Fig 2: Loss of Cdc42 leads to disruptions in Sertoli cell polarity and BTB function in cKO juvenile testesImmunofluorescent images of P24 control Dhh-Cre;Cdc42flox/+ (A, C, E, G, I, and K), P30 control (M), P24 cKO (B, D, F, H, J, and L), and P30 cKO (N) testes. (M’) and (N’) are higher-magnification images of the boxed regions in (M) and (N).(A–H) While control (A, C, E, and G) testes showed compartment-specific enrichment of CLDN11, CTNNB1, PARD3, SCRIB, and phalloidin staining (arrowheads), cKO testes (B, D, F, and H) exhibited aberrant localization of these factors (arrows).(I–L) Both control (I and K) and cKO (J and L) testes displayed strong expression of ITGA6 and ITGB1 in the tubule basement membrane; however, cKO testes regularly displayed ectopic expression of ITGB1 in the central portion of tubules.(M and N) While P30 control (M) testes showed only the interstitial and basal tubular presence of biotin (detected by streptavidin staining) in a tracer assay, cKO testes (N) showed widespread presence of biotin deep within tubules (arrows in N’) (n = 3 testes each for controls and cKO). Dashed lines indicate tubule boundaries.Scale bars, 50 µm.

Fig 3: Identification of aEC1 as arterial ECs around the circumference of branch point orifices.(A) Barplots of scRNA-seq read counts for Nog, Itga6, Alpl, Cldn5, S100a4, Thbs1, and Vcam1 in arterial EC clusters (aEC1-6). Each bar represents a single cell, either GFPhigh (green) or GFPlow (blue). (B–H) Confocal images of whole-mount en face preparations of mouse thoracic aortae centered on intercostal branch points. Immunostaining of S1PR1-GS mouse aortae for noggin (NOG) and CD31 (B), or integrin alpha-6 (ITGA6) and VE-Cadherin (VEC) (C). Immunostaining of C57BL/6J mouse aortae for CD31 and alkaline phosphatase, tissue-nonspecific isozyme (ALPL) (D) or claudin-5 (CLDN5), FSP1 (S100A4), and VEC (E). (F) Immunostaining of S1PR1-GS mouse aorta for ITGA6, thrombospondin 1 (TSP1), and VEC. Yellow arrows indicate TSP1+ ECs. (G) Immunostaining of an S1PR1-GS mouse aorta for FSP1 and TSP1. The circumference of an intercostal branch point harbors FSP1+GFP+ cells, wereas TSP1+GFP+ cells (cyan arrows) are distal from the circumference of the branch point orifice. (H) Immunostaining of a mouse aorta for vascular cell adhesion molecule 1 (VCAM1) and VEC. Two rows of cells exhibiting the morphology and VEC-localization of cells around branch point orifices are outlined. Images are representative of observations from 3 (B, C, E–H) and 2 (D) mice. Scale bars are 50 µM.

Fig 4: Spatio-temporal and molecular differences between branch point-specific arterial EC cluster aEC1 and non-branch point ECs.(A) Heatmap of selected transcription factors enriched and depleted in aEC1 cells (see also Figure 7—figure supplement 1A). (B) Immunostaining of an S1PR1-GS mouse thoracic aorta for lymphoid enhancer-binding factor 1 (LEF1), ITGA6, and VEC. Image is representative of observations from two mice. Scale bar is 100 µM. (C) Immunostaining of postnatal day 6 (P6) (n = 2) and 12 week (n = 3) S1PR1-GS mice thoracic aortae for ITGA6 and VEC. Arrows indicate non-branch point GFP+ EC. Scale bars are 100 µM. (D) Quantification of non-branch point GFP+ EC in thoracic aorta en face preparations from P6 (n = 2), P12 (n = 3), 3–4 month-old (n = 3), and 15 month old (n = 2) mice. Each dot is a field. Representative fields are shown in Figure 7—figure supplement 2. Non-branch point GFP pixels were normalized to total VEC pixels per field. One-way ANOVA followed by Bonferroni’s post hoc test, *P = 0.04; **P = 0.04; ***P = 0.0008. (E) Representative images of TSP1 and VEC immunostaining of thoracic aorta en face preparations from P6, 3 month old, 15 month old mice. Scale bars are 200 µM. (F) Quantification of TSP1 pixels normalized to total VEC pixels per field from P6 (n = 3), 3 month old (n = 2), and 15 month old (n = 2) mice. One-way ANOVA followed by Bonferroni’s post hoc test, **P = 0.001; ****P < 0.0001. (G) Sagittal cryosection (14 µM) of an E11.5 S1PR1-GS mouse embryo immunostained for CD31, ITGA6, and TSP1. Arrows indicate the dorsal aorta. Scale bar is 500 µM. (H) The dorsal aorta at higher magnification with CD31, ITGA6, and GFP channels shown. Scale bar is 100 µM. (G) and (H) are representative of observations from two embryos. (I–J) Genome browser image of GFPhigh and GFPlow MAECs ATAC-seq signal (RPGC normalized) at Itga6 and Thbs1 (TSP1) loci. Peaks with increased accessibility in GFPhigh MAECs (GFPhigh DAPs) are indicated in (I). All GFPhigh and GFPlow MAECs peaks are shown in (J). All LEF1, nuclear receptor subfamily four group A member 1 (NUR77), transcription factor SOX-17 (SOX17), and transcription factor GATA-6 (GATA6) motifs identified in consensus peaks (Figure 2A) are also shown. Orange bars in (I) highlight GFPhigh MAECs DAPs containing LEF1, SOX17, and/or NUR77 motifs. Yellow bars in (J) highlight peaks containing GATA6 motifs.

Fig 5: Identification of TNBC stromal compartment. (A) t-SNE plot showing pooled stromal cells from 24 patients colored by FlowSOM clusters. (B) t-SNE plot colored with Ki-67 + LNR (left), with depicted Ki-67 + LNR-high Cluster 4 (blue) and -low Cluster 9 (orange) populations in right panel. (C) Histograms of selected stromal markers showing their expression in Cluster 4 (blue) and Cluster 9 (orange). (D) Subpopulations of CD49c-, CD49f-, and CD97-positive stromal cells from 24 patients, visualized as dot plots (top panel) and as histograms across all 10 stromal clusters (bottom panel). (E) t-SNE maps of stromal cells colored by expression profile of selected surface antigens and signaling molecules (npß-catenin – active, non-phospho-beta catenin).