Lack of Genetic Interaction between Tbx18 and Tbx2/Tbx20 in Mouse Epicardial Development

The epicardium, the outermost layer of the heart, is an essential source of cells and signals for the formation of the cardiac fibrous skeleton and the coronary vasculature, and for the maturation of the myocardium during embryonic development. The molecular factors that control epicardial mobilization and differentiation, and direct the epicardial-myocardial cross-talk are, however, insufficiently understood. The T-box transcription factor gene Tbx18 is specifically expressed in the epicardium of vertebrate embryos. Loss of Tbx18 is dispensable for epicardial development, but may influence coronary vessel maturation. In contrast, over-expression of an activator version of TBX18 severely impairs epicardial development by premature differentiation of epicardial cells into SMCs indicating a potential redundancy of Tbx18 with other repressors of the T-box gene family. Here, we show that Tbx2 and Tbx20 are co-expressed with Tbx18 at different stages of epicardial development. Using a conditional gene targeting approach we find that neither the epicardial loss of Tbx2 nor the combined loss of Tbx2 and Tbx18 affects epicardial development. Similarly, we observed that the heterozygous loss of Tbx20 with and without additional loss of Tbx18 does not impact on epicardial integrity and mobilization in mouse embryos. Thus, Tbx18 does not function redundantly with Tbx2 or Tbx20 in epicardial development.


Introduction
The epicardium is an epithelial monolayer that completely covers the outer surface of the heart. It protects the underlying myocardium and allows mobility of the heart within the pericardial cavity. In addition to this structural role in homeostasis, the epicardium has been recognized as an important source of cells and signals directing and modulating myocardial growth and vascularization both in development and under injury conditions (for recent reviews see [1,2]).
Epicardial development in the mouse starts at embryonic day (E) 9.5 with the formation of the proepicardium, a cauliflower-like mesothelial cell aggregate at the venous pole of the heart [3,4]. Cells of the proepicardium delaminate and attach to the adjacent myocardium. At E10.5, a contiguous epithelial epicardial layer surrounds the heart tube. Between E11.5 and E14.5, individual epicardial cells undergo an epithelial-mesenchymal transition (EMT), invade the underlying myocardium and largely differentiate into smooth muscle cells (SMCs) and cardiac fibroblasts [5][6][7][8][9][10]. Concomitantly, the epicardium acts as a source of signals that nurture the myocardium and promote the in-growth of the coronary plexus and vascularization of the cardiac muscle [10,11]. Intriguingly, it has been uncovered in recent years both in zebrafish and mouse that the adult epicardium can reactivate an embryonic gene program upon injury conditions [12]. As a consequence, the epicardium secretes factors that promote neovascularization of the myocardium, and provides cells that upon differentiation into fibroblasts and SMCs contribute to scar formation [13,14].
Although several signaling pathways and transcription factors have been implicated in the distinct subprograms of epicardial development, namely proepicardium formation, epicardial EMT, fate decision or epicardial-myocardial crosstalk [11,15], we are far from understanding the tight regulatory networks orchestrating all of these processes in time and space, and using them for regenerative purposes.
T-box (Tbx) genes encode a large family of transcription factors that regulate a variety of developmental processes in both vertebrates and invertebrates. They are characterized by a common DNA-binding motif, the T-box that recognizes and binds conserved DNA-elements in the genome to mediate transcriptional activation and/or repression of target genes. Tbox genes often act in a combinatorial or hierarchical fashion and frequently exhibit an exquisite dose-sensitivity (for reviews see [16,17]). In the developing mammalian heart, expression of six of the 17 murine family members have been detected and related to different subprograms of myocardial patterning and differentiation (for a review see [18]). Tbx5 and Tbx20 act in the early heart tube, and independently activate the chamber myocardial gene program [19][20][21][22][23] whereas Tbx2 and Tbx3 act together to locally repress this program to favor valvuloseptal and conduction system development [24][25][26]. Tbx1 acts in the pharyngeal mesoderm to maintain proliferation of mesenchymal precursor cells for formation of a myocardialized and septated outflow tract [27]. Tbx18 is expressed in the sinus venosus region at the posterior pole of the heart and is required for myocardialization of the caval veins and formation of a large portion of the sinoatrial node [28,29].
Additional roles of these Tbx genes in epicardial development have been suggested. Tbx5 expression was detected in a heterogenous fashion in the proepicardium at E9.5 and the nascent epicardium at E10.5. Epicardial expression strongly declined after this stage. Conditional deletion of Tbx5 from the (pro-)epicardium led to reduced attachment of proepicardial cells to the myocardium and epicardial blebbing that are probably causative for the reduced epicardial EMT, fibroblast and SMC formation, and defective myocardial and coronary vessel maturation [30].
Tbx18 is strongly expressed in the proepicardium at E9.5 and is maintained in the epicardium until birth in all vertebrate models analyzed to date [9,[31][32][33][34]. We have recently reported that Tbx18-deficient mice that were maintained on an outbred background do not exhibit epicardial defects whereas mice with epicardial overexpression of an activating form of TBX18 (a VP16-fusion protein) show loss of epicardial EMT due to premature SMC differentiation of epicardial cells [35]. Since TBX18 possesses transcriptional repressor activity via an eh1-motif near the N-terminus that recruits Groucho corepressors [36], these findings point to a possible redundancy with another repressing member of this gene family in maintaining the progenitor character of epicardial cells. This hypothesis is supported by a recent study in which mice deficient for another null allele of Tbx18 exhibit epicardial blebbing and coronary defects when maintained on an inbred background [37].
We here aimed to decipher a functional redundancy of Tbx18 with other transcriptional repressors of the Tbx gene family in epicardial development. We identify Tbx2 and Tbx20 as being coexpressed with Tbx18 in the developing (pro-)epicardium and subsequently test for genetic interaction of Tbx18 and Tbx2/Tbx20 in this tissue.

Epicardial explant cultures
Explant cultures of primary epicardial cells were obtained as described before [9].

RNA isolation, reverse transcription and PCR analysis
RNA of epicardial explants was obtained using PeqGold RNApure (Peqlab) according to the manufacturer's manual and subsequently transcribed using the RevertAid Reverse Transcriptase (Fermentas). 3 μl of undiluted epicardial cDNA or 1 μl of control cDNA (prepared from different embryonic tissues) were used in the PCR reaction. Primers, PCR conditions and controls are depicted in S1 Table. Histological analysis For histological stainings embryos were fixed in 4% paraformaldehyde overnight, transferred to PBS, paraffin embedded, and sectioned to 5-μm. Sections were stained with hematoxylin and eosin following standard procedures.

In situ hybridization analysis
Nonradioactive in situ hybridization analysis with digoxigenin-labeled antisense riboprobes was performed on 10 μm sections of paraffin-embedded embryos as described [43].

Documentation, Quantification and Statistics
Sections were photographed using a Leica DM5000 microscope with Leica DFC300FX digital camera. All images were processed in ImageJ and Adobe Photoshop CS4. Statistical analyses were performed using the 2-tailed Student's t-test. Data were expressed as mean±SD. Differences were considered not significant when the P-value was higher than 0.05. At least two specimens were analyzed per stage, genotype and assay.

Expression of T-box genes during epicardial development
To determine whether the members of the cardiac T-box gene family Tbx1, Tbx2, Tbx3, Tbx5, Tbx20 are coexpressed with Tbx18 in epicardial development, we performed a comparative expression analysis in mouse embryos. As a sensitive assay we first performed qualitative RT-PCR analysis from E11.5 epicardial explant cultures grown for three days under serum-free conditions. This assay revealed the expression of Tbx18, Tbx20, Tbx2 and Tbx5 in undifferentiated epicardial cells whereas Tbx3 and Tbx1 were not detected ( Fig 1A). To resolve epicardial expression in time and space we subsequently performed section in situ hybridization of E9.5 to E14.5 hearts (Fig 1B). At E9.5, expression of Tbx18 was strong in the proepicardium. Expression of Tbx20, Tbx5 and Tbx2 was markedly weaker but still above background levels. From E10.5 to E14.5, Tbx18 expression was detected in the epicardium but not in subepicardial cells in the right ventricle. (Due to endogenous expression of Tbx18 in the myocardium of the left ventricle and the interventricular septum [44], we restricted our analysis on the right ventricle). Expression of Tbx5, Tbx3 and Tbx1 could not be detected in the epicardium at any of these stages. In contrast, we found expression of Tbx20 in epicardial cells at E10.5 (Fig 1B, arrow). Due to the myocardial expression of Tbx20 (Fig 1B, Ã ), Tbx20-expressing epicardial and epicardium-derived cells could not be distinguished by this method. Tbx2 transcripts were detected in a subset of epicardial cells (Fig 1B, arrow) at E12.5 and at E14.5, and in coronary arteries at E14.5 ( Fig 1B, black arrowheads).
For further clarification, we also analyzed TBX2 and TBX3 protein expression by immunofluorescence. At E9.5, Wilms tumor 1 (WT1) and TBX18 were found in the entire proepicardium as previously reported [44,45] whereas TBX2 protein was confined to the caudal part of this tissue. Lineage analysis using a Tbx2 cre line [42] and a Rosa26 mTmG/+ reporter line [41] showed that the proepicardium itself was completely derived from cells formerly expressing Tbx2 (Fig 2A and 2B). At E13.5, a subset of epicardial and subepicardial cells expressed TBX2 protein as shown by double immunofluorescence against TBX2 and GFP, that in this case marked the epicardial lineage by epicardium-specific recombination under the control of the Tbx18 promoter [9] (Tbx18 cre/+ ; R26 mTmG/+ mice) ( Fig 2C). Expression of TBX3 was neither detected in the proepicardium at E9.5 nor in the epicardium at E13.5 ( Fig 2B and 2C), confirming as in the case of TBX2 our mRNA expression analysis. We conclude that Tbx18 is co-expressed with Tbx5, Tbx20 and Tbx2 in a subset of cells in the proepicardium at E9.5 and in a subset of epicardial cells until E14.5.

Combined loss of Tbx18 and Tbx2 does not affect epicardial development
Since Tbx2 is co-expressed with Tbx18 in a subset of proepicardial and epicardial cells and encodes a transcriptional repressor like Tbx18 [36,46], we tested for functional redundancy of the two genes in epicardial development by a conditional gene targeting approach. (Pro)epicardial deletion of Tbx2 was achieved using a floxed allele of Tbx2 and the Tbx18 cre mouse line that mediates robust recombination in the proepicardium, and in the epicardium and its descendants [9,47]. Absence of TBX18 protein in the epicardium/pericardium of Tbx18-null embryos, and of TBX2 protein in Tbx18 cre/+ ;Tbx2 fl/fl embryos at E10.5 confirmed the suitability of this genetic approach (S1 Fig).
We focused our analysis on E14.5 embryos to be able to compare our findings with that of a previous study on mice with epicardial overexpression of an activator version of TBX18 [35]. Hematoxylin and eosin staining of transverse sections through the heart region did not reveal any difference in the histological appearance of the cardiac chambers in compound mutants (Tbx18 cre/GFP ;Tbx2 fl/+ and Tbx18 cre/+ ;Tbx2 fl/fl ), and double mutants (Tbx18 cre/GFP ;Tbx2 fl/fl ) compared to control embryos (Tbx18 cre/+ ); septa and valves were formed normally and the ventricular myocardium was of normal thickness. In Tbx18-deficient mice pleuropericardial membranes were not completely resolved from the body wall but remained laterally attached in agreement with our previous report on the role of Tbx18 in the development of this tissue [48] (Fig 3A, arrows). Higher magnification of the histologically stained right ventricle demonstrated that the epicardium was correctly attached in double and compound mutant embryos ( Fig 3B). To visualize the epicardium and its descendants on a cellular level, we analyzed GFP expression from the R26 mTmG reporter allele in the different mutant combinations. GFP-positive cells localized to the subepicardial space and intermingled with cardiomyocytes in double and compound mutants as in control mice; and visual inspection of transverse sections did not reveal gross changes in the number of immigrating cells (Fig 3C). Similarly, we did not detect  S1 Table). (B) In situ hybridization analysis of Tbx18, Tbx5, Tbx20, Tbx2, Tbx3 and Tbx1 expression on sagital (E9.5) and transverse (E10.5, E12.5, E14.5) sections through the heart. Shown are higher magnifications of the proepicardium (E9.5) and of the right ventricle (E10.5 to E14.5). Black arrows indicate proepicardial and epicardial expression of Tbx18, Tbx5, Tbx20 and Tbx2, asterisks point to known expression domains of Tbx5 and Tbx20 in the atrium, and of Tbx2 and Tbx3 in the liver primordium at E9.5. Black arrowheads indicate coronary artery expression of Tbx2 at E14.5. Scale bars are 50 μm. CA, common atrium; CAr, coronary artery; Epi, epicardium; PE, proepicardium; RV, right ventricle; SV, sinus venosus.
doi:10.1371/journal.pone.0156787.g001 differences in the expression of Wilms tumor 1 (WT1), a marker for epicardial and epicardium-derived cells, and more weakly for endothelial cells [45,49] in double and compound mutant embryos compared to the control (Fig 3C). Expression of endomucin (EMCN), a marker of venous and capillary endothelial cells and of the endocardium [50], was indistinguishable in double and compound mutant and control hearts indicating that formation of the coronary plexus occurred normally (Fig 3C). We conclude that epicardial signaling is unaffected by loss of Tbx18 and/or Tbx2. To clarify whether loss of Tbx18 and Tbx2 impairs SMC differentiation, expression of SMC proteins NOTCH3 and Transgelin (TAGLN) was analyzed. Epicardial cells were delineated by costaining for collagen IV (COLIV) expression in the basal lamina [9]. TAGLN protein expression was found in subepicardial cells and cardiomyocytes but not in epicardial cells in any of the analyzed genotypes at this stage ( Fig 3C). NOTCH3 protein was detectable in up to 70% of epicardial cells of Tbx18 cre/GFP ;Tbx2 fl/fl and Tbx18 cre/GFP ; Tbx2 fl/+ hearts but only in 50% of control or Tbx18 cre/+ ;Tbx2 fl/fl epicardial cells indicating that the loss of Tbx18 accounts for this effect (Fig 3C, S2 Fig).
At E18.5, epicardium-derived cells have fully differentiated into fibroblasts and SMCs, constituting the cardiac fibrous skeleton and completing the formation of the coronary vasculature. Hematoxylin and eosin staining of transverse sections through the heart at this stage did not reveal histological defects in addition to the pericardial changes observed in Tbx18-deficient embryos at E14.5 (Fig 4A). Coronary arteries developed normally in Tbx18/Tbx2   (Fig 4B). We conclude, that Tbx2 and Tbx18 are neither individually nor combinatorially required in the epicardium.

Misexpression of human TBX2 in epicardial cells and their progeny does not affect cardiac development
Although the loss of Tbx2 does not effect epicardial development, its localized expression in a subset of epicardial cells may indicate an involvement of the gene in the specification of distinct epicardial sublineages. To study the potential effect of Tbx2 on lineage segregation in the epicardium, we generated mice ectopically expressing human TBX2 in the whole epicardium and its descendants. For this purpose Tbx18 cre/+ ;R26 mTmG/mTmG males were mated to females homozygous for an integration of a cre-inducible TBX2 expression cassette in the X-chromosomal Hprt locus (Hprt CAG::TBX2/CAG::TBX2 ) [26]. Since female Tbx18 cre/+ ;R26 mTmG/+ ;Hprt CAG::TBX2/+ embryos express the transgene in a mosaic fashion due to X-chromosome inactivation, we subsequently only analyzed male Tbx18 cre/+ ;R26 mTmG/+ ;Hprt CAG::TBX2/y embryos that express the transgene homogenously. Tbx18 cre/+ ;R26 mTmG/+ ;Hprt CAG::TBX2/y embryos were present in the expected Mendelian ratio at E18.5 (n = 10/42). Histological analysis revealed that the pleuropericardial membranes were not fully detached from the body wall. However, septa and valves were unaffected and the ventricular walls exhibited normal thickness and trabeculation (Fig 5A). Immunofluorescence analysis confirmed expression of human TBX2 protein in the epicardium and epicardium-derived cells at levels similar to that of endogenous mouse TBX2 in coronary SMCs. Weak ectopic expression of TAGLN was associated with human TBX2 protein in the epicardium and the myocardium in Tbx18 cre/+ ;R26 mTmG/+ ;Hprt CAG::TBX2/y mice (Fig 5B). Formation of coronary arteries was unaffected although the surrounding SMC layer appeared thinned in Tbx18 cre/+ ; R26 mTmG/+ ;Hprt CAG::TBX2/y hearts as indicated by staining for the SM proteins ACTA2 and TAGLN (Fig 5C). However, the contribution of epicardium-derived cells to the SM lineage surrounding coronary arteries was unaffected in Tbx18 cre/+ ;R26 mTmG/+ ;Hprt CAG::TBX2/y hearts (S4 Fig). In contrast to epicardium-derived cells in control hearts, TAGLN but not ACTA2 was ectopically expressed in epicardial cells and in a majority of epicardium-derived cells of Tbx18 cre/+ ;R26 mTmG/+ ;Hprt CAG::TBX2/y hearts at E18.5 (Fig 5A and 5B, S4 Fig: red arrow heads). EMCN expression in mutants was unchanged from the control indicating the formation of a normal venous and capillary network in the coronary vasculature. Finally, the formation and distribution of POSTN-positive fibroblasts in the myocardium was normal in Tbx18 cre/+ ;R26 mTmG/+ ; Hprt CAG::TBX2/y hearts at this stage (Fig 5D, S4 Fig). Together, this suggests that homogenous epicardial misexpression of human TBX2 does not impair formation, migration and differentiation of the epicardial and epicardium-derived cells. Although ectopic expression of TAGLN in epicardial and epicardium-derived cells of Tbx18 cre/+ ;R26 mTmG/+ ;Hprt CAG::TBX2/y hearts was observed, the differentiation potential of those cells was maintained.
Histological stainings of transverse sections of Tbx18 cre/cre ;Tbx20 LacZ/+ hearts revealed normal chamber architecture and septa formation but pericardial defects similar to Tbx18-null mice (black arrows in Fig 6A). Higher magnification of the right ventricular epicardium revealed a monolayer of flattened cells covering the myocardium in all mutants; a thin subepicardial space was formed in Tbx18 cre/cre ;Tbx20 LacZ/+ hearts as in Tbx18 cre/LacZ and control hearts (Fig 6B). Blood filled vessels visualized by the presence of eosin-positive red blood cells grew into the subepicardial space in all mutants. Section in situ hybridization of transverse sections against Aldh1a2 confirmed the structural integrity of the epicardium (Fig 6C), and immunofluorescence of EMCN expression demonstrated that the coronary plexus was recruited correctly (Fig 6D). Both findings indicate that the epicardial-myocardial crosstalk is undisturbed. Immunofluorescence of the lineage label GFP and the epicardial marker WT1 further proved the formation of a complete epicardium that was tightly attached to the surface of the heart (Fig 6D). Epicardium-derived cells were present in the subepicardium and myocardium in mutants as in controls as visualized by expression of GFP and/or WT1. The amount of GFPand WT1-positive cells entering the myocardium and their distribution between subepicardial and myocardial compartments was unaltered indicating proper EMT and immigration patterns in Tbx18 cre/cre ;Tbx20 LacZ/+ , Tbx18 cre/LacZ , Tbx18 cre/+ ;Tbx20 LacZ/+ and control hearts ( Fig  6D). Expression of TAGLN was restricted to the subepicardial space in Tbx18 cre/cre ;Tbx20 LacZ/+ hearts (Fig 6D) arguing against premature SMC differentiation of epicardial cells. Expression of NOTCH3 was observed in some epicardial cells of Tbx18-null mutant embryos but was not enhanced by the additional loss of Tbx20 (S2 Fig). Hence, the loss of one Tbx20 allele does not alter epicardial development in wild-type or Tbx18-deficient mice.

Discussion
Here, we found that Tbx2, Tbx20 and Tbx5 are co-expressed with Tbx18 at different stages of epicardial development. Our genetic experiments showed that Tbx2 is dispensable for epicardial development, and that neither Tbx2 nor Tbx20 redundantly interact with Tbx18 in any of the subprograms important for formation, migration and differentiation of epicardial cells in mouse embryos.

Tbx18 and combinatorial interaction with Tbx genes in epicardial development
Our previous analysis demonstrated that loss of Tbx18 does not affect epicardial function whereas epicardial-specific misexpression of an activator version of TBX18 (TBX18VP16) led to premature SMC differentiation of epicardial cells [35]. This suggested that TBX18 possibly together with a related transcriptional repressor maintains the precursor character of epicardial cells by repressing SMC differentiation. Our sensitive PCR and in situ hybridization methods detected expression of three additional cardiac Tbx genes, Tbx5, Tbx20 and Tbx2 in the proepicardium and epicardium. Expression of Tbx5 was detected in the E9.5 proepicardium, and in the E10.5-11.5 epicardium but was subsequently down-regulated. A recent report found a similar pattern of expression both on the level of Tbx5 mRNA and TBX5 protein, and uncovered that epicardial-specific loss of Tbx5 is associated with blebbing and reduced EMT but not with premature SMC differentiation of epicardial cells. Augmented expression of Tbx5 in the proepicardium led to reduced proepicardial migration and enhanced apoptosis in chick embryos [30]. Together, this suggests that precise levels of Tbx5 are important for formation and To date, TBX5 has only been characterized as a transcriptional activator in cardiac development [30,53] whereas a Groucho-dependent role as a transcriptional repressor was assigned to TBX18 [36]. Although it cannot be formally excluded that both factors behave in a biochemically equivalent fashion (as activators or repressors) in epicardial development, the phenotypic differences between Tbx5-and Tbx18-deficient embryos argue against a redundant function. Given our earlier finding that TBX18 can compete with TBX5 for DNA-binding sites in vitro [36], it cannot be excluded, however, that TBX18 is important to fine-tune the transcriptional responses to TBX5 by selectively repressing some of the TBX5 target genes. More detailed transcriptional profiling of Tbx5-and Tbx18-deficient (pro-)epicardial cells may help to test such a scenario.
We also detected expression of Tbx20 in the proepicardium and early epicardium albeit unambiguous assignment to this tissue was hampered by strong myocardial expression of the gene. Tbx20-deficient embryos form a proepicardium but an analysis of subsequent epicardial development is impossible due to the lethality at E9.5 [20]. Mice heterozygous for a Tbx20 null allele survive into adulthood with diverse cardiac pathologies, including defects of septation and valvulogenesis and cardiomyopathy that however, do not correspond to an epicardial requirement of the gene [22,54]. Here, we have shown that mice compound mutant for Tbx18 and Tbx20 do not exhibit defects in epicardial development. Despite the fact that TBX20 can act as a repressor in developing and mature hearts [22,51,52,55], these genetic findings argue that Tbx20 neither on its own nor in combination with Tbx18 plays an essential role in the development of the epicardium. Since TBX20 can also act as a transcriptional activator [55], it may cooperate with TBX5 in regulating proepicardial and early epicardial development. Such a possibility may be addressed by proepicardial-specific deletion of both Tbx5 and Tbx20 in the mouse.
Finally, we detected expression of Tbx2 (but not of the closely related Tbx3 gene) in a subset of proepicardial and epicardial cells in the developing mouse heart. Since Tbx2 like Tbx18 encodes a strong transcriptional repressor [36,46], we focused our genetic studies on the role of Tbx2 and its possible redundancy with Tbx18 in epicardial development. However, neither embryos with an epicardial deletion of Tbx2 nor with combined deficiency of Tbx18 and Tbx2 exhibited epicardial defects, strongly arguing against an individual or combined role for Tbx2 in epicardial development. Similar to Tbx18 [35], misexpression of TBX2 in the epicardial lineage did not affect epicardial function nor did it prevent SMC differentiation of epicardiumderived cells. Both for TBX2 and TBX18, this may reflect the lack of cofactors necessary to exert this function. Since TBX2 (and TBX3) have been characterized as strong competitive inhibitors of TBX5-activated gene programs in the atrioventricular canal and outflow tract [26,56], the possibility again exists, that TBX2 represses TBX5 target genes in individual proepicardial and epicardial cells.
While TBX5, TBX20 and TBX2 do not hold promise as cooperation partners of TBX18 in maintenance of epicardial integrity, complex dose-dependent antagonistic and synergistic interactions between the different TBX family members may exist that play an important role in generating a molecular heterogeneity in fate decisions in proepicardial and epicardial cells as in other developmental contexts [57]. Other T-box proteins that have not yet been associated with cardiac development might additionally be expressed in the epicardium to feed into this regulatory network. cells in Tbx18 cre/LacZ and Tbx18 cre/cre ;Tbx20 LacZ/+ mutants whereas none are observed in the depicted sections of Tbx20 LacZ/+ or Tbx18 cre/+ ;Tbx20 LacZ/+ mutants. These results are confirmed by double immunofluorescence against NOTCH3 and COLIV. White arrowheads point toward NOTCH3-positive cells within the epicardium. Scale bars are 50

TBX18 function in repression of SMC differentiation may depend on additional cofactors
While the identity of a TBX protein acting redundantly with TBX18 in repression of SMC differentiation of epicardial cells remains enigmatic, the maintenance of epicardial precursor character in Tbx18-deficient embryos may also reflect lack of an activator of the SMC program in these cells. Interestingly, Wu and colleagues recently analyzed the potential of Tbx18 to inhibit a SMC differentiation program in vitro. They used C3H10T1/2 cells, multipotent mesenchymal progenitors that initiate the SMC pathway when exposed to TGFß1, and showed that cells transfected with a Tbx18 expression vector exhibit a markedly reduced expression of SMC markers. This result was not due to increased apoptosis or reduced proliferation indicating that TBX18 is indeed able to suppress a SMC differentiation pathway. Moreover, they found in transactivation assays that TBX18 inhibits Serum response factor (SRF)-CArGbox dependent activation of the promotors of the SMC-specific genes Tagln, Fos and Actg2 [37]. Lack of SMC differentiation in Tbx18-deficient epicardial cells may therefore simply reflect the lack of expression of the master activators of the SMC program, Myocardin (Myocd) and/or Srf [58] in the epicardium. TBX18VP16, an activator version of TBX18, on the other hand may be able to activate target genes usually repressed by TBX18 independently of co-factors leading to premature differentiation of the epicardium into SMCs [35].
It is worth to note that NOTCH3 expression was upregulated in epicardial cells overexpressing TBX18VP16 as well as in cells of Tbx18-null epicardia. Notch3 contains functional T-sites in the zebrafish [59] suggesting that Notch3 expression is activated independently from MYOCD/SRF but is directly repressed by TBX18. Although epicardial overexpression of the intracellular fragment of NOTCH is sufficient to induce SMC differentiation [9], lack of the appropriate ligand in Tbx18-deficient epicardial cells will prevent activation of Notch3 and induction of the SMC pathway. Tbx2 or Tbx20. In order to quantify NOTCH3-expressing epicardial cells, immunofluorescent stainings against NOTCH3 were analyzed. (A) Quantification of NOTCH3-positive epicardial cells in Tbx18 cre/GFP ;Tbx2 fl/fl (70.0±4.4%), Tbx18 cre/GFP ;Tbx2 fl/+ (67.9±8.2%), Tbx18 cre/+ ;Tbx2 fl/fl (52.2±13.8%) and Tbx18 cre/+ hearts. Two specimens of each genotype were analyzed and the ratio of NOTCH3-positive cells within the right ventricular epicardium was determined and displayed as percentage. Error bars indicate the standard deviation. (B) Ratio of NOTCH3-expressing cells within the epicardium of Tbx18 cre/cre ;Tbx20 LacZ/+ (74.3±10.4%, n = 3), Tbx18 cre/LacZ (82.9±9.0%, n = 5), Tbx18 cre/+ ;Tbx20 LacZ/+ (61.7±5.3%, n = 2) mutant hearts and Tbx18 cre/+ (51.2±14.2%, n = 6) as well as Tbx20 LacZ/+ (55.0±11.8%, n = 2) control hearts is displayed as percentage. Number of specimens per genotype as indicated and error bars represent the standard deviation. (C) Direct comparison of the ratio of NOTCH3-positive cells in Tbx18 cre/LacZ (82.9±9.0%) and Tbx18 cre/+ (54.5±13.0%) mutants. Five specimens per genotype were analyzed and the standard deviation blotted as error bar. Student's t-test confirmed the significance of these results. Epicardial cells stained for the lineage label GFP enter the myocardium of Tbx18 cre/+ ; R26 mTmG/+ ;Hprt CAG::TBX2/y hearts as in Tbx18 cre/+ ;R26 mTmG/+ ;Hprt +/+ controls and contribute to coronary SMCs as indicated by double-immunofluorescence staining against the SMC marker proteins ACTA2 or TAGLN and GFP. Besides, intermyocardial epicardium-derived cells express TAGLN but not ACTA2 in Tbx18 cre/+ ;R26 mTmG/+ ;Hprt CAG::TBX2/y hearts which was not observed in control hearts at E18.5 (red arrowheads). As in control hearts, epicardium-derived cells of mutant hearts contribute partially to cardiac fibroblasts as indicated by double staining for the epicardial lineage marker GFP and the fibroblast marker protein POSTN. The arrows point towards interstitial epicardium-derived fibroblasts whereas the arrowheads mark coronary fibroblasts derived from the epicardium. The number of analyzed specimen is two and the error bar represents 40 μm. CAr, coronary artery. (TIF) S1 Table. RT-PCR primer and conditions. The listed RT-PCR primer and conditions were used for qualitative RT-PCR experiments. (XLSX)