Spatiotemporal expression patterns of zebrafish perlecan mRNA and protein. (A–G) Whole mount ISH with a digoxigenin-labeled perlecan domain V antisense probe for the localization of perlecan mRNA (blue/purple staining). Perlecan mRNA can be detected at the 20-somite stage in regions of the developing brain (A) and along both sides of the notochord (B, arrows). At 1 dpf, perlecan expression is seen in the head region (C), in the developing somites (C, black arrows), and within the developing axial vasculature (D, arrows). Also note perlecan expression localized within the duct of Cuvier (C, red arrows). By 5 dpf (F and G [magnified image of the region boxed in F]), perlecan expression significantly increases throughout the trunk musculature (G, arrows), the major trunk/tail vessels including the DLAV, DA, and PCV, and in the developing gastrointestinal tract and future fin regions. (E) ISH with a digoxigenin-labeled perlecan domain V sense probe. ISH was performed in groups of two to five samples, and representative images are shown. (H–N) Whole mount immunohistochemistry with an affinity-purified anti–mouse perlecan antibody. Perlecan can be detected as early as the 64- and 1,000-cell stage of embryonic development (2 and 3 hpf, respectively) throughout the cell mound (I and J). At 1 dpf, perlecan protein (L and M) is detected throughout the head (L, arrow), trunk, and tail and is specifically localized to the developing muscle myoseptae (M, arrows) and the developing vasculature. By 2 dpf (N), perlecan protein is specifically detected in the trunk vasculature, including the DLAV (N, black arrows) and axial vessels (N, white arrows). (H and K) Immunohistochemistry images in which the primary antibody was omitted. Bars, 500 μm.