Fig. 4: GATA2 is required at the mitosis-to-G1 transition in vivo for embryonic development.
From: GATA2 mitotic bookmarking is required for definitive haematopoiesis
a Western blot analysis of GATA2 and cyclin B1 proteins in HEK 293T expressing GATA2 fused to the mitotic degradation (MD) domain of cyclin B1 or an inactive form (MDmut) before (0 h) and 2, 4 and 6 hours (h) after release from nocodazole arrest. Actin shown as loading control. Async – Asynchronous cells. Western blots were performed twice. kDA, kilodaltons. b Direct reprogramming strategy to convert human dermal fibroblast (HDFs) into induced hemogenic cells (iHem). HDFs were transduced with lentivirus encoding MD- or MDmut-GATA2, plus GFI1B and FOS factors, and the kinetics of CD9 activation was evaluated by flow cytometry. c Quantification of CD9 expression from day (d) 4 to d12. M2rtTA (M2) was used as control. d Volcano plots showing differential gene expression of GATA2 bookmarked genes at day 4 and 6 of reprogramming with MD-GATA2 and MDmut-GATA2. Relevant genes downregulated in MD-GATA2 condition (left) are highlighted in red. e Number of bookmarked differentially expressed genes between MD-GATA2 and MDmut-GATA2 conditions at d4 and d6, with binding sites for HSPC regulators. Upregulated genes in MDmut-GATA2 condition at d4 and d6 (d4_MDmut and d6_MDmut, respectively), and upregulated genes in MD-GATA2 at d4 and d6 (d4_MD and d6_MD, respectively) are shown. f Schematic representation of the mouse model developed to assess mitotic degradation of GATA2 in vivo by inserting the MD domain upstream Gata2 gene. g Frequency of homozygous (MD/MD), heterozygous (MD/WT) and wild-type (WT/WT) embryos at embryonic day (E) 9.5, E10.5, E11.5, E13.5 and pups, after crossing heterozygous mice. h Representative images of MD-Gata2 embryos at E10.5 and E11.5, and control MDmut-Gata2 embryos at E11.5. Scale bars, 1 mm. i, j Flow cytometry quantification of E10.5 (i) and E11.5 (j) erythroblasts after whole-embryo bleeding. Graphs show percentage of total erythroblasts (Erythro) or immature (type I) to mature (Type III) erythroblasts gated within lineage negative (Lin-) live single cell (SC) population. n(E10.5 WT/WT) = 3, n(E10.5 MD/WT) = 5, n(E10.5 MD/MD) = 3 n(E11.5 WT/WT) = 4, n(E11.5 MD/WT) = 5, n(E11.5 Mut/Mut) = 9, n(E11.5 MD/MD) = 5. Mean ± SD is shown. Source data are provided as Source Data file.
