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Hematopoietic regulatory domain of gata1 gene is positively regulated by GATA1 protein in zebrafish embryos

Makoto Kobayashi, Keizo Nishikawa and Masayuki Yamamoto*

Center for Tsukuba Advanced Research Alliance and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8577, Japan



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  		Fig. 1. Identification of the first intron in the 5'-UTR of the zebrafish gata1 gene. (A) Structure of the zebrafish gata1 gene. Gray and white boxes denote the coding and noncoding region of the GATA1 cDNA, respectively. E, X and S indicate enzyme sites for EcoRI, XbaI and SpeI, respectively. (B) Southern blot analysis. Zebrafish genomic DNA (lanes 1-3) or GATA1 phage clone DNA (lanes 4-6) was digested with XbaI (lanes 1 and 4), SpeI (lanes 2 and 5) or both enzymes (lanes 3 and 6). After Southern blotting, membrane was hybridized with a probe corresponding to the first intron (black bar in panel A). (C) PCR analysis. PCR was performed using zebrafish genomic DNA (lane 1), GATA1 phage clone DNA (lane 2) and GATA1 cDNA (lane 3) as templates. No template DNA was added in lane 4. Primers are indicated by arrows in A.

 


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  		Fig. 2. Activity of the first intron in GFP reporter gene expression. (A) Construction of reporter genes. Zebrafish gata1 locus is shown at the top, where open and striped bars denote the upstream region and the first intron, respectively. (B) GFP expression of the 8.1kG1-eGFP and 8.1kG1dI1-eGFP constructs. Top panels indicate the endogenous gata1 expression analyzed by whole mount in situ hybridization. ICM, intermediate cell mass. (C) Region for counting GFP-expressing cells. (D) Ratios of GFP-expressing cell numbers between the 8.1kG1-eGFP- and 8.1kG1dI1-eGFP-injected embryos. Embryos that did express GFP in the ICM at 24 hours were selected and further analyzed at the embryonic days indicated. Concentration of the injected DNA was 10, 25 or 50 µg/ml, as indicating by white circles, black circles and white squares, respectively. Five pictures of each embryo were taken at a 30 msecond video rate, and numbers of cells expressing GFP in the square shown in C were counted and averaged. Percentages were calculated from the mean of positive cell numbers in examined embryos (n=21 and n=37 for 8.1kG1-eGFP and 8.1kG1dI1-eGFP, respectively).

 


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  		Fig. 3. Template activities of the various gata1 constructs. Values in the left column show the percentages of embryos that contained more than 21 GFP-positive cells in the ICM at 22 hours after injection of indicated reporter constructs. Values in the right column indicates that the percentages of embryos that contained more than six GFP-positive cells in the ICM. The numbers of embryos examined for each construct are indicated in the parentheses. dGATA and CACCC denote the double GATA motif and CACCC box, respectively.

 


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  		Fig. 4. GATA1 overexpression analyses using 8.1kG1-eGFP germline transgenic fish. (A) GFP expression in the F2 embryos. Embryos were fixed at 15 hours or 7 days. Dorsal views with anterior towards the top (left panel) or a lateral view with anterior towards the left (right panel). LPM, lateral plate mesoderm. (B) Ectopic GFP expression in the GATA1 overexpressed embryos. Dorsal views with anterior towards the bottom of the living 8.1kG1-eGFP embryo at 18 hours, in which GATA1 and DsRed RNAs were co-injected. Pictures were taken without filters (left), using the green filters (middle) and using GFP plus filters (right). Arrows indicate cells in which GATA1 was overexpressed. (C) GATA1 deletion mutants used in the analyses. (D) Percentages of ectopic GFP-expressing embryos at 15 hours among embryos that were injected with wild-type or mutated GATA1. Each mRNA was injected into more than 30 embryos. Results for wild-type GATA1 (white circles), GATA1dCF (black circles), GATA1dNF (white triangles), GATA1dN56 (black squares) and GATA1dN80 (white squares). (E) Immunoblot analysis of nuclear proteins of zebrafish embryos injected with mRNA for HA-tagged wild-type GATA1 (WT), NF-deleted GATA1 (dNF), or CF-deleted GATA1 (dCF). In vitro translated proteins of these GATA1 mutant constructs were also analyzed. Black and white arrowheads denote migration positions of the wild-type and zinc finger-deleted GATA1, respectively.

 


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  		Fig. 5. Induction of the GFP expression directed from gata1-HRD by GATA1 overexpression. (A) A successive-injection system for the gata1 mRNA and the GFP reporter constructs. Embryos that were injected with template DNA into a blastomere at early one-cell stage were again injected with the GATA1 mRNA together with tetramethyl-rhodamine dextran into a single blastomere at the two- to eight-cell stage. (B) Ectopic GFP expression in the GATA1 mRNA-injected embryo. Embryos were fixed at 15 hours and were flattened on the slides with dorsal side upwards after removing the yolk. Dorsal views of embryos with anterior towards the left that were injected with GATA1 mRNA plus tetramethyl-rhodamine dextran (top, thick arrows) or with only tetramethyl-rhodamine dextran (bottom, thin arrows). Pictures in the left, middle and right columns were taken using the GFP plus, green or both filters, respectively. Arrowheads indicate the GFP expression in the LPM. (C) Difference in GFP induction by GATA1 among various gata1 constructs. Values indicate the percentages of embryos that showed ectopic GFP expression after injecting both the GATA1 mRNA and GFP reporter constructs. The numbers of embryos observed for each construct are indicated in parentheses.

 

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