Fig 1: Endothelins Promote Hematopoiesis in the Mouse and Human Model Systems(A) CD31, cKit, and EDN1 immunostaining in the Ao of an E10.5 mouse embryo. Arrowheads indicate cKit+EDN1+ IAHCs or individual emerging Hem cells. The images in (A’’) show a magnification of the boxed region in (A’). Scale bars: 50 µm.(B) Experimental strategies for mouse and human model systems. MC, methocult.(C) Production of CFU-Cs in E9.5 caudal tissues cultured with each of the test factors (REN1, EDN1, and EDN2) at 10 ng/µL and 100 ng/µL. n = 4 independent experiments.(D) Proportion of CFU-Mac and CFU-GEMM from (B), normalized to 1; n = 4 independent experiments.(E) Repopulation with E9.5 caudal part cells cultured with the test factors (REN1, EDN1, and EDN2) and 18 weeks following transplantation (*p < 0.05, paired t test, n = 3 independent experiments). The shape on the graph indicates the experiment. The red line shows the upper limit of control repopulation levels). The numbers of long-term repopulating mice (18 weeks) higher than 1% are as follows: CTRL, 0/11; REN1_10, 3/11; REN1_100, 5/12; EDN1_10, 3/11; EDN1_100, 3/12; EDN2_10, 2/11; EDN2_100, 6/12.(F) Effect of test factors on production of CFU-Cs by human ES cells at 12 days of differentiation. The number of day 14 CFU-Cs per 5,000 plated cells is shown; factors were added at day 8 of culture (**p < 0.01, paired t test; error bars, ± SEM, n = 4).(G) Production of CFU-GEMM from (E) (**p < 0.01, paired t test; error bars, ± SEM).
Fig 2: Model of ET-1 signaling in the postnatal SVZ.a In the developing postnatal SVZ, RGCs secrete ET-1, which binds to Ednrb receptors on RGCs in an autocrine manner to activate Notch signaling. This leads to upregulation of stem cell genes and downregulation of proneural factors, resulting in RGC maintenance and proliferation. ET-1 also binds to Ednrb receptors on OPCs within the SVZ to upregulate the transcription factor Gsx1 and downregulate OL maturation factors S100b and Ust, thereby promoting OPC proliferation and blocking maturation. b Following demyelination of the SCWM, Type B cells in the adult mouse SVZ upregulate ET-1. This induces upregulation of Notch signaling components Jag1 and Hes5 in Type B cells, leading to increased proliferation. ET-1 also induces upregulation of Gsx1 and downregulation of S100b in SVZ OPCs, increasing their proliferation as well.
Fig 3: Basal expression of ET-1 mRNA on kidney sections of control mice. Details showing RNAscope for ET-1 mRNA (red) in cortex (A), outer (B), and inner medulla (C) on a control kidney section. ET-1 mRNA was detected within glomeruli (glom) and renal vessels (arrows). Merged details of the co-hybridization of ET-1 with the endothelial marker CD31 (green) (D, E, F) revealed endothelial cells as the only expression site of ET-1 synthesis in the healthy kidney. Scale bars = 50μm
Fig 4: Caveolin-1 overexpression attenuates insulin mediated cellular actions. Representative images (a) and graph (b) showing NO generation (green fluorescence) in basal and insulin stimulated conditions in control (adnull) and caveolin-1 overexpressing (adcav-1) cells. Graph (c) showing stimulation of ET-1 mRNA expression by insulin (100 nM) in control (ad-null) and cav-1 overexpressing cells. Data are mean ± SEM. Overall P values were determined using Wilcoxon Rank Sums test. For pairwise comparison, Wilcoxon method was used. *p < 0.05 compared to adnull cells w/o insulin, #p < 0.05 compared to adcav-1 cells w/o insulin and ^p < 0.05 compared to adnull cells with insulin.
Fig 5: Identification of downstream molecular targets of ET-1 signaling in OPCs.a Experimental procedure. b Volcano plot displaying differentially expressed genes (DEGs) between control and ET-1-treated OPCs. The y-axis corresponds to the mean expression value of log10 (p value) and the x-axis displays the log2 fold change value. The red dots represent the transcripts that have a minimum log2 fold change of 0.05 and an adjusted p value of <0.1 (Wald test with Benjamini−Hochberg post hoc). c Predicted upstream regulators from IPA analysis of DEGs that are highlighted in red in b. Yellow arrow points to Edn1 as a predicted upstream regulator. d Venn diagram displaying the number of shared and distinct DEGs identified from the neurosphere and OPC RNAseq datasets. e Heatmap of the 37 DEGs shared by the neurosphere and OPC RNAseq datasets. The heatmap depicts the read counts from the OPC RNAseq data. Only 1 gene (Sgk1*) exhibited the opposite change in the neurosphere RNAseq dataset. f Heatmap of the 41 DEGs specific to the OPC RNAseq dataset. g Representative images of Yfp+ recombined OPCs in the dorsal SVZ of WT and Ednrb OPC-cKO mice at P10. Arrows point to the presence of S100b, Ust, or Gsx1 mRNA puncta within the Yfp+ cells. Scale bar = 10 μm. h Quantification of the percentage of Yfp+ OPCs that contain S100b, Ust, or Gsx1+ mRNA puncta within the dorsal SVZ of WT and Ednrb cKO mice at P10. (n = 3 WT, 3 Ednrb OPC-cKO mice). **p value = 0.007214 (S100b and Gsx1); **p value = 0.006335 (Ust) (Multiple t-tests with Holm–Sidak multiple comparisons correction). Data are presented as mean values ± SEM. Source data are provided as a Source Data file.
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