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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

Culture on rDLL1 decreases the number of single megakaryocytes and increases the size of megakaryocytic colonies generated by CD9High MEPs.CD9High MEPs were cultured for two days on either IgGs or rDLL1 before analysis of their progeny by colony assays as described in Fig 3 except that single megakaryocytes and megakaryocytic colonies displaying different numbers of megakaryocytes were numbered separately. A: Piled histograms showing the numbers (left panel) and percentages (right panel) of single megakaryocytes (MK1), megakaryocytic colonies containing at least 2 megakaryocytes (MK ≥2) as well as few erythroid (Ery), myeloid (Myelo) and mixed erythro-megakaryocytic (Ery/Mk) colonies generated after the 2 days culture on either IgGs or rDLL1 (mean and standard deviations from 3 independent MEP preparations). Table on the right displays 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 table and by asterisks on the right histogram. B: Histograms showing the percentages of different sizes of pure megakaryocytic colonies (from single to 8 and more megakaryocytes) on IgGs or rDLL1 (means and standard deviations from the same 3 independent MEP preparations as in A). Table on the right displays 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 Table and by full braces and asterisks on histogram.
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pone.0153860.g004: Culture on rDLL1 decreases the number of single megakaryocytes and increases the size of megakaryocytic colonies generated by CD9High MEPs.CD9High MEPs were cultured for two days on either IgGs or rDLL1 before analysis of their progeny by colony assays as described in Fig 3 except that single megakaryocytes and megakaryocytic colonies displaying different numbers of megakaryocytes were numbered separately. A: Piled histograms showing the numbers (left panel) and percentages (right panel) of single megakaryocytes (MK1), megakaryocytic colonies containing at least 2 megakaryocytes (MK ≥2) as well as few erythroid (Ery), myeloid (Myelo) and mixed erythro-megakaryocytic (Ery/Mk) colonies generated after the 2 days culture on either IgGs or rDLL1 (mean and standard deviations from 3 independent MEP preparations). Table on the right displays 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 table and by asterisks on the right histogram. B: Histograms showing the percentages of different sizes of pure megakaryocytic colonies (from single to 8 and more megakaryocytes) on IgGs or rDLL1 (means and standard deviations from the same 3 independent MEP preparations as in A). Table on the right displays 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 Table and by full braces and asterisks on histogram.

Mentions: Before investigating the origin of the increase of erythroid and bipotent colonies by CD9High MEPs, we decided to characterize the effect of rDLL1 on the size and maturation of pure megakaryocytic colonies. We noticed that up to 90% of the viable progeny generated by CD9High MEPs after a 2 days culture on IgGs were actually exclusively composed of megakaryocytic colonies and up to 60% of single megakaryocytes (Fig 4A). While the total number of this viable progeny did not significantly change on rDLL1 culture (Fig 4A, left panel), the proportion of single megakaryocytes significantly decreased for the benefit of a significantly increased proportion of megakaryocytic and bipotent colonies (Fig 4A, right panel). Moreover, by counting the relative proportions of megakaryocytic colonies with various numbers of megakaryocytes, we found a slight (-10%) but significant decrease in single megakaryocytes (Fig 4B, MK1) and a significant increase in colonies displaying 3, 4 or more than 8 megakaryocytes generated by CD9High MEPs cultured on rDLL1 versus IgGs (Fig 4B). Importantly, this increase in the size of megakaryocytic colonies as well as the increase in bipotent colonies induced by rDLL1 were abrogated in the presence of either DAPT or Notch2 neutralizing antibody thus indicating that these effects are specifically induced by Notch2 receptor activation (S5 Fig). These results thus indicated that most (>90%) of CD9High MEPs were indeed strongly biased towards megakaryocytic differentiation and that their culture on rDLL1 induced them to undertake additional divisions before terminal differentiation.


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)

Culture on rDLL1 decreases the number of single megakaryocytes and increases the size of megakaryocytic colonies generated by CD9High MEPs.CD9High MEPs were cultured for two days on either IgGs or rDLL1 before analysis of their progeny by colony assays as described in Fig 3 except that single megakaryocytes and megakaryocytic colonies displaying different numbers of megakaryocytes were numbered separately. A: Piled histograms showing the numbers (left panel) and percentages (right panel) of single megakaryocytes (MK1), megakaryocytic colonies containing at least 2 megakaryocytes (MK ≥2) as well as few erythroid (Ery), myeloid (Myelo) and mixed erythro-megakaryocytic (Ery/Mk) colonies generated after the 2 days culture on either IgGs or rDLL1 (mean and standard deviations from 3 independent MEP preparations). Table on the right displays 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 table and by asterisks on the right histogram. B: Histograms showing the percentages of different sizes of pure megakaryocytic colonies (from single to 8 and more megakaryocytes) on IgGs or rDLL1 (means and standard deviations from the same 3 independent MEP preparations as in A). Table on the right displays 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 Table and by full braces and asterisks on histogram.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153860.g004: Culture on rDLL1 decreases the number of single megakaryocytes and increases the size of megakaryocytic colonies generated by CD9High MEPs.CD9High MEPs were cultured for two days on either IgGs or rDLL1 before analysis of their progeny by colony assays as described in Fig 3 except that single megakaryocytes and megakaryocytic colonies displaying different numbers of megakaryocytes were numbered separately. A: Piled histograms showing the numbers (left panel) and percentages (right panel) of single megakaryocytes (MK1), megakaryocytic colonies containing at least 2 megakaryocytes (MK ≥2) as well as few erythroid (Ery), myeloid (Myelo) and mixed erythro-megakaryocytic (Ery/Mk) colonies generated after the 2 days culture on either IgGs or rDLL1 (mean and standard deviations from 3 independent MEP preparations). Table on the right displays 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 table and by asterisks on the right histogram. B: Histograms showing the percentages of different sizes of pure megakaryocytic colonies (from single to 8 and more megakaryocytes) on IgGs or rDLL1 (means and standard deviations from the same 3 independent MEP preparations as in A). Table on the right displays 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 Table and by full braces and asterisks on histogram.
Mentions: Before investigating the origin of the increase of erythroid and bipotent colonies by CD9High MEPs, we decided to characterize the effect of rDLL1 on the size and maturation of pure megakaryocytic colonies. We noticed that up to 90% of the viable progeny generated by CD9High MEPs after a 2 days culture on IgGs were actually exclusively composed of megakaryocytic colonies and up to 60% of single megakaryocytes (Fig 4A). While the total number of this viable progeny did not significantly change on rDLL1 culture (Fig 4A, left panel), the proportion of single megakaryocytes significantly decreased for the benefit of a significantly increased proportion of megakaryocytic and bipotent colonies (Fig 4A, right panel). Moreover, by counting the relative proportions of megakaryocytic colonies with various numbers of megakaryocytes, we found a slight (-10%) but significant decrease in single megakaryocytes (Fig 4B, MK1) and a significant increase in colonies displaying 3, 4 or more than 8 megakaryocytes generated by CD9High MEPs cultured on rDLL1 versus IgGs (Fig 4B). Importantly, this increase in the size of megakaryocytic colonies as well as the increase in bipotent colonies induced by rDLL1 were abrogated in the presence of either DAPT or Notch2 neutralizing antibody thus indicating that these effects are specifically induced by Notch2 receptor activation (S5 Fig). These results thus indicated that most (>90%) of CD9High MEPs were indeed strongly biased towards megakaryocytic differentiation and that their culture on rDLL1 induced them to undertake additional divisions before terminal differentiation.

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