Integrin αIIb (CD41) plays a role in the maintenance of hematopoietic stem cell activity in the mouse embryonic aorta

Abstract

Integrins are transmembrane receptors that play important roles as modulators of cell behaviour through their adhesion properties and the initiation of signaling cascades. The αIIb integrin subunit (CD41) is one of the first cell surface markers indicative of hematopoietic commitment. αIIb pairs exclusively with β3 to form the αIIbβ3 integrin. β3 (CD61) also pairs with αv (CD51) to form the αvβ3 integrin. The expression and putative role of these integrins during mouse hematopoietic development is as yet unknown. We show here that hematopoietic stem cells (HSCs) differentially express αIIbβ3 and αvβ3 integrins throughout development. Whereas the first HSCs generated in the aorta at mid-gestation express both integrins, HSCs from the placenta only express αvβ3, and most fetal liver HSCs do not express either integrin. By using αIIb deficient embryos, we show that αIIb is not only a reliable HSC marker but it also plays an important and specific function in maintaining the HSC activity in the mouse embryonic aorta.

Keywords: Aorta; CD41; Fetal liver; Hematopoietic stem cells; Integrin; Mouse development; Placenta.

Fig. 1.. Phenotypic and functional analyses of integrin-based sorted cell fractions.

(A,C,E,G) Flow cytometric analyses of E11 Aorta–Gonad–Mesonephros (AGM), E12 yolk sac (YS), E12 placenta (PL) and E14 fetal liver (FL). Representative sorting gates of each population are indicated. (B,D,F,H) Hematopoietic repopulation analyses after injection of integrin-based sorted fractions of AGM (n = 2), YS (n = 4), PL (n = 2) and FL (n = 3). Numbers above columns indicate number of mice repopulated/number of mice injected. Dose of injected cells is indicated as embryo equivalent (ee).

Fig. 2.. Location of phenotypically defined HSCs in AGM and placenta.

(A) Intra-aortic hematopoietic clusters (IAHCs) of E10.5 wild-type embryos. Non-fixed embryo slices were stained with antibodies against CD51 (αv), CD41 (αIIb) and CD61 (β3). Scale bars: 10 µm. (B) Non-fixed placenta slices (E12) were stained with antibodies against Tie-2. Transmitted light and fluorescent images are merged. Scale bar: 100 µm. (C) Confocal stack image of an E12 non-fixed placenta slice stained with antibodies against CD51, CD61 and Tie-2. Close up of the boxed area (left panel) shows a group of labelled cells (single and merged fluorescent channels are shown). Scale bars: 10 µm. UA: umbilical artery, C: chorionic plate, L: labyrinth, S: spongiotrophoblast layer, FV: fetal vessel.

Fig. 3.. Intra-aortic hematopoietic clusters and in vitro clonogenic progenitor activity of AGM cells isolated from E11 CD41 (α_IIb) deficient embryos.

(A) In vitro clonogenic analyses. AGMs were isolated from E11 wild-type (α_IIb^+/+), heterozygous (α_IIb^+/tk) or homozygous deficient embryos (α_IIb^tk/tk). Error bars: standard deviations for n = 3 independent experiments. CFU-GEMM: CFU-Granulocyte–Erythroid–Macrophage–Megakaryocyte; CFU-GM: CFU-Granulocyte–Macrophage; CFU-M: CFU-Macrophage; CFU-G: CFU-Granulocyte; BFU-E: Burst-Forming Unit-Erythroid. (B) Number of c-kit⁺ cells per E11 α_IIb^+/+ (n = 5 embryos), α_IIb^+/tk (n = 5 embryos) and α_IIb^tk/tk (n = 3 embryos) aorta. Error bars: standard deviations. Confocal stack images of the mouse aorta region of E11 α_IIb^+/+ (C) and α_IIb^tk/tk (D) embryos after whole mount staining for c-kit and CD31. Scale bars: 50 µm. ns: not statistically significant. Error bars: standard deviations.

Fig. 4.. Functional analyses of AGM, yolk sac and fetal liver cells isolated from E11 CD41 (α_IIb) deficient embryos.

Hematopoietic repopulation analysis of mice after injection of (A,B) AGM, (C) yolk sac or (D) fetal liver cells isolated from E11 wild-type (α_IIb^+/+), heterozygous (α_IIb^+/tk) or homozygous deficient embryos (α_IIb^tk/tk). (A,B) Percentage of chimerism in peripheral blood for each injected mice. Lozenge: one transplanted mouse. Red lozenge: mice used to perform secondary transplantations. Dashed line: limit of positivity (>10% of chimerism). Red line: chimerism average. (A) AGM cells were injected directly (n = 3) or (B) after explant culture (n = 3). The genotype of embryos, the number of mice repopulated/number of mice transplanted (# Rep./# Inj.) and the percentage of repopulated mice (% Rep.) are indicated below the graphs. 1 embryo equivalent (ee) of AGM cells was injected per recipient (n = 3). p-values are indicated. (C,D) Percentage of repopulated mice. Numbers below columns: number of mice repopulated/number of mice transplanted. (C) 1 ee of yolk sac cells (n = 3) and (D) 3 to 4.5 ee of fetal liver cells (n = 4) were injected per recipient. Due to the very low number of HSCs in E11 FL, α_IIb^+/tk and α_IIb^tk/tk cells were pooled. ns: not statistically significant.

Fig. 5.. Functional analyses of E14 fetal liver cells isolated from CD41 (α_IIb) deficient embryos.

Hematopoietic repopulation analysis of mice after the injection of fetal liver cells isolated from E14 wild-type (α_IIb^+/+; grey bars), heterozygous (α_IIb^+/tk; dark grey bars), or homozygous deficient (α_IIb^tk/tk; black bars embryos. Numbers above columns: number of mice repopulated/number of mice transplanted.