MicroRNA regulation of cell lineages in mouse and human embryonic stem cells

Abstract

Cell fate decisions of pluripotent embryonic stem (ES) cells are dictated by activation and repression of lineage-specific genes. Numerous signaling and transcriptional networks progressively narrow and specify the potential of ES cells. Whether specific microRNAs help refine and limit gene expression and, thereby, could be used to manipulate ES cell differentiation has largely been unexplored. Here, we show that two serum response factor (SRF)-dependent muscle-specific microRNAs, miR-1 and miR-133, promote mesoderm formation from ES cells but have opposing functions during further differentiation into cardiac muscle progenitors. Furthermore, miR-1 and miR-133 were potent repressors of nonmuscle gene expression and cell fate during mouse and human ES cell differentiation. miR-1's effects were in part mediated by translational repression of the Notch ligand Delta-like 1 (Dll-1). Our findings indicate that muscle-specific miRNAs reinforce the silencing of nonmuscle genes during cell lineage commitment and suggest that miRNAs may have general utility in regulating cell-fate decisions from pluripotent ES cells.

Figure 1. Identification of miRNAs expressed in ES cell–derived cardiomyocytes

(A) mES cells carrying a GFP transgene under control of the cardiomyocyte-specific β-myosin heavy chain promoter were differentiated for 13 days as embryoid bodies (EBs), sorted by GFP expression, and analyzed by miRNA microarray. miRNAs enriched at least threefold in the GFP⁺ compared to GFP⁻ cell populations are listed along with their fold enrichment and whether they were detected in ES cells. (B, C) Quantitative RT-PCR (qRT-PCR) showing enrichment of miR-1 and miR-133 inflow-sorted Nkx2.5-GFP⁺ cardiac progenitors from day 4 EBs (B) but not in Flk-1⁺ vascular progenitors, which are enriched for the endothelial-specific miRNA, miR-126 (C). (D) qRT-PCR showing expression kinetics of miR-1 and miR-133 during days 4–10 of EB differentiation.

Figure 2. Effects of miR-1 and miR-133 on mesoderm differentiation

(A) Schematic of methods used to express miRNAs in mES cells. mES cells were infected with lentiviruses expressing miR-1 or miR-133 under control of a heterologous EF-1 promoter. Stably infected cells were selected based on their resistance to blasticidin in order to generate stable miRNA-expressing mES cell lines (mES^miR-1 and mES^miR-133). (B) qRT-PCR results confirmed the expression of miR-1 and miR-133; expression of the unintroduced miRNA was unchanged. miR-1 and miR-133 were expressed at levels comparable to those in the adult mouse heart. (C) The population doubling times of mES^miR-1 and mES^miR-133 cells were similar to those of wild-type mES cells. (D) qRT-PCR analyzing expression of Bry, an early mesoderm marker, in control, mES^miR-1, and mES^miR-133 EBs collected on day 4 of differentiation. Expression of miR-1 or miR-133 increased expression of Bry. (E, F) qRT-PCR analysis of Nkx2.5 (E) and Myogenin (F) expression from day 4, 6, or 10 EBs formed from control, mES^miR-1, or mES^miR-133 cells. Control EBs displayed an induction of Nkx2.5 expression over time that was enhanced by miR-1 and suppressed by miR-133. Induction of Myogenin expression was enhanced by miR-1, but not by miR-133. (G) Differences in Nkx2.5 expression (green fluorescent cells) were also visualized at day 10 of differentiation by expressing the miRNAs in an Nkx2.5-GFP transgenic mES cell line. (H) Expression of miR-1 and miR-133 was undetectable in day 10 SRF^−/− EBs by qRT-PCR. (I) Overexpression of miR-1 and to a lesser extent, miR-133, in SRF^−/− EBs restored the Bry and Mesp1 downregulation in day 10 EBs typical of wild-type cells. (J) Expression of Cd53, Cxcl4, and Thbs1, which mark hematopoietic lineages, and of Mef2c, which encodes a major regulator of muscle differentiation, was partially rescued in SRF^−/− EBs upon expression of miR-1 or miR-133.

Figure 3. Both miR-1 and miR-133 suppress endoderm and neuroectoderm differentiation in mES cells

(A, B) qRT-PCR analysis of the endoderm markers Afp (A) or Hnf4α (B) from day 4, 6, or 10 nodal-treated EBs formed from control, mES^miR-1 or mES^miR-133 cells. Induction of Afp and Hnf4α expression normally observed during differentiation in the presence of nodal was suppressed by expression of miR-1 or miR-133. (C) qRT-PCR analysis of the neural marker Ncam1 from day 4, 6, or 10 RA-treated EBs formed from control, mES^miR-1 or mES^miR-133 cells. Expression of miR-1 or miR-133 suppressed the induction of Ncam normally observed during differentiation in the presence of RA. (D) qRT-PCR analysis of the neural progenitor marker Nestin in day 4, 8, or 10 RA-treated EBs formed from control, mES^miR-1 or mES^miR-133 cells. Nestin expression declined in wild-type EBs by day 10 as neurons differentiated, but was maintained in mES^miR-1 and mES^miR-133 EBs. (E) Plot comparing results from mRNA expression microarray analyses of day 10 control, mES^miR-1, and mES^miR-133 EBs. Plot shows that most genes were coordinately regulated. (F) Examples of genes that were coordinately regulated in mES^miR-1 and mES^miR-133 EBs compared to controls.

Figure 4. Differentiation of neural cells is suppressed by miR-1 or miR-133 in teratomas

Teratomas were generated by injecting control, mES^miR-1, or mES^miR-133 cells into the rear flank of SCID mice. After 6 weeks, hematoxylin/eosin-stained teratomas derived from control ES cells were strikingly homogeneous (A) and composed mostly of βIII-tubulin-immunoreactive neural cells (D). Teratomas from mES^miR-1 or mES^miR-133 cells were more heterogeneous (B,C) and contained fewer βIII-tubulin-positive cells (E,F). An accumulation of nestin-positive neural precursors was observed in miR-1 or miR-133 expressing teratomas compared to control (G-I). Expression of miR-1 or miR-133 enhanced muscle specification, as shown by immunostaining with smooth muscle α-actin antibody (J-L). Quantification of areas immunostained with each antibody is indicated as percentages with standard deviation. Scale bars represent ~2mm.

Figure 5. Dll-1 protein levels are negatively regulated by miR-1 in mES cells, and knockdown of Dll-1 expression promotes cardiac mesoderm and suppresses non-mesodermal gene expression

(A) Dll-1 and Dll-4 mRNA levels, assessed by qRT-PCR, were somewhat higher in mES^miR-1 and mES^miR-133 cells than in controls. (B) Immunostaining with Dll-4 or Dll-1 antibody showed equivalent Dll-4 protein levels in mES^miR-1, mES^miR-133 cells, and control mES cells; Dll-1 protein levels were lower in mES^miR-1 cells and higher in mES^miR-133 cells than wild–type mES cells. (C) miR-1 expression caused a dose-dependent decrease in epitope (V5)-tagged Dll-1 protein levels by Western blot without affecting RNA expression of Dll-1 assessed by qRT-PCR (graph). Gapdh protein levels reflect equal loading of protein. (D) Dll-1 mRNA levels, assessed by qRT-PCR, were 62% and 40% lower in response to two distinct short hairpin RNAs targeting Dll-1 mRNA (Dll-1^shRNA-1 and Dll-1^shRNA-2), compared to control cell line. (E) EBs formed from Dll-1^control, Dll-1^shRNA-1 and Dll-1^shRNA-2 ES cells were scored for beating cardiomyocytes on days 8, 10, and 12 of differentiation. Beating cardiomyocytes appeared earlier and were more numerous in EBs from Dll-1^shRNA cell lines than in EBs from the control line. (F) qRT-PCR analyses of Nkx2.5, Myogenin, Afp, and Nestin expression in EBs generated from Dll-1^control, Dll-1^shRNA-1 and Dll-1^shRNA-2 ES cells. Knocking down Dll-1 increased Myogenin expression, decreased Afp expression and sustained Nestin expression compared to controls.

Figure 6. Effects of miR-1 or miR-133 expression in hES cells

(A) Lentivirus-mediated expression of miR-1 or miR-133 in human ES (hES) cells was verified by qRT-PCR. (B) NKX2.5 mRNA expression assessed by qRT-PCR in hEBs collected on days 4, 6, and 8. Overexpression of miR-1 in hES cells increased NKX2.5 expression compared to wild type, while miR-133 expression led to decreased NKX2.5 induction. (C) Human EBs were scored for beating on day 18 of differentiation. Expression of miR-1 increased the number of beating human EBs, while expression of miR-133 did not. (D) Day 18 human EBs were immunostained with antibodies to nestin or βIII-tubulin.

Figure 7. Model of miR-1/miR-133 effects during ES cell differentiation

miR-1 and miR-133 promotion of mesoderm and inhibition of endoderm and ectoderm differentiation at specific stages are indicated. Opposing effects of the two miRNAs in later steps of muscle differentiation are also shown. miR-1 inhibition of Dll-1 translation, along with yet unknown targets, likely contribute to the some of the observed effects of miR-1.