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Runx1 is required for progression of CD41+ embryonic precursors into HSCs but not prior to this.

Liakhovitskaia A, Rybtsov S, Smith T, Batsivari A, Rybtsova N, Rode C, de Bruijn M, Buchholz F, Gordon-Keylock S, Zhao S, Medvinsky A - Development (2014)

Bottom Line: Although largely dispensable at later stages, the Runx1 transcription factor is crucially important during developmental maturation of HSCs; however, exact points of crucial involvement of Runx1 in this multi-step developmental maturation process remain unclear.We report that Runx1 deficiency does not preclude formation of VE-cad+CD45-CD41+ cells, which are phenotypically equivalent to precursors of definitive HSCs (pre-HSC Type I) but blocks transition to the subsequent CD45+ stage (pre-HSC Type II).These data emphasise that developmental progression of HSCs during a very short period of time is regulated by precise stage-specific molecular mechanisms.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh EH16 4UU, UK.

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HSCs are rescued in [CD41-Cre::Runx1LacZ/Δ] embryos. (A) Experimental design: left, transplantation of E14.5 foetal livers; right, transplantation of fresh and cultured E11.5 AGM regions. (B-D) Long-term donor-derived haematopoietic repopulation with (B) E14.5 foetal livers from control Runx1wt/Δ and rescued Runx1Re/Δ embryos; (C) uncultured E11.5 AGM region cells; and (D) cultured E11.5 AGM region cells. The donor cell contribution (%) into the peripheral blood of recipient mice is shown (for details of culture, transplantation and analysis, see Materials and Methods). Each symbol represents one recipient mouse. Data obtained from three independent experiments. (E) Representative examples of long-term multilineage donor-derived haematopoietic repopulation ([CD41-Cre::Runx1LacZ/Δ] E14.5 foetal liver, 14 weeks post-transplantation). Gating was carried out on 7AAD-Ly5.2+ cells.
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DEV110841F3: HSCs are rescued in [CD41-Cre::Runx1LacZ/Δ] embryos. (A) Experimental design: left, transplantation of E14.5 foetal livers; right, transplantation of fresh and cultured E11.5 AGM regions. (B-D) Long-term donor-derived haematopoietic repopulation with (B) E14.5 foetal livers from control Runx1wt/Δ and rescued Runx1Re/Δ embryos; (C) uncultured E11.5 AGM region cells; and (D) cultured E11.5 AGM region cells. The donor cell contribution (%) into the peripheral blood of recipient mice is shown (for details of culture, transplantation and analysis, see Materials and Methods). Each symbol represents one recipient mouse. Data obtained from three independent experiments. (E) Representative examples of long-term multilineage donor-derived haematopoietic repopulation ([CD41-Cre::Runx1LacZ/Δ] E14.5 foetal liver, 14 weeks post-transplantation). Gating was carried out on 7AAD-Ly5.2+ cells.

Mentions: To test whether development of HSCs was rescued, foetal liver cells from E14.5 Runx1Re/Δ embryos were transplanted into irradiated recipients. This led to successful long-term multi-lineage donor-derived engraftment, with only one exception (Fig. 3B). All donor-derived lymphoid and myeloid lineages were represented similar to control Runx1 heterozygous transplants (Fig. 3E). Transplantations into secondary recipients also gave multi-lineage donor-derived haematopoietic engraftment (data not shown). However, when we tested whether HSCs are rescued in the AGM region, we found that, in contrast to Runx1 heterozygous AGM regions, transplantation of E11.5 Runx1Re/Δ AGM regions did not produce haematopoietic repopulation (Fig. 3C). One out of five yolk sacs and one of six placentas were able to repopulate irradiated recipients (not shown). To test the possibility of delayed HSC development in rescued embryos, AGM region explants were cultured for 4 days in conditions supporting HSC development followed by transplantation into irradiated recipients (Fig. 3D). All four recipients transplanted showed high levels of donor-derived multi-lineage haematopoietic engraftment, thus demonstrating the presence of rescued pre-HSCs in the AGM region of Runx1Re/Δ embryos (Fig. 3D). None of the five Runx1LacZ/Δ AGM explants, which did not harbour the Cre transgene, were able to repopulate recipient mice.Fig. 3.


Runx1 is required for progression of CD41+ embryonic precursors into HSCs but not prior to this.

Liakhovitskaia A, Rybtsov S, Smith T, Batsivari A, Rybtsova N, Rode C, de Bruijn M, Buchholz F, Gordon-Keylock S, Zhao S, Medvinsky A - Development (2014)

HSCs are rescued in [CD41-Cre::Runx1LacZ/Δ] embryos. (A) Experimental design: left, transplantation of E14.5 foetal livers; right, transplantation of fresh and cultured E11.5 AGM regions. (B-D) Long-term donor-derived haematopoietic repopulation with (B) E14.5 foetal livers from control Runx1wt/Δ and rescued Runx1Re/Δ embryos; (C) uncultured E11.5 AGM region cells; and (D) cultured E11.5 AGM region cells. The donor cell contribution (%) into the peripheral blood of recipient mice is shown (for details of culture, transplantation and analysis, see Materials and Methods). Each symbol represents one recipient mouse. Data obtained from three independent experiments. (E) Representative examples of long-term multilineage donor-derived haematopoietic repopulation ([CD41-Cre::Runx1LacZ/Δ] E14.5 foetal liver, 14 weeks post-transplantation). Gating was carried out on 7AAD-Ly5.2+ cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199125&req=5

DEV110841F3: HSCs are rescued in [CD41-Cre::Runx1LacZ/Δ] embryos. (A) Experimental design: left, transplantation of E14.5 foetal livers; right, transplantation of fresh and cultured E11.5 AGM regions. (B-D) Long-term donor-derived haematopoietic repopulation with (B) E14.5 foetal livers from control Runx1wt/Δ and rescued Runx1Re/Δ embryos; (C) uncultured E11.5 AGM region cells; and (D) cultured E11.5 AGM region cells. The donor cell contribution (%) into the peripheral blood of recipient mice is shown (for details of culture, transplantation and analysis, see Materials and Methods). Each symbol represents one recipient mouse. Data obtained from three independent experiments. (E) Representative examples of long-term multilineage donor-derived haematopoietic repopulation ([CD41-Cre::Runx1LacZ/Δ] E14.5 foetal liver, 14 weeks post-transplantation). Gating was carried out on 7AAD-Ly5.2+ cells.
Mentions: To test whether development of HSCs was rescued, foetal liver cells from E14.5 Runx1Re/Δ embryos were transplanted into irradiated recipients. This led to successful long-term multi-lineage donor-derived engraftment, with only one exception (Fig. 3B). All donor-derived lymphoid and myeloid lineages were represented similar to control Runx1 heterozygous transplants (Fig. 3E). Transplantations into secondary recipients also gave multi-lineage donor-derived haematopoietic engraftment (data not shown). However, when we tested whether HSCs are rescued in the AGM region, we found that, in contrast to Runx1 heterozygous AGM regions, transplantation of E11.5 Runx1Re/Δ AGM regions did not produce haematopoietic repopulation (Fig. 3C). One out of five yolk sacs and one of six placentas were able to repopulate irradiated recipients (not shown). To test the possibility of delayed HSC development in rescued embryos, AGM region explants were cultured for 4 days in conditions supporting HSC development followed by transplantation into irradiated recipients (Fig. 3D). All four recipients transplanted showed high levels of donor-derived multi-lineage haematopoietic engraftment, thus demonstrating the presence of rescued pre-HSCs in the AGM region of Runx1Re/Δ embryos (Fig. 3D). None of the five Runx1LacZ/Δ AGM explants, which did not harbour the Cre transgene, were able to repopulate recipient mice.Fig. 3.

Bottom Line: Although largely dispensable at later stages, the Runx1 transcription factor is crucially important during developmental maturation of HSCs; however, exact points of crucial involvement of Runx1 in this multi-step developmental maturation process remain unclear.We report that Runx1 deficiency does not preclude formation of VE-cad+CD45-CD41+ cells, which are phenotypically equivalent to precursors of definitive HSCs (pre-HSC Type I) but blocks transition to the subsequent CD45+ stage (pre-HSC Type II).These data emphasise that developmental progression of HSCs during a very short period of time is regulated by precise stage-specific molecular mechanisms.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh EH16 4UU, UK.

Show MeSH
Related in: MedlinePlus