Expression of the miR-144/451 locus in wild-type embryonic and adult zebrafish erythroid cells. (A) miR144/451 locus expression (22 hours postfertilization [hpf]) by RT-PCR using primers confirming that both miRNAs originate from a single precursor transcript. Schema shows primer binding sites in green. RT indicates reverse transcriptase; Gen, genomic DNA control. (B) Whole mount in situ hybridization (WISH) analysis of miR-144 at the 3 embryonic ages shown. miR-144 expression (blue) initiates at 18 hpf in the hematopoietic intermediate cell mass (ICM), becomes confluent across all cells of the ICM (20 hpf), and disperses with erythrocytes at the onset of circulation, pooling over the yolk during fixation for WISH (30 hpf). Lateral views, anterior to left; inset (bottom panel) is ventral view. (C) Sagittal section of 24-hpf WISH embryos showing miR-144 expression (blue) in cytoplasm of erythrocytes of the axial ICM. NC indicates notochord, Y, yolk. Scale bar = 20 μm. (D) Double WISH showing concordant miR-144 (blue) and hbbe3 (red) expression. Left panel, bright field; right panel, fluorescence microscopy highlighting hbbe3 expression. (E) miR-451 and gata1 expression (blue) in WT and clo embryos at 24 hpf, showing concordant loss of gata1 and miR-451 expression in clo. (F) miR-144 expression (blue) in hematopoietic cells nestled between renal tubules (RT) of adult kidney (i, white arrowheads) and the red pulp of the spleen (ii, white arrowheads) in nuclear fast red-counterstained sections cut from tissue fragment in situ hybridization. Detail (iii) shows miR-144 expression localized to the cytoplasm of circulating adult erythrocytes. Scale bar = 10 μm.