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globin expression by in situ hybridization through 5 dpf. Expression patterns of the α- (A) and β-globin (B) genes. For detected genes, expression is seen in bilateral stripes at the 16ss, in the ICM at the 25ss and 24 hpf stage. With the onset of circulation at 24 hpf, globin positive cells can be seen throughout the vasculature with probes for expressed globin genes, particularly in the vascular plexus of the caudal hematopoietic tissue.

Generation and characterization of LCR-GFP transgenic zebrafish line: (A) sequence and location of the HS-26 DNase I hypersensitivity site (HS-26; yellow rectangle) and locus control region (LCR; red rectangle) within the major globin locus. The proximal globin promoter used is indicated with a red star. Shaded boxes indicate binding motifs for canonical erythrocyte transcription factors. (B) Representation of the vectors assembled from genomic fragments and the resulting GFP expression patterns. Fluorescent images of LCR-GFP transgenic zebrafish embryos at 16ss, 22 hpf and 36 hpf respectively (C–E). (F) Fluorescent image of the flank of an adult LCR-GFP transgenic zebrafish. (G) Red blood cell gate as determined by forward and side scatter for peripheral blood, and the analysis of the percent of GFP positive red blood cells in an LCR-GFP transgenic adult (green) versus in a wild-type adult (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

GFP mRNA expression in LCR-GFP transgenic zebrafish line. In situ hybridization was performed to detect GFP RNA expression in the LCR-GFP transgenic zebrafish line as RNA expression precedes visible GFP fluorescence. This was performed at the 12 s.s. (A), 13 s.s. (B), 16 s.s. (C), 18 s.s. (D), 24 hpf (E) and 48 hpf (F) stages.

Functional characterization of the LCR in transiently injected embryos. (A) Summary of the transgenic constructs injected including the enhancer element, promoter and vector backbone. All vectors listed contain a GFP reporter open reading frame. Bright field, GFP and composite images of representative Gata1Peak-LCR-MinPro-GFP (B) and DNaseIPeak-LCR-MinPro-GFP (C) embryos. ¹The # Observed refers to all embryos observed from the injected clutch. ²The # Observed refers to the number of productively injected embryos as determined by expression by an mCherry reporter plasmid.