Since the discovery of the origin of cVSMC and cardiac fibroblasts over 18 years ago, multiple signaling pathways have been identified that affect the formation, attachment, or EMT of the epicardium. However, few genes have been identified that are essential for cardiac fibroblast formation. Here, we have not only identified a unique requirement for PDGF receptor signaling in regulating epicardial EMT and EPDC formation but also have identified an essential role for, PDGFRα, specifically in cardiac fibroblast formation.
Several growth factor signaling pathways have been implicated in the induction of EMT during development and various pathological states, and PDGF has been linked to the EMT process during cancer progression, organ fibrosis48–50
, smooth muscle cell generation from the chicken proepicardium51
, and in the regenerating zebrafish heart52
. Some suggested mechanisms for PDGF’s role in EMT include stabilization of βcatenin53
or induction of transcriptional activators of EMT, such as ZEB1/2 and Snai254
. However, we do not observe these specific effects in epicardial cells after PDGF stimulation. We have identified the transcription factor, Sox9, as a downstream target of PDGF stimulated EMT. Our data show that Sox9 could rescue the EMT defect seen in PDGF receptor mutants and that Sox9 expression was up-regulated upon PDGF stimulation of epicardial cells. These data are consistent with the known role for Sox9 in avian neural crest cell EMT27
. Sox9 is a member of the SRY related HMG-box family of transcription factors that is important for the development of many tissues and cell types. Cardiovascular defects have been reported in Sox9
, but no epicardial phenotype has been described. Our data suggest that Sox9 could be an important component of PDGF receptor signaling during epicardial EMT, but further investigation is necessary to determine the mechanistic link between PDGF and Sox9.
Our data show that loss of both PDGF receptors led to defective EMT and failure to form any epicardial derivatives. Interestingly, individual deletion of the PDGF receptors also led to reduced epicardial EMT and a loss of only a subpopulation of EPDCs. There are two potential scenarios to explain when PDGF receptor function is required. In the first scenario, epicardial cells are heterogeneous, and each epicardial cell would only give rise to a specific lineage of EPDCs, either VSMC or fibroblast. Here, PDGF signaling might regulate a lineage specific EMT. Consistent with this possibility, experiments using limiting amounts of retrovirus to transduce the proepicardial organ57
revealed that tagged cells contributed only to the VSMC lineage. However, there have been no reports suggesting differential gene expression in the epicardium. For example, Tbx18, Tcf21, Raldh2, and both of the PDGF receptors seem to be uniformly expressed in the epicardium prior to EMT. A second possibility for PDGF function is that PDGF signaling by each receptor is redundant in regards to the EMT process, but after EMT, PDGFRα is expressed in fibroblast progenitors, while PDGFRβ is in cVSMC. In this scenario, the most likely role for PDGF receptor signal transduction is expansion and migration of the progenitor population17, 58
. These unanswered questions require further investigation using temporal deletion to identify the window of epicardial fate specification.
The role of cardiac fibroblasts in heart pathogenesis is well appreciated, but the function of these cells during development is poorly understood. It has been proposed that cardiac fibroblasts perform a variety of essential duties during heart formation. These include stimulation of cardiomyocyte proliferation43
, isolation of the ventricular from the atrial conduction system59
, distribution of mechanical forces60
, and, of course, deposition and degradation of ECM. Recent estimates are that cardiac fibroblasts comprise about 27% of the cells within the murine heart61
, but our data demonstrates that these cells are dispensable for heart development. Under non-pathological conditions, mice without epicardial derived fibroblasts lack adventitial collagen, but heart function is normal.
Because many cell populations have been proposed to contribute to fibrosis formation during pathological circumstances, there is the possibility that another source of fibroblasts fills the void. Proposed origins for this substitute fibroblast population include endothelial cells, fibrocytes, monocytes, and mural cells60
. A complete functional substitution by these cells in the absence of epicardial derived fibroblasts is unlikely as mutant hearts that were 8–10 weeks old continued to lack adventitial ECM. Comparison of PDGFRαEKO
hearts did suggest a partial rescue of the ECM, presumably by the existing cVSMC, although the levels of matrix never appear to reach wild type levels. While our initial examination suggests no major deficits in mutant animals, further studies are warranted to investigate cardiac homeostasis and other functional parameters such as conduction and stress response.
In summary, we demonstrate that cardiac fibroblast and cVSMC development is mediated by a combined role of the PDGF receptors in controlling epicardial EMT. This process appears to be linked to a lineage specific requirement of the receptors. Specifically, PDGFRα, is essential for cardiac fibroblast development. Finally, we establish a novel role for Sox9 as a critical downstream component of PDGF signaling in regulating epicardial EMT.
Novelty and Significance
What is known?
- Cardiac fibroblasts and coronary vascular smooth muscle cells (cVSMC) are epicardial derived cells (EPDC) that arise after an epithelial-to-mesenchymal transition (EMT).
- Platelet derived growth factor receptor (PDGFR) β is required for coronary vascular smooth muscle cell development.
- PDGF (Platelet derived growth factor) signaling plays a role in coronary vessel remodeling during cardiac zebrafish regeneration.
What new information does this article contribute?
- PDGF receptors are required for epicardial EMT and failure of this process leads to animals lacking EPDCs.
- Animals lacking EPDCs have no overt phenotype and are viable.
- PDGFRα is the first receptor identified to be required for cardiac fibroblast formation, but PDGFRα is dispensable for cVSMC development.
- The PDGF receptor genes are required in a lineage specific manner for the formation of the two EPDC cell populations, cVSMC and cardiac fibroblasts.
Cardiac fibrosis is a major consequence of long-term cardiac disease, and the epicardium is the major source of resident cardiac fibroblasts that potentially contribute to this disease. Here, we report that PDGF receptor signaling is required for epicardial EMT. Expression of the transcription factor, Sox9, is reduced in epicardial cells lacking PDGF receptors, and expression of Sox9 rescues EMT in the absence of PDGF receptor signaling. We also report that disruption of the epicardial EMT process leads to the inability to generate cardiac fibroblasts and cVSMC. Additionally, the loss of PDGFRα leads to a defect exclusively in cardiac fibroblast formation. This work is the first example of a lineage specific disruption of epicardial derivatives. Our findings show a previously unidentified role for PDGF receptor signaling in epicardial EMT and EPDC fate specification and provide a novel model to investigate the role of cardiac fibroblasts during embryogenesis as well as in the adult.