Identification of 2 putative zebrafish npm1 genes. (A) Clustal W alignment of human NPM1 (top row), zebrafish Npm1a (middle row), and Npm1b (bottom row) proteins. * indicates identical residues; colon (:), highly similar residues; and period (.), similar residues. Critical tryptophan residues necessary for nucleolar localization are highlighted in blue in the boxed area. (B) The genomic loci surrounding human NPM1 on chromosome 5q35.1 (top row) are syntenic with the regions where npm1a (on chromosome 10) and npm1b (on chromosome 14) are located in the zebrafish genome (bottom row left and right, respectively). (C) RT-PCRs showing npm1a (top row) and npm1b (middle row) expression levels. β-actin expression levels are used as a loading control (bottom row). Left: npm1a and npm1b embryonic expression was assessed from whole embryos at the indicated time points. Right: RT-PCRs from precursor, lymphoid, myeloid, and erythroid cells sorted from adult kidney marrow (gating strategy based on forward- and side-scatter plots²⁸ ). (D,F,G,I) npm1a or npm1b WISH assays in 24-hpf or 48-hpf embryos, lateral view, anterior to the left, dorsal upwards. (E,H) Close-up dorsal view (anterior to the left) of the brain and anterior trunk in flat-mounted, deyolked 24-hpf embryos stained with npm1a or npm1b probes. Digoxigenin-labeled RNA probes encoding the full-length npm1a and npm1b sequences were transcribed from linearized cDNA constructs using the DIG RNA labeling kit (Roche) following manufacturer instructions.