Final Report Summary - WOUNDHEALINGEGF (The role of EGF/ERK signaling pathway during the wound healing response in Drosophila and zebrafish epithelia)
Epithelia act as physical barriers that protect living organisms from the surrounding environment. The organisation and homeostasis in epithelia requires robust mechanisms that assure their integrity in a variety of biological situations, such as normal cell turnover, inflammation and injury. Simple epithelial tissues, such as those of a developing embryo, have the extraordinary capacity to resolve wounds in a rapid and efficient manner by means of a resealing mechanism. Remarkably, this process occurs without scar formation and inflammatory response, in contrast to adult wounds. The molecular machinery that controls this process seems to be conserved across species. It involves dramatic cellular rearrangements, which together with the assembly of a contractile supracellular cable formed by actin and myosin, lead to resealing of the epithelium.
Albeit the importance of studying epithelial wound healing, the current knowledge of this process comes mostly from in vitro studies. Only in recent years more integrated and multidisciplinary approaches have started to be used. The key cellular events and signalling pathways that regulate this process have now started to be identified, but many open questions still remain about how they are integrated and eventually lead to the closure of an epithelial hole.
Thus, the main goal of this project was to uncover the molecular mechanisms that regulate the epithelial wound response in the context of a developing organism. For this we mainly used the simple epithelium of the Drosophila embryo. This model system allowed us to combine advanced live imaging techniques and powerful genetic tools in order to answer our questions. Furthermore, we took advantage of a laser-wounding assay established by our lab that allowed us to precisely ablate cells in the embryonic epithelium.
Recent studies have shown that extracellular-regulated kinase (ERK) is strongly activated upon wounding in the Drosophila embryonic epithelium. However, it is still unclear how activation of this pathway contributes to a proper wound response and what are the molecular mechanisms that regulate its activation. Our first goal was to investigate the function of the ERK pathway during wound healing in Drosophila. We found that ERK activation is essential for proper wound healing to occur. The epidermal growth factor receptor (EGFR) is also required for this process, suggesting that the EGFR/ERK pathway is one of the first pathways being activated upon injury. We have also characterised a novel gene, which we named hole-in-one (holn1) that is required for a proper wound healing response and for the induction of several wound response genes at the wound site. These wound response genes are known to be regulated by ERK, suggesting that Holn1 acts in the same pathway. We also found that Holn1 may be involved in the Ras/ERK signalling pathway by acting downstream of Ras and ERK. In addition, considering that the mammalian homologue of Holn1 is involved in pre-mRNA splicing, we propose a model whereby Holn1 contributes to efficient wound healing by acting in the nucleus to promote the splicing of components involved in the ERK pathway.
In the second part of this project, we aimed at determining what regulates wound closure at the cellular level, in particular at its early stages. Here, we hypothesised that this early response is mediated by cell-cell junctions, which are important for the transmission of mechanical forces between cells. We mainly focused on the role septate junctions (SJs), the functional analogues of vertebrate tight junctions (TJs), as we had evidence from our previous work that this type of junctions are involved in wound closure both in Drosophila and in zebrafish embryos. SJs and TJs are essential structures for the maintenance of the epithelial barrier function by sealing intercellular spaces and simultaneously allowing selective permeability of an epithelium. In vertebrates, TJ components have been linked to pathological conditions, such as cancer and inflammatory bowel diseases, constituting current targets for drug development. Studies of Drosophila embryos have also revealed that SJs regulate cell shape changes and adhesion during embryonic development independently of their barrier function. Surprisingly, the potential function of SJs, and also of vertebrate TJs in wound healing has never been addressed. Therefore, the main goal of this task was to determine whether SJs are important during wound healing in Drosophila. We found that the localisation of these junctions during wound closure is dynamic, as they are excluded from the leading edge of the wound shortly after wounding and only recover after the hole is closed. Furthermore, we discovered that Ly6 family proteins, which are known to be involved in the formation of SJs, are essential for wound healing to occur. These results suggest that SJs need to be remodelled during wounding closure. In addition, by detailed mutant analysis, we identified several key components of SJs that are crucial for wound healing. In conclusion, we discovered that SJs are essential structures for wound healing in Drosophila epithelia. Altogether, these results constitute a major step in the understanding of epithelial repair and SJ function. In the future, we plan to further explore the cellular mechanisms by which SJs regulate wound closure and how the assembly of these junctions is controlled.
We have also started to investigate to what extent these molecular mechanisms are conserved in a vertebrate model, the zebrafish embryonic epithelium. Our data suggests that the ERK pathway is also activated upon wounding in zebrafish. In addition, work in our lab indicates that TJs are important for wound healing in the zebrafish embryo. Therefore, this project has provided us with strong clues about the essential mechanisms that regulate epithelial wound healing, establishing a solid framework for future studies and potential clinical applications.
Albeit the importance of studying epithelial wound healing, the current knowledge of this process comes mostly from in vitro studies. Only in recent years more integrated and multidisciplinary approaches have started to be used. The key cellular events and signalling pathways that regulate this process have now started to be identified, but many open questions still remain about how they are integrated and eventually lead to the closure of an epithelial hole.
Thus, the main goal of this project was to uncover the molecular mechanisms that regulate the epithelial wound response in the context of a developing organism. For this we mainly used the simple epithelium of the Drosophila embryo. This model system allowed us to combine advanced live imaging techniques and powerful genetic tools in order to answer our questions. Furthermore, we took advantage of a laser-wounding assay established by our lab that allowed us to precisely ablate cells in the embryonic epithelium.
Recent studies have shown that extracellular-regulated kinase (ERK) is strongly activated upon wounding in the Drosophila embryonic epithelium. However, it is still unclear how activation of this pathway contributes to a proper wound response and what are the molecular mechanisms that regulate its activation. Our first goal was to investigate the function of the ERK pathway during wound healing in Drosophila. We found that ERK activation is essential for proper wound healing to occur. The epidermal growth factor receptor (EGFR) is also required for this process, suggesting that the EGFR/ERK pathway is one of the first pathways being activated upon injury. We have also characterised a novel gene, which we named hole-in-one (holn1) that is required for a proper wound healing response and for the induction of several wound response genes at the wound site. These wound response genes are known to be regulated by ERK, suggesting that Holn1 acts in the same pathway. We also found that Holn1 may be involved in the Ras/ERK signalling pathway by acting downstream of Ras and ERK. In addition, considering that the mammalian homologue of Holn1 is involved in pre-mRNA splicing, we propose a model whereby Holn1 contributes to efficient wound healing by acting in the nucleus to promote the splicing of components involved in the ERK pathway.
In the second part of this project, we aimed at determining what regulates wound closure at the cellular level, in particular at its early stages. Here, we hypothesised that this early response is mediated by cell-cell junctions, which are important for the transmission of mechanical forces between cells. We mainly focused on the role septate junctions (SJs), the functional analogues of vertebrate tight junctions (TJs), as we had evidence from our previous work that this type of junctions are involved in wound closure both in Drosophila and in zebrafish embryos. SJs and TJs are essential structures for the maintenance of the epithelial barrier function by sealing intercellular spaces and simultaneously allowing selective permeability of an epithelium. In vertebrates, TJ components have been linked to pathological conditions, such as cancer and inflammatory bowel diseases, constituting current targets for drug development. Studies of Drosophila embryos have also revealed that SJs regulate cell shape changes and adhesion during embryonic development independently of their barrier function. Surprisingly, the potential function of SJs, and also of vertebrate TJs in wound healing has never been addressed. Therefore, the main goal of this task was to determine whether SJs are important during wound healing in Drosophila. We found that the localisation of these junctions during wound closure is dynamic, as they are excluded from the leading edge of the wound shortly after wounding and only recover after the hole is closed. Furthermore, we discovered that Ly6 family proteins, which are known to be involved in the formation of SJs, are essential for wound healing to occur. These results suggest that SJs need to be remodelled during wounding closure. In addition, by detailed mutant analysis, we identified several key components of SJs that are crucial for wound healing. In conclusion, we discovered that SJs are essential structures for wound healing in Drosophila epithelia. Altogether, these results constitute a major step in the understanding of epithelial repair and SJ function. In the future, we plan to further explore the cellular mechanisms by which SJs regulate wound closure and how the assembly of these junctions is controlled.
We have also started to investigate to what extent these molecular mechanisms are conserved in a vertebrate model, the zebrafish embryonic epithelium. Our data suggests that the ERK pathway is also activated upon wounding in zebrafish. In addition, work in our lab indicates that TJs are important for wound healing in the zebrafish embryo. Therefore, this project has provided us with strong clues about the essential mechanisms that regulate epithelial wound healing, establishing a solid framework for future studies and potential clinical applications.