Reduced megakaryocyte (Mk)-vessel interactions in adult Lama4^−/− mouse BM and the effect of LAMA4 protein on Mk maturation. (A) Confocal images showing fluorescence staining of Mks and blood vessels in Lama4^+/+ and Lama4^−/− femur sections 6 weeks (6wk) after the irradiation (IR). The enlarged images show the MKs (CD41⁺) associated with vessels stained by pan-laminin antibody (laminin-111), and arterioles were stained with SCA1. Mks were identified by CD41 staining. The yellow arrows point to Mks that are not adjacent to vessels, and white arrows point to Mks associated with vessels. (B-C) The total number (B) and vessel-associated Mks (C) in Lama4^+/+ and Lama4^−/− femurs at steady-state and 6 weeks after IR. Each dot represents the average value of 2 measurements from 2 bone sections from 1 sample. The Mks in 2 different microscopic areas (metaphysis and diaphysis) in each section were counted and calculated. The horizontal bars represent mean ± SD. Data were from 3 to 4 mice of each genotype. The P values were determined by unpaired parametric t test. (D) Experimental setup for assessing the impact of recombinant LAMA4 protein for Mk maturation in in vitro culture. Lama4^−/− marrow from femurs and sorted MkP (LIN⁻SCA1⁻CD41⁺CD150⁺) were cultured with LAMA4 proteins or vehicle (PBS) for 4 days in the presence of SCF and THPO. Mk-counting and FACS were performed on day 3 and day 4, respectively. (E) Representative histograms showing size distribution (FSC-A) of total live cells from Lama4^−/− marrow cultured with LAMA4 proteins or PBS. (F) The percent of cells with high intensity of SSC-A after stimulation with LAMA4 proteins or PBS. The cells were first gated from PI⁻LIN⁻ cells. (G) Representative FACS profiles showing gating of total CD41⁺ cells from Lama4^−/− marrow cultured with LAMA4 proteins or PBS (left) and histograms showing the cell size distribution indicated by the intensity of FSC-A (right). (H) The percent of CD41⁺ and CD41⁺CD150⁺ cells from the marrow after culture with LAMA4 proteins or PBS. The cells were first gated from PI⁻LIN⁻SCA1⁻ cells. (I) Normalized mature Mk counts 3 days after culture with LAMA4 or PBS. The numbers of Mks counted in each experiment were normalized to that in PBS-treated culture due to variations among independent experiments. (J) May Grunwald-Giemsa staining showing the morphology of CD41⁺ Mks sorted from the cultures on day 4. The data in D-J were from 3 independent experiments with 3 Lama4^−/− mice (2 female and 1 male). See also supplemental Figure 7