Characterization of the zebrafish mmp9 gene. (A) Sequence Analysis. Full-length nucleotide sequence and deduced protein sequence (bold) of zebrafish mmp9 with residue numbers indicated. The conserved domains of the encoded Mmp9 protein were analysed with the Conserved Domain program at the GenBank website (www.ncbi.nlm.nih.gov). These include a matrix metalloprotease catalytic domain (red), a zinc catalytic domain (blue), three fibronectin domains (green), and a hemopexin-like domain (pink). (B) Phylogenetic tree of matrix metalloproteinases. Radial phylogenetic tree constructed using TreeView (Page, 1996) based on a sequence alignment produced with ClustalX (Heringa, 1999). Bootstrap values shown were calculated from 100 predicted trees. The following sequences from GenBank were used: Dr Mmp9, (this study NP_998288); Bt Mmp2, NP_777170; Bt Mmp9, CAA55127; Cc Mmp9, BAB39390; Dr Mmp2, AAP74482; Gg Mmp2, NP_989751; Gg Mmp9, AAG47650; Hs MMP2, AAH02576; Hs MMP9, CAC10459; Hs MMP-12, P39900; Mm Mmp2, NP_032636; Mm Mmp9, BAB23442; Ol Mmp2 BAA85769; Ol Mmp9, BAA85770; Om Mmp2 BAA78479; Om Mmp9, CAC85923; Po Mmp9, BAB68366; Rn Mmp9, AAA90911; Xl Mmp9, AAD41624. Abbreviations: Bt, Bos taurus (cow); Cc, Cyprinus carpio (carp); Dr, Danio rerio (zebrafish); Gg, Gallus gallus (chicken); Hs, Homo sapiens (human); Mm, Mus musculus (mouse); Ol, Oryzias latipes (medaka fish); Om, Oncorhynchus mykiss (rainbow trout); Po, Paralichthys olivaceus (Japanese flounder); Rn, Rattus norvegicus (Norway rat); Xl, Xenopus laevis (African clawed frog). Scale bar indicates 0.1 nucleotide substitutions per site.

Analysis of mmp9 expression during zebrafish development. (A) RT-PCR analysis. RNA was prepared from unfertilized eggs (0) or from embryos at the indicated times (hours (h) or days (d)) post fertilization with (+) or without (-) RT using primers specific for (i) mmp9 and (ii) β-actin. Controls are cloned mmp9 cDNA (positive control, p) and water (negative control, n). (B–L) Whole mount in situ hybridization. Staged zebrafish embryos were analyzed using a DIG-labeled mmp9 probe at the times indicated, with site of mmp9 expression shown with arrows. In (B, E, and H–L) embryos are orientated in a lateral view with the anterior to the left and the dorsal side up and mmp9 expression indicated with arrows. The embryo in (C) is orientated in a dorsal view with the anterior to the left. (D and F) Represent cross sections of embryos in (C and E), respectively, along the plane indicated with the dotted line in each case. (G) Represents a flat-mounted embryo with the yolk removed viewed dorsally with anterior to the left. The outline highlights the body of the embryo.

Further analysis of mmp9-positive cell populations. (A and B) Adult spleen. Whole mount in situ hybridization using a DIG-labeled mmp9 probe of adult zebrafish spleen, either whole (A) or in transverse cross-section (B), showing mmp9 expression (arrow) in the splenic cords of the red pulp of the zebrafish spleen. The inset shows the whole spleen in cross-section. (C–H) Response to trauma. A trauma assay was performed on 48 hpf zebrafish embryos. Embryos either unclipped (t = 0) or 8 h after being tail clipped (t = 8) were subjected to whole mount in situ hybridization with lyz (C and D), mmp9 (E and F), or both lyz and mmp9 (G and H, lyz: blue staining and mmp9: red staining). Embryos are orientated in a lateral view with the anterior to the left and the dorsal side is up. Arrows indicate the migration of cells to the site of trauma. (I and J) Comparison to mmp13⁺ cell population. Two-color whole mount in situ hybridization performed on wild-type 4 dpf embryos, with both mmp9 (red staining) and mmp13 (blue staining). (J) is an enlargement of the area boxed in (I). Embryos are orientated in a lateral view with the anterior to the left and the dorsal side uppermost, with colored arrows indicating the position of cells expressing each gene.

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