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Notch Stimulates Both Self-Renewal and Lineage Plasticity in a Subset of Murine CD9High Committed Megakaryocytic Progenitors.

Weiss-Gayet M, Starck J, Chaabouni A, Chazaud B, Morlé F - PLoS ONE (2016)

Bottom Line: We showed that Notch activation stimulated the SCF-dependent and preferential amplification of Kit+ erythroid and bipotent progenitors while favoring commitment towards the erythroid at the expense of megakaryocytic lineage.Altogether, these results indicate that Notch activation is able to extend the number of divisions of MK-committed CD9High MEPs before terminal maturation while allowing a fraction of them to generate alternative lineages.This unexpected plasticity of MK-committed progenitors revealed upon Notch activation helps to better understand the functional promiscuity between megakaryocytic lineage and hematopoietic stem cells.

View Article: PubMed Central - PubMed

Affiliation: Institut NeuroMyoGène (INMG), Université Claude Bernard Lyon1, Villeurbanne, France.

ABSTRACT
This study aimed at reinvestigating the controversial contribution of Notch signaling to megakaryocytic lineage development. For that purpose, we combined colony assays and single cells progeny analyses of purified megakaryocyte-erythroid progenitors (MEP) after short-term cultures on recombinant Notch ligand rDLL1. We showed that Notch activation stimulated the SCF-dependent and preferential amplification of Kit+ erythroid and bipotent progenitors while favoring commitment towards the erythroid at the expense of megakaryocytic lineage. Interestingly, we also identified a CD9High MEP subset that spontaneously generated almost exclusively megakaryocytic progeny mainly composed of single megakaryocytes. We showed that Notch activation decreased the extent of polyploidization and maturation of megakaryocytes, increased the size of megakaryocytic colonies and surprisingly restored the generation of erythroid and mixed colonies by this CD9High MEP subset. Importantly, the size increase of megakaryocytic colonies occurred at the expense of the production of single megakaryocytes and the restoration of colonies of alternative lineages occurred at the expense of the whole megakaryocytic progeny. Altogether, these results indicate that Notch activation is able to extend the number of divisions of MK-committed CD9High MEPs before terminal maturation while allowing a fraction of them to generate alternative lineages. This unexpected plasticity of MK-committed progenitors revealed upon Notch activation helps to better understand the functional promiscuity between megakaryocytic lineage and hematopoietic stem cells.

No MeSH data available.


Related in: MedlinePlus

Single cell progeny analyses of CD9Med and CD9High MEPs with or without Notch activation.Single CD9Med or CD9High MEP were individually seeded in 96 wells culture plates that have been coated with either IgGs or rDLL1 and containing medium supplemented with a complete cocktail of myeloid cytokines. The different types of developed colonies were numbered at day 7 by visual inspection under bright light microscope as illustrated in S8 Fig. A, B: Repartition of the indicated type of colony as a percentage of all single seeded CD9Med MEP (A) and as a percentage of CD9Med MEP raising colonies (B) (Means and standard deviations from 3 independent experiments). C, D: Repartition of the indicated type of colony as a percentage of all single seeded CD9High MEP (C) and as a percentage of CD9High MEP raising colonies (D) (Means and standard deviations from 3 independent experiments). Tables on the right display p-values in Tukey’s test post ANOVA and in Student t-test analyses of the variations in the proportions of different types of colonies between IgG and rDLL1 conditions. Statistically significant variations are indicated by grey boxes in Tables and by asterisks on right histograms.
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pone.0153860.g007: Single cell progeny analyses of CD9Med and CD9High MEPs with or without Notch activation.Single CD9Med or CD9High MEP were individually seeded in 96 wells culture plates that have been coated with either IgGs or rDLL1 and containing medium supplemented with a complete cocktail of myeloid cytokines. The different types of developed colonies were numbered at day 7 by visual inspection under bright light microscope as illustrated in S8 Fig. A, B: Repartition of the indicated type of colony as a percentage of all single seeded CD9Med MEP (A) and as a percentage of CD9Med MEP raising colonies (B) (Means and standard deviations from 3 independent experiments). C, D: Repartition of the indicated type of colony as a percentage of all single seeded CD9High MEP (C) and as a percentage of CD9High MEP raising colonies (D) (Means and standard deviations from 3 independent experiments). Tables on the right display p-values in Tukey’s test post ANOVA and in Student t-test analyses of the variations in the proportions of different types of colonies between IgG and rDLL1 conditions. Statistically significant variations are indicated by grey boxes in Tables and by asterisks on right histograms.

Mentions: Based on the above results (Figs 2 and 3), the question remained of whether, in addition to its proliferative effect on committed erythroid progenitors, Notch activation could also favor the preferential commitment of bipotent progenitors towards the erythroid lineage. To directly address this question, single CD9Med MEP cells were individually seeded in wells coated with either IgGs or rDLL1 and their progeny was characterized after a 9 days culture by morphological examination as illustrated in S8 Fig. As expected, single CD9Med MEPs generated colonies containing either only large megakaryocytic cells, only small erythroid cells, or both erythroid and megakaryocytic cells (Fig 7A). A few number of mixed colonies also contained granulo-monocytic cells identified by their intermediate size and expression of CD11b (Fig 7A). While the proportion of single CD9Med MEPs generating colonies did not significantly change when cultured on either IgGs or rDLL1 (Fig 7A), the proportion of CD9Med MEPs that led to erythroid colonies increased at the expense of those giving rise to megakaryocytic colonies (only colonies containing over 4 cells were scored) in the presence of rDLL1 as compared with IgGs (Fig 7B). These results thus indicated that Notch activation indeed favored the commitment of CD9Med MEP towards erythroid lineage at the expense of megakaryocytic lineage.


Notch Stimulates Both Self-Renewal and Lineage Plasticity in a Subset of Murine CD9High Committed Megakaryocytic Progenitors.

Weiss-Gayet M, Starck J, Chaabouni A, Chazaud B, Morlé F - PLoS ONE (2016)

Single cell progeny analyses of CD9Med and CD9High MEPs with or without Notch activation.Single CD9Med or CD9High MEP were individually seeded in 96 wells culture plates that have been coated with either IgGs or rDLL1 and containing medium supplemented with a complete cocktail of myeloid cytokines. The different types of developed colonies were numbered at day 7 by visual inspection under bright light microscope as illustrated in S8 Fig. A, B: Repartition of the indicated type of colony as a percentage of all single seeded CD9Med MEP (A) and as a percentage of CD9Med MEP raising colonies (B) (Means and standard deviations from 3 independent experiments). C, D: Repartition of the indicated type of colony as a percentage of all single seeded CD9High MEP (C) and as a percentage of CD9High MEP raising colonies (D) (Means and standard deviations from 3 independent experiments). Tables on the right display p-values in Tukey’s test post ANOVA and in Student t-test analyses of the variations in the proportions of different types of colonies between IgG and rDLL1 conditions. Statistically significant variations are indicated by grey boxes in Tables and by asterisks on right histograms.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4835090&req=5

pone.0153860.g007: Single cell progeny analyses of CD9Med and CD9High MEPs with or without Notch activation.Single CD9Med or CD9High MEP were individually seeded in 96 wells culture plates that have been coated with either IgGs or rDLL1 and containing medium supplemented with a complete cocktail of myeloid cytokines. The different types of developed colonies were numbered at day 7 by visual inspection under bright light microscope as illustrated in S8 Fig. A, B: Repartition of the indicated type of colony as a percentage of all single seeded CD9Med MEP (A) and as a percentage of CD9Med MEP raising colonies (B) (Means and standard deviations from 3 independent experiments). C, D: Repartition of the indicated type of colony as a percentage of all single seeded CD9High MEP (C) and as a percentage of CD9High MEP raising colonies (D) (Means and standard deviations from 3 independent experiments). Tables on the right display p-values in Tukey’s test post ANOVA and in Student t-test analyses of the variations in the proportions of different types of colonies between IgG and rDLL1 conditions. Statistically significant variations are indicated by grey boxes in Tables and by asterisks on right histograms.
Mentions: Based on the above results (Figs 2 and 3), the question remained of whether, in addition to its proliferative effect on committed erythroid progenitors, Notch activation could also favor the preferential commitment of bipotent progenitors towards the erythroid lineage. To directly address this question, single CD9Med MEP cells were individually seeded in wells coated with either IgGs or rDLL1 and their progeny was characterized after a 9 days culture by morphological examination as illustrated in S8 Fig. As expected, single CD9Med MEPs generated colonies containing either only large megakaryocytic cells, only small erythroid cells, or both erythroid and megakaryocytic cells (Fig 7A). A few number of mixed colonies also contained granulo-monocytic cells identified by their intermediate size and expression of CD11b (Fig 7A). While the proportion of single CD9Med MEPs generating colonies did not significantly change when cultured on either IgGs or rDLL1 (Fig 7A), the proportion of CD9Med MEPs that led to erythroid colonies increased at the expense of those giving rise to megakaryocytic colonies (only colonies containing over 4 cells were scored) in the presence of rDLL1 as compared with IgGs (Fig 7B). These results thus indicated that Notch activation indeed favored the commitment of CD9Med MEP towards erythroid lineage at the expense of megakaryocytic lineage.

Bottom Line: We showed that Notch activation stimulated the SCF-dependent and preferential amplification of Kit+ erythroid and bipotent progenitors while favoring commitment towards the erythroid at the expense of megakaryocytic lineage.Altogether, these results indicate that Notch activation is able to extend the number of divisions of MK-committed CD9High MEPs before terminal maturation while allowing a fraction of them to generate alternative lineages.This unexpected plasticity of MK-committed progenitors revealed upon Notch activation helps to better understand the functional promiscuity between megakaryocytic lineage and hematopoietic stem cells.

View Article: PubMed Central - PubMed

Affiliation: Institut NeuroMyoGène (INMG), Université Claude Bernard Lyon1, Villeurbanne, France.

ABSTRACT
This study aimed at reinvestigating the controversial contribution of Notch signaling to megakaryocytic lineage development. For that purpose, we combined colony assays and single cells progeny analyses of purified megakaryocyte-erythroid progenitors (MEP) after short-term cultures on recombinant Notch ligand rDLL1. We showed that Notch activation stimulated the SCF-dependent and preferential amplification of Kit+ erythroid and bipotent progenitors while favoring commitment towards the erythroid at the expense of megakaryocytic lineage. Interestingly, we also identified a CD9High MEP subset that spontaneously generated almost exclusively megakaryocytic progeny mainly composed of single megakaryocytes. We showed that Notch activation decreased the extent of polyploidization and maturation of megakaryocytes, increased the size of megakaryocytic colonies and surprisingly restored the generation of erythroid and mixed colonies by this CD9High MEP subset. Importantly, the size increase of megakaryocytic colonies occurred at the expense of the production of single megakaryocytes and the restoration of colonies of alternative lineages occurred at the expense of the whole megakaryocytic progeny. Altogether, these results indicate that Notch activation is able to extend the number of divisions of MK-committed CD9High MEPs before terminal maturation while allowing a fraction of them to generate alternative lineages. This unexpected plasticity of MK-committed progenitors revealed upon Notch activation helps to better understand the functional promiscuity between megakaryocytic lineage and hematopoietic stem cells.

No MeSH data available.


Related in: MedlinePlus