Etv2 rescues Flk1 mutant embryoid bodies

Abstract

Independent mouse knockouts of Etv2 and Flk1 are embryonic lethal and lack hematopoietic and endothelial lineages. We previously reported that Flk1 activates Etv2 in the initiation of hematopoiesis and vasculogenesis. However, Flk1 and its ligand VEGF are expressed throughout development, from E7.0 to adulthood, whereas Etv2 is expressed only transiently during embryogenesis. These observations suggest a complex regulatory interaction between Flk1 and Etv2. To further examine the Flk1 and Etv2 regulatory interaction, we transduced Etv2 and Flk1 mutant ES cells with viral integrants that inducibly overexpress Flk1 or Etv2. We demonstrated that forced expression of Etv2 rescued the hematopoietic and endothelial potential of differentiating Flk1 and Etv2 mutant cells. We further discovered that forced expression of Flk1 can rescue that of the Flk1, but not Etv2 mutant cells. Therefore, we conclude that the requirement for Flk1 can be bypassed by expressing Etv2, supporting the notion that disruption of Etv2 expression is responsible for the early phenotypes of the Etv2 and Flk1 mutant embryos.

Keywords: development; hematopoiesis; vasculogenesis.

Figure 1. Transduced cell lines overexpress Etv2 or Flk1

Etv2 mutant (A) or Flk1 mutant (B) ESCs stably transduced with Flk1-pSam2 express mCherry in response to 1μg/ml doxycycline as detected by flow cytometric analysis. Both cell lines transduced with Flk1-pSam2 express Flk1 protein only in the presence of doxycycline as shown by western blot analysis (C). Etv2 mutant (D) or Flk1 mutant (E) embryonic stem cells stably transduced with Etv2-pSam2 express mCherry in response to 1μg/ml doxycycline by flow cytometric analysis. Both cell lines transduced with Etv2-pSam2 express HA-tagged Etv2 protein only in the presence of doxycline as shown by western blot analysis (F). The control lanes contain cell extract from 293T cells transiently transfected with either a Flk1 expression construct (C) or an Etv2-HA expression construct (F).

Figure 2. Etv2, but not Flk1, rescues the hematopoietic and endothelial compartments of Etv2 mutant embryoid bodies

(A-C) Flow cytometric analysis of a representative differentiation experiment comparing Wildtype 2.1 EBs (A), Etv2 mutant:Etv2-pSam2 (B) and Etv2 mutant:Flk1-pSam2 (C) at EB D6. Wildtype and mutant cells were analyzed for endothelial (CD31, Flk1) (upper panel) and hematopoietic markers (CD41, CD45) (lower panel) in multiple doxycycline induction schemes: no doxycycline (A, B left panels, C left panels), 1μg/ml doxycycline from EB D2-4 (B middle panels, C middle panels), and 1μg/ml doxycycline from EB D4-6 (B right panels, C right panels). (D-F) Quantification of the FACS profiles is graphically displayed for CD31+/Flk1+ endothelial cells (D) CD41+/CD45- hematopoietic progenitor cells (E), and CD41+/CD45+ hematopoietic cells (F). Results from the wildtype control are shown in red and results from Etv2 mutant cells are shown in black. Error bars indicate SEM, (n equals at least 3, see Table S1).

Figure 3. Etv2 or Flk1 rescues the phenotype of Flk1 mutant embryoid bodies

(A-C) Flow cytometric analysis of a representative differentiation experiment comparing Flk1 wildtype (R1) EBs (A), Flk1 mutant:Flk1-pSam2 (B) and Flk1 mutant:Etv2-pSam2 (C) at EB D6. Wildtype and mutant cells were analyzed for endothelial (CD31, Flk1) (upper panels) and hematopoietic markers (CD41, CD45) (lower panels) in multiple doxycycline induction schemes: no doxycycline (A, B left panels, C left panels), 1μg/ml doxycycline from D2-4 (B middle panels, C middle panels), and 1μg/ml doxycycline from D4-6 (B right panels, C right panels). (D-F) Data from all experiments are graphically displayed for CD31+/Flk1- (D, left panel) cells and CD31+/Flk1+ endothelial cells (D, right panel), CD41+/CD45- hematopoietic progenitor cells (E), and CD41+/CD45+ hematopoietic cells (F). Results from the wildtype control are shown in red and results from mutant cells are shown in black. Error bars indicate SEM (n equals at least 3, see Table S2).

Figure 4. Etv2 expression rescues the expression of hematopoietic and endothelial transcripts in Etv2 and Flk1 mutant embryoid bodies

(A-C) Expression levels vWF (A), Cdh5 (B), and CD41 (C) in Wildtype 2.1 (grey) and transduced Etv2 mutant D6 EBs (black). (D-F) Expression levels of vWF (D), Cdh5 (E), and CD41 (F) in Wildtype R1 (grey) and transduced Flk1 mutant D6 EBs (black). All results were normalized to the respective wildtype cells. The doxycycline induction scheme, transduced virus, and cell lines are indicated under each graph. (n= 3), Error bars represent SEM.

Figure 5. Etv2 rescues hematopoietic progenitors in the Etv2 mutant and Flk1 mutant EBs

Cells were dissociated from Wildtype 2.1 (red bar) and Etv2 mutant D6 EBs (A-C) transduced with Etv2-pSam2 (white bars) or Flk1-pSam2 (grey bars) or Wildtype R1 and Flk1 mutant EBs (D-F) transduced with Etv2-pSam2 (white bars) or Flk1-pSam2 (grey bars). 20,000 cells were plated in methocult M3434 in triplicate and cultured for 7 days. Colonies were counted and identified as either GM (granulocyte or macrophage containing) (A,D), BFU (erythrocyte burst forming units) (B,E), or GEMM (granulocyte, erythrocyte and macrophage and megakaryocyte mixed) colonies (C,F). n=3, error bars represent SEM.