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. 2010 Aug 10;107(32):14199-204.
doi: 10.1073/pnas.1002494107. Epub 2010 Jul 26.

Immune-related zinc finger gene ZFAT is an essential transcriptional regulator for hematopoietic differentiation in blood islands

Toshiyuki Tsunoda  1 , Yasuo Takashima, Yoko Tanaka, Takahiro Fujimoto, Keiko Doi, Yumiko Hirose, Midori Koyanagi, Yasuhiro Yoshida, Tadashi Okamura, Masahide Kuroki, Takehiko Sasazuki, Senji Shirasawa
Affiliations

Immune-related zinc finger gene ZFAT is an essential transcriptional regulator for hematopoietic differentiation in blood islands

Toshiyuki Tsunoda et al. Proc Natl Acad Sci U S A. .

Abstract

TAL1 plays pivotal roles in vascular and hematopoietic developments through the complex with LMO2 and GATA1. Hemangioblasts, which have a differentiation potential for both endothelial and hematopoietic lineages, arise in the primitive streak and migrate into the yolk sac to form blood islands, where primitive hematopoiesis occurs. ZFAT (a zinc-finger gene in autoimmune thyroid disease susceptibility region/an immune-related transcriptional regulator containing 18 C(2)H(2)-type zinc-finger domains and one AT-hook) was originally identified as an immune-related transcriptional regulator containing 18 C(2)H(2)-type zinc-finger domains and one AT-hook, and is highly conserved among species. ZFAT is thought to be a critical transcription factor involved in immune-regulation and apoptosis; however, developmental roles for ZFAT remain unknown. Here we show that Zfat-deficient (Zfat(-/-)) mice are embryonic-lethal, with impaired differentiation of hematopoietic progenitor cells in blood islands, where ZFAT is exactly expressed. Expression levels of Tal1, Lmo2, and Gata1 in Zfat(-/-) yolk sacs are much reduced compared with those of wild-type mice, and ChIP-PCR analysis revealed that ZFAT binds promoter regions for these genes in vivo. Furthermore, profound reduction in TAL1, LMO2, and GATA1 protein expressions are observed in Zfat(-/-) blood islands. Taken together, these results suggest that ZFAT is indispensable for mouse embryonic development and functions as a critical transcription factor for primitive hematopoiesis through direct-regulation of Tal1, Lmo2, and Gata1. Elucidation of ZFAT functions in hematopoiesis might lead to a better understanding of transcriptional networks in differentiation and cellular programs of hematopoietic lineage and provide useful information for applied medicine in stem cell therapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Zfat is indispensable for mouse embryonic development. (A) Targeting disruption of the Zfat gene. WT, wild-type; HR, homologous recombinant; B, BglII site; closed box: E, exon; neo, neomycin resistance cassette; D, DTA (diphtheria-toxin A fragment); shaded box: external probe. (B) Southern blotting of BglII-digested DNA using 3′ external probe (Left). PCR-based genotyping of Zfat+/− progeny (Right). (C) Genotyping statistics of progeny from Zfat+/− mice. The number and ratio of embryos showing normal development are shown. (D) Typical phenotype of Zfat−/− embryos at E9.5. (E) ZFAT expression during early developmental stage. Thy, thymocyte; Spl, splenocyte; ERK1, loading control.
Fig. 2.
Fig. 2.
Impaired differentiation of hematopoietic progenitor cells in Zfat−/− blood islands. (A) Embryos with yolk sacs from Zfat+/+ or Zfat−/− mice at E9.5. (Scale bars, 500 μm.) (B) H&E-stained sections of Zfat+/+ and Zfat−/− placentas at E8.0. Region surrounded by the dotted line represents spongiotrophoblast layer. (Scale bars, 100 μm.) (C) H&E-stained sections of blood islands of Zfat+/+ and Zfat−/− yolk sacs at E8.0 (Upper) and E8.5 (Lower). Region surrounded by the dotted line represents hematopoietic progenitor cells. Arrows, endothelial cells; asterisks, visceral endodermal cells. (Scale bars, 50 μm.) (D) ZFAT protein expression in endothelial and hematopoietic progenitor cells in Zfat+/+ blood islands at E8.0. The region surrounded by the dotted line represents hematopoietic progenitor cells. Arrows, endothelial cells. (Scale bars, 10 μm.)
Fig. 3.
Fig. 3.
ZFAT directly regulates expressions of Tal1, Lmo2, and Gata1 genes in blood islands. (A) MicroRNA expression levels for Zfat, Tal1, Lmo2, Gata1, Cd41, Runx1, Flk-1, Gata2, Kit, and Gapdh genes in Zfat−/− (black bar) and Zfat+/+ (white bar) yolk sacs at E7.5. *P < 0.001. (B) Luciferase assay using 1-kb probes (Upper) and 200-bp probes (Lower) for detection of ZFAT-DNA binding. Activation domain (AD)-ZFAT-binding activity, black bar; AD-binding activity, white bar. *P < 0.05; **P < 0.01. (C) ChIP-PCR assay for detection of the bindings of ZFAT with the DNA elements (region for 200-bp probe in B) in yolk sacs at E7.5 and adult kidney as a control tissue. End-point PCR products at 35- and 42-cycled PCR. YS, yolk sac; Kid, kidney. (D) Quantification of ChIP DNA (region for 200-bp probe in B). Quantities of the ChIP DNA in yolk sacs at E7.5 with M16 anti-ZFAT antibody (red bar) and control IgG (blue bar). Quantities of the ChIP DNA in kidney with M16 (green bar) and control IgG (yellow bar). Bar indicates the total amount of ChIP DNA normalized by M16-ChIP DNA for Kifap3 promoter in kidney as 1.0 unit. *P < 0.05; **P < 0.01. (E) ZFAT-binding regions in the Tal1, Lmo2, and Gata1 promoters. Closed box, ZFAT binding regions; blue circle, CCAAT element; red circle, GATA binding site; yellow circle, E-box; green circle, Ets binding site; black circle, CACCC element; pP, proximal promoter; blue bar, known regulatory region for each gene expression; arrow, transcriptional start site for each gene.
Fig. 4.
Fig. 4.
Reduced expressions of TAL1, LMO2, and GATA1 in Zfat−/− blood islands at E8.0. Expressions were detected by immunohistochemical staining using each antibody. The region surrounded by the dotted line represents hematopoietic progenitor cells. Arrows, endothelial cells. (Scale bars, 50 μm.)
Fig. 5.
Fig. 5.
Reduced expression of RUNX1, FLK-1, and CD41 in Zfat−/− blood islands. Expressions of RUNX1 at E8.0 (Top), FLK-1 at E8.0 (Middle), and CD41 at E8.25 (Bottom) in the blood islands. Expressions were detected by immunohistochemical staining using each antibody. The region surrounded by the dotted line represents hematopoietic progenitor cells. (Scale bars, 50 μm.)
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