Retinoic acid enhances skeletal muscle progenitor formation and bypasses inhibition by bone morphogenetic protein 4 but not dominant negative beta-catenin

Abstract

Background: Understanding stem cell differentiation is essential for the future design of cell therapies. While retinoic acid (RA) is the most potent small molecule enhancer of skeletal myogenesis in stem cells, the stage and mechanism of its function has not yet been elucidated. Further, the intersection of RA with other signalling pathways that stimulate or inhibit myogenesis (such as Wnt and BMP4, respectively) is unknown. Thus, the purpose of this study is to examine the molecular mechanisms by which RA enhances skeletal myogenesis and interacts with Wnt and BMP4 signalling during P19 or mouse embryonic stem (ES) cell differentiation.

Results: Treatment of P19 or mouse ES cells with low levels of RA led to an enhancement of skeletal myogenesis by upregulating the expression of the mesodermal marker, Wnt3a, the skeletal muscle progenitor factors Pax3 and Meox1, and the myogenic regulatory factors (MRFs) MyoD and myogenin. By chromatin immunoprecipitation, RA receptors (RARs) bound directly to regulatory regions in the Wnt3a, Pax3, and Meox1 genes and RA activated a beta-catenin-responsive promoter in aggregated P19 cells. In the presence of a dominant negative beta-catenin/engrailed repressor fusion protein, RA could not bypass the inhibition of skeletal myogenesis nor upregulate Meox1 or MyoD. Thus, RA functions both upstream and downstream of Wnt signalling. In contrast, it functions downstream of BMP4, as it abrogates BMP4 inhibition of myogenesis and Meox1, Pax3, and MyoD expression. Furthermore, RA downregulated BMP4 expression and upregulated the BMP4 inhibitor, Tob1. Finally, RA inhibited cardiomyogenesis but not in the presence of BMP4.

Conclusion: RA can enhance skeletal myogenesis in stem cells at the muscle specification/progenitor stage by activating RARs bound directly to mesoderm and skeletal muscle progenitor genes, activating beta-catenin function and inhibiting bone morphogenetic protein (BMP) signalling. Thus, a signalling pathway can function at multiple levels to positively regulate a developmental program and can function by abrogating inhibitory pathways. Finally, since RA enhances skeletal muscle progenitor formation, it will be a valuable tool for designing future stem cell therapies.

Figures

Figure 1

Retinoic acid inhibits cardiomyogenesis and enhances skeletal myogenesis in P19 cells. P19 cells were aggregated with 0.8% dimethylsulfoxide (DMSO) in the presence and absence of 30 nM RA. Panel I: Cells were fixed on day 9 for immunofluorescence with MF20 antibody (A-D) and counter stained with Hoechst dye (E-H). Magnification is 160x. Panel II: Cardiac (n = 3) and skeletal (n = 4) myogenesis were quantified by counting the number of MHC+ve myocytes as a percentage of the total. Average +/- standard error of mean (SEM) is shown and statistics were Student's t-test, *P < 0.05. Panel III: Total RNA was harvested for northern blot analysis on the days indicated and hybridized to the cDNAs on the right. Lanes are spliced from the same autoradiogram. Panel IV: Quantitative polymerase chain reaction (PCR) analysis was performed on day 4 of differentiation for Pax3 and Wnt3a transcript levels (n = 2) and on day 9 for MyoD levels (n = 4). Results were expressed as fold change of transcript levels in the presence compared to the absence of RA treatment. Panel V: Chromatin immunoprecipitation experiments were performed on day 2 P19 aggregates treated with DMSO/retinoic acid and analysed by real-time PCR using primers for sites within regulatory regions of the genes indicated. Average +/-SEM is shown, relative to IgG, and statistics were Student's t-test of each region compared to IgG, n = 3-4, *P < 0.05. Panel VI: The position and conservation of the Meox1-2 and the Pax3-2 retinoic acid response elements are shown.

Figure 2

Mouse embryonic stem (mES) cells differentiate into skeletal muscle in response to retinoic acid (RA). mES cells were aggregated in hanging drops for 2 days, cultured in suspension for a further 5 days with increasing concentrations of RA, and transferred to tissue culture dishes. Panel I: RNA was harvested from day 7 cultures and subjected to reverse transcriptase- polymerase chain reaction (PCR) followed by Southern blot analysis with the probes indicated on the left. Panel II: RNA was harvested from days 7 and 15 after differentiation with or without 25nM retinoic acid and examined by quantitative PCR analysis. The expression levels are expressed as fold increase in the presence, compared to the absence of RA, as the mean and standard error, n = 3. Statistics were Student's t-test, *P < 0.05. Panel III: On day 20 of differentiation, cells were fixed and reacted with Hoechst dye to detect nuclei (A and C) and with MF20 antibody to detect muscle (B and D). Magnification is 400×.

Figure 3

Retinoic acid (RA) cannot override the inhibition of skeletal myogenesis by β-Cat/EnR. P19[control] (A, B, E, F) and P19[β-Cat/EnR] (C, D, G, H) cells were aggregated in the presence of 0.8% dimethylsulfoxide (DMSO) with (E-H) and without (A-D) 10 nM RA. Cells were fixed on day 9 of differentiation for immunofluorescence with MF20 antibody (B, D, F, H) and counter stained with Hoechst dye (A, C, E, G). Magnification is 160×.

Figure 4

Retinoic acid (RA) enhances the expression of Pax3/7 but not MyoD or Meox1 in the presence of β-Cat/EnR. Panel I: P19[control] and P19[β-Cat/EnR] cultures were differentiated with 0.8% dimethylsulfoxide in the presence of 0, 3, and 10 nM RA. Total RNA was harvested and hybridized with the probes as indicated. Panel II: quantitative polymerase chain reaction analysis of Pax3 and Pax7 transcript levels, for each condition in Panel I, shown as one representative experiment performed in triplicate. Panel III: RA activates β-catenin in P19 aggregate but not monolayer cultures. P19 cells were transfected with the TOPFlash or FOPFlash reporter and treated with the compounds indicated in aggregated or monolayer cultures. Cells were harvested 24 hours later for luciferase assays (n = 2). Numbers represent the average +/- standard error of mean and statistics were Student's t-test, *P < 0.05.

Figure 5

PAX3/7 expression in P19 cultures treated with dimethylsulfoxide (DMSO) and 10 nM retinoic acid (RA) is indicative of skeletal myogenesis and not neurogenesis. Panel I: P19[control] cells, undifferentiated in monolayer cultures (control) or differentiated with 1% DMSO, 1% DMSO+ 10 nM RA, or 1% DMSO + 1 μM RA for 9 days, were immunolabelled with anti-TuJ1 (green) to detect terminally differentiated neurons and stained with the nuclear marker Hoechst (blue). Panel II: P19[β-Cat/EnR] cells were treated as in Panel I. Panel III: Quantitative analysis of the percentage of Tuj1-positive cells was established, expressed as the percentage of the total cell number (n = 6-10). Panel IV: Immunofluorescent staining of PAX3/7 protein (green), committed neuronal precursors (doublecortin-positive, red), and terminally differentiated neurons (TuJ1-positive, blue) in triple-labelled P19[control] cultures treated with DMSO + 10 nM RA, demonstrating that PAX3/7-positive cells (arrow) are not neuronal precursors or neurons. Panel V: Quantitative analysis indicated that the overwhelming majority of PAX3/7-positive cells in all treatments were non-neuronal. Statistics were analysis of variance, post-hoc Bonferroni, *P < 0.05, Scale bars, 50 μm.

Figure 6

BMP4 inhibits skeletal but not cardiac myogenesis. Panel I: P19[BMP4] and P19[control] cells were aggregated in the presence of 0.8% dimethylsulfoxide (DMSO). Cells were fixed on day 9, stained with MF20 antibody (A-D), and counter-stained with Hoechst dye to show the nuclei (E-H). Magnification is 400x. Panel II: The number of MHC+ve cells were counted and the average +/- standard error of mean shown. Statistics were Student's t-test, *P < 0.05, n = 3.

Figure 7

BMP4 inhibits skeletal muscle specification. Panels I and II: P19[BMP4] and P19[control] cells were aggregated in the presence of 0.8% dimethylsulfoxide (DMSO). P19[control] cells were also aggregated in the absence of DMSO to serve as negative controls. On days 0, 6 and 9 (Panel I) and days 1-5 (Panel II), total RNA was harvested for northern blot analysis and hybridized with the cDNAs indicated on the right. Panel III: P19 cells were transfected with the TOPFlash reporter, aggregated, and treated with the compounds indicated. Cells were harvested 24 hours later for luciferase assays (n = 2). Numbers represent the average +/- standard error of mean and statistics were Student's t-test, *P < 0.05.

Figure 8

Retinoic acid (RA) and BMP4 counteract each other's inhibition of skeletal myogenesis or cardiomyogenesis. P19 cells were mixed with P19[BMP4] or P19[control] cells in the presence of 1% dimethylsulfoxide (DMSO), with or without RA. Panel I: P19[BMP4] cultures treated with RA were fixed on day 9 for immunofluorescence with MF20 antibody (A, B) and counter stained with Hoechst dye (C, D). Magnification is 160x. Panel II: Skeletal myogenesis was quantified for each condition by counting the number of myosin heavy chain+ve bipolar skeletal myocytes, expressed as the percentage of total cells (white bars) and their standard errors (n = 3). MyoD transcript levels (black bars) were quantified by quantitative polymerase chain reaction and expressed relative to control cultures (n = 2), *P < 0.05. Panel III: Cardiomyogenesis was quantified as described for Panel II, n = 4. Panel IV: On days 6 and 9 total RNA was harvested for northern blot analysis and probed with the cDNAs indicated on the right. Panel V: A time course of P19[BMP4] and P19[control] cells aggregated in the presence of DMSO and RA. Total RNA was harvested for northern blot analysis on days 1-4, 6, and 9 and probed with the cDNAs indicated on the right.

Figure 9

Model of the intersection of retinoic acid (RA), Wnt, and BMP4 signalling during cardiac and skeletal muscle development. BMP4 upregulates Wnt/β-catenin during mesoderm induction (green arrow) and blocks skeletal myogenesis by downregulation of Meox1, Pax3 and myogenic regulatory factor expression (red inhibition arrow). This inhibition can be reversed by RA, which enhances Tob1, Wnt3a, Pax3 and Meox1 expression, activates β-catenin and inhibits BMP4 expression (green arrows). RA receptors bind directly to the Wnt3a, Meox1, and Pax3 regulatory regions (bold green arrows). RA inhibits GATA-4 expression and cardiomyogenesis, likely by inhibiting BMP4 expression and function. Grey arrows indicate previous work [references [6,8-10,35,38,40,44,102,103].