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Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse yolk sac: a comparative study between in situ electroporation and retroviral transduction.

Giroux SJ, Alves-Leiva C, Lécluse Y, Martin P, Albagli O, Godin I - BMC Dev. Biol. (2007)

Bottom Line: Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated.We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U790, Institut Gustave Roussy-PR1, Villejuif, France. Sebastien.Giroux@u-bourgogne.fr <Sebastien.Giroux@u-bourgogne.fr>

ABSTRACT

Background: Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.

Results: One transduction protocol involves transient modification of gene expression through in situ electroporation of the prospective blood islands, which allows the evolution of transfected mesodermal cells in their "normal" environment, upon organ culture. Following in situ electroporation of a GFP reporter construct into the YS cavity of embryos at post-streak (mesodermal/pre-hematopoietic precursors) or early somite (hematopoietic precursors) stages, high GFP expression levels as well as a good preservation of cell viability is observed in YS explants. Moreover, the erythro-myeloid progeny typical of the YS arises from GFP+ mesodermal cells or hematopoietic precursors, even if the number of targeted precursors is low. The second approach, based on retroviral transduction allows a very efficient transduction of large precursor numbers, but may only be used to target 8 dpc YS hematopoietic precursors. Again, transduced cells generate a progeny quantitatively and qualitatively similar to that of control YS.

Conclusion: We thus provide two protocols whose combination may allow a thorough study of both early and late events of hematopoietic development in the murine YS. In situ electroporation constitutes the only possible gene transfer method to transduce mesodermal/pre-hematopoietic precursors and analyze the earliest steps of hematopoietic development. Both in situ electroporation and retroviral transduction may be used to target early hematopoietic precursors, but the latter appears more convenient if a large pool of stably transduced cells is required. We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints.

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Determination of the development stages enriched in immature precursors. Upper panel: Temporal evolution of lmo2 expression from the OB to LHF stages: initially expressed by most extra-embryonic mesodermal cells (A), lmo2 expression rapidly restricts to a smaller cell number at the EB stage (B). Lmo2 expression subsequently expands with the development of blood islands endothelial and hematopoietic cells from LB (C) to LHF (D), and subsequent stages. Middle panel: Transcripts of the embryonic globin β-H1 are present in a minute cell number at the OB/EB stages (E, F). During the following stages (LB: G to LHF: H, and subsequent stages), β-H1 expressing erythroid cells rapidly expand. The arrows in the upper and middle panels point to equivalent zone of the blood islands. Lower panel: The comparative evolution of lmo2 and β-H1 expressions points to embryos at the OB/EB stages as enriched in immature precursors. Scale bar: 100 μm.
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Figure 1: Determination of the development stages enriched in immature precursors. Upper panel: Temporal evolution of lmo2 expression from the OB to LHF stages: initially expressed by most extra-embryonic mesodermal cells (A), lmo2 expression rapidly restricts to a smaller cell number at the EB stage (B). Lmo2 expression subsequently expands with the development of blood islands endothelial and hematopoietic cells from LB (C) to LHF (D), and subsequent stages. Middle panel: Transcripts of the embryonic globin β-H1 are present in a minute cell number at the OB/EB stages (E, F). During the following stages (LB: G to LHF: H, and subsequent stages), β-H1 expressing erythroid cells rapidly expand. The arrows in the upper and middle panels point to equivalent zone of the blood islands. Lower panel: The comparative evolution of lmo2 and β-H1 expressions points to embryos at the OB/EB stages as enriched in immature precursors. Scale bar: 100 μm.

Mentions: Lmo2 transcripts are first detected at the OB stage in the whole extra-embryonic mesoderm (Fig. 1A) and are subsequently restricted, at the EB stage (Fig. 1B), to a subset of mesodermal cells, which might correspond to blood islands precursors. Upon proliferation of these precursors, lmo2 expression expends to encompass all hematopoietic derivatives of the blood islands, from the LB to LHF stages (Fig. 1C, D) [17].


Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse yolk sac: a comparative study between in situ electroporation and retroviral transduction.

Giroux SJ, Alves-Leiva C, Lécluse Y, Martin P, Albagli O, Godin I - BMC Dev. Biol. (2007)

Determination of the development stages enriched in immature precursors. Upper panel: Temporal evolution of lmo2 expression from the OB to LHF stages: initially expressed by most extra-embryonic mesodermal cells (A), lmo2 expression rapidly restricts to a smaller cell number at the EB stage (B). Lmo2 expression subsequently expands with the development of blood islands endothelial and hematopoietic cells from LB (C) to LHF (D), and subsequent stages. Middle panel: Transcripts of the embryonic globin β-H1 are present in a minute cell number at the OB/EB stages (E, F). During the following stages (LB: G to LHF: H, and subsequent stages), β-H1 expressing erythroid cells rapidly expand. The arrows in the upper and middle panels point to equivalent zone of the blood islands. Lower panel: The comparative evolution of lmo2 and β-H1 expressions points to embryos at the OB/EB stages as enriched in immature precursors. Scale bar: 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1941737&req=5

Figure 1: Determination of the development stages enriched in immature precursors. Upper panel: Temporal evolution of lmo2 expression from the OB to LHF stages: initially expressed by most extra-embryonic mesodermal cells (A), lmo2 expression rapidly restricts to a smaller cell number at the EB stage (B). Lmo2 expression subsequently expands with the development of blood islands endothelial and hematopoietic cells from LB (C) to LHF (D), and subsequent stages. Middle panel: Transcripts of the embryonic globin β-H1 are present in a minute cell number at the OB/EB stages (E, F). During the following stages (LB: G to LHF: H, and subsequent stages), β-H1 expressing erythroid cells rapidly expand. The arrows in the upper and middle panels point to equivalent zone of the blood islands. Lower panel: The comparative evolution of lmo2 and β-H1 expressions points to embryos at the OB/EB stages as enriched in immature precursors. Scale bar: 100 μm.
Mentions: Lmo2 transcripts are first detected at the OB stage in the whole extra-embryonic mesoderm (Fig. 1A) and are subsequently restricted, at the EB stage (Fig. 1B), to a subset of mesodermal cells, which might correspond to blood islands precursors. Upon proliferation of these precursors, lmo2 expression expends to encompass all hematopoietic derivatives of the blood islands, from the LB to LHF stages (Fig. 1C, D) [17].

Bottom Line: Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated.We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U790, Institut Gustave Roussy-PR1, Villejuif, France. Sebastien.Giroux@u-bourgogne.fr <Sebastien.Giroux@u-bourgogne.fr>

ABSTRACT

Background: Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.

Results: One transduction protocol involves transient modification of gene expression through in situ electroporation of the prospective blood islands, which allows the evolution of transfected mesodermal cells in their "normal" environment, upon organ culture. Following in situ electroporation of a GFP reporter construct into the YS cavity of embryos at post-streak (mesodermal/pre-hematopoietic precursors) or early somite (hematopoietic precursors) stages, high GFP expression levels as well as a good preservation of cell viability is observed in YS explants. Moreover, the erythro-myeloid progeny typical of the YS arises from GFP+ mesodermal cells or hematopoietic precursors, even if the number of targeted precursors is low. The second approach, based on retroviral transduction allows a very efficient transduction of large precursor numbers, but may only be used to target 8 dpc YS hematopoietic precursors. Again, transduced cells generate a progeny quantitatively and qualitatively similar to that of control YS.

Conclusion: We thus provide two protocols whose combination may allow a thorough study of both early and late events of hematopoietic development in the murine YS. In situ electroporation constitutes the only possible gene transfer method to transduce mesodermal/pre-hematopoietic precursors and analyze the earliest steps of hematopoietic development. Both in situ electroporation and retroviral transduction may be used to target early hematopoietic precursors, but the latter appears more convenient if a large pool of stably transduced cells is required. We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints.

Show MeSH