FIGURE SUMMARY
Title

A highly conserved regulatory element controls hematopoietic expression of GATA-2 in zebrafish

Authors
Yang, Z., Jiang, H., Zhao, F., Shankar, D.B., Sakamoto, K.M., Zhang, M.Q., and Lin, S.
Source
Full text @ BMC Dev. Biol.

GFP expression in GATA-2 transgenic zebrafish. (A) Whole mount in situ hybridization shows GATA-2 mRNA expressed in zebrafish embryonic brain, ganglia cells, motor neurons and hematopoietic ICM region at 24 hpf stage. Arrows indicate the ICM. (B) Transgenic zebrafish carrying a modified BAC construct containing a 20 kb genomic sequence upstream of, and adjacent to the GATA-2 start codon show GFP expression in the ICM, brain and spinal cord at 24 hpf stage. (C) Transgenic zebrafish embryos carrying a GFP reporter gene construct linked to a 7.3 kb genomic sequence upstream of and adjacent to the GATA-2 start codon have strong GFP expression in neuronal cells, but not in the ICM at 24 hpf. (D) Transgenic zebrafish embryos carrying BACΔUp1/GFP have strong GFP expression in the ICM at 24 hpf stage. (E) Transgenic zebrafish embryos carrying BACΔUp2/GFP and (F) BACΔUp1ΔUp2/GFP have GFP expression in neuronal tissue but not in the ICM. (G) Transgenic zebrafish embryos carrying Up1-mp/GFP have strong GFP expression in the heart at 24 phf stage. (H) Stable transgenic zebrafish embryos containing the Up2 element linked with mp/GFP have strong GFP expression in the ICM and weak GFP expression in the spinal cord at 24 hpf stage. (I) Transgenic zebrafish embryos carrying Up2a-mp/GFP have GFP expression in ICM region at 24 hpf stage; non-specific GFP expression was observed the in notochord. (J-L) Transgenic zebrafish embryos carrying base change mutation in Up2 element derived from construct Up2-mp/GFP. (J) Mutation in the binding site of ARP1 shows no effect on the GFP hematopoietic expression. (K) Mutation in the binding site of E2F-1 results in weaker GFP expression in ICM and strong non-specific expression in muscle and notochord. (L) Mutation in HOXA3-A binding site shows significantly reduced GFP expression in ICM. Similar reduction of GFP in ICM was seen in LMO2 mutation (see Additional file 3, Supplementary Figure S2). White arrows indicate ICM expression of GFP.

Conservation of Up2 enhancer activity and transacting factors. (A). Transient transgenic assays of hematopoietic GFP expression in embryos injected with wild type and mutant constructs identify HOXA3, LMO2 and E2F-1 as potential transcription factors. Also see Additional file 2, Supplementary Table S for more information about the results of hematopoietic transient expression assay. (B). Typical transient GFP expression patterns in injected embryos at 22 hpf. Left, GFP expression is detected in hematopoietic cells (arrow); Right, non-specific GFP expression was observed in muscle, heart and neuron tissue but not in hematopoietic cells (C). EMSA shows transcription factor E2F-1 (left) and HOXA3 (right) specifically bind to the probe corresponding to Up2 sequence, respectively. Probe containing HOXA3-A binding site was analyzed in EMSA. (D). Human myeloid leukemia cell lines, KG1 and K562, and mouse fibroblast cell line, NIH3T3 were transiently transfected with the mp/GFP, Up2-mp/GFP and Up2a-mp/GFP constructs. GFP expression was stronger in myeloid cells than in the fibroblast cell line. In the myeloid cell lines, enhancer activity of Up2 and Up2a was found to be statistically significant (*p < 0.002, ^p < 0.05, # p < 0.035, ** p < 0.032). The enhancer activity of Up2a/mp was less than that of Up2/mp. This figure represents the average of three independent experiments performed in duplicate.

Single RT-PCR product was observed after the nested PCR with commercial 5′RACE primer and zebrafish GATA-2 inner 3′ reversed primer. Sequencing results shown that the RT-PCT product is as same as that previously reported. 1, 100bp ladder from New England Biolabs, Inc. 2, RT-PCR product after the 2and round nested PCR. 3, Negative control for RT-PCR.

Transgenic zebrafish embryos carrying base change mutation in the binding site of LMO2 significantly reduced GFP hematopoietic expression.

Acknowledgments
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