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Blood, Vol. 113, Issue 6, 1241-1249, February 5, 2009
Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors
Blood Yi Ni Lam et al.
113: 1241
Supplemental materials for: Lam et al
Files in this Data Supplement:
- Figure S1. Expression of EGFP mRNA in Tg(runx1P1:EGFP) embryos (JPG, 58.4 KB)
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(A) Lateral view and (B) dorsal view of 16 hpf flat-mounted Tg(runx1P1:EGFP) embryo following in situ hybridization for EGFP. EGFP mRNA is expressed in the posterior end of the bilateral stripes of the lateral plate mesoderm (arrowheads).
- Figure S2. FACS analysis of fluorescent cells from runx1 P1 and P2 transgenic embryos (JPG, 88 KB)
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To confirm that expression of EGFP driven by the two runx1 promoters recapitulates the endogenous expression of the two runx1 isoforms, EGFP-expressing cells were isolated from transgenic embryos by fluorescence activated cell sorting (FACS). As both transgenic lines have strong EGFP expression in neuronal tissues, embryos were dissected prior to FACS to remove anterior neuronal cells. The posterior parts containing the PBI of 22 hpf runx1P1 transgenic embryos (boxed) were dissected for cell dissociation. For 48hpf runx1P2 transgenic embryos, the part of the embryo posterior to the yolk ball was used (boxed). The dissected tissues were dissociated by treatment with trypsin. Cells expressing EGFP were sorted by FACS and analysed for forward and side scatter characteristics. Expression of EGFP was confirmed by examining the sorted cells under fluorescence microscopy. RNA was extracted from the sorted EGFP-positive cells and used for cDNA synthesis. RT− controls without reverse transcriptase in the cDNA synthesis reaction were used to check for contamination of the RNA. As expected, using primers that amplify a region common to both isoforms, fluorescent cells isolated from both transgenics expressed runx1. EGFP-positive cells from the runx1P1, but not the runx1P2 transgenic, express the runx1P1 isoform. Expression of the runx1P2 isoform was detected in the fluorescent cells isolated from both transgenics, with stronger expression observed in the runx1P2 transgenic. It is possible that some of the neuronal and notochord tissues dissected with the PBI of runx1P1 transgenics express EGFP and runx1P2.
- Figure S3. VISTA analysis of the +23 enhancer sequence in mouse, human, Xenopus, zebrafish, and Fugu runx1 (JPG, 79 KB)
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A recent study on the transcriptional regulation of runx1, by comparative analysis of runx1 genomic sequences from human, mouse, dog, opossum, chicken, and Xenopus, identified a highly conserved enhancer element within the first intron, located 23.5 kb downstream of the runx1P1 ATG translational start site.36 This +23 intronic enhancer element was found to drive transgenic expression in all runx1-expressing hematopoietic domains in mouse embryos, while the P1 and P2 promoters were found to lack tissue specificity.37 We undertook a similar in silico analysis with VISTA,46 aligning the 130 kb mouse runx1 intron 1 sequences with those of the same intron in human, Xenopus, zebrafish, and the pufferfish Takifugu rubripes. The percentage conservation with the mouse sequence is indicated by the height of the peaks. Sequences over 100 bp with >70% identity with the mouse genome are coloured in pink. The +23 enhancer was not found in the runx1 sequences of zebrafish or Fugu. While it is possible that the genomic region containing the intronic enhancer may not be fully sequenced in the fish genomes, the P1 and P2 promoters are capable of driving expression in definitive hematopoietic domains. This raises the possibility of differences between species in the contribution of various transcriptional elements in driving runx1 hematopoietic expression.
- Figure S4. Lineage tracing of runx1P1:EGFP+ cells from the PBI (JPG, 154 KB)
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Because the hematopoietic EGFP expression in the Tg(runx1P1:EGFP) transgenic in the PBI precedes that of Tg(runx1P2:EGFP) in the AGM, it is possible that these two populations of hematopoietic cells are related. We performed lineage tracing of the EGFP-positive cells in the PBI in the runx1P1 transgenic. One-cell stage runx1P1 transgenic embryos were injected with photoactivatable rhodamine dextran (Molecular Probes) and allowed to develop to 20 hpf, protected from light. (A,B) Caged rhodamine in green fluorescent cells in the PBI was uncaged at 20 hpf, resulting in red fluorescence in the PBI cells. The embryos were grown to 48 hpf, and the AGM and the PBI regions were imaged. (C) No red fluorescence was observed in the AGM region of uncaged runx1P1 transgenic embryos (dashed oval). The erythrocytes in the circulation showed weak red fluorescence. (D) Some of the vasculature in the PBI region also fluoresced red (arrowhead), suggesting some angioblasts, which develop in close association with hematopoietic progenitors were also uncaged in the PBI region. These results suggest the hematopoietic populations in the PBI and the AGM labeled in the runx1P1 and runx1P2 transgenic, respectively, arise independently.
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