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Gradual Rarefaction of Hematopoietic Precursors and Atrophy in a Depleted microRNA 29a, b and c Environment.

Kauffman L, Balatti V, Cascione L, Fadda P, Racke F, Santhanam R, Costinean S - PLoS ONE (2015)

Bottom Line: The self-renewing ability of HSCs is fundamental for the maintenance of a pool of bone marrow precursors throughout the life of an individual.The genetic mechanisms underlying such a complex process are still poorly understood.Here, we show that constitutive in vivo deletion of miR29ab1 leads to reduced number of HSCs and that miR29ab1 deficient bone marrow cannot repopulate the bone marrow of irradiated mice.

View Article: PubMed Central - PubMed

Affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, United States of America.

ABSTRACT

Background: The self-renewing ability of HSCs is fundamental for the maintenance of a pool of bone marrow precursors throughout the life of an individual. The genetic mechanisms underlying such a complex process are still poorly understood.

Results and significance: Here, we show that constitutive in vivo deletion of miR29ab1 leads to reduced number of HSCs and that miR29ab1 deficient bone marrow cannot repopulate the bone marrow of irradiated mice. An Affymetrix analysis of the miR29ab1 knockout mice identifies key proteins that could be responsible for this phenotype, as DNMT3a and b. Moreover, our findings reveal that whereas miR29b2c knockout mice do not exhibit any spontaneous abnormality, the double knock out--miR29ab1b2c--has marked generalized atrophy, raising the possibility that the two bi-cistrons might cooperate in order to maintain the stem cell number in general, not only limited to the bone marrow.

No MeSH data available.


Related in: MedlinePlus

Northern Blot for expression of miR29a in the miR29ab1 hetero and homozygous mice (1 –spleen; 2 –liver).
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pone.0131981.g001: Northern Blot for expression of miR29a in the miR29ab1 hetero and homozygous mice (1 –spleen; 2 –liver).

Mentions: We generated two distinct miR29ab1 and miR29b2c loxP conditional ko mice without deletion. These were afterwards crossed with EIIA cre mice, which led to the deletion of the two 29 microRNAs clusters during the first couple of weeks of embryogenesis [6]. The heterozygous were then crossed again and pups were checked for complete absence of expression of the genes (S1A and S1B File). Northern Blots and RT-PCRs performed to check for the absence of miR expression in the homozygous for both miR29ab1 and b2c found total absence of miR29a and b in the 29ab1 knockout and lack of miR29c in the miR29b2c knockouts confirming that miR29b transcription comes mostly from the 29b1 gene (chromosome 7) and not the b2 (chromosome 1) [7, 8] (Fig 1 and S1C, S1D and S1E File). In order to generate the mir29ab1 and b2c double knockout we crossed the two individual knockouts. The pups were once again tested for gene and mature sequence lack of expression. MiR29ab1 knockout mice were normal at birth. Over a 2-year span, miR29ab1 homozygous mice were generated at a rate of 10% and had a lifespan decreased to approximately one year (n = 125) versus 2–2 1/2 years for their wild type littermates (n = 392) (Fig 2). These mice exhibited a decrease of spleen size and weight compared to their littermate wild types (S1F File). Histological examination of the spleens and bone marrows showed a decrease of the white pulp, especially granulocytic lineage (Fig 3). The histological data led us to think that miR29ab1 might play a role in the self-renewal and maintenance of HSCs. To test this hypothesis, we designed an in vitro colony forming assay experiment. We harvested the bone marrows of mice at 2, 6 and 10 months of age and plated the HSCs. We then counted the colonies (colony forming units—CFU) formed and noticed that CFUs for all types of precursors (erythroid, granulocytes, macrophages and megakaryocytes) were numerically less represented in the knockouts compared to the wild types (by a ratio of 0.2–0.5 knockouts vs wild types–Fig 4–p = 0.04, n = 4;Fig 5–p = 0.01, n = 8 and Fig 6–p = 0.006, n = 8). Then, we checked the influence age might have on the number of colonies and discovered that the knockout bone marrows were able to partially compensate for the impact of the miR29a and b deficit with time, i.e. the older mice had only a 1–2 fold less CFU-GM compared to the wild types whereas the younger mice had a 5–10 fold difference. Flow cytometry performed on bone marrow hematopoietic cells show a decrease for CD3, CD117, CD19, IgM, Lys6g, B220 but not for CD11b (S1G File, n-10; p = 0.0001). Hence, there seems to be a reduction of precursor and mature forms in the bone marrow for all lineages. We cannot tell whether this is due to miR29a or b as it has already been shown that the mature sequence of mIR29b is transcribed mainly from the 29b1 gene and not b2 [8]. Complete blood counts (CBC) performed on both the knockout and wild type mice showed a slight decrease the white blood cell count (WBC) by a factor of 0.47 and a decrease of total lymphocyte count by a factor of 0.4 (S1H File; n = 3; p = 0.02 for WBC and lymphocyte counts; neutrophil count difference was not statistically relevant). Cytogenetic analysis of several miR29ab1 knockout mice did not reveal any abnormality (S1I File). Affymetrix analysis of mRNA expression in the miR29ab1 knockouts compared to the wild types showed a significant increase in expression of both DNMT3a and 3b (1.06 and 1.04 log fold respectively) together with other WISP1 (a Wnt pathway related mRNA) and Pak6 (Cdc42 related kinase) (S2A File). To ascertain the role of miR29a and b in maintaining the HSCs bone marrow population, we performed a bone marrow transplant experiment where we transplanted the bone marrow of 5 knockouts mice into 2 irradiated mice. We clearly saw a lack of engraftment of the bone marrows of the miR29ab1 ko as compared to the wild types. MiR 29b2c homozygous mice were born at a normal rate of approximately 25%, and had a normal life span (n = 120). They did not exhibit any histological abnormality. Mir29 double knockouts were markedly atrophic with an almost absent white pulp in the spleen (n = 4). Spleens as well as the rest of their internal organs were markedly decreased in size. The homozygous double knockouts were born at a rate of 1% and had a lifespan of approximately one month (S1K File). Histology showed almost absence of the myeloid lineage (S1L File).


Gradual Rarefaction of Hematopoietic Precursors and Atrophy in a Depleted microRNA 29a, b and c Environment.

Kauffman L, Balatti V, Cascione L, Fadda P, Racke F, Santhanam R, Costinean S - PLoS ONE (2015)

Northern Blot for expression of miR29a in the miR29ab1 hetero and homozygous mice (1 –spleen; 2 –liver).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492741&req=5

pone.0131981.g001: Northern Blot for expression of miR29a in the miR29ab1 hetero and homozygous mice (1 –spleen; 2 –liver).
Mentions: We generated two distinct miR29ab1 and miR29b2c loxP conditional ko mice without deletion. These were afterwards crossed with EIIA cre mice, which led to the deletion of the two 29 microRNAs clusters during the first couple of weeks of embryogenesis [6]. The heterozygous were then crossed again and pups were checked for complete absence of expression of the genes (S1A and S1B File). Northern Blots and RT-PCRs performed to check for the absence of miR expression in the homozygous for both miR29ab1 and b2c found total absence of miR29a and b in the 29ab1 knockout and lack of miR29c in the miR29b2c knockouts confirming that miR29b transcription comes mostly from the 29b1 gene (chromosome 7) and not the b2 (chromosome 1) [7, 8] (Fig 1 and S1C, S1D and S1E File). In order to generate the mir29ab1 and b2c double knockout we crossed the two individual knockouts. The pups were once again tested for gene and mature sequence lack of expression. MiR29ab1 knockout mice were normal at birth. Over a 2-year span, miR29ab1 homozygous mice were generated at a rate of 10% and had a lifespan decreased to approximately one year (n = 125) versus 2–2 1/2 years for their wild type littermates (n = 392) (Fig 2). These mice exhibited a decrease of spleen size and weight compared to their littermate wild types (S1F File). Histological examination of the spleens and bone marrows showed a decrease of the white pulp, especially granulocytic lineage (Fig 3). The histological data led us to think that miR29ab1 might play a role in the self-renewal and maintenance of HSCs. To test this hypothesis, we designed an in vitro colony forming assay experiment. We harvested the bone marrows of mice at 2, 6 and 10 months of age and plated the HSCs. We then counted the colonies (colony forming units—CFU) formed and noticed that CFUs for all types of precursors (erythroid, granulocytes, macrophages and megakaryocytes) were numerically less represented in the knockouts compared to the wild types (by a ratio of 0.2–0.5 knockouts vs wild types–Fig 4–p = 0.04, n = 4;Fig 5–p = 0.01, n = 8 and Fig 6–p = 0.006, n = 8). Then, we checked the influence age might have on the number of colonies and discovered that the knockout bone marrows were able to partially compensate for the impact of the miR29a and b deficit with time, i.e. the older mice had only a 1–2 fold less CFU-GM compared to the wild types whereas the younger mice had a 5–10 fold difference. Flow cytometry performed on bone marrow hematopoietic cells show a decrease for CD3, CD117, CD19, IgM, Lys6g, B220 but not for CD11b (S1G File, n-10; p = 0.0001). Hence, there seems to be a reduction of precursor and mature forms in the bone marrow for all lineages. We cannot tell whether this is due to miR29a or b as it has already been shown that the mature sequence of mIR29b is transcribed mainly from the 29b1 gene and not b2 [8]. Complete blood counts (CBC) performed on both the knockout and wild type mice showed a slight decrease the white blood cell count (WBC) by a factor of 0.47 and a decrease of total lymphocyte count by a factor of 0.4 (S1H File; n = 3; p = 0.02 for WBC and lymphocyte counts; neutrophil count difference was not statistically relevant). Cytogenetic analysis of several miR29ab1 knockout mice did not reveal any abnormality (S1I File). Affymetrix analysis of mRNA expression in the miR29ab1 knockouts compared to the wild types showed a significant increase in expression of both DNMT3a and 3b (1.06 and 1.04 log fold respectively) together with other WISP1 (a Wnt pathway related mRNA) and Pak6 (Cdc42 related kinase) (S2A File). To ascertain the role of miR29a and b in maintaining the HSCs bone marrow population, we performed a bone marrow transplant experiment where we transplanted the bone marrow of 5 knockouts mice into 2 irradiated mice. We clearly saw a lack of engraftment of the bone marrows of the miR29ab1 ko as compared to the wild types. MiR 29b2c homozygous mice were born at a normal rate of approximately 25%, and had a normal life span (n = 120). They did not exhibit any histological abnormality. Mir29 double knockouts were markedly atrophic with an almost absent white pulp in the spleen (n = 4). Spleens as well as the rest of their internal organs were markedly decreased in size. The homozygous double knockouts were born at a rate of 1% and had a lifespan of approximately one month (S1K File). Histology showed almost absence of the myeloid lineage (S1L File).

Bottom Line: The self-renewing ability of HSCs is fundamental for the maintenance of a pool of bone marrow precursors throughout the life of an individual.The genetic mechanisms underlying such a complex process are still poorly understood.Here, we show that constitutive in vivo deletion of miR29ab1 leads to reduced number of HSCs and that miR29ab1 deficient bone marrow cannot repopulate the bone marrow of irradiated mice.

View Article: PubMed Central - PubMed

Affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, United States of America.

ABSTRACT

Background: The self-renewing ability of HSCs is fundamental for the maintenance of a pool of bone marrow precursors throughout the life of an individual. The genetic mechanisms underlying such a complex process are still poorly understood.

Results and significance: Here, we show that constitutive in vivo deletion of miR29ab1 leads to reduced number of HSCs and that miR29ab1 deficient bone marrow cannot repopulate the bone marrow of irradiated mice. An Affymetrix analysis of the miR29ab1 knockout mice identifies key proteins that could be responsible for this phenotype, as DNMT3a and b. Moreover, our findings reveal that whereas miR29b2c knockout mice do not exhibit any spontaneous abnormality, the double knock out--miR29ab1b2c--has marked generalized atrophy, raising the possibility that the two bi-cistrons might cooperate in order to maintain the stem cell number in general, not only limited to the bone marrow.

No MeSH data available.


Related in: MedlinePlus