In situ cytologic alterations of MKs in WASp-deficient bone marrow.(A) Semithin toluidine blue staining sections of wild-type and WASp-deficient bone marrows. Epon-embedded sections of wild-type bone marrow MKs examined by light microscopy show centrally located nuclei (i, arrows), whereas MKs in WASp-deficient bone marrow (ii, arrows) display eccentric moon crescent-like nucleus attesting for the cell structure disorganization (original magnification, × 200). (B) Immunohistochemical staining of VWF in wild-type (i,iii,v) and WASp-deficient (ii,iv,vi) bone marrows. Indirect immunolabeling of VWF by immunohistochemistry (described in “Materials and methods”) shows quantitative and qualitative alterations of WASp-deficient MKs and the abnormal presence of platelets in femoral bone marrow obtained from WASp-deficient mice in comparison with wild-type 129Sv mice. (i-ii) Normal distribution of MKs in mouse bone marrow is shown, whereas WASp-deficient bone marrow MKs appear more numerous (original magnification, × 200). (iii-iv) Typical morphology of MKs in wild-type bone marrow. Some WASp-deficient bone marrow MKs have lost their round shape, and they appear stretched with elongated cytoplasmic extensions resembling proplatelets (arrow) (original magnification, × 400). (v-vi) At a higher magnification, the MK appears well limited, with round shape. No labeling is detected in the extramegakaryocytic space. In contrast, WASp-deficient bone marrow MKs often exhibit an atypical shape with an eccentric position of the nucleus. Some labeling (arrow) is detected in the extramegakaryocytic space, suggesting the presence of in situ platelet production (original magnification, × 600).