miRNA Expression in Mouse ES Cells and ES Cell–Derived Cardiomyocytes
To determine which miRNAs are enriched during differentiation of mouse ES (mES) cells into cardiomyocytes, we used a mES cell line carrying a green fluorescent protein (GFP) transgene under control of the β-myosin heavy chain promoter, which is uniquely expressed in differentiated cardiomyocytes. We isolated RNA from GFP+ and GFP− cells by fluorescence-activated cell sorting after 13 days of EB differentiation and profiled miRNA expression by microarray analysis. Seventeen miRNAs were enriched at least 3-fold in the GFP+ population (). Approximately half of the miRNAs that were enriched in mES cell-derived cardiomyocytes, including the muscle-specific miRNAs miR-1 and miR-133, were undetectable in undifferentiated mES cells, indicating that they were unique to differentiating cells ().
Identification of miRNAs expressed in ES cell–derived cardiomyocytes
To determine whether miR-1 and miR-133 were present and enriched in early cardiac progenitors, we utilized a mES cell line carrying a GFP
transgene under transcriptional control of a recombinant bacterial artificial chromosome containing the Nkx2.5
enhancer (B. Conklin and E. Hsiao, unpublished results). This line effectively marks the early emergence of pre-cardiac mesoderm. Sorting of GFP-positive cells in day 4 EBs followed by quantitative RT-PCR (qRT-PCR) revealed that the muscle-specific miRNAs were expressed specifically in the early pre-cardiac mesoderm at this early stage (), while the vascular endothelium-enriched miRNA, miR-126, was absent (Kuehbacher et al., 2007
). Conversely, when we sorted vascular progenitors from day 4 EBs based on their cell surface expression of Flk-1, miR-1 and miR-133 were absent from the Flk-1+
mesoderm population in which miR-126 was highly expressed (). We also examined the kinetics of miR-1/miR-133 expression in differentiating whole EBs (). Both were detectable as early as day 4 and their expression increased until day 6 after which their relative abundance in the growing EBs diminished other cell types emerged.
miR-1 and miR-133 Can Promote Mesoderm Differentiation in mES Cells
Since miR-1 and miR-133 were not expressed in undifferentiated mES cells, but were specifically enriched in pre-cardiac mesoderm, we hypothesized that their introduction into mES cells might bias cells toward a muscle lineage. Lentiviruses were used to infect and select ES cell lines expressing miR-1 (mESmiR-1) or miR-133 (mESmiR-133) (). The levels of introduced miRNAs approximated those of the endogenous miRNAs in the mouse heart (). The morphology (data not shown) and doubling time of the cell lines in LIF-containing medium were unaltered (), and the pluripotency markers Oct-4 and Nanog were expressed at normal levels (data not shown).
Effects of miR-1 and miR-133 on mesoderm differentiation
To assess the lineage potential of mES cells expressing miR-1 and miR-133, we differentiated control, mESmiR-1, and mESmiR-133 cells by the hanging drop method, collected the resulting EBs on days 4, 6, and 10 of differentiation, and examined the expression of lineage markers by qRT-PCR. Since miR-1 and miR-133 were normally expressed in day 4 pre-cardiac mesoderm, we examined expression of the early mesoderm marker, Brachyury (Bry). Bry expression was detected transiently in control EBs at day 4 and then rapidly declined ( and data not shown). In day 4 EBs expressing miR-1 or miR-133, Bry expression was dramatically enhanced (), suggesting that both can promote mesodermal gene expression in pluripotent mES cells.
To determine the effects of miR-1 and miR-133 on further differentiation, we examined expression of Nkx2.5, a transcription factor that is one of the earliest cardiac markers (). In control EBs, Nkx2.5 expression was detected by day 6 and was maintained at day 10. Expression of miR-1 increased Nkx2.5 expression at day 6; by day 10, it was ~7-fold greater than in control EBs. Strikingly, expression of miR-133 blocked induction of Nkx2.5 at both time points. We performed a similar expression analysis of Myogenin, an early skeletal muscle marker, to determine the effects of miR-1 and miR-133 on skeletal muscle differentiation. qRT-PCR analysis of Myogenin expression in day 4, 6, or 10 EBs revealed that miR-1, but not miR-133, markedly enhanced Myogenin expression ().
The increase in Nkx2.5 expression, as assessed by qRT-PCR, may represent either an increase in the amount of Nkx2.5 expressed per cell or in the number of cells expressing Nkx2.5. To distinguish between these two possibilities, we infected the Nkx2.5-GFP mES line with control, miR-1-, or miR-133-expressing lentivirus, selected with antibiotic, and differentiated these cells for 10 days. GFP was expressed in more miR-1-expressing EBs, and at higher levels per cell, than in wild-type EBs, and was almost undetectable in miR-133 expressing cells (). Thus, miR-1 appears to promote the emergence of both cardiac and skeletal progenitors in mES cells, while miR-133 does not enhance further differentiation of mesoderm precursors into either lineage.
miR-1 or miR-133 Can Rescue Mesoderm Gene Expression in SRF−/− EBs
Efficient methods for stable miRNA knockdown studies in differentiating EBs are not yet available due to the rapid doubling time of ES cells. However, we previously showed that expression of the miR-1/miR-133 locus in embryonic mouse hearts is directly dependent on SRF (Zhao et al., 2005
) and we therefore sought to use SRF
-null ES cells as a model for complementation experiments that might reveal the specific contribution of these miRNAs within SRF
-null cells (Zhao et al., 2005
). We found that SRF
-null EBs failed to activate miR-1 or miR-133 (), confirming the SRF-dependency in the ES cell system, consistent with in vivo observations. Differentiation of mesodermal progenitors in EBs lacking SRF
is weak and delayed (Weinhold et al., 2000
). To our surprise, however, we found that Bry
expression persisted in SRF
-null EBs, even after 10 days of differentiation, reflecting delayed or arrested differentiation of mesodermal progenitors that normally downregulate Bry
by day 5 (). Despite the many genes dysregulated in SRF
-null EBs, re-introduction of miR-1 in SRF
-null ES cells rescued the abnormal accumulation of Bry+
progenitors at day 10 of differentiation, with Bry
levels returning close to wild-type levels. Introduction of miR-133 had an intermediate effect on the level of Bry
expression at day 10, but Bry levels were still significantly elevated. SRF-/-
ES cells also displayed elevated expression of Mesp1
, a marker of nascent cardiac mesoderm that is usually downregulated as differentiation progresses (Saga et al., 1996
) and this was similarly corrected by reintroduction of miR-1 or miR-133 (). These data suggest miR-1, and to a lesser degree, miR-133, can promote the progression of mesodermal progenitors and that the arrest of mesodermal progenitors in the absence of SRF may be largely due to the absence of this family of miRNAs.
Consistent with the changes in Bry
expression, expression of miR-1 or miR-133 restored the expression of a number of mesodermal genes in day 10 SRF
-null EBs (). Blood cell–specific genes, such as Cd53
, and Thbs1
, were dramatically downregulated in SRF−/−
EBs, reflecting the loss of hematopoeitic lineages in the absence of SRF. However, their expression was reinitiated upon reintroduction of miR-1 or miR-133, likely representing relief of the block to mesodermal differentiation. Even expression of Mef2c
, a major regulator of muscle lineages (Li et al., 1997
), was restored by miR-1 and, to a lesser extent, by miR-133.
miR-1 and miR-133 Suppress Endoderm Differentiation in mES Cells
It has been proposed that in some contexts miRNAs function in a “fail-safe” mechanism to clear latent gene expression by targeting pathways that should not be activated in a particular cell type (Hornstein et al., 2005
). We therefore investigated whether miR-1 and miR-133 might not only promote muscle lineage decisions, but also reinforce them by repressing nonmuscle gene expression. First, we differentiated control, mESmiR-1
, and mESmiR-133
ES cells in the presence of recombinant nodal, a potent inducer of endoderm differentiation in mES cells (Vallier et al., 2004
; Pfendler et al., 2005
). As expected, nodal stimulated expression of the endoderm markers α-Fetoprotein
) and Hnf4α
in control EBs (). These markers were expressed at dramatically lower levels in mESmiR-1
EBs than in control EBs, indicating that miR-1
can each function as potent repressors of endoderm gene expression during differentiation of pluripotent mES cells ().
Both miR-1 and miR-133 suppress endoderm and neuroectoderm differentiation in mES cells
miR-1 and miR-133 Suppress Neural Differentiation From mES Cells
Next, we asked whether miR-1 or miR-133 could also suppress neuroectoderm gene expression from pluripotent mES cells. Control, mESmiR-1
, and mESmiR-133
ES cells were differentiated in the presence of retinoic acid (RA), a potent inducer of neural differentiation (Bain et al., 1995
; Bain et al., 1996
). RA-treated, control EBs expressed high levels of neural cell adhesion molecule 1 (Ncam1
), a marker of mature neurons, by day 10 of differentiation, but Ncam1
induction was suppressed in both mESmiR-1
EBs (). We also examined expression of Nestin
, which is restricted largely to neural progenitor cells and is downregulated upon further neural differentiation (Hockfield and McKay, 1985
expression persisted beyond day 10 in mESmiR-1
EBs, well after its decline in control EBs, suggesting an accumulation of neural progenitors (). Suppression of endoderm or neuroectoderm differentiation was not observed when an endothelial-enriched microRNA, miR-126, was similarly introduced into mES cells (Supp. Fig. 1
), indicating specificity of miR-1 and miR-133 effects. These data indicate that both miR-1 and miR-133 can curtail the differentiation of pluripotent cells into mature neurons, even as cells are pushed toward that lineage by timed administration of RA.
Coordinate Dysregulation of Gene Expression in mESmiR-1 and mESmiR-133 EBs
To more broadly assess the influence of miR-1 or miR-133 on lineage specification and gene expression, we performed mRNA expression microarray analyses on day 10 control, mESmiR-1, and mESmiR-133 EBs. Consistent with the similar effects of miR-1 and miR-133 on repression of nonmuscle gene expression, the vast majority of genes were coordinately regulated between mESmiR-1 and mESmiR-133 EBs (). Among the most highly downregulated genes in both the mESmiR-1 and mESmiR-133 EBs were the early endoderm markers, Afp and Hnf4α, consistent with our qRT-PCR results from EBs treated with nodal (). Expression of other genes normally enriched in endodermal structures, such as those encoding apolipoproteins, was also downregulated in both mESmiR-1 and mESmiR-133 EBs (). These results support the idea that miR-1 and miR-133 can suppress endoderm specification and differentiation.
Among the most highly upregulated genes in both mESmiR-1 and mESmiR-133 EBs were those associated with neuroectoderm specification and early neural differentiation These included the early neurogenic transcription factors, Neurod4, Phox2b, and Myt1 and a number of Hox genes involved in neural specification (). This is consistent with our observation of persistent Nestin expression in mESmiR-1- and mESmiR-133-derived EBs and the apparent disruption of late-stage neuronal differentiation by these miRNAs.
A number of mesodermal genes were also commonly dysregulated in both mESmiR-1
EBs (). Runx2 and Twist1, which are highly expressed in developing bone (Ducy et al., 1997
; Bialek et al., 2004
), were both upregulated, further supporting our conclusion that mesoderm specification is increased in miR-1- or miR-133-expressing EBs. However, a number of genes encoding sarcomeric proteins found in differentiated muscle cells were decreased in both mESmiR-1
EBs. The mechanism for diminished sarcomeric gene expression in EBs may differ in the two cells lines: mesodermal progenitors in the mESmiR-133
EBs likely fail to differentiate into muscle, remaining in the progenitor state, while differentiating muscle cells in mESmiR-1
EBs may prematurely exit the cell cycle resulting in fewer cardiac cells, as was observed upon overexpression of miR-1 in the mouse heart (Zhao et al., 2005
). Both would result in underrepresented muscle gene expression and each is consistent with our current understanding of miR-1 and miR-133 function.
miR-1 and miR-133 Suppress Neural Differentiation during Teratoma Formation
To examine the ability of miR-1 and miR-133 to suppress nonmesodermal lineages in a more in vivo setting, we injected wild-type or miRNA-expressing mES cells subcutaneously into SCID mice and monitored their differentiation in vivo. Transplanted cells of each line formed teratomas in the recipients and were analyzed 6 weeks after inoculation. Teratomas from control, mESmiR-1, or mESmiR-133 cells included derivatives of all three embryonic germ layers, but the control teratomas were much more homogeneous (). As shown by immunostaining with βIII-tubulin antibodies, teratomas from control mES cells were composed mostly of differentiated neurons (). In contrast, teratomas formed from mESmiR-1 or mESmiR-133 cells had far fewer differentiated neuronal cells ().
Differentiation of neural cells is suppressed by miR-1 or miR-133 in teratomas
Based on our analyses of neural differentiation in EBs, we also immunostained teratomas using an antibody to nestin. Control teratomas were fully differentiated and contained only rare pockets of nestin-positive neural progenitors, as expected (). However, mESmiR-1 and mESmiR-133 teratomas contained abundant nestin-positive cells even after 6 weeks of development, suggesting an arrest of neural differentiation at the progenitor stage (). The accumulation of nestin-positive progenitors in these teratomas further supports the idea that miR-1 and miR-133 permit specification of the ectodermal lineage from pluripotent mES cells, but inhibit complete differentiation of neural progenitor cells into neurons.
We also immunostained teratomas using an antibody to smooth muscle α-actin, a marker of smooth muscle and immature striated muscle cells (cardiac and skeletal). Consistent with the promesodermal effects of miR-1 and miR-133 in EBs, teratomas derived from mESmiR-1
- and mESmiR-133
-derived teratomas had more cells on average expressing smooth muscle α-actin () than control (). High magnification views of immunostained sections demonstrate the specificity of each antibody (Supp.Fig. 2
The Notch Ligand, Delta-like 1, is Translationally Repressed by miR-1
miRNAs likely function by regulating numerous pathways, but in some cases a subset serve as the “major” effectors. Since Notch signaling can promote neural differentiation and inhibit muscle differentiation in ES cells (Nemir et al., 2006
; Lowell at al., 2006
), which is opposite of miR-1’s effects, we hypothesized that miR-1-mediated repression of Notch signaling may contribute to the observed effects of miR-1 in mES cells. Indeed, we had previously shown that miR-1 directly targets the Notch ligand delta
for repression (Kwon et al., 2005
). Three orthologs of Drosophila delta
have been identified in mice—Dll-1
, and Dll-4
, but not Dll-3
, contained putative miR-1 or miR-133 binding sites in their 3’ UTR. As shown by qRT-PCR analysis, mRNA expression of Dll-1
was similar in mESmiR-1
cells and somewhat higher than in control mES cells ().
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
Since miRNAs can block the translation of target mRNAs, we examined Dll-1 and Dll-4 protein levels in all three mES cell lines (). mESmiR-1
, and control cells had similar levels of Dll-4 by immunocytochemistry () and Western analysis (data not shown). Quantitative analysis of endogenous Dll-1 protein was not possible due to the lack of published Dll-1 antibodies that function in Western blots. However, mESmiR-1
cells had consistently decreased Dll-1 protein levels by immunocytochemistry despite having normal levels of Dll-1
mRNA, consistent with translational inhibition of Dll-1 by miR-1. Although a potential miR-1 binding site in the Dll-1 3’-UTR has extensive, conserved sequence matching (Supp. Fig. 3a
) and is present in an accessible region with little secondary structure (data not shown), repression through this site was not transferable to the luciferase 3’-UTR in the surrogate assay commonly employed to test specific binding sites (Supp. Fig. 3b
). However, miR-1 potently repressed protein, but not mRNA expression of an epitope-tagged Dll-1 containing the full 3’UTR in a dose-dependent manner indicating translational inhibition of Dll-1 in mammalian cells ().
Dll-1 Knockdown in mES Cells Promotes Cardiac Mesoderm and Suppresses Non-mesoderm Gene Expression
To determine whether downregulation of Dll-1 protein by miR-1 could account for a subset of the effects of miR-1 on cell lineage decisions, we used short hairpin RNA (shRNA) constructs directed against distinct regions of Dll-1 to generate two different Dll-1shRNA cell lines (Dll-1shRNA-1 and Dll-1shRNA-2). The Dll-1 mRNA level was about 62% lower in Dll-1shRNA-1 cells and 40% lower in Dll-1shRNA-2 cells than in a control line expressing a scrambled shRNA construct (). Oct3/4 levels and cell morphology were unaltered (data not shown). EBs formed from Dll-1shRNA cells had a much greater propensity toward cardiomyocyte differentiation and formed beating cardiomyocytes earlier than control EBs (). By day 12 of differentiation, 89% of EBs formed from Dll-1shRNA-1 cells and 97% of EBs from Dll-1shRNA-2 cells contained beating cardiomyocytes compared to 48% of Dll-1control EBs. Nkx2.5 expression, marking cardiac progenitors, was also more highly induced in Dll-1shRNA than in control EBs (), as were Nkx2.5-GFP-positive cells (data not shown). In addition, Myogenin expression was higher in Dll-1shRNA EBs compared to controls (). Although the effect of Dll-1 knockdown on Nkx2.5 and myogenin expression was not as robust as miR-1 expression, the trends were similar. These results indicate that depletion of Dll-1 increases muscle differentiation from mES cells and suggest that miR-1 may promote cardiac differentiation, in part, by downregulating Dll-1 protein.
We also performed qRT-PCR analyses on EBs formed from Dll-1shRNA cell lines to determine if suppression of ectodermal and endodermal lineages by miR-1 might also involve Dll-1 downregulation. Expression of the endoderm markers Afp () and Hnf4α (data not shown) was lower in Dll-1shRNA EBs than in Dll-1control EBs. Moreover, expression of Nestin, which decreased between days 10 and 12 as neurons differentiated in Dll-1control EBs, was increased during this period in both lines of Dll-1shRNA EBs (). Thus, loss of Dll-1 also repressesendoderm differentiation and and results in persistence of neural progenitor gene expression.
Effects of miR-1 or miR-133 in Human ES Cells
Human ES (hES) cells often behave differently than mES cells. To investigate whether miR-1 or miR-133 function similarly in the two cell types, we infected the H9 hES cell line with the same lentiviruses encoding either miR-1 or miR-133. Expression was verified by qRT-PCR (). The resulting hESmiR-1 and hESmiR-133 cell lines were differentiated as EBs in suspension and collected on days 4, 6, and 8. NKX2.5 expression was detectable by qRT-PCR in control human EBs by day 6 and decreased overall by day 8 (). As in the mouse EBs, hESmiR-1 EBs had higher levels of NKX2.5 expression than controls, while hESmiR-133 EBs failed to induce NKX2.5 expression to the levels observed in controls (). Consistent with this, we also found that the percentage of hESmiR-1 EBs with beating cardiac cells on day 18 of differentiation was more than 3-fold higher than in wild-type EBs, while hESmiR-133 EBs did not display enhanced cardiomyocyte formation (). Thus, regulation of cardiac differentiation by miR-1 and miR-133 appears to be grossly similar in hES and mES cells.
Effects of miR-1 or miR-133 expression in hES cells
To examine the effects of miR-1 or miR-133 expression on neuroectoderm differentiation in hES cells, we also immunostained day 18 control, hESmiR-1, and hESmiR-133 EBs with antibodies recognizing nestin or βIII-tubulin (). Like miRNA-expressing mouse EBs, hESmiR-1 and hESmiR-133 EBs accumulated more nestin-positive progenitors than control human EBs. As in our mouse ES cells studies, there were fewer βIII-tubulin positive neural cells in hESmiR-133 EBs compared to controls, although this effect was not consistent for hESmiR-1 cells. These results demonstrate that the muscle-specific miRNAs miR-1 and miR-133 have similar, but somewhat unique effects on the differentiation of hES and mES cells, and suggest that miRNAs may be useful for coaxing and repressing differentiation of human or mouse ES cells into particular lineages.