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First published online 18 July 2007
doi: 10.1242/dev.001818

Development 134, 3031-3040 (2007)
Published by The Company of Biologists 2007
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Dual role of Mpl receptor during the establishment of definitive hematopoiesis

Laurence Petit-Cocault1,2,3, Cécile Volle-Challier1,3,4, Maud Fleury2,3, Bruno Péault1,3,5 and Michèle Souyri1,2,3,*

1 Institut National de la Santé et de la Recherche Médicale U506, Villejuif, F-94807, France.
2 U602, Hôpital Paul Brousse, Villejuif, F-94807, France.
3 Université Paris-Sud, Villejuif, F-94807, France.
4 Sanofi-Synthelabo Recherche, Département Cardiovasculaire Thrombose, Toulouse, F-31036, France.
5 Children's Hospital of Pittsburgh, Pittsburgh, PA, USA.


Figure 1
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  		Fig. 1. Mpl expression in hematopoietic organs of control C57Bl6 embryos. Mpl expression was assessed by RT-PCR analysis in the mouse fetal liver (A), AGM region (B), spleen and thymus (C) from individual embryos. PCR of Actb (Actin) and Mpl were performed in parallel, in order to normalize Mpl signal after 35 cycles of amplification.

 

Figure 2
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  		Fig. 2. Detection of Mpl expression in the AGM and FL by in situ hybridization. Transverse sections of E10.5 (A,B,D,H), E11.5 (C,E,I,J), E12.5 (F) and E16.5 (G) mouse embryos were hybridized with Mpl antisense riboprobes. (A,B,C) Mpl expression in the hematopoietic cells emerging from the ventral wall of the aorta in the AGM region. (D,E,F,G) Mpl expression in the liver at various times of development. (H,I,J) Mpl expression in the yolk sac. The boxed areas are shown at high magnification to the right of each panel.

 

Figure 3
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  		Fig. 3. Mpl and TPO levels assessed by q-PCR. (A) Mpl expression was assessed in mouse early yolk sacs (E8.0, E9.0, E9.5) and embryos (E8.0, E9.0, PSP E9.5, AGM E10.5 and E11.5) by relative q-PCR. (B) TPO expression was also evaluated by relative q-PCR in E10.5 and E11.5 AGMs and fetal livers. Each column represents an average of Mpl or TPO level normalized to the Gapdh internal control±s.d.

 

Figure 4
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  		Fig. 4. Experimental design of in vitro clonogenic and in vivo long-term reconstitution assays. Single-cell suspensions were prepared from the AGM region (E11.5 and E12.5) and liver (E11.5, E12.5 and E14.5) of control C57Bl6 and Mpl-/- embryos (expressing the Ly5.2 allele of CD45), and analyzed in vitro in clonogenic assays, or in vivo for long-term reconstitution (LTR) of lethally irradiated C57Bl6 mice expressing the Ly5.1 allele of CD45. At E12.5, single-cell suspensions of yolk sac or peripheral blood were also tested. The engraftment and repopulating activity of the injected cells were assessed at different times after injection (5, 10 and 20 weeks) by FACS analysis on the basis of the detection of Ly5.2 in the peripheral blood (minimal level of reconstitution considered for positive reconstitution was 5%). Twenty weeks after injection, LTR ability (LTRA) was evaluated (FACS analysis) in positive mice by the percentage of Ly5.2+ cells present in the bone marrow (BM). The HSC content was thereafter determined by secondary transplantation of bone marrow cell suspensions from primary recipients into lethally irradiated B6-Ly5.1 mice.

 

Figure 5
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  		Fig. 5. Comparison of the CFC content of Mpl-/- and C57Bl6 mouse embryos at E11.5, E12.5 and E14.5. Total CFCs and CFU-GM, BFU-E, CFU-GEMM and CFU with MK content of (A) E11.5 AGM and (B) E11.5, E12.5 and E14.5 FL are presented. Half an AGM suspension and 5x104 FL cells were used in each Petri dish. The results of a single experiment representative of two other independent experiments are shown. For each experiment, three independent Petri dishes were prepared and scored, and the results shown represent the mean±s.e. *, P<0.05.

 

Figure 6
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  		Fig. 6. Flow cytometric analysis of bone marrow, spleen, thymus and blood from mice reconstituted with Mpl-/- E11.5 AGM. Twenty weeks after injection, primary reconstituted mice were sacrificed and cell suspensions prepared from femoral marrow, spleen and thymus. A peripheral blood sample was also taken and treated as described in Materials and methods. Bone marrow and spleen cells were co-incubated with Ly5.2 antibody coupled to phycoerythrin (PE) and CD34, Sca1, Thy1, Ter119, Gr1 or B220 antibodies directly or indirectly coupled to FITC. Thymus cells were co-incubated with Ly5.2-PE and CD4-FITC or CD8-FITC. Blood cells were co-incubated with Ly5.2 and B220. Percentages of double-positive cells are indicated in the upper right quadrant.

 

Figure 7
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  		Fig. 7. Different impacts of the absence of Mpl signaling during establishment of definitive hematopoiesis in the mouse embryo. The anatomical locations involved in the generation of the pool of HSCs are shown, together with the timing of their activity on a timescale of embryonic days. AGM and placenta are the sites of production of the first HSCs (Emergence). These cells (red) migrate to the FL, where they mature further and expand (Maturation/expansion). The FL is also colonized by HSCs coming from the YS (yellow) and placenta (purple). Ultimately, HSCs establish steady-state levels in the bone marrow, where balance between self-renewal, quiescence and differentiation is tightly regulated. The absence of Mpl leads to a delayed and defective production of HSCs by the AGM region, and to a defect in amplification and self-renewal/survival of HSCs in the FL, which in turn results in the HSC defect described for the adult bone marrow.

 

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