The specification of tissue size during development remains one of the most challenging questions in biology. In order to achieve consistent organ and body size in individuals of the same species, tight control must be applied to cell growth and cell number at the end of development. Since the genes that restrict organ size are likely to be targets of tumour-promoting mutations, the study of these mechanisms is relevant to both developmental and cancer biology.
The Hippo (Hpo) pathway comprises the kinases Hpo and Warts, the adaptors Salvador and Mats, the cytoskeletal proteins Expanded and Merlin, the atypical cadherin Fat and the transcriptional co-factor Yorkie (Harvey and Tapon. 2007). This pathway has been shown to restrict tissue size through the control of cell division and apoptosis during development in Drosophila. We aim to elucidate the role of Hpo signalling in growth control using a combination of genetics, cell biology and biochemistry. In addition, we also study other regulators of tissue growth or apoptosis, such as the mitochondrial protease HtrA2 and the Src proto-oncogene.
The Hippo pathway regulates apical domain size independently of its growth control function
In addition to their well-characterised over proliferation phenotype, adult epithelial cells mutant for the kinases Hippo and Warts present a hypertrophy of the apical domain. we examined the molecular basis of this apical hypertrophy and its impact on cell proliferation. In the wing imaginal disc epithelium, we observe increased staining for members of the apical polarity complexes aPKC and Crumbs as well as adherens junction components when Hippo activity is compromised, while baso-lateral markers are not affected. This increase in apical proteins is correlated with a hypertrophy of the apical domain and adherens junctions. The cell surface localisation of the Notch receptor is also increased in mutant clones, opening the possibility that aberrant receptor signalling may participate in overgrowth of hpo-deficient tissue. Interestingly however, while the polarity determinant Crumbs is required for the accumulation of apical proteins, this does not appear to significantly contribute to the over proliferation defect elicited by loss of Hippo signalling (Figure 1). Therefore, Hippo signalling controls growth and apical domain size via distinct mechanisms (Harvey and Tapon Nat Rev Cancer 2007; 7: 182-191).
Kibra is a new regulator of the Salvador/Warts/Hippo signalling network
In collaboration with the Thompson lab (LRI) we identified the WW-domain-containing protein Kibra (Kib), Drosophila orthologue of mammalian KIBRA, as a new member of the SWH network. Kib, which colocalises and physically interacts with Mer and Ex, also promotes the Mer/Ex association. Furthermore, the Kib/Mer interaction is conserved in human cells. Loss of kib induces a hpo-like phenotype and genetic experiments place it upstream of the core kinase cassette. Finally, Kib binds to Wts and kib depletion in tissue culture cells induces a marked reduction in Yki phosphorylation without affecting the Yki/Wts interaction. We suggest that Kib is part of an apical scaffold that promotes SWH pathway activity.
The dASPP-dRASSF8 complex regulates cell-cell adhesion during Drosophila retinal morphogenesis
Adherens junctions (AJs) provide structure to epithelial tissues by connecting adjacent cells through homophilic E-Cadherin interactions, and are linked to the actin cytoskeleton via the intermediate binding proteins b-Catenin and a-Catenin. Rather than being static structures, AJs are extensively remodelled during development, allowing the cell rearrangements required for morphogenesis.
We had previously identified dASPP as a positive regulator of dCsk (Drosophila C-terminal Src kinase). We recently showed that dRASSF8, the Drosophila RASSF8 homolog, binds to dASPP and that this interaction is required for normal dASPP levels. Our genetic and biochemical data suggests that dRASSF8 acts in concert with dASPP to promote dCsk activity. Both proteins specifically localize to AJs and are mutually required for each other¿s localization. Furthermore, we observe abnormal E-Cadherin localization in mutant pupal retinas, correlating with aberrant cellular arrangements. Loss of dCsk or over expression of Src elicits similar AJ defects. Since Src is known to regulate AJs in both Drosophila and mammals, we propose that dASPP and dRASSF8 fine-tune cell-cell adhesion during development by directing dCsk and Src activity (Langton et al, Curr Biol; in press).
Drosophila HtrA2 maintains mitochondrial integrity downstream of PINK1
High Temperature Requirement A2 (HtrA2/Omi) is a mitochondrial protease that exhibits proapoptotic and cell protective properties and has been linked to Parkinson disease (PD). Impaired mitochondrial function is a common trait in PD patients, and is likely to play a significant role in pathogenesis of parkinsonism, but the molecular mechanisms remain poorly understood. Genetic studies in Drosophila have provided valuable insight into the function of other PD-linked genes, in particular PINK1 and parkin, and their role in maintaining mitochondrial integrity. Recently, HtrA2 was shown to be phosphorylated in a PINK1-dependent manner, suggesting it might act in the PINK1 pathway.
In collaboration with the Whitworth (Sheffield University), Martins (Leicester University) and Downward (LRI) labs, we generated and characterized mutations in Drosophila HtrA2, and analysed its function with PINK1 and parkin. Interestingly, we found HtrA2 appears to be dispensable for developmental or stress-induced apoptosis. In addition, HtrA2 mutants share some phenotypic similarities with parkin and PINK1 mutants, suggesting that it may function in maintaining mitochondrial integrity. Our genetic studies suggest HtrA2 acts downstream of PINK1 but in a pathway parallel to Parkin (Tain et al. Cell Death Differ 2009; 16: 1118-1125).
Figure 1. Crumbs (Crb) is dispensable for overgrowth of wts mutant tissue. Mosaic third instar wing imaginal discs stained with Hoechst (in blue) to label nuclei. Mutant clones of the indicated genotypes (negative for the GFP marker in green) were generated using the hsFLP/FRT sytem. A: Wild type clones. B: Warts mutant clones (wts) overgrow compared with wild-type (A) or crb-mutant clones (C). C: Crumbs mutant clones (crb). D: Loss of crb fails to rescue the wts overgrowth phenotype.
For a list of refereed research papers, see Publications (in navigation on left).
