Runx1 function in hematopoiesis is required in cells that express Tek

Abstract

Runx1 expression marks the putative hemogenic endothelium between embryonic days (E) 8.5 to 11.5 of mouse gestation and is required for the formation of intra-aortic hematopoietic clusters, leading to the hypothesis that Runx1 is required for the transition from endothelial to hematopoietic cell. To address this hypothesis, we ablated the Runx1 gene by Cre-recombinase-mediated excision, with Cre expression under the control of the Tek promoter and enhancer. Most embryos died between E12.5 and E13.5 with a phenotype almost identical to Runx1 deficiency. We conclude that Runx1 function in establishing definitive hematopoiesis is required in a Tek+ cell.

Figure 1.

Hematopoietic analyses. (A) Gross appearance of E12.5 fetuses. Arrows indicate hemorrhages. (B) Expression from the Runx1^lz allele in E11.5 Runx1^f/lz and Runx1^f/lz Tg(Tek-cre) littermates. The individual Runx1⁺ cells distributed throughout the Runx1^f/lz fetus are absent in the Runx1^f/lz Tg(Tek-cre) fetus (regions in head indicated by black arrows are enlarged in panels to the right). White arrows indicate fetal livers. (C) Transverse section through the AGM region and fetal liver of E10.5 fetuses illustrating the lack of Runx1⁺ progenitors in the center of the fetal liver (fl) in Runx1^f/lz Tg(Tek-cre) animals. (D) C-kit⁺ hematopoietic clusters (hc's) in the umbilical artery, whole mount. (E) Colony-forming assays represented as total colonies per organ. Error bars represent 95% confidence intervals. Yolk sac (E9.5): n_experiments = 3, n_f/+ = 6, n_f/rd = 6, n_{f/+; Tek-cre} = 5, n_{f/rd; Tek-cre} = 5. Fetal liver (E11.5): n_experiments = 3, n_f/+ = 5, n_f/rd = 11, n_{f/+; Tek-cre} = 3, n_{f/rd; Tek-cre} = 8. Dorsal aorta and mesenchyme (E11.5): n_experiments = 4, n_f/+ = 6, n_f/rd = 11, n_{f/+; Tek-cre} = 11, n_{f/rd; Tek-cre} = 8. The difference between Runx1^f/+ and all other genotypes is significant at P < .001. (F) FACS analysis of the yolk sac (E9.5), fetal liver (E11.5), and AGM region plus vitelline and umbilical (V + U) arteries (E11.5). The average percentage ± SD of c-kit⁺CD41⁺ cells in the yolk sac is 5.8 ± 1.1 Runx1^f/+; 0.5 ± 0.5 Runx1^f/lz Tg(Tek-cre), and of CD45⁺CD41⁺ cells is 4.4 ± 0.6 Runx1^f/+; 0.5 ± 0.3 Runx1^f/lz Tg(Tek-cre). The average percentage of c-kit⁺CD41⁺ cells in the fetal liver is 5.0 ± 0.8 Runx1^f/+; 0.4 ± 0.4 Runx1^f/lz Tg(Tek-cre), and CD45⁺CD41⁺ cells is 6.3 ± 2.0 Runx1^f/+; 1.4 ± 1.4 Runx1^f/lz Tg(Tek-cre). Average c-kit⁺CD41⁺ cells in the AGM, V + U is 1.1 ± 0.7 Runx1^f/+; 0.0 ± 0.0 Runx1^f/lz Tg(Tek-cre), and of CD45⁺CD41⁺ cells is 1.2 ± 1.0 Runx1^f/+; 0.1 ± 0.1 Runx1^f/lz Tg(Tek-cre). (G) Assessment of excision efficiency by Tek-cre, determined by comparing the percentage of β-gal⁺ fetal liver cells in ROSA26 and R26R Tg(Tek-cre) mice. Representative plots and histograms are shown. Histograms indicate the percentage of β-gal⁺ and β-gal^- cells expressing (or not expressing, in the case of Ter119) the indicated cell-surface markers.

Figure 2.

Sites of Runx1 excision. (A) Tek expression in the para-aortic splanchnopleure of E8.5 Tg(Tek-lacZ) embryos. (B) Dorsal aorta from boxed region in panel A, illustrating that β-gal⁺ cells are confined to the endothelium. (C) Vitelline artery, from boxed region in panel A, showing Tek-lacZ expression in endothelial and mesenchymal cells. (D) Section through the AGM region of an E10.5 Tg(Tek-lacZ) embryo. (E) Ventral aspect of the dorsal aorta from the region boxed in panel D. (F) Vitelline artery from boxed region in panel D (40 ×). (G) Transverse section through the AGM region of an E10.5 R26R Tg(Tek-cre) fetus. (H) Detailed view of the dorsal aorta from the region boxed in panel G. Examination of approximately 1000 endothelial cells from 30 sections determined that 52% were β-gal⁺ and had therefore undergone R26R excision by Tek-cre. (I) Vitelline artery of an E10.5 R26R Tg(Tek-cre) fetus, showing that excision occurred in both mesenchymal and endothelial cells. (J) Dorsal aortae of an E8.5 Runxl ^lz/+ embryo, showing β-gal⁺ cells are confined to the endothelium. By E10.5 many ventral para-aortic mesenchymal cells are also Runx1⁺. (K) Vitelline artery, E8.5 Runxl ^lz/+ embryo showing β-gal⁺ cells confined to the endothelium. (L) Vitelline artery, E10.5 Runxl ^lz/+ embryo (40 ×). Da indicates dorsal aorta; v, vitelline artery; m, mesenchymal cell; e, endothelial cell; hc, hematopoietic cluster.