In embryonic development, EMT occurs when epithelial cells become migratory, invade new microenvironments and acquire mesenchymal characteristics such as elongated morphology, rearranged actin cytoskeleton, increased production of lytic enzymes, loss of apical/basal polarity, and junction organization (Thiery, 2002
During heart development a subset of endocardial cells, located in the AV and cono-ventricular regions, detaches from the endocardial sheet and invades the underlying cardiac jelly to form the endocardial cushions, which give rise to valves and septa. This appears to be a typical EMT of a subset of endocardial cells. TGFβ plays an important role in this phenomenon but the molecular mechanisms controlling the process have only been partially clarified.
In this paper, we provide evidence that induction of EMT in the endocardial cells requires β-catenin transcriptional activity. The requirement for β-catenin was demonstrated by the observation that in mice, in which the β-catenin gene was selectively inactivated in endothelial/endocardial cells, the heart cushion fails to develop. In these embryos, endocardial cells do not undergo EMT. This was demonstrated in vivo by a strong reduction of cellularity in the cardiac jelly and ex vivo in AV explants by the failure of endocardial cells to acquire mesenchymal characteristics and to invade the collagen gel. In addition, cultured endothelial cells deficient for β-catenin exhibit an impaired ability to undergo transformation and to express αSMA upon stimulation with TGFβ2.
These defects are most likely due to a lack of β-catenin transcriptional activity because in reporter mice, cells invading the cardiac jelly or growing out into the collagen gel in AV explants, express the reporter gene LacZ.
These results are in agreement with a recent publication by Hurlstone et al. (2003)
in zebrafish, showing that a lack of function mutation in the adenomatous polyposis coli
gene, leads to increased β-catenin signaling and results in excessive and misplaced endocardial cushion formation. Conversely, overexpression of adenomatous polyposis coli
or inhibition of Wnt by Dickkopf 1 inhibited this phenomenon. These findings support the idea of a prominent role of the Wnt/β-catenin pathway in determining endocardial cell fate.
Interestingly, in vivo and in the AV explants only a limited number of cells were β-galactosidase positive suggesting that activation of β-catenin transcriptional activity is a transient phenomenon, which likely follows a specific temporal pattern. We demonstrated that single cells expressed both endothelial markers and β-galactosidase in explants from BAT-gal reporter mice. This suggests that β-catenin and/or its transcriptional activity likely acts at early/intermediate steps of cell transformation when cells have not yet acquired full mesenchymal characteristics, while losing endothelial characteristics.
Using explants from ROSA26 mice crossed with Tie2-Cre transgenics, in which Tie2-Cre induces LacZ expression in endothelial cells in an irreversible way, we demonstrated that virtually all cells in AV explants, undergoing EMT are of endothelial origin. These data fully support previous work in vivo on mouse heart development (Kisanuki et al., 2001
In cultured cells, TGFβ2 induced αSMA expression in the endothelium in a β-catenin–dependent way. αSMA is the most frequently used marker of EMT in the endocardium (Paranya et al., 2001
) and, with the limitations of a culture system, these results support the data in vivo and in organ culture. In addition, the observations on cultured cells suggest that the effect of TGFβ2 is cell-autonomous, not requiring the interaction with other contiguous cells types present in vivo. Our data support a model in which both β-catenin/TCF/Lef transcriptional activity and TGFβ signaling are required for EMT, but whether these two pathways directly interact, or whether one or the other is upstream, remains to be elucidated. Interestingly, from the data reported here, TGFβ signaling through smads is not significantly altered by the absence of β-catenin, arguing in favor of a role of β-catenin in parallel or downstream of smads. It has been reported that members of the Wnt-signaling pathway like β-catenin and TCF/Lef can independently interact with smad2, smad3, and smad4 (Labbe et al., 2000
; Nishita et al., 2000
; Tian and Phillips, 2002
). Nishita et al. (2000)
and Labbe et al. (2000)
also show that during development of Xenopus laevis
a number of target genes exhibit responsiveness to both Wnt and TGFβ signals.
A recent paper demonstrates that TGFβ3 can up-regulate Lef-1 transcription during EMT in mouse palatal development (Nawshad and Hay, 2003
). When smad2 and smad4 are present in the nucleus, Lef-1 is activated without β-catenin. Smad2/4 would therefore interact preferentially with Lef-1 if β-catenin is absent. However, Nawshad and Hay (2003)
found that palatal EMT would be promoted in the absence of β-catenin, likely by a Lef–smad2 complex, which is neither the case in the present work in mouse, nor in a zebrafish model (Hurlstone et al., 2003
). Therefore, it is conceivable that the mechanism through which TGFβ induces EMT in endocardial cells is not identical to those in the palatal epithelium. Most likely, this might depend on the endothelial-specific expression of TGFβ and/or Wnt receptors.
Other signaling pathways, like EGF, VEGF, and BMP, have been implicated in heart cushion/valve formation (Brown et al., 1999
; Dor et al., 2001
; Kim et al., 2001
; Iwamoto et al., 2003
). It was recently reported that also Notch activity promotes EMT during cardiac development via transcriptional induction of the snail1 repressor, which facilitates VE-cadherin down-regulation (Noseda et al., 2004
; Timmerman et al., 2004
). The authors, using endothelial cells induced to transform by activated Notch, could not find evidence for TGFβ activation. This suggests that Notch may act independently from TGFβ. To understand if in endothelial cells genes of the Notch signaling pathway are altered in the absence of β-catenin, we analyzed the expression of Notch1, delta-like 4, but we could not find any significant regulation neither in vivo, nor in vitro (). Interestingly, upon stimulation with TGFβ2, snail1 is equally up-regulated in cells with and without β-catenin, suggesting on the one hand that β-catenin is not upstream of snail1, on the other hand that snail1 expression is not sufficient to promote control levels of EMT in the absence of β-catenin. As a late target of EMT, protein expression levels of VE-cadherin were analyzed, but we could not find any significant regulation neither in vivo, nor in vitro, between WT and KO (). However, upon TGFβ2 stimulation of cells in vitro, only a slight decrease of protein levels for VE-cadherin could be detected in both cell lines ( B). This observation is supported by immunostaining, in which the majority of cells positive for αSMA also express VE-cadherin ( C). Overall these data suggest that β-catenin may act to a large extent at early stages of EMT, before VE-cadherin down-regulation.
However, keeping in mind that cadherins may indirectly regulate the free pool of β-catenin (Gumbiner, 1995
; Nelson and Nusse, 2004
), we cannot exclude that the slight down-regulation of VE-cadherin observed upon TGFβ2 might contribute to β-catenin signaling level, thus providing a possible link between Wnt and TGFβ signaling.
In conclusion, endocardial EMT leading to cell invasion of the cardiac jelly is a complex phenomenon, in which different signaling pathways may interplay. There is growing evidence from different in vivo and in vitro systems that vascular endothelial cells may acquire smooth muscle cell markers and can contribute to vessel wall formation during development as well as during pathological conditions, such as artherosclerosis and restenosis (DeRuiter et al., 1997
; Frid et al., 2002
; Iurlaro et al., 2003
; Yurugi-Kobayashi et al., 2003
). Therefore, the comprehension of the mechanisms underlying endothelial/endocardial transformation will be crucial not only for the understanding of diseases like congenital heart defects, but also of artherosclerotic degenerations of the vessel wall.