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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

Comparison of the Notch response of sorted MEP cells expressing different levels of CD9.Progeny analyses of equal numbers of sorted CD9Med and CD9High MEP subsets were performed by colony assays before (Day 0) and after a two days culture either on IgG or rDLL1 with or without DAPT as described in Fig 2. The numbers of different types of colonies generated by either CD9Med or CD9High MEP are presented on left (A, B, C, D, E) and right (F, G, H, I, J) histograms respectively (means and standard deviations from 3 independent MEP preparations). Results of ANOVA analyses performed on each dataset are indicated above each corresponding histogram. A, B: Piled histograms showing the cumulated numbers of the different types of colonies generated by CD9Med (A) or CD9High MEP (F). Results of ANOVA analysis performed on the total numbers of colonies are indicated above the histogram. B, G: Histograms showing the numbers of erythroid colonies generated by CD9Med (B) or CD9High MEP (G). C, H: Histograms showing the numbers of mixed colonies generated by CD9Med (C) or CD9High MEP (H). D, I: Histograms showing the numbers of megakaryocytic colonies generated by CD9Med (D) or CD9High MEP (I). E, J: Histograms showing the numbers of myeloid colonies generated by CD9Med (E) or CD9High MEP (J). Statistically significant differences between conditions validated by either ANOVA analyses followed by Tukey’s test or by Student t-test are indicated by full and dotted braces respectively associated with corresponding p values.
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pone.0153860.g003: Comparison of the Notch response of sorted MEP cells expressing different levels of CD9.Progeny analyses of equal numbers of sorted CD9Med and CD9High MEP subsets were performed by colony assays before (Day 0) and after a two days culture either on IgG or rDLL1 with or without DAPT as described in Fig 2. The numbers of different types of colonies generated by either CD9Med or CD9High MEP are presented on left (A, B, C, D, E) and right (F, G, H, I, J) histograms respectively (means and standard deviations from 3 independent MEP preparations). Results of ANOVA analyses performed on each dataset are indicated above each corresponding histogram. A, B: Piled histograms showing the cumulated numbers of the different types of colonies generated by CD9Med (A) or CD9High MEP (F). Results of ANOVA analysis performed on the total numbers of colonies are indicated above the histogram. B, G: Histograms showing the numbers of erythroid colonies generated by CD9Med (B) or CD9High MEP (G). C, H: Histograms showing the numbers of mixed colonies generated by CD9Med (C) or CD9High MEP (H). D, I: Histograms showing the numbers of megakaryocytic colonies generated by CD9Med (D) or CD9High MEP (I). E, J: Histograms showing the numbers of myeloid colonies generated by CD9Med (E) or CD9High MEP (J). Statistically significant differences between conditions validated by either ANOVA analyses followed by Tukey’s test or by Student t-test are indicated by full and dotted braces respectively associated with corresponding p values.

Mentions: The next step of our study was to reinvestigate the controversial effect of Notch pathway on megakaryocytic differentiation using purified megakaryocytic progenitors that are present in the MEP population. Based on several recent studies [40, 41], we reasoned that committed megakaryocytic progenitors present in the MEP population should be purified based on their high level of CD9 expression. As shown in S4 Fig, MEP cells displayed a roughly bimodal distribution of CD9 expression level allowing the sorting of three different subsets according to their CD9 expression level (CD9Low, CD9Med and CD9High). Colony assays showed that the proportion of megakaryocytic colonies positively correlated with the expression level of CD9 reaching more than 90% of megakaryocytic colonies generated by CD9High subset in association with a reduced clonogenicity (S4 Fig). Since the CD9Low subset appeared to be slightly contaminated by a few proportion of GMPs (S4 Fig), we focused our next analyses on the comparison of the Notch response between CD9Med and CD9High subsets that contained most of the erythroid-megakaryocytic potential with minimal GMP contamination. For that purpose, we followed the same protocol described in Fig 2 and the results obtained with three independent preparations of CD9Med or CD9High cells are presented in Fig 3A–3E and 3F–3J respectively. As expected, CD9Med MEPs (representing about 50% of total MEP cells), roughly reproduced results obtained with the unfractionated MEP population notably the significant increase of bipotent colonies (Fig 3C) together with a slight but not significant decrease of megakaryocytic colonies after the 2 days culture on rDLL1 instead of IgG (Fig 3D). In contrast, the very low numbers of erythroid and bipotent colonies generated by CD9High MEPs at day 0 or after the 2 days culture on IgG were markedly enhanced (up to 10 and 40 fold respectively) after the 2 days culture on rDLL1 (Fig 3G and 3H). These results thus indicated that Notch activation unexpectedly induced the generation of mixed colonies by CD9High MEPs that otherwise spontaneously generate only megakaryocytic colonies.


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)

Comparison of the Notch response of sorted MEP cells expressing different levels of CD9.Progeny analyses of equal numbers of sorted CD9Med and CD9High MEP subsets were performed by colony assays before (Day 0) and after a two days culture either on IgG or rDLL1 with or without DAPT as described in Fig 2. The numbers of different types of colonies generated by either CD9Med or CD9High MEP are presented on left (A, B, C, D, E) and right (F, G, H, I, J) histograms respectively (means and standard deviations from 3 independent MEP preparations). Results of ANOVA analyses performed on each dataset are indicated above each corresponding histogram. A, B: Piled histograms showing the cumulated numbers of the different types of colonies generated by CD9Med (A) or CD9High MEP (F). Results of ANOVA analysis performed on the total numbers of colonies are indicated above the histogram. B, G: Histograms showing the numbers of erythroid colonies generated by CD9Med (B) or CD9High MEP (G). C, H: Histograms showing the numbers of mixed colonies generated by CD9Med (C) or CD9High MEP (H). D, I: Histograms showing the numbers of megakaryocytic colonies generated by CD9Med (D) or CD9High MEP (I). E, J: Histograms showing the numbers of myeloid colonies generated by CD9Med (E) or CD9High MEP (J). Statistically significant differences between conditions validated by either ANOVA analyses followed by Tukey’s test or by Student t-test are indicated by full and dotted braces respectively associated with corresponding p values.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153860.g003: Comparison of the Notch response of sorted MEP cells expressing different levels of CD9.Progeny analyses of equal numbers of sorted CD9Med and CD9High MEP subsets were performed by colony assays before (Day 0) and after a two days culture either on IgG or rDLL1 with or without DAPT as described in Fig 2. The numbers of different types of colonies generated by either CD9Med or CD9High MEP are presented on left (A, B, C, D, E) and right (F, G, H, I, J) histograms respectively (means and standard deviations from 3 independent MEP preparations). Results of ANOVA analyses performed on each dataset are indicated above each corresponding histogram. A, B: Piled histograms showing the cumulated numbers of the different types of colonies generated by CD9Med (A) or CD9High MEP (F). Results of ANOVA analysis performed on the total numbers of colonies are indicated above the histogram. B, G: Histograms showing the numbers of erythroid colonies generated by CD9Med (B) or CD9High MEP (G). C, H: Histograms showing the numbers of mixed colonies generated by CD9Med (C) or CD9High MEP (H). D, I: Histograms showing the numbers of megakaryocytic colonies generated by CD9Med (D) or CD9High MEP (I). E, J: Histograms showing the numbers of myeloid colonies generated by CD9Med (E) or CD9High MEP (J). Statistically significant differences between conditions validated by either ANOVA analyses followed by Tukey’s test or by Student t-test are indicated by full and dotted braces respectively associated with corresponding p values.
Mentions: The next step of our study was to reinvestigate the controversial effect of Notch pathway on megakaryocytic differentiation using purified megakaryocytic progenitors that are present in the MEP population. Based on several recent studies [40, 41], we reasoned that committed megakaryocytic progenitors present in the MEP population should be purified based on their high level of CD9 expression. As shown in S4 Fig, MEP cells displayed a roughly bimodal distribution of CD9 expression level allowing the sorting of three different subsets according to their CD9 expression level (CD9Low, CD9Med and CD9High). Colony assays showed that the proportion of megakaryocytic colonies positively correlated with the expression level of CD9 reaching more than 90% of megakaryocytic colonies generated by CD9High subset in association with a reduced clonogenicity (S4 Fig). Since the CD9Low subset appeared to be slightly contaminated by a few proportion of GMPs (S4 Fig), we focused our next analyses on the comparison of the Notch response between CD9Med and CD9High subsets that contained most of the erythroid-megakaryocytic potential with minimal GMP contamination. For that purpose, we followed the same protocol described in Fig 2 and the results obtained with three independent preparations of CD9Med or CD9High cells are presented in Fig 3A–3E and 3F–3J respectively. As expected, CD9Med MEPs (representing about 50% of total MEP cells), roughly reproduced results obtained with the unfractionated MEP population notably the significant increase of bipotent colonies (Fig 3C) together with a slight but not significant decrease of megakaryocytic colonies after the 2 days culture on rDLL1 instead of IgG (Fig 3D). In contrast, the very low numbers of erythroid and bipotent colonies generated by CD9High MEPs at day 0 or after the 2 days culture on IgG were markedly enhanced (up to 10 and 40 fold respectively) after the 2 days culture on rDLL1 (Fig 3G and 3H). These results thus indicated that Notch activation unexpectedly induced the generation of mixed colonies by CD9High MEPs that otherwise spontaneously generate only megakaryocytic colonies.

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