Opn and HA are highly expressed in the metaphysis and endosteal regions. Has3-synthesized HA significantly affects the ability of HSPC to transendothelially migrate into the metaphysis region at very early time points after transplantation. (A) Immunohistochemical staining of longitudinal femoral sections reveals strong expression of Opn around trabeculae in the metaphysis as evidenced at higher magnification (boxed region to the right of the image). (B) HA binding protein reveals strong expression of HA around trabeculae in the metaphysis as evidenced at higher magnification (boxed region to the left of the image). Scale bar represents 1 mm (low magnification images) and 100 μm (higher magnification images). Micrographs captured using a 10× (UPLSApo NA 0.4) objective on an Olympus BX51 microscope fitted with a SPOT RT-SE6 camera. Montages constructed with Photoshop CS2. (C) Homing of transplanted HSPC to the extravascular BM in recipients devoid of either Has3-synthesized HA or Opn. Data are corrected for the proportion of HSPC in BVs. (D) The distribution of HSPCs within the femur of recipients devoid of Has3-synthesized HA reveals an increased percentage in the BVs of Has3^−/− recipients compared with WT at 15 minutes after transplantation. (E) At 45 minutes, there is a statistically significant increase in the percentage of HSPCs within the BVs of Has3^−/− recipients, with a concomitant decrease in HSPCs within the cellular marrow. There is also a significantly higher percentage of HSPC crossing the BVs in Has3^−/− recipients: WT 15 minutes, n = 7; Opn^−/− 15 minutes, n = 4; Has3^−/− 15 minutes, n = 3. WT 45 minutes, n = 8; Opn^−/− 45 minutes, n = 4; Has3^−/− 45 minutes, n = 9. Experimental repeats more than or equal to 3. *P = .01 (in BV). *P = .03 (crossing BVs). **P = .005 (in extravascular BM). (F) Homing of HSPCs in Has3^−/− recipients exhibited an aberrant distribution with significantly less HSPC homing to the metaphysis. ***P < .001. (G) The spatial distribution defect was still evident after 45 minutes. *P = .02. (F-G) Symbols represent individual recipients. Experimental repeats ≥ 3. Data are mean ± SEM. See also supplemental Figures 1-3.