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Tcf7l1 directly regulates cardiomyocyte differentiation in embryonic stem cells
© The Author(s). 2018
- Received: 20 July 2018
- Accepted: 21 September 2018
- Published: 11 October 2018
The T-cell factor/lymphoid enhancer factor (TCF/LEF) family protein Tcf7l1 is highly abundant in embryonic stem cells (ESCs), regulating pluripotency and preparing epiblasts for further differentiation. Defects in the cardiovascular system in Tcf7l1-null mouse were considered secondary to mesoderm malformation. Here, we used temporally controlled Tcf7l1 expression in Tcf7l1-null ESCs to address whether Tcf7l1 directly contributes to cardiac forward programming. Tcf7l1 knockout during differentiation impaired cardiomyocyte formation but did not affect mesoderm formation. Tcf7l1-null ESCs showed delay in mesoderm formation, but once completed, ectopic Tcf7l1 augmented cardiomyocyte differentiation. Further, Tcf7l1-VP16 and Tcf7l1dN showed procardiac activity whereas Tcf7l1-En was ineffective. Our results support that Tcf7l1 contributes to cardiac lineage development as a β-catenin-independent transactivator of cardiac genes.
- T-cell factor/lymphoid enhancer factor
- Cardiac myocytes
The Wnt/ β-catenin signaling pathway is critical in stem cell pluripotency, differentiation and homeostasis [1, 2]. In the absence of WNT ligand, β-catenin is phosphorylated by a destruction complex composed of adenomatous polyposis coli (APC), glycogen synthase kinase 3 (GSK3), and kinases casein kinase 1 (CK1) . Phosphorylated β-catenin is ubiquitinated and degraded by proteasomes. WNT ligand binding disaggregates the destruction complex, and in turn stabilizes β-catenin. Next, β-catenin is translocated into the nucleus where it binds T-cell factor/lymphoid enhancer factor (TCF/LEF) family proteins to transactivate downstream genes . The Wnt/ β-catenin pathway plays a biphasic role in cardiogenesis: an initial activation phase in which Wnt/β-catenin promotes mesoderm formation, followed by an inhibitory phase in which the pathway is shut off to allow cardiac gene expression.
There are four TCF/LEF family proteins in mammals: TCF7, LEF1, TCF7l1, and TCF7l2 . They bind the consensus DNA element 5′-(A/T)(A/T)CAAAG-3′ [3, 4]. Their interactions with both β-catenin and the transregulatory element are necessary for activating target genes in response to WNT signaling [6–8]. In mouse genetic studies, only Tcf7l1 deletion led to severe embryonic defects and lethality. The defects are related to delayed mesoderm specification, axis mesoderm duplication, and impaired lateral mesoderm formation. Some severely affected embryos display enlarged cardiac sacs, missing hearts, and multiple large blood vessels . In embryonic stem cells, Tcf7l1 negatively modulates the expression of pluripotent genes, and prepares the epiblast for transition to lineage specification [10–12]. It has been reported that Tcf7l1 can function independently of β-catenin during gastrulation and hypothalamopituitary (HP) axis formation [3, 4, 13]. Because of defective mesoderm formation, whether Tcf7l1 intrinsically contributes to cardiac development has not been determined in Tcf7l1−/− embryos.
Herein, based on a Tcf7l1−/− background, we conducted temporally controlled Tcf7l1 rescuing experiments, and demonstrate that Tcf7l1 acts as an activator-like transcription factor and regulates cardiac lineage development independent of β-catenin.
Tcf7l1+/+ and Tcf7l1−/− ESC lines were provided by Dr Bradley J. Merrill (University of Illinois at Chicago, USA). ESCs were propagated in 0.1% gelatin-coated dishes and cultured with feeder-free ESC medium (DMEM (Gibco) supplemented with 15% FBS (Atlanta Biologicals), 100 U/ml penicillin G, 100 μg/ml streptomycin sulfate, 2 mM l-glutamine, 0.1 mM β-mercaptoethanol, 1× 103 U/ml murine leukemia inhibitory factor (LIF; Global Stem)). The medium was changed daily. To induce EB formation and differentiation, the ESCs were grown as 20 μl hanging droplets (2 × 104 cells/ml) in SFDM without LIF . EBs were collected as indicated and the medium was replaced every 2 days. 293FT cells were cultured in DMEM (Gibco) supplemented with 20% FBS (Atlanta Biologicals), 100 U/ml penicillin G, 100 μg/ml streptomycin sulfate, and 2 mM l-glutamine.
Construction of the inducible expression vector, preparation of lentiviral vectors, and selection of stable expression clones
We used the Tet-On advanced lentiviral vector system (Clontech) and the Tet-Off advanced lentiviral vector system (Clontech) for inducible gene expression. Tcf7l1 and Tcf7l1dN (N-ter 73 amino acid deletion) genes were amplified by PCR from Homo sapiens transcription factor 7-like 1 cDNA clone (OriGene Technologies) using Pfx DNA Polymerase (Invitrogen). Tcf7l1-VP16 was prepared by fusing aa 314–471 of Tcf7l1 to the VP16 activation domain. Tcf7l1-En was prepared by fusing aa 314–471 of Tcf7l1 to the repressor domain of Engrailed 1.
Additional materials and methods are presented in Additional file 1: Supplemental information.
Next, we addressed whether the transcription repressor or activator role of Tcf7l1 is involved in activating the cardiomyocyte program. Into Tcf7l1−/− ESCs, we introduced three versions of Tcf7l1 transgene: wildtype; Tcf7l1-VP16, a fusion between the Tcf7l1 DNA-binding domain and the VP16 transactivation domain; and Tcf7l1-En, a fusion between the Tcf7l1 DNA-binding domain and the Engrailed repression domain (Additional file 3: Figure S2A). The differentiation timing of the ESCs receiving these transgenes varied, but it was consistent that ESCs expressing Tcf7l1-VP16 showed significantly increased mesodermal markers (T and Mesp1) compared to ESCs expressing Tcf7l1-En (Additional file 3: Figure S2B). Slightly increased expression of Nkx2–5 but little effect on Sox17 was also present. Earlier work in our laboratory  established that TCF/LEF proteins cooperate with Oct4 to drive the transcription of Mesp1. The significant upregulation of Mesp1 by Tcf7l1-VP16 suggests that Tcf7l1 may be the responsible TCF/LEF protein.
We chose to activate ectopic Tcf7l1 expression at day 7, when Tcf7l1−/− cells have passed the stage of mesoderm formation (Fig. 3c–f). This allowed us to evaluate the effect of Tcf7l1 transgenes on cardiomyocyte differentiation. By assaying the cardiac gene Nkx2–5, only Tcf7l1dN and Tcf7l1-VP16 activated the cardiomyocyte program (Fig. 3f). Tcf7l1-En downregulated Nkx2–5. In immunostaining of α-Actinin, Tcf7l1dN and Tcf7l1-VP16 boosted formation of sarcomeric structures but not Tcf7l1-En (Fig. 3d, e). Ectopic wildtype Tcf7l1 did not activate Nkx2–5 expression, consistent with the notion that Tcf7l1 is a weaker transactivator compared to Tcf7 and Lef1 [15, 16]. Both Tcf7l1dN and Tcf7l1-VP16 showed more pronounced effects, perhaps because these variants have acquired higher transactivating capacity. These data support our hypothesis that transactivating activity of Tcf7l1 directly contributes to cardiac linage development. Moreover, the de-novo function of Tcf7l1 does not require its interaction with β-catenin in cardiomyocyte differentiation.
The development of the cardiovascular system requires precisely regulated canonical WNT signaling [19, 20]. As an important downstream factor of WNT, Tcf7l1 is critical in maintaining pluripotency as well as preparing ESCs for gastrulation [21, 22]. However, the function of Tcf7l1 in cardiomyocyte differentiation was unknown, mainly because KO of Tcf7l1 impairs prerequisite steps. In this study, we demonstrate that Tcf7l1 is intrinsically required for the establishment of the cardiomyocyte linage. Our data support that Tcf7l1 contributes to cardiac lineage development as a β-catenin-independent transactivator for Mesp1 and other cardiac lineage-determining genes.
The TCF/LEF family members play important but distinctive roles in embryonic development. Tcf7 is essential for thymocyte differentiation. Homologous deletion of Lef1 led to missing teeth, mammary glands, whiskers, and hair. Tcf7l2 is obligatory for formation of epithelial stem cells in the small intestine. The role and underlying mechanisms of Tcf7l1 are stage dependent and very enigmatic. Tcf7l1 is important for pluripotency maintenance, mesoderm induction, and further specification. It may be specifically required for heart formation: mildly affected Tcf7l1 null mutants had enlarged hearts, while severely affected mice fail to develop the heart. Our work provides a first-degree approximation of how Tcf7l1 may affect cardiomyocyte formation. The ESC models established in this study may be useful in conditionally manipulating Tcf7l1 expression at the organism level. It was previously reported that Tcf7l1 restricts cardiomyocytes while promoting endothelial specification in zebrafish . Although Tcf7l1 may play different roles in these two species, it is more likely that the loss of Tcf7l1 has triggered compensation by other TCF/LEF factors and the phenotypes reflect varied overall effects. To this end, Moreira et al.  demonstrated that a single TCF/LEF factor is sufficient for trilineage differentiation in ESCs, but how the stoichiometry of TCF/LEF factors contributes to cell fate specification and organogenesis warrants additional investigation.
Previous studies have found that Tcf7l1 protein mostly act as a transcriptional repressor, in the absence of β-catenin [9, 22, 25–28]. β-catenin binding releases the repression activity of Tcf7l1, thus maintaining pluripotent cell renewal and triggering gastrulation. However, β-catenin binding seems unessential for gastrulation, as knockin Tcf7l1∆N mutant mice gastrulate normally . In this study, Tcf7l1 worked as a β-catenin-independent transactivator because only Tcf7l1dN and Tcf7l1-VP16 rescued Tcf7l1−/− cells for cardiomyocyte differentiation. Although current literature leans heavily toward repressor activity of Tcf7l1, emerging evidence supports that it can also function as a transactivator. It induces LCN2 expression in a β-catenin-independent fashion and drives skin carcinogenesis . Whether the transactivator function of Tcf7l1 requires other cofactors remains unknown.
Both opposite and compensatory effects among the TCF/LEF family members exist in developmental processes, but we were unable to address such effects in this study. Further study is needed to investigate the function of other individual TCF/LEF members, as well as the mechanism of their balanced relationships during cardiomyocyte differentiation.
The authors thank Bradley Merrill for providing Tcf7l1–/– embryonic stem cells, and David Stewart and Robert Schwartz for helpful discussions.
Supported by research funds from the University of Houston (to YL), and grants from the American Heart Association (11SDG5260033 and 16GRNT27760164 to YL) and the US Department of Defense Congressionally Directed Medical Research Programs (PR162075 to YL). The funding bodies do not have roles in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
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All data generated or analyzed during this study are included in this published article and its Additional files.
RL was responsible for collection and/or assembly of data, data analysis and interpretation, and manuscript writing. YL was responsible for collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript. Both authors read and approved the final manuscript.
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