P < 0.0001. Error bars ± s.e.m. Graphics in j (mammary, bone and calcium) were reproduced or adapted from BioRender ( https://www.biorender.com ). Graphic in j (dam and litter) was reproduced from Mind the Graph ( https://mindthegraph.com ) under a Creative Commons licence CC BY-SA 4.0 .

Article Snippet: Blots were then probed overnight at 4 °C with anti-CCN3 antibody (R&D Systems, AF1976; 1:3,000) in TBS-T with 5% serum.

Techniques: Staining, Control, Mutagenesis, Knockdown, Injection, shRNA, Derivative Assay, Two Tailed Test, One-tailed Test

Journal: Nature

Article Title: A maternal brain hormone that builds bone

doi: 10.1038/s41586-024-07634-3

Figure Lengend Snippet: a , Representative images of brain sections and CCN3 staining quantification from dams injected with shControl (upper) or shRNA-m Ccn3 (lower) and collected at 12DPP. Yellow border defines area in which CCN3 immunostaining intensity was quantified. Scale bars = 500 µm. b , Percent reduction in ARC CCN3 immunostaining intensity of shRNA-m Ccn3 dams fed SD (N = 9) or LCD (N = 2) as compared to shRNA control dams fed SD (N = 5). c , The time interval between mating (day 0) and observation of copulatory plug (Control N = 5, shRNA-m Ccn3 N = 9). d , Milk consumption in litters from shCtrl (N = 3) and sh Ccn3 (N = 9) as measured by weight recovery following 3-hour separation from lactating dams fed SD. e , Mean body weights for litters (N = 4 litters, N = 6 pups/litter) nursed by shCtrl mothers at 4, 8, and 12DPP (grey) or switched to an sh Ccn3 mother beginning at 8DPP (blue and grey). Unpaired 2-tailed Student’s T-test in panel c. Two-way ANOVA with repeated measures (Holm-Šidák’s multiple-comparisons test) in panel d. Error Bars ± SEM.

Article Snippet: Blots were then probed overnight at 4 °C with anti-CCN3 antibody (R&D Systems, AF1976; 1:3,000) in TBS-T with 5% serum.

Techniques: Staining, Injection, shRNA, Immunostaining, Control

Journal: Nature Communications

Article Title: Subcortical serotonin 5HT 2c receptor-containing neurons sex-specifically regulate binge-like alcohol consumption, social, and arousal behaviors in mice

doi: 10.1038/s41467-023-36808-2

Figure Lengend Snippet: A Representative CtB 647 infection in BNST, females. Similar images were obtained in n = 3 mice B Representative CtB 555 infection, females. Similar images were obtained in n = 3 mice. C Representative CtB 647 infection in BNST, males. Similar images were obtained in n = 4 mice. D Representative CtB 555 infection in LHb, males. Similar images were obtained in n = 4 mice. E Representative CtB 647 labeling in DRN, females. Similar images were obtained in n = 3 mice. F Representative CtB 555 labeling in DRN, females. Similar images were obtained in n = 3 mice. G Representative CtB 647 infection in BNST, males. Similar images were obtained in n = 4 mice. H Representative CtB 555 infection, males. Similar images were obtained in n = 4 mice. I Representative 5HT labeling in DRN, females. Similar images were obtained in n = 3 mice. J Representative labeling in DRN for CtB 647, CtB 555, and 5HT, females. Similar images were obtained in n = 3 mice. K Representative 5HT labeling in DRN, males. Similar images were obtained in n = 4 mice. L Representative labeling in DRN for CtB 647, CtB 555, and 5HT, males. Similar images were obtained in n = 4 mice. M Percent of DRN-LHb neurons positive for 5HT N Percent of DRN-BNST neurons positive for 5HT. O Percent of CtB 555 neurons that co-express CtB 647 in DRN. P Percent of CtB 647 neurons that co-express CtB 555 in DRN. Q In-situ hybridization for 5HT 2c , vGAT, and vGlut2 in BNST, females. Similar images were obtained in n = 3 mice. R In-situ hybridization for 5HT 2c , vGAT, and vGlut2 in BNST, males. Similar images were obtained in n = 4 mice. S , In-situ hybridization for 5HT 2c , vGAT, and vGlut2 in LHb, females. Similar images were obtained in n = 3 mice. T In-situ hybridization for 5HT 2c , vGAT, and vGlut2 in LHb, males. Similar images were obtained in n = 4 mice. U Dorsal BNST 5HT2c neuron overlaps with vGAT or vGlut2. V Ventral BNST 5HT2c neuron overlaps with vGAT or vGlut2. W LHb 5HT2c neuron overlap with vGAT or vGlut2. n = 3 males, n = 4 females, 2 slices/mouse. No statistical comparisons were performed on this data as they are intended to be descriptive. All data are represented as mean ± SEM. Source data are provided as a file. Created with Biorender.com .

Article Snippet: ISH was performed to fluorescently label mRNA for mouse serotonin receptor 2c (Mm-Htr 2c , probe#: 401001), vesicular GABA transporter (Mm-Slc32a1, probe#:319191), and vesicular glutamate transporter 2 (Mm-Slc17a6, probe#: 319171) using the RNAscope Fluorescence Multiplex Assay Kit (Advanced Cell Diagnostics) according to the manufacturer’s instructions.

Techniques: Infection, Labeling, In Situ Hybridization

Journal: bioRxiv

Article Title: Decoding the activated stem cell phenotype of the vividly maturing neonatal pituitary

doi: 10.1101/2022.01.18.476723

Figure Lengend Snippet: Neonatal pituitary stem cells show a pronounced WNT profile. (A) DEG-based GSEA plots of indicated WNT-related hallmarks in neonatal versus adult stem cell clusters (SC1, SC2, Prolif SC). Normalized enrichment score (NES), and P - and FDR-values are listed. (B) Heatmaps displaying scaled expression of several WNT ligand and receptor genes in adult and neonatal AP. (C) Left: Violin plots displaying mRNA expression level of indicated genes in SC1, SC2, MC and Prolif SC of adult and neonatal AP. Right: Bar graphs displaying relative gene expression of indicated genes in neonatal AP, as determined by RT-qPCR (relative to expression in adult AP, set as 1 (dashed line)) (mean ± SEM). Data points represent biological replicates. ** P ≤ 0.01. (D) Left: Projection on UMAP plot of selected WNT-associated genes’ expression in neonatal stem cell (SC1, SC2, Prolif SC) and MC clusters, with indication of cell clusters. Right: RNAscope in situ hybridization analysis of neonatal pituitary for Sox2 (red), Lgr4, Lgr6 and Rspo3 (all green). Nuclei are stained with DAPI (blue). Arrowheads indicate the marginal zone (MZ). (Scale bar, 100 μm). (E) Bar graph displaying relative gene expression level of indicated genes in neonatal AP-derived organoids (mean ± SEM). Data points represent biological replicates. (F) Organoid development from neonatal AP cells, cultured and exposed to WNT ligands as indicated (P0). Top: Representative brightfield pictures of organoid cultures. (Scale bar, 500 μm). Bottom: Bar graph showing number of organoids formed under conditions as indicated (mean ± SEM). Data points represent biological replicates. (G) Top: Immunofluorescence staining of Ki67 (red) in neonatal AP organoids formed under conditions as indicated (P0). Nuclei are stained with Hoechst33342 (blue). Arrowheads indicate Ki67 + cells. (Scale bar, 100 µm). Bottom: Bar graphs showing percentage of Ki67 + cells in organoids as indicated (relative to DMSO, set as 1 (dashed line)) (mean ± SEM). Data points represent individual organoids from 3 biological replicates. * P ≤ 0.05. (H) Top: Projection on UMAP plot of Rspo1 gene expression in neonatal stem cell (SC1, SC2, Prolif SC) and MC clusters, with indication of cell clusters. Bottom: Organoid development from neonatal AP cells formed in standard RSpECT medium (with RSPO1) or RSpECT medium in which RSPO1 was replaced with RSPO3 (P0). Representative brightfield images are shown. (Scale bar, 500 μm). (I) Organoid development from adult AP cells formed under conditions as indicated (P0). Top left: Representative brightfield pictures of organoid cultures. (Scale bar, 500 μm). Bottom left: Immunofluorescence staining of Ki67 (green) in adult AP organoids, formed under conditions as indicated. Nuclei are stained with Hoechst33342 (blue). (Scale bar, 100 µm). Middle: Bar graph showing percentage of Ki67 + cells in organoids as indicated (relative to PitOM, set as 1 (dashed line)) (mean ± SEM). Right: Violin plot showing diameter of organoids developed in conditions as indicated. Data points represent biological replicates. * P ≤ 0.05. (J) Left: UMAP plot of fetal human and neonatal mouse AP combined. Middle and right: UMAP plot of annotated cell clusters in human and mouse AP, respectively. Somato, somatotropes; Lacto, lactotropes; Cortico, corticotropes; Gonado, gonadotropes; Thyro, thyrotropes; Melano, melanotropes; SC1 and SC2, stem cell cluster 1 and 2; EC, endothelial cells; IC, immune cells; CT, connective tissue cells; PL, posterior lobe (pituicyte) cells; Gonado Prog, gonado progenitor cells; Gonado Prec, gonadotrope precursor cells; CC, cell cycle cells; RBC, red blood cells, Pro.PIT1, progenitor cells of PIT1 lineage; Pre.Gonado, precursor cells of gonadotropes. (K) Projection of Il6 gene expression on human fetal pituitary UMAP plot, with indication of cell clusters.

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2) and Lgr6 (404961- C2).

Techniques: Expressing, Quantitative RT-PCR, In Situ Hybridization, Staining, Derivative Assay, Cell Culture, Immunofluorescence

Journal: Research Square

Article Title: Prolonged activation of EP3 receptor-expressing preoptic neurons underlies torpor responses

doi: 10.21203/rs.3.rs-2861253/v1

Figure Lengend Snippet: RNA-scope in situ hybridization (ISH) showed the overlap of EP3R (red, Ptger3 gene) and PACAP (blue, Adcyap1 gene) in the MnPO of a Vglut2-IRES-Cre-L10 mouse (green, Slc17a6 ) (a). The colocalization of Ptger3, Adcyap1 and Slc17a6 is clear at a higher magnification (b-e). We found about 45% of the MnPO PACAP neurons co-expressing EP3R and 61% of EP3R neurons expressing PACAP. We crossed Ptger3 flox/flox mice with Adcyap1 Cre mice to cause deletion of EP3R from PACAP+ neurons (PACAP+EP3R-null mice) (f). ISH showed successful deletion of EP3R (green) from MnPO PACAP (red) neurons, and a remaining subset of MnPO EP3R+/PACAP- neurons (white arrows) (g-i). In WT mice, after the first peak of Tb elevation caused by the stress, mice showed a typical fever response 1–3h after LPS injection. However, in PACAP+EP3R-null mice LPS caused a small hypothermic response 1–3 hr after injection (0.4 °C ± 0.03 WT SAL vs. 1.1 °C ± 0.04 WT LPS vs. 0.03 °C ± 0.04 PACAP+EP3R-null mice SAL vs. −0.4 °C ± 0.04 PACAP+EP3R-null mice LPS, F(3,92)=195.1 One-way ANOVA, followed by Bonferroni’s post hoc test, p<0.0001) (j-k). A schematic figure of the protocol for calcium imaging (left panel) and a representative image of GCAMP6s native fluorescence expression and fiber placement in a Adcyap1 Cre male mouse is shown (right panel) (l). A recording of calcium signaling (green) and Tb (black) of a Adcyap1 Cre mouse illustrates a reduction in fluorescence during fever relative to the baseline (DF/F) (m). Data analyses of 1-h bins of the DF/F before and after LPS injection normalized by the baseline condition show a robust reduction in the DF/F of calcium fluorescence in MnPO PACAP neurons during fever (n=3), and standard variance (SD, i.e., spikiness) of the signal peaks before fever is reduced after the LPS injection (n-o). These results suggest that fever may be caused by the inhibition of hypothermic MnPO PACAP+ neurons. To test this, we photoinhibited MnPO PACAP neurons (p). Photoinhibiting MnPO PACAP cell bodies for 15 min (10 stimuli of 60 s on followed by 30 s off) caused a hyperthermic effect that lasted for ~ 1 h (n=4) compared to sham stimulation (0.6 ± 0.09°C SEM MnPO STIM vs 0.06 ± 0.04 °C SEM SHAM STIM, paired t-test, p<0.0001) (q). ArchT-GFP+ expression and optical fiber placement in MnPO neurons of a Adcyap1 Cre mouse (r).

Article Snippet: POA sections were used for Ptger3 (EP3R) mRNA labelling (RNAscope Probe- Mm- Ptger3 ; catalog #504481, Advanced Cell Diagnostics) or Adcyap1 (PACAP) mRNA labelling (RNAscope Probe-Mm-Adcyap1; catalog #405911-C2, Advanced Cell Diagnostics, CA).

Techniques: RNAscope, In Situ Hybridization, Expressing, Injection, Imaging, Fluorescence, Inhibition

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: ( A ) Differentially expressed gene (DEG)-based GSEA plots of indicated WNT-related hallmarks in neonatal versus adult stem cell clusters (SC1, SC2, and Prolif SC). Normalized enrichment score (NES), and p and FDR values are listed. ( B ) Heatmaps displaying scaled expression of several WNT ligand and receptor genes in adult and neonatal anterior pituitary (AP). ( C ) Left : Violin plots displaying mRNA expression level of indicated genes in SC1, SC2, MC, and Prolif SC of adult and neonatal AP. Right : Bar graphs displaying relative gene expression of indicated genes in neonatal AP, as determined by RT-qPCR (relative to expression in adult AP, set as 1 [dashed line]) (mean ± standard error of the mean [SEM]). Data points represent biological replicates ( n = 3; unpaired t -test). ( D ) Left : Projection on UMAP plot of selected WNT-associated genes’ expression in neonatal stem cell (SC1, SC2, and Prolif SC) and MC clusters, with indication of cell clusters. Right : RNAscope in situ hybridization analysis of neonatal pituitary for Sox2 (red), Lgr4 , Lgr6 , and Rspo3 (all green). Nuclei are stained with DAPI (blue). Arrowheads indicate the marginal zone (MZ) (scale bar, 100 μm).

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, RNAscope, In Situ Hybridization, Staining

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet:

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques: Isolation, Sequencing, RNAscope, Recombinant, Multiplex Assay, SYBR Green Assay, Software

Journal: Science Advances

Article Title: A neural substrate of compulsive alcohol use

doi: 10.1126/sciadv.abg9045

Figure Lengend Snippet: ( A ) Representative images of CeA photomicrographs showing Fos (green) and PKCδ (red) immunoreactivity colabeling [scale bars, 200 μm (left) and 50 μm (right)] in punishment-resistant ( n = 10) and punishment-sensitive ( n = 9) rats. ( B to D ) Mean number of cells (±SEM) positive for Fos, PKCδ, and double-labeled cells/mm 2 . * P < 0.001, * P < 0.05, * P < 0.01. ( E ) Mean fold change in Prkcd mRNA levels in punishment-resistant ( n = 8) and punishment-sensitive ( n = 6) rats, measured by qPCR. * P < 0.01.

Article Snippet: Inventoried TaqMan gene expression assay probes ( Prkcd : Rn00440891 and Gapdh : Rn4308313; Life Technologies, Carlsbad, CA) were used to assess the expression of the target gene on an ABI 7900HT Fast Real-time PCR system (Life Technologies, Carlsbad, CA).

Techniques: Labeling

Journal: Science Advances

Article Title: A neural substrate of compulsive alcohol use

doi: 10.1126/sciadv.abg9045

Figure Lengend Snippet: ( A ) Experimental design. ( B ) Virus injection site (scale bar, 2 mm) and mean fold change in Prkcd mRNA levels following a viral-mediated knockdown ( n = 8 per group). * P < 0.05. ( C ) Expression of Prkcd in the CeA measured by RNAscope. Brown dots represent the expression of Prkcd [scale bars, 2 mm (left), 50 μm (right), and 5 μm (inset)] ( n = 8 per group). * P < 0.05. ( D ) Representative images of CeA photomicrographs (scale bar, 50 μm) showing Fos (blue), PKCδ (magenta) immunoreactivity, and yellow fluorescent protein (YFP) colabeling. Mean number of cells positive (±SEM) for Fos, PKCδ, and double-labeled cells in punishment-resistant ( n = 6 per group) and punishment-sensitive ( n = 5 per group) rats/mm 2 . # P < 0.001, * P < 0.001, * P < 0.05, * P < 0.01. ( E ) Mean number of alcohol-reinforced lever presses (±SEM) during the 30-min punishment session control in punishment-resistant ( n = 19) and punishment-sensitive rats ( n = 19) receiving shCtrl, or shRNA knockdown of PKCδ ( n = 18; n = 19). # P < 0.001. ( F ) Mean resistance score (±SEM). # P < 0.001.

Article Snippet: Inventoried TaqMan gene expression assay probes ( Prkcd : Rn00440891 and Gapdh : Rn4308313; Life Technologies, Carlsbad, CA) were used to assess the expression of the target gene on an ABI 7900HT Fast Real-time PCR system (Life Technologies, Carlsbad, CA).

Techniques: Virus, Injection, Knockdown, Expressing, RNAscope, Labeling, Control, shRNA