cpa5 identifies zebrafish MC progenitors. (A) Double whole-mount ISH using a digoxigenin-labeled RNA antisense probe to zebrafish cpa5 (blue) and FITC-labeled RNA anti-sense probe (red) to pu.1, mpo, l-plastin, and lysozyme C (see Figure S3) demonstrate coexpression of cpa5 in a proportion of cells: (i) tail, (ii) head/yolk sac (5× objective). Evidence of coexpression is shown by colocalization observed in higher-magnification images of selected cells: (iii) brightfield, (iv) fluorescence (10× objective). No colocalization is observed for fms and cebp-α: (i) brightfield, (ii) fluorescence (8× objective). (B) gata-2 and pu.1 are both required for the development of early MCs as evidenced by the absence of cpa5 expression in gata-2 and pu.1 morphants, whereas gata-1 morphants paradoxically demonstrate increased numbers of cpa5⁺ cells. Fog-1 is dispensable for early MC development as evidenced by wild-type cpa5 expression in fog-1 morphants. Compound gata-1/pu.1 morphants demonstrate an absence of cpa5 expression, whereas compound gata-1/fog-1 morphants show a dramatic increase in numbers of cpa5⁺ cells. Lateral views, anterior left and dorsal at the top (28 hpf, 5× objective). Inset boxes demonstrate a higher-magnification view of the tail and the region around the intermediate cell mass. (C) Proposed model of transcription factor interactions required for early MC development. Established interactions are represented by —, and potential interactions by …. All images obtained with Leica application suite version 2.4.OR (Leica Microsystems, Heerbrugg, Switzerland); figure panels assembled using Adobe Photoshop CS3 Extended version 10.0 (Adobe Systems).