Figure 3.

Niche formation is dependent on endochondral ossification. a-c, Suppression of SLF expression in fb cells did not alter osteogenesis or niche formation. 14.5 dpc fb cells were transduced with lentivirus as indicated, and 2000 sorted GFP+ cells were injected under the renal capsule. a, Paraffin-embedded sections were obtained 32 days after transplantation and stained with pentachrome. b, Representative FACS profiles of pre-gated, live CD45⁺lineage^- cells harvested from intact 15.5 dpc normal (WT) or mutant (Sl/Sl) fetal bones 40 days after transplantation (n=4). c, Frequency of LT-HSC in different ectopic niches shown in mean values ± Standard error of the mean (SEM) (WT bone n=3, Sl/Sl bone n=4, sh-Mock n=6, sh-SLF n=3). d, Knockdown efficiency of sh-SLF and sh-Osterix was determined by qRT-PCR in 1A5 osteoblast cell line. Relative expression percentage shown in mean values ± Standard Deviation (SD) (n=3). e, Suppression of osterix expression disrupted osteogenesis and niche formation. Fetal bone cells from 13.5 dpc (upper panel) or 14.5 dpc (lower panel) were transduced with the lentivirus indicated and 2,000 sorted GFP+ cells were transplanted (n=4). f, VEGF is required for formation of bone marrow cavity. Adult C57Bl/6 mice were intravenously injected with 10⁸ m.o.i of the adenovirus constructs expressing either mouse Fc (Ad-Fc) or soluble ectodomain of VEGFR1 (Ad-solVEGFR1) (n=3). Two days after virus injection, 13.5 dpc fb elements were transplanted under the renal capsule. Paraffin-embedded sections were obtained 25 days after transplantation and stained with pentachrome. (Scale bars in brightfield images = 500 μM, in pentachrome-stained images = 100 μM).