Distinct niche signatures between 3-week-old preswitch and 5- and 12-week-old postswitch HSC^α-cats. (A) Representation of the age groups used for isolation of α-catulin^GFP/+ femurs. (B-E) Three-week-old (wo) HSC^α-cats were less associated with MKs (B; 3 wo, n = 8; 5 wo, n = 7; 12 wo, n = 12) and CD105⁺ sinusoids (C; 3 wo, n = 8; 5 wo, n = 6; 12 wo, n = 12), compared with postswitch HSC^α-cats, but were preferentially associated with Cxcl12 stroma (D; 3 wo, n = 4; 5 wo, n = 4; 12 wo, n = 5) and showed a marginally higher preference toward Col.1/Opn⁺ osteoblasts and bone matrix (E; 3 wo, n = 17; 5 wo, n = 10; 12 wo, n = 13). (F) Percentage of 3-wo HSC^α-cats found within 5 μm of the trabecular or cortical bone surfaces (n = 12). (G-H) Comparison of BM volume occupied by MKs (G; 3 wo, n = 7; 5 wo, n = 7; 12 wo, n = 11) and Cxcl12 stroma (H; 3 wo, n = 3; 5 wo, n = 3; 12 wo, n = 4) in juvenile and adult α-catulin^GFP/+ femurs. (I-J) Scatterplots showing 2-dimensional (2D) distance quantification of single 3-wo HSC^α-cat in relation to CD105⁺ sinusoids and Cxlc12 stroma (I; 192 HSCs and 414 RDs, n = 4), as well as entire vasculature and bone surfaces (J; 197 HSCs and 427 RDs, n = 4). (K) Representative image of a single 3-wo HSC^α-cat that was adjacent to bone surface and transition zone (TZ) vessels simultaneously. (L-M) Quantification of HSC frequency occupying niches with distinct cellular composition in 3-wo α-catulin^GFP/+ femurs. Graphic depiction of 3-wo HSC^α-cat localization in relation to triple niches, such as entire vasculature, bone, and MKs (L; n = 4) and sinusoids, bone, and Cxcl12 stroma (M; n = 4). Data in panels B-H represent mean ± standard deviation. The 2-tailed nonparametric Mann-Whitney U test was used to assess statistical significance in panels A-D (for the 0-5-μm bin) and H-I. Statistical significance for panels E-G was assessed by 1-tailed Mann-Whitney U test. *P < .05; **P < .01; ***P < .001; ****P < .0001.