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Induction of Pax3 gene expression impedes cardiac differentiation.

Li Q, Le May M, Lacroix N, Chen J - Sci Rep (2013)

Bottom Line: Interestingly, the inhibitory effect of small molecules on cardiac differentiation depends on the function of Pax3, but not the mesoderm factor Meox1.Thus Pax3 is an inhibitor of cardiac differentiation in lineage specification.Our studies reveal the dual roles of Pax3 in stem cell fate determinations and provide new molecular insights into small molecule-enhanced lineage specification.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada. qiaoli@uottawa.ca

ABSTRACT
Cell-based therapies using pluripotent stem cells hold great promise as regenerative approaches to treat many types of diseases. Nevertheless many challenges remain and, perhaps foremost, is the issue of how to direct and enhance the specification and differentiation of a desired cell type for potential therapeutics. We have examined the molecular basis for the inverse correlation of cardiac and skeletal myogenesis in small molecule-enhanced stem cell differentiation. Our study shows that activation of premyogenic factor Pax3 coincides with inhibiting gene expression of early cardiac factor GATA4. Interestingly, the inhibitory effect of small molecules on cardiac differentiation depends on the function of Pax3, but not the mesoderm factor Meox1. Thus Pax3 is an inhibitor of cardiac differentiation in lineage specification. Our studies reveal the dual roles of Pax3 in stem cell fate determinations and provide new molecular insights into small molecule-enhanced lineage specification.

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Role of Pax3 in cardiac differentiation.(A) Clones of P19 cells expressing either a dominant negative Meox1 (Meox1/EnR) or Pax3 (Pax3/EnR) were treated with bexarotene (BEX, 100 nM) or RA (10 nM) during EB formation. The cells were then maintained on coverslips without any treatments for an additional 5 days and stained with specific antibodies for MyoD, myosin heavy chain and Hoechst for the nuclei. Cells harbouring the empty vector were used as controls. Quantification is plotted as the fraction of cardiac myocytes (car, dark grey) and skeletal myocytes (sk, light grey) in relation to the total cell population (n = 5). (B)–(G) Real-time RT-PCR was used to determine the transcript levels of EnR, Meox1, Myf5, GATA4, Nkx2.5 and Tbx5 on day 4 differentiation in the same batch of cDNA from the cells harboring the dominant negative Pax3. Quantification is presented as fold changes in reference to the untreated EBs (n = 3).
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f3: Role of Pax3 in cardiac differentiation.(A) Clones of P19 cells expressing either a dominant negative Meox1 (Meox1/EnR) or Pax3 (Pax3/EnR) were treated with bexarotene (BEX, 100 nM) or RA (10 nM) during EB formation. The cells were then maintained on coverslips without any treatments for an additional 5 days and stained with specific antibodies for MyoD, myosin heavy chain and Hoechst for the nuclei. Cells harbouring the empty vector were used as controls. Quantification is plotted as the fraction of cardiac myocytes (car, dark grey) and skeletal myocytes (sk, light grey) in relation to the total cell population (n = 5). (B)–(G) Real-time RT-PCR was used to determine the transcript levels of EnR, Meox1, Myf5, GATA4, Nkx2.5 and Tbx5 on day 4 differentiation in the same batch of cDNA from the cells harboring the dominant negative Pax3. Quantification is presented as fold changes in reference to the untreated EBs (n = 3).

Mentions: Similar to the wild-type cells, the control cells harboring the empty vector were converted into cardiac and skeletal myocytes, about 7% and 6% respectively, following treatment with DMSO, but failed to produce cardiac myocytes after treatment with bexarotene or RA (Fig. 3A). On the other hand, the dominant negative Meox1 cells generated only about 2% and 1% of cardiac and skeletal myocytes after DMSO treatments, and cotreatment with bexarotene and RA enhanced the generation of skeletal myocytes only to about 5% and 6% respectively (Fig. 3A). This is consistent with previous studies, because Meox1 is important for skeletal myogenesis2326. Regardless, bexarotene and RA impeded cardiac differentiation in the dominant Meox1 cells (Fig. 3A), suggesting that the inhibitory effects of bexarotene and RA on cardiac differentiation are not dependent on the function of Meox1.


Induction of Pax3 gene expression impedes cardiac differentiation.

Li Q, Le May M, Lacroix N, Chen J - Sci Rep (2013)

Role of Pax3 in cardiac differentiation.(A) Clones of P19 cells expressing either a dominant negative Meox1 (Meox1/EnR) or Pax3 (Pax3/EnR) were treated with bexarotene (BEX, 100 nM) or RA (10 nM) during EB formation. The cells were then maintained on coverslips without any treatments for an additional 5 days and stained with specific antibodies for MyoD, myosin heavy chain and Hoechst for the nuclei. Cells harbouring the empty vector were used as controls. Quantification is plotted as the fraction of cardiac myocytes (car, dark grey) and skeletal myocytes (sk, light grey) in relation to the total cell population (n = 5). (B)–(G) Real-time RT-PCR was used to determine the transcript levels of EnR, Meox1, Myf5, GATA4, Nkx2.5 and Tbx5 on day 4 differentiation in the same batch of cDNA from the cells harboring the dominant negative Pax3. Quantification is presented as fold changes in reference to the untreated EBs (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750538&req=5

f3: Role of Pax3 in cardiac differentiation.(A) Clones of P19 cells expressing either a dominant negative Meox1 (Meox1/EnR) or Pax3 (Pax3/EnR) were treated with bexarotene (BEX, 100 nM) or RA (10 nM) during EB formation. The cells were then maintained on coverslips without any treatments for an additional 5 days and stained with specific antibodies for MyoD, myosin heavy chain and Hoechst for the nuclei. Cells harbouring the empty vector were used as controls. Quantification is plotted as the fraction of cardiac myocytes (car, dark grey) and skeletal myocytes (sk, light grey) in relation to the total cell population (n = 5). (B)–(G) Real-time RT-PCR was used to determine the transcript levels of EnR, Meox1, Myf5, GATA4, Nkx2.5 and Tbx5 on day 4 differentiation in the same batch of cDNA from the cells harboring the dominant negative Pax3. Quantification is presented as fold changes in reference to the untreated EBs (n = 3).
Mentions: Similar to the wild-type cells, the control cells harboring the empty vector were converted into cardiac and skeletal myocytes, about 7% and 6% respectively, following treatment with DMSO, but failed to produce cardiac myocytes after treatment with bexarotene or RA (Fig. 3A). On the other hand, the dominant negative Meox1 cells generated only about 2% and 1% of cardiac and skeletal myocytes after DMSO treatments, and cotreatment with bexarotene and RA enhanced the generation of skeletal myocytes only to about 5% and 6% respectively (Fig. 3A). This is consistent with previous studies, because Meox1 is important for skeletal myogenesis2326. Regardless, bexarotene and RA impeded cardiac differentiation in the dominant Meox1 cells (Fig. 3A), suggesting that the inhibitory effects of bexarotene and RA on cardiac differentiation are not dependent on the function of Meox1.

Bottom Line: Interestingly, the inhibitory effect of small molecules on cardiac differentiation depends on the function of Pax3, but not the mesoderm factor Meox1.Thus Pax3 is an inhibitor of cardiac differentiation in lineage specification.Our studies reveal the dual roles of Pax3 in stem cell fate determinations and provide new molecular insights into small molecule-enhanced lineage specification.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada. qiaoli@uottawa.ca

ABSTRACT
Cell-based therapies using pluripotent stem cells hold great promise as regenerative approaches to treat many types of diseases. Nevertheless many challenges remain and, perhaps foremost, is the issue of how to direct and enhance the specification and differentiation of a desired cell type for potential therapeutics. We have examined the molecular basis for the inverse correlation of cardiac and skeletal myogenesis in small molecule-enhanced stem cell differentiation. Our study shows that activation of premyogenic factor Pax3 coincides with inhibiting gene expression of early cardiac factor GATA4. Interestingly, the inhibitory effect of small molecules on cardiac differentiation depends on the function of Pax3, but not the mesoderm factor Meox1. Thus Pax3 is an inhibitor of cardiac differentiation in lineage specification. Our studies reveal the dual roles of Pax3 in stem cell fate determinations and provide new molecular insights into small molecule-enhanced lineage specification.

Show MeSH