Here, we describe expression of Scx
, two markers of proepicardial and epicardial development. Our in situ
and immunohistochemical characterization shows that each of these markers identifies a population of cells largely distinct from Tbx18
. These results indicate that, based on gene expression, there are several molecularly distinguishable cell populations within the developing PEO. We next used recombinase based fate mapping under the control of two independent Cre driver mouse lines to study the cell types derived from the Scx
expressing PEO cells. The differences in cell fates between the Tbx18
and WT-1, Scx
, and Sema3D
cells indicate that these represent distinct, functionally significant, subpopulations. Moreover, the finding that both the Scx
populations contribute to the endothelial lineage reconciles previous chick and mouse fate mapping data that has until now been contradictory. In particular, studies in avian species have suggested that proepicardial cells may give rise to endothelial cells (Gittenberger-de Groot et al., 1998
; Reese et al., 2002
) but the previous fate maps in mice have failed to confirm this data (Cai et al., 2008
; Zhou et al., 2008
). This seeming disparity arose because prior avian studies, by the techniques utilized, labeled cells throughout the proepicardium while the previous murine studies focused on what we now conclude to be restricted sub-populations that provide little or no endothelial contribution.
The cardiac endocardium and the venous endothelium of the sinus venosus could potentially represent independent sources of coronary endothelial cells to those identified in our studies. However, our fate mapping data indicate that by E10.5 Sema3D
lineage traced cells contribute to the sinus venosus, while Scx
lineage traced cells contribute to the cardiac endocardium by E11 in the absence of any active expression of either of these markers within these tissues. Previous studies have timed the contribution from the sinus venosus beginning primarily at E11.5 and that of the endocardium at E12.5 and later (Red-Horse et al., 2010
), 24-48 hours later than Scx
proepicardial cells have begun to contribute to these tissues. Therefore, our data are consistent with the possibility that some Scx/Sema3D
precursors traverse through the sinus venosus endothelium en route to the heart and/or transiently contribute to the endocardium before entering the coronary vascular endothelial lineage. It is possible that previous studies of the sinus venosus and endocardium may have unknowingly included cells that originate in the proepicardium while inadvertently overlooking proepicardial derivatives that failed to express Tbx18
. At the same time, our observation that Scx
lineage traced cells express endothelial markers on the epicardial surface of the heart at E11, prior to any sinus venosus contribution, suggests that some coronary endothelium derived from the proepicardium arises via traditional routes of proepicardial migration (Cosette and Misra, 2011
; Hiruma and Hirakow, 1989
; Komiyama et al., 1987
; Nahirney et al., 2003
; Viragh and Challice, 1981
In any recombinase-based fate mapping strategy, the conclusion reached depends upon the assumption that Cre is expressed only in the tissue being mapped. The possibility always remains that there is leaky expression of Cre below detectable levels in other locations relevant to the mapping, in our case within the heart itself or alternative tissues that contribute to the heart. Despite careful expression analysis by ISH, IHC, and qPCR, we cannot rule out the formal possibility that Sema3D or Scx are expressed at sub-detectable, yet nonetheless significant, levels within relevant tissues. Additionally, at later time points, both lines express in other tissues within the embryo and we cannot technically eliminate the possibility that some other source of Scx or Sema3D expressing cells gives rise to a population in the heart. However, the pattern of expression that we observe in innumerable sections from staged embryos strongly suggests an origin from the epicardial surface with subsequent epicardial-to-endocardial migration. If the cells originated in pharyngeal mesenchyme, for example (as do second heart progenitors or those expressing Isl1Cre or Mef2c-AHF-Cre), then they would be expected to migrate to the ventricles via the anterior or posterior poles of the heart, and we would first identify these indelibly labeled cells in the outflow or inflow tracts. But this is not the pattern that we see. If the labeled cells originated via the circulation from a distant site, then we would expect to see them first on the endocardial surface, or adjacent to coronary vessels, but we do not. The earliest endothelial cells that we mark are first noted directly on the epicardial surface of the heart at E11 and derive from lineage traced epicardial cells. For these reasons, we believe our data strongly supports the conclusion that the labeled cells arise from the PEO.
Because an endothelial lineage had not previously been reported to arise from the PEO in mice, we sought to verify the competence of murine PEO cells to contribute to coronary endothelium in two ways: mouse to chick proepicardial transplants as well as an in vitro cell culture assay. In both experiments we utilized PEO cells that actively express Scx or Sema3D at E9.5, hence in each case all endothelial cells that arose must have originated from E9.5 mouse PEO cells. These experiments confirm the competence of PEO lineage traced cells to give rise to endothelial cells both in vivo and in vitro. Furthermore, mouse to chick proepicardial transplants utilizing WT-1 expressing cells failed to give rise to endothelial cells but were able to give rise to smooth muscle cells as marked by smooth muscle markers. This important control supports the notion that the PEO is comprised of genetically distinct subcompartments that differ in their downstream fates.
In addition to observing endothelial cells in our in vitro cultures arising from the ScxGFP expressing fractions, we also saw smaller numbers of endothelial cells in cultures of GFP- cells. A likely explanation is that, given that the Scx and Sema3D expressing PEO populations only partially overlap, the endothelial cells in the GFP- fraction arise from the Sema3D cells that were excluded during FACS. However, our results do not rule out the possibility that there exist additional, as yet uncharacterized, subcompartments of the PEO that also have the potential to give rise to endothelial cells. It is also possible that known proepicardial populations (such as Tbx18 or WT-1) are not restricted from the endothelial lineage while in the PEO or in culture though their descendants become restricted from the endothelial lineage in vivo.
These studies demonstrate and begin to define complex inhomogeneous populations of cardiac precursor cells within the PEO. These genetically distinct subcompartments differ in both the routes and the timing of their migration and also give rise to distinct albeit overlapping cell fates, including contributions to the vascular endothelium. Our results establish the complexity of the PEO as a source of multiple progenitor populations while simultaneously offering a more complete understanding of the diversity of tissues that give rise to the coronary vascular endothelium.