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Conditional Gata2 inactivation results in HSC loss and lymphatic mispatterning
Kim-Chew Lim, … , Masayuki Yamamoto, James Douglas Engel
Kim-Chew Lim, … , Masayuki Yamamoto, James Douglas Engel
Published September 10, 2012
Citation Information: J Clin Invest. 2012;122(10):3705-3717. https://doi.org/10.1172/JCI61619.
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Research Article Article has an altmetric score of 20

Conditional Gata2 inactivation results in HSC loss and lymphatic mispatterning

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Abstract

The transcription factor GATA-2 plays vital roles in quite diverse developmental programs, including hematopoietic stem cell (HSC) survival and proliferation. We previously identified a vascular endothelial (VE) enhancer that regulates GATA-2 activity in pan-endothelial cells. To more thoroughly define the in vivo regulatory properties of this enhancer, we generated a tamoxifen-inducible Cre transgenic mouse line using the Gata2 VE enhancer (Gata2 VECre) and utilized it to temporally direct tissue-specific conditional loss of Gata2. Here, we report that Gata2 VECre–mediated loss of GATA-2 led to anemia, hemorrhage, and eventual death in edematous embryos. We further determined that the etiology of anemia in conditional Gata2 mutant embryos involved HSC loss in the fetal liver, as demonstrated by in vitro colony-forming and immunophenotypic as well as in vivo long-term competitive repopulation experiments. We further documented that the edema and hemorrhage in conditional Gata2 mutant embryos were due to defective lymphatic development. Thus, we unexpectedly discovered that in addition to its contribution to endothelial cell development, the VE enhancer also regulates GATA-2 expression in definitive fetal liver and adult BM HSCs, and that GATA-2 function is required for proper lymphatic vascular development during embryogenesis.

Authors

Kim-Chew Lim, Tomonori Hosoya, William Brandt, Chia-Jui Ku, Sakie Hosoya-Ohmura, Sally A. Camper, Masayuki Yamamoto, James Douglas Engel

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Figure 8

Tx-treated TgVE:Gata2–/fl embryos exhibit anemia, edema, hemorrhage, and late-embryonic lethality.

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Tx-treated TgVE:Gata2–/fl embryos exhibit anemia, edema, hemorrhage, and...
(A) Gata2+/fl female mice were intercrossed with TgVE:Gata2+/– compound transgenic males. Embryos were collected from pregnant dams that were gavaged with Tx between E9 and E11. Anemia, edema, and hemorrhage were detected in Tx-treated TgVE:Gata2–/fl (Tg:–/f) embryos, but not in any other genotype control littermates, of which some are shown here (Gata2–/fl, TgVE:Gata2+/–, TgVE:Gata2+/fl [–/f, Tg:+/–, Tg:+/f, respectively]), beginning around E13.5 and progressively increasing in severity before they succumbed to lethality and necrosis by E15.5–E16.5. Arrows: subcutaneous edema in TgVE:Gata2–/fl embryos. Note the visibly paler FLs (magenta asterisks) in Tx-treated E13.5 and E15.5 TgVE:Gata2–/fl embryos in comparison to Tx-treated controls. (A patch of hemorrhage in the E14.5 TgVE:Gata2–/fl embryo visually obscured its pallid liver). These phenotypes were reproducible using either of two transgenic lines (TgVE56: E14.5 embryos; TgVE62: E13.5 and E15.5 embryos). Further experiments performed with only a single Tx delivery on E9 (data not shown) or after 3 consecutive doses from E11 to E13 instead of E9 to E11 generated the same phenotypes (anemia, edema, hemorrhaging) in TgVE:Gata2–/fl embryos. Scale bar: 0.5 mm. (B) Genotyping of a (large) representative E13.5 litter collected from a Tx-gavaged Gata2–/fl female intercrossed with a TgVE:Gata2+/fl adult male. Yolk sac genomic DNAs were used in separate PCR reactions to specifically detect the Cre and mCh transgenes (top), 3 Gata2 alleles (fl, wild-type [+], or knockout [–]; middle) and the Cre-mediated Gata2 exon 5–deleted allele (Δ; bottom). The Gata2Δ amplicon was only detected in embryos bearing both the Gata2fl allele and TgVE. Since CreERT2 expression is restricted to endothelial cells in the yolk sac, the PCR amplicon derived from the unrecombined Gata2fl allele was detected in total yolk sac genomic DNA of Tx-treated TgVE:Gata2–/fl embryos. To generate the Gata2Δ allele as a positive control, the Gata2+/fl mouse was interbred with the ubiquitously expressed AyuI-Cre transgenic mouse (21). Sequences of primers used for PCR genotyping are listed in Supplemental Table 1.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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