. _:b12702174 "Crb acts through Ex [32-34], and Ft acts through both Ex [14,15,19] and the unconventional myosin Dachs (D) [>>12<<]. The overexpression of ex can promote ectopic Hippo signaling, and when expressed in otherwise wild type clones can restrain clonal growth in a Hpo and Wts-dependent manner [13,17,41]. Significantly, the overexpression of ex in scrib" . _:b473776858 . . _:b12702149 "In contrast, Sd is largely dispensable for normal eye disc growth and proliferation and specifically mediates Hippo pathway mutant overgrowth [>>4<<,5]. Therefore, to determine if the ectopic cell proliferation and altered cell morphology of scrib mutant cells were due to loss of Hippo pathway signaling we utilized RNAi-mediated knockdown of sd function in scrib mutant eye disc clones" . _:b473777101 "2"^^ . _:b473776869 . _:b473777100 "2"^^ . _:b473776868 . _:b473776987 . . _:b473777103 "2"^^ . _:b473776871 . _:b12702171 . _:b473777102 "2"^^ . . _:b473776870 . _:b473776792 . _:b12702172 . _:b473777097 "2"^^ . _:b473776865 . _:b473777006 . _:b12702249 "Furthermore, increasing evidence links Hippo pathway deregulation to tumorigenesis [reviewed in [>>21<<]]." . _:b473777096 "2"^^ . _:b473776864 . _:b473777099 "2"^^ . _:b12702210 "(UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12 [75]; UAS-scrib19.2 [38]; FRT82B, scrib1 [76]; scrib3 [77]; UAS-scribRNAi (VDRC #27424); UAS-sdRNAi (NIG #8544R-2); wtsX1 [78]; UAS-wtsRNAi (NIG #12072R-1); ykiB5 [7]; UAS-ykiRNAi [>>5<<]." . _:b473776923 . _:b473776867 . . _:b473777098 "2"^^ . _:b473776866 . . _:b473777109 "2"^^ . _:b473776877 . _:b473777108 "2"^^ . . _:b473776876 . _:b473776990 . _:b473776878 . _:b473777111 "2"^^ . _:b473776875 . _:b12702241 "apico-basal epithelial polarity regulators such as Scrib and aPKC, since the Hippo pathway components ft, fj and ds also participate in planar cell polarity pathways [reviewed in [56]], as do scrib [57], lgl [58] and aPKC [59,>>60<<]. The emerging picture is therefore one of a complex network of interactions whereby multiple components regulating cellular architecture are employed by cells to read their position within a morphogenetic field and respond with" . _:b473776879 . _:b473777110 "2"^^ . _:b473776878 . _:b473777105 "2"^^ . _:b12702201 "UAS-bskDN [67]; UAS-bskRNAi (NIG #5680R-1); d1 [68]; dGC13 [12]; exe1 [69]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [>>26<<]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12 [75]; UAS-scrib19.2 [38]; FRT82B, scrib1 [76]; scrib3 [77]; UAS-scribRNAi (VDRC #27424); UAS-sdRNAi (NIG #8544R-2); wtsX1 [78]; UAS-wtsRNAi (NIG #12072R-1); ykiB5 [7];" . _:b12702217 "Indeed, a number of recent reports indicating that increased levels of F-actin are sufficient to inhibit Hippo pathway activity [47,>>48<<] are also suggestive, since the aPKC-dependent loss of apico-basal cell polarity in scrib mutants is often associated with F-actin accumulations (data not shown)." . . _:b473776873 . _:b473777104 "2"^^ . _:b12702222 "In lgl mutants, the cell polarity defects in the wing disc are also JNK-dependent [>>37<<], and while we did not analyze cell polarity markers upon scrib knockdown in the wing, one possibility is that loss of scrib is associated with JNK-independent cell polarity defects that impact upon the Hippo pathway in a similar manner" . _:b473776826 . _:b473776872 . . _:b473777107 "2"^^ . _:b473776875 . _:b12702216 "Indeed, a number of recent reports indicating that increased levels of F-actin are sufficient to inhibit Hippo pathway activity [>>47<<,48] are also suggestive, since the aPKC-dependent loss of apico-basal cell polarity in scrib mutants is often associated with F-actin accumulations (data not shown)." . _:b473777106 "2"^^ . _:b473776874 . . _:b473776885 . _:b473777116 "2"^^ . . _:b473776884 . _:b12702212 _:b12702240 . _:b12702212 _:b12702241 . _:b473776847 . _:b473776887 . _:b473776886 . _:b473777113 "2"^^ . . _:b12702150 . _:b473776881 . _:b12702212 _:b12702228 . _:b12702212 _:b12702229 . _:b473777112 "2"^^ . . _:b12702212 _:b12702230 . _:b473776880 . _:b12702149 . . _:b12702212 _:b12702231 . _:b12702212 _:b12702224 . _:b473776976 . _:b12702212 _:b12702225 . _:b473777115 "2"^^ . _:b473776853 . _:b12702212 _:b12702226 . _:b473776883 . _:b12702214 "Indeed, in zebrafish Scrib binds to, and functionally cooperates with, Ft [45]; and as this interaction could be conserved in Drosophila [>>45<<] very direct points of intersection between Scrib and the upstream regulators of Hippo signaling can be envisaged." . _:b12702212 _:b12702227 . _:b12702212 _:b12702236 . _:b12702212 _:b12702237 . _:b473777114 "2"^^ . _:b12702212 _:b12702238 . _:b473776882 . _:b12702207 . _:b12702212 _:b12702239 . _:b12702212 _:b12702232 . _:b473776913 . _:b12702212 _:b12702233 . _:b12702212 _:b12702234 . _:b473776893 . _:b12702212 _:b12702235 . _:b473776874 . _:b12702212 _:b12702213 . _:b12702212 _:b12702214 . _:b473776892 . _:b473776789 . _:b12702212 _:b12702215 . . _:b12702216 . _:b473776895 . _:b12702212 _:b12702220 . _:b12702212 _:b12702221 . _:b12702212 _:b12702222 . _:b473776894 . _:b12702212 _:b12702223 . _:b12702212 _:b12702216 . _:b12702212 _:b12702217 . _:b12702212 _:b12702218 . _:b473776889 . . _:b12702212 _:b12702219 . _:b473776961 . _:b12702144 . _:b473776888 . _:b12702251 . . _:b473776891 . _:b12702173 "Crb acts through Ex [32-34], and Ft acts through both Ex [14,15,>>19<<] and the unconventional myosin Dachs (D) [12]." . _:b473777060 . _:b473776890 . "10.1186%2F1471-213X-11-57" . . _:b12702220 "A recent report indicates that the impaired Hippo pathway signaling in lgl mutant wing discs is dependent upon JNK signaling, and that ectopic aPKC signaling in the wing disc also acts through JNK to promote Yki activity [>>37<<]. In the eye disc, however, we show that Hippo pathway impairment in scrib mutants, as well as upon ectopic activation of aPKC signaling, cannot be rescued by blocking JNK, and this is also likely to be the case for lgl mutants [49]." . . _:b473776914 . . . _:b473776819 . . _:b12702249 . . _:b12702230 "signaling was blocked in the mutant clones, and whilst the rescue of some non-cell autonomous effects would be consistent with the role that JNK can play in promoting non-cell autonomous compensatory proliferation through Yki activity [>>37<<], it also remains possible that JNK may act in opposite ways to downregulate DIAP1 or other reporters in mutant cells destined to die." . . . _:b473777018 . _:b12702155 "scrib mutants was dependent upon impaired Hippo signaling we could not remove Sd function, as was done in the eye disc, since Sd is required for normal wing disc growth through association with the wing determination factor Vestigial [>>42<<,43]. Therefore, we utilized a yki null allele, ykiB5, to halve the gene dosage of yki in a scrib1/scrib3 mutant background." . _:b12702207 "9J which is exe1) [69]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12 [75]; UAS-scrib19.2 [38]; FRT82B, scrib1 [76]; scrib3 [>>77<<]; UAS-scribRNAi (VDRC #27424); UAS-sdRNAi (NIG #8544R-2); wtsX1 [78]; UAS-wtsRNAi (NIG #12072R-1); ykiB5 [7]; UAS-ykiRNAi [5]." . _:b473777063 . _:b12702160 . _:b12702227 "Indeed, the role of JNK in neoplasia is proving to be complex, since in some contexts it functions as an oncogene to promote neoplasia, whilst in different contexts it acts as a tumor suppressor through the induction of apoptosis [38,>>39<<,51-53]. Its effects upon Hippo pathway regulation may therefore also be context dependent." . . _:b12702261 "Furthermore, loss of lgl has been shown to impair Hippo signaling in the eye disc in an aPKC signaling-dependent manner [>>35<<]. Indeed in the wing disc both the loss of lgl or ectopic aPKC activity promotes Yki activity through an upregulation of Jun N-terminal kinase (JNK) signaling [37]. Whether scrib regulates the Hippo pathway in the eye or wing disc is not" . _:b473776824 . . _:b12702186 "Mammalian Scrib also functions within planar cell polarity pathways [>>63<<], and although links with the Hippo pathway have not yet been described, the connection is likely to be conserved since studies in the zebrafish indicate that zScrib binds to zFat1 and also promotes Hippo pathway activation [44]." . _:b473776970 . . _:b473777055 . . _:b12702139 . _:b473776950 . . _:b473777027 . . _:b473776815 . _:b473777057 . _:b12702260 "dlg, lgl or scrib mutant follicle cells surrounding the female ovary have elevated levels of Yki targets Cyclin E and DIAP1, and exhibit strong genetic interactions with wts [>>36<<]. Furthermore, loss of lgl has been shown to impair Hippo signaling in the eye disc in an aPKC signaling-dependent manner [35]. Indeed in the wing disc both the loss of lgl or ectopic aPKC activity promotes Yki activity through an" . _:b473776891 . . _:b12702211 . _:b12702230 . _:b12702178 "Ft can act in parallel to Ex, and indeed the overexpression of ex in ft mutant clones is unable to restrain ft mutant tissue overgrowth [>>16<<]. We therefore next determined if impaired Ft signaling could be responsible for downregulating the Hippo pathway in scrib mutants." . _:b473776834 . . _:b473777066 . _:b473776919 . _:b12702188 . . _:b473776881 . _:b473777013 . . _:b12702181 . _:b473776949 . _:b12702177 . _:b12702220 . _:b473776922 . . . _:b12702222 . _:b12702251 "phenotypes of a number of these mutants, including scrib, lgl, avl and TSG101 are dependent upon atypical protein kinase C (aPKC) activity, since mutant phenotypes can be rescued by reducing atypical protein kinase C function [>>23<<-25]. Direct and mutual antagonism between the junctional tumor suppressors and aPKC has been demonstrated by the ability of aPKC to associate with and phosphorylate Lgl, thereby releasing Lgl from the cell cortex and thus potentially" . _:b12702251 . . _:b473776806 . . . . . _:b12702247 . _:b473776981 . _:b12702140 . . _:b473777112 . _:b473776991 . _:b473776918 . _:b12702208 . . _:b12702235 . . _:b12702232 . . . _:b473776813 . _:b12702233 . . . _:b473777078 . _:b12702236 . _:b12702252 . _:b12702213 . . _:b473776791 . _:b12702214 . _:b12702206 . _:b473776823 . _:b473776928 . _:b12702199 "(VDRC #2907); bsk2 [66]; UAS-bskDN [67]; UAS-bskRNAi (NIG #5680R-1); d1 [68]; dGC13 [12]; exe1 [69]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [>>72<<]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12 [75]; UAS-scrib19.2 [38]; FRT82B, scrib1 [76]; scrib3 [77]; UAS-scribRNAi (VDRC #27424); UAS-sdRNAi (NIG #8544R-2); wtsX1 [78]; UAS-wtsRNAi (NIG" . . . . _:b12702262 . . _:b12702210 . . _:b12702195 "UAS-mCD8-GFP;;Tub-GAL4, FRT82B, Tub-GAL80 [64]; y, w, hs-FLP; FRT82B, Ubi-GFP; en-GAL4; UAS-DaPKC\u0394N [26]; UAS-DaPKCCAAXDN [65]; UAS-aPKCRNAi (VDRC #2907); bsk2 [66]; UAS-bskDN [67]; UAS-bskRNAi (NIG #5680R-1); d1 [68]; dGC13 [12]; exe1 [>>69<<]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12 [75];" . . _:b473776886 . _:b473777032 . _:b12702243 . . _:b12702200 "bsk2 [66]; UAS-bskDN [67]; UAS-bskRNAi (NIG #5680R-1); d1 [68]; dGC13 [12]; exe1 [69]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [>>26<<]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12 [75]; UAS-scrib19.2 [38]; FRT82B, scrib1 [76]; scrib3 [77]; UAS-scribRNAi (VDRC #27424); UAS-sdRNAi (NIG #8544R-2); wtsX1 [78]; UAS-wtsRNAi (NIG #12072R-1);" . . _:b473776979 . . _:b473777026 . _:b473777012 . . _:b473777002 . _:b473776958 . _:b12702256 . _:b473777031 . _:b12702257 . _:b12702237 . . _:b473777074 . . _:b473776936 . _:b473776900 . . _:b12702208 "#9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12 [75]; UAS-scrib19.2 [38]; FRT82B, scrib1 [76]; scrib3 [77]; UAS-scribRNAi (VDRC #27424); UAS-sdRNAi (NIG #8544R-2); wtsX1 [>>78<<]; UAS-wtsRNAi (NIG #12072R-1); ykiB5 [7]; UAS-ykiRNAi [5]." . . _:b473776851 . _:b473776867 . _:b12702175 . . _:b473776880 . _:b473776985 . . _:b473776957 . _:b473777044 . . . _:b12702209 . . _:b473777087 . . . _:b12702246 . _:b473776852 . _:b12702246 . _:b12702240 "traditional apico-basal epithelial polarity regulators such as Scrib and aPKC, since the Hippo pathway components ft, fj and ds also participate in planar cell polarity pathways [reviewed in [56]], as do scrib [57], lgl [58] and aPKC [>>59<<,60]. The emerging picture is therefore one of a complex network of interactions whereby multiple components regulating cellular architecture are employed by cells to read their position within a morphogenetic field and respond with" . _:b473776926 . _:b473777051 . . . _:b12702189 . . . . _:b12702139 "results" . . . _:b12702239 . . . . . _:b473776807 . _:b12702187 "[63], and although links with the Hippo pathway have not yet been described, the connection is likely to be conserved since studies in the zebrafish indicate that zScrib binds to zFat1 and also promotes Hippo pathway activation [>>44<<]. The uniting of these two powerful tumor suppressor pathways clearly has important implications for human carcinogenesis." . _:b473777025 . _:b12702229 "effects, with increased expression in wild type cells surrounding mutant clones, were also sometimes observed (data not shown), consistent with the involvement of the Hippo pathway in regenerative proliferation around dying tissue [>>54<<]. Much of this variability appeared to be reduced when JNK signaling was blocked in the mutant clones, and whilst the rescue of some non-cell autonomous effects would be consistent with the role that JNK can play in promoting non-cell" . _:b473777082 . _:b12702228 . . . . _:b12702262 "Indeed in the wing disc both the loss of lgl or ectopic aPKC activity promotes Yki activity through an upregulation of Jun N-terminal kinase (JNK) signaling [>>37<<]. Whether scrib regulates the Hippo pathway in the eye or wing disc is not yet clear. In contrast to lgl, reduced levels of Scrib in the eye disc to levels where apico-basal cell polarity is only mildly affected does not impair Hippo" . _:b473776915 . . _:b473776901 . . . _:b473776893 . . _:b473776900 . . _:b473777099 . . _:b473777015 . . _:b473776903 . _:b473776902 . . _:b12702219 "Furthermore, Ex and Ft localization were unaffected in the absence of lgl, whilst both Hpo and Ras-associated domain family protein (RASSF) were co-mislocalized, consistent with the Hippo pathway being impaired downstream of Ft and Ex [>>35<<]. Whether a common aPKC-dependent mechanism of Hippo pathway inhibition is operative in both lgl and scrib mutant eye discs will require further investigation." . _:b473776873 . _:b473776825 . _:b473776848 . . . _:b473776897 . _:b12702156 "mutants was dependent upon impaired Hippo signaling we could not remove Sd function, as was done in the eye disc, since Sd is required for normal wing disc growth through association with the wing determination factor Vestigial [42,>>43<<]. Therefore, we utilized a yki null allele, ykiB5, to halve the gene dosage of yki in a scrib1/scrib3 mutant background." . _:b473776947 . _:b473776896 . _:b473776995 . . . _:b473776899 . . _:b473777064 . _:b473776898 . . _:b473776909 . _:b12702189 "The following Drosophila stocks were used: ey-FLP1, UAS-mCD8-GFP;;Tub-GAL4, FRT82B, Tub-GAL80 [>>64<<]; y, w, hs-FLP; FRT82B, Ubi-GFP; en-GAL4; UAS-DaPKC\u0394N [26]; UAS-DaPKCCAAXDN [65]; UAS-aPKCRNAi (VDRC #2907); bsk2 [66]; UAS-bskDN [67]; UAS-bskRNAi (NIG #5680R-1); d1 [68]; dGC13 [12]; exe1 [69]; ex697 (ex-lacZ in all figures except" . _:b473776908 . _:b12702204 "exe1 [69]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12 [>>75<<]; UAS-scrib19.2 [38]; FRT82B, scrib1 [76]; scrib3 [77]; UAS-scribRNAi (VDRC #27424); UAS-sdRNAi (NIG #8544R-2); wtsX1 [78]; UAS-wtsRNAi (NIG #12072R-1); ykiB5 [7]; UAS-ykiRNAi [5]." . _:b473777076 . _:b12702229 . _:b473776911 . _:b473776897 . _:b473776910 . _:b473777071 . . . _:b473776822 . _:b473776905 . . _:b473776799 . _:b473776816 . . _:b473776904 . _:b473776814 . . _:b12702151 "are apically localized within the pseudo-stratified columnar epithelium (Figure 3A), however, in scrib mutant clones, cells are often extruded basally and Elav-positive nuclei are aberrantly localized basally within the epithelium [>>38<<]. In scrib mutants (data not shown), or in scrib mutants protected from cell death by the expression of bskDN, knockdown of sd function with sdRNAi failed to restore normal cell morphology to the mutant tissue (Figure 3B, C)." . _:b473776907 . _:b12702218 "however, lgl mutant eye disc clones are not associated with the severe alterations in cell morphology characteristic of scrib mutant cells, yet the impairment to Hippo signaling in lgl mutant clones is also dependent upon aPKC activity [>>35<<]. Furthermore, Ex and Ft localization were unaffected in the absence of lgl, whilst both Hpo and Ras-associated domain family protein (RASSF) were co-mislocalized, consistent with the Hippo pathway being impaired downstream of Ft and Ex" . _:b473776906 . _:b473776917 . . _:b473776916 . . . _:b473776919 . _:b473776843 . _:b473777022 . _:b12702246 . . _:b473776918 . _:b473776913 . _:b473776956 . _:b12702181 _:b12702182 . _:b12702181 _:b12702183 . _:b473777108 . _:b12702181 _:b12702184 . _:b12702181 _:b12702185 . _:b473776912 . _:b12702181 _:b12702186 . _:b12702145 "As we had previously shown that scrib mutant cells continue to ectopically proliferate in the eye disc even when JNK signaling is blocked [>>38<<], it seemed likely that this impairment to Hippo signaling would not depend upon JNK activation." . _:b12702181 _:b12702187 . _:b12702233 "Loss of wts is capable of eliciting neoplastic overgrowth [55], and Hippo pathway mutants induce apical hypertrophy with increased levels of apical cell determinants such as Crb, aPKC [30,>>31<<], and F-actin [48], although, interestingly, despite the potential for increased Crb, aPKC and F-actin to promote tissue hyperplasia, the overgrowth in Hippo pathway mutants is independent of the apical hypertrophy [30,31]." . _:b473776915 . _:b473777061 . _:b12702188 _:b12702196 . _:b12702188 _:b12702197 . . _:b473776914 . . _:b12702188 _:b12702198 . _:b12702188 _:b12702199 . _:b12702188 _:b12702192 . _:b12702188 _:b12702193 . _:b473776925 . _:b12702188 _:b12702194 . _:b473776971 . . _:b12702188 _:b12702195 . . _:b12702188 _:b12702204 . _:b12702188 _:b12702205 . _:b473776924 . _:b12702188 _:b12702206 . _:b12702188 _:b12702207 . . _:b12702188 _:b12702200 . . _:b473776927 . _:b12702188 _:b12702201 . _:b12702188 _:b12702202 . . _:b12702188 _:b12702203 . _:b473776805 . _:b473776926 . _:b12702228 . _:b473776840 . _:b12702169 . _:b12702239 "not confined to traditional apico-basal epithelial polarity regulators such as Scrib and aPKC, since the Hippo pathway components ft, fj and ds also participate in planar cell polarity pathways [reviewed in [56]], as do scrib [57], lgl [>>58<<] and aPKC [59,60]." . _:b473776921 . _:b12702247 . . _:b12702188 _:b12702189 . _:b473776920 . _:b12702188 _:b12702190 . _:b12702188 _:b12702191 . . . _:b473776923 . _:b12702173 . . . _:b473776922 . . _:b473777021 . _:b473776933 . . . _:b473776932 . _:b473776978 . _:b473776935 . . _:b473776934 . _:b12702159 "mutant eye clones die via JNK-mediated apoptosis, if RasACT or its downstream effector, Rafgof, is expressed in the mutant clones, cell death is prevented and massive and invasive tumors develop throughout an extended larval stage [>>39<<]. To determine if downregulation of Hippo pathway signaling is also an important mediator of these overgrowths we examined the expression of the Hippo pathway reporters, ex-lacZ and fj-lacZ, in scrib- + Rafgof tumors." . _:b473776812 . _:b473776929 . . _:b12702238 "is not confined to traditional apico-basal epithelial polarity regulators such as Scrib and aPKC, since the Hippo pathway components ft, fj and ds also participate in planar cell polarity pathways [reviewed in [56]], as do scrib [>>57<<], lgl [58] and aPKC [59,60]." . . . _:b473776928 . _:b12702246 . _:b473776931 . _:b473776902 . _:b473776930 . . _:b473776941 . _:b473776972 . . _:b473777058 . _:b473776940 . _:b473777047 . _:b473776943 . . _:b12702183 "Similarly, the mammalian Scrib module is increasingly implicated in tumorigenesis [reviewed in [>>28<<]], and mammalian Scrib can restrain tissue transformation by oncogenic Ras [61], and Myc [62]." . . _:b473776942 . _:b473777053 . _:b473776937 . _:b12702232 "Loss of wts is capable of eliciting neoplastic overgrowth [55], and Hippo pathway mutants induce apical hypertrophy with increased levels of apical cell determinants such as Crb, aPKC [>>30<<,31], and F-actin [48], although, interestingly, despite the potential for increased Crb, aPKC and F-actin to promote tissue hyperplasia, the overgrowth in Hippo pathway mutants is independent of the apical hypertrophy [30,31]." . . . . _:b473776936 . _:b473776939 . _:b473777103 . _:b473776938 . _:b473776837 . _:b473777041 . _:b473776949 . _:b473776948 . _:b473776951 . _:b12702166 "however, we have previously shown that although the expression of a truncated activated allele of aPKC (aPKC\u0394N) in eye disc clones induces JNK-dependent cell death, if JNK signaling is blocked, ectopic cell proliferation still ensues [>>38<<]. Indeed, the Hippo pathway reporters fj-lacZ and ex-lacZ were ectopically expressed in aPKC\u0394N and bskDN coexpressing eye disc clones (Figure 7A-D)." . _:b473776950 . _:b473776844 . _:b473776861 . . . _:b473776927 . _:b473776945 . _:b12702244 "Traditionally two distinct classes of tumor suppressor mutants have been described, the loss of which cause either hyperplastic or neoplastic overgrowth [reviewed in [>>2<<]]. Hyperplastic overgrowth is characterized by excessive cell proliferation that is eventually restrained by terminal differentiation, while neoplastic overgrowth exhibits impaired differentiation, defects in cell polarity and the" . . _:b12702255 "Indeed, wts mutants were originally identified based upon mutant cell morphology [>>29<<], and this is now known to be a phenotype associated with other Hippo pathway mutants and due to Yki-dependent upregulation of the apical cell polarity determinant Crumbs (Crb) and apical hypertrophy [30,31]." . _:b473776907 . _:b473776944 . . _:b473776947 . _:b473776946 . _:b473776957 . . . _:b473776841 . _:b473776956 . . _:b473776959 . . _:b473776958 . _:b473776953 . . _:b473776952 . _:b12702188 "methods" . _:b473776896 . _:b473776955 . _:b473776954 . _:b12702195 . _:b473776965 . . _:b473776964 . _:b473776884 . . _:b473776967 . _:b473776966 . _:b473776961 . . _:b12702196 . _:b473776960 . _:b473776894 . _:b473776963 . _:b473776855 . _:b473776818 . _:b473776962 . _:b473776920 . _:b473776973 . _:b473776972 . _:b12702187 . _:b473776975 . _:b12702176 "The overexpression of ex can promote ectopic Hippo signaling, and when expressed in otherwise wild type clones can restrain clonal growth in a Hpo and Wts-dependent manner [13,>>17<<,41]. Significantly, the overexpression of ex in scrib mutant eye disc clones also restrained clonal growth, and this was the case even if JNK-mediated apoptosis of the clonal tissue was prevented (Figure 9A-F). Indeed, scrib clones" . _:b473777049 . _:b473776975 . _:b473777016 . . _:b12702247 . _:b473776795 . _:b473776974 . _:b12702197 "UAS-DaPKC\u0394N [26]; UAS-DaPKCCAAXDN [65]; UAS-aPKCRNAi (VDRC #2907); bsk2 [66]; UAS-bskDN [67]; UAS-bskRNAi (NIG #5680R-1); d1 [68]; dGC13 [12]; exe1 [69]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [69]; UAS-ex [>>70<<]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12 [75]; UAS-scrib19.2 [38]; FRT82B, scrib1 [76]; scrib3 [77]; UAS-scribRNAi (VDRC #27424);" . _:b473776856 . _:b473776969 . _:b473776983 . _:b473776968 . _:b473776966 . _:b473776971 . _:b12702194 "ey-FLP1, UAS-mCD8-GFP;;Tub-GAL4, FRT82B, Tub-GAL80 [64]; y, w, hs-FLP; FRT82B, Ubi-GFP; en-GAL4; UAS-DaPKC\u0394N [26]; UAS-DaPKCCAAXDN [65]; UAS-aPKCRNAi (VDRC #2907); bsk2 [66]; UAS-bskDN [67]; UAS-bskRNAi (NIG #5680R-1); d1 [68]; dGC13 [>>12<<]; exe1 [69]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12" . _:b473776857 . . _:b473776970 . . _:b473776981 . _:b473776982 . _:b473776984 . _:b473777092 . . _:b473776985 . _:b473776980 . _:b473776986 . _:b473777059 . _:b473776987 . _:b473776988 . _:b473776989 . _:b12702221 "In the eye disc, however, we show that Hippo pathway impairment in scrib mutants, as well as upon ectopic activation of aPKC signaling, cannot be rescued by blocking JNK, and this is also likely to be the case for lgl mutants [>>49<<]. Furthermore, in scrib mutant wing discs, although we demonstrate that JNK signaling is ectopically activated upon scrib knockdown, we also show that Hippo pathway impairment in the wing occurs, at least in part, even when JNK signaling" . _:b473776983 . _:b473776990 . _:b473776991 . . _:b473776976 . . . _:b473776982 . _:b473776977 . _:b473776978 . _:b473776979 . _:b473776980 . . _:b473776981 . _:b12702257 "upon mutant cell morphology [29], and this is now known to be a phenotype associated with other Hippo pathway mutants and due to Yki-dependent upregulation of the apical cell polarity determinant Crumbs (Crb) and apical hypertrophy [30,>>31<<]. Crb itself acts to regulate Hippo signaling by binding to Ex [32], and either excessive Crb activity or loss of Crb results in deregulation of Ex and an impairment to Hippo signaling resulting in tissue overgrowth [32-35]." . _:b473776977 . _:b473776982 . _:b12702192 "The following Drosophila stocks were used: ey-FLP1, UAS-mCD8-GFP;;Tub-GAL4, FRT82B, Tub-GAL80 [64]; y, w, hs-FLP; FRT82B, Ubi-GFP; en-GAL4; UAS-DaPKC\u0394N [26]; UAS-DaPKCCAAXDN [65]; UAS-aPKCRNAi (VDRC #2907); bsk2 [66]; UAS-bskDN [>>67<<]; UAS-bskRNAi (NIG #5680R-1); d1 [68]; dGC13 [12]; exe1 [69]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof" . _:b473776983 . _:b473776968 . _:b473776969 . _:b473776976 . _:b473776970 . _:b473776971 . . _:b473776972 . _:b473776973 . _:b473776979 . _:b473776974 . . _:b473776975 . _:b473776960 . . _:b473776978 . _:b473776961 . _:b473776962 . _:b473776963 . _:b473776964 . _:b473776842 . _:b473776989 . _:b473776965 . . _:b473776966 . . _:b473776967 . . _:b473777016 . _:b473777017 . _:b473776988 . _:b473777018 . _:b473777091 . _:b473777019 . _:b473777020 . _:b473777021 . _:b473776991 . _:b473777022 . _:b473777023 . _:b473777054 . _:b473777008 . _:b473777009 . _:b473776990 . _:b473777010 . _:b473777011 . _:b12702206 "except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12 [75]; UAS-scrib19.2 [38]; FRT82B, scrib1 [>>76<<]; scrib3 [77]; UAS-scribRNAi (VDRC #27424); UAS-sdRNAi (NIG #8544R-2); wtsX1 [78]; UAS-wtsRNAi (NIG #12072R-1); ykiB5 [7]; UAS-ykiRNAi [5]." . . _:b12702139 _:b12702140 . _:b473777012 . _:b12702139 _:b12702141 . _:b473777097 . _:b473776985 . _:b473777013 . _:b12702139 _:b12702142 . _:b473777014 . _:b12702139 _:b12702143 . _:b473777015 . _:b473777000 . _:b473777001 . _:b473776984 . _:b473777002 . _:b473777003 . . _:b473776800 . _:b473777004 . _:b473777005 . _:b473776987 . _:b473777006 . _:b473777007 . . _:b12702139 _:b12702152 . _:b12702203 "d1 [68]; dGC13 [12]; exe1 [69]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [>>74<<]; UAS-dRas1V12 [75]; UAS-scrib19.2 [38]; FRT82B, scrib1 [76]; scrib3 [77]; UAS-scribRNAi (VDRC #27424); UAS-sdRNAi (NIG #8544R-2); wtsX1 [78]; UAS-wtsRNAi (NIG #12072R-1); ykiB5 [7]; UAS-ykiRNAi [5]." . _:b473776992 . _:b12702139 _:b12702153 . _:b473776986 . _:b473776993 . _:b12702139 _:b12702154 . _:b473776994 . _:b12702139 _:b12702155 . _:b473776995 . . _:b473776996 . _:b12702139 _:b12702156 . _:b12702139 _:b12702157 . _:b473776997 . _:b473776997 . _:b12702139 _:b12702158 . _:b473776998 . _:b12702139 _:b12702159 . _:b473776999 . _:b12702139 _:b12702144 . _:b473776920 . _:b12702139 _:b12702145 . _:b473776996 . _:b473776921 . _:b12702139 _:b12702146 . _:b473776922 . _:b12702139 _:b12702147 . _:b473776923 . _:b12702139 _:b12702148 . _:b473777034 . _:b473776924 . _:b12702139 _:b12702149 . _:b473776999 . _:b473776925 . _:b12702139 _:b12702150 . _:b473776926 . . _:b473777046 . _:b473776927 . _:b12702139 _:b12702151 . _:b12702139 _:b12702168 . _:b473776912 . _:b12702139 _:b12702169 . _:b473776998 . . _:b473776913 . _:b12702139 _:b12702170 . _:b473776914 . _:b12702139 _:b12702171 . _:b473776915 . _:b473776811 . _:b12702139 _:b12702172 . _:b12702166 . _:b473776916 . _:b12702139 _:b12702173 . _:b473776993 . _:b473776917 . _:b12702139 _:b12702174 . _:b473776918 . _:b12702139 _:b12702175 . _:b473776919 . _:b12702139 _:b12702160 . _:b473776904 . _:b12702139 _:b12702161 . _:b473776992 . . _:b473776905 . _:b12702139 _:b12702162 . . _:b473776906 . _:b12702139 _:b12702163 . _:b473776907 . _:b473776924 . _:b12702139 _:b12702164 . _:b473776860 . _:b473776908 . _:b12702139 _:b12702165 . _:b473776995 . . _:b473776909 . _:b12702139 _:b12702166 . _:b12702179 "To do this we generated scrib clones in a d mutant animal background, since Ft acts through inhibiting D and loss of d fully abrogates ft mutant overgrowth [>>12<<] (see Figure 10)." . _:b473776910 . _:b12702139 _:b12702167 . _:b473776911 . . _:b473776896 . _:b473776897 . _:b473776994 . _:b473776898 . _:b473776899 . _:b473776900 . _:b12702185 . _:b12702162 . _:b473776901 . _:b473777005 . _:b473776902 . . _:b473776969 . _:b473776903 . . _:b12702161 . _:b473777109 . _:b473776952 . _:b12702139 _:b12702176 . _:b12702139 _:b12702177 . _:b473777004 . _:b473776953 . _:b12702139 _:b12702178 . _:b473776954 . _:b12702139 _:b12702179 . _:b473776955 . _:b12702139 _:b12702180 . . _:b473776956 . _:b473776957 . _:b473777007 . _:b473776958 . . _:b473776959 . _:b473776944 . . _:b473776945 . _:b473777006 . . . _:b473776946 . _:b473776947 . . _:b473776948 . . _:b473777001 . _:b473776839 . _:b473776949 . _:b473776950 . _:b473776951 . _:b473776936 . . _:b473777000 . _:b473776937 . . _:b12702256 "upon mutant cell morphology [29], and this is now known to be a phenotype associated with other Hippo pathway mutants and due to Yki-dependent upregulation of the apical cell polarity determinant Crumbs (Crb) and apical hypertrophy [>>30<<,31]. Crb itself acts to regulate Hippo signaling by binding to Ex [32], and either excessive Crb activity or loss of Crb results in deregulation of Ex and an impairment to Hippo signaling resulting in tissue overgrowth [32-35]." . _:b473776938 . _:b473776939 . . _:b473776940 . . _:b473777003 . . _:b473776941 . _:b473776942 . _:b473776943 . _:b473776928 . _:b473776929 . _:b473777002 . _:b473776930 . _:b473777101 . _:b473776931 . _:b473776932 . _:b473776933 . _:b473777013 . _:b473776934 . _:b473776935 . _:b473777112 . _:b12702153 . _:b473777113 . _:b473777107 . _:b473777012 . _:b473777114 . _:b473777115 . _:b473777116 . _:b473777015 . _:b12702178 . _:b473777089 . _:b473777104 . _:b12702151 . _:b473777105 . _:b473777014 . _:b473777106 . . _:b473777107 . _:b473777108 . _:b473777109 . _:b473777009 . . . _:b473777110 . _:b473777111 . _:b473777096 . _:b473777097 . _:b473777008 . _:b473776912 . _:b473777098 . _:b473777099 . _:b473777100 . _:b473777101 . _:b473777011 . _:b12702242 "background" . _:b473777095 . _:b473777102 . _:b473777103 . _:b473777088 . _:b12702147 . _:b473777089 . _:b473777010 . _:b473777090 . . _:b473776963 . _:b473777091 . _:b473777092 . . _:b473777093 . _:b473777021 . _:b473777094 . _:b473777095 . . _:b12702145 . _:b473777020 . . . . _:b12702185 "Similarly, the mammalian Scrib module is increasingly implicated in tumorigenesis [reviewed in [28]], and mammalian Scrib can restrain tissue transformation by oncogenic Ras [61], and Myc [>>62<<]. Mammalian Scrib also functions within planar cell polarity pathways [63], and although links with the Hippo pathway have not yet been described, the connection is likely to be conserved since studies in the zebrafish indicate that" . . _:b473777105 . _:b473777023 . . _:b473777022 . _:b473776831 . _:b473776833 . . _:b473776973 . _:b473777017 . _:b473777086 . _:b12702217 . _:b12702231 "Loss of wts is capable of eliciting neoplastic overgrowth [>>55<<], and Hippo pathway mutants induce apical hypertrophy with increased levels of apical cell determinants such as Crb, aPKC [30,31], and F-actin [48], although, interestingly, despite the potential for increased Crb, aPKC and F-actin to" . _:b12702205 . _:b473777016 . _:b473776916 . _:b473777024 . _:b12702212 . _:b473777019 . _:b473777008 . . _:b12702213 "Indeed, in zebrafish Scrib binds to, and functionally cooperates with, Ft [>>45<<]; and as this interaction could be conserved in Drosophila [45] very direct points of intersection between Scrib and the upstream regulators of Hippo signaling can be envisaged." . _:b473777018 . _:b473777029 . _:b473777048 . _:b473777094 . _:b473777049 . _:b473777113 . _:b473777028 . _:b473777050 . _:b473777051 . _:b473777052 . _:b473777053 . _:b473777031 . _:b473777054 . _:b473777055 . _:b473777040 . _:b473777041 . _:b473777030 . _:b473777042 . . _:b473777043 . _:b473777044 . . _:b473777025 . _:b473777045 . _:b473777046 . _:b12702247 "The pathway is regulated through input from upstream components including Merlin and Expanded (Ex), and the transmembrane proteins Fat (Ft) and Dachsous (Ds) [>>12<<-19]. It is proposed that the primary function of the Hippo pathway is to incorporate positional cues within an epithelial field to dictate the ultimate size of organ development [20]. The pathway is highly conserved and also functions to" . . _:b473777047 . _:b473777032 . _:b473777033 . _:b473777024 . _:b473777034 . . _:b473777035 . _:b12702242 . _:b473777036 . _:b473777037 . _:b473777042 . _:b473777027 . _:b473777038 . _:b473777039 . _:b473777024 . _:b473777025 . _:b473777026 . _:b473777026 . _:b473777027 . _:b473776901 . _:b12702170 . _:b473777028 . _:b473777029 . _:b473777037 . _:b473777030 . . _:b473777031 . _:b473777080 . _:b473777081 . _:b473777036 . _:b473777082 . _:b12702147 "The bskDN transgene is highly effective at blocking JNK signaling since it completely abrogates both the ectopic expression of the JNK pathway reporter, msn-lacZ, and the JNK pathway target, Paxillin, in scrib mutant clones [>>38<<]. However, to confirm that JNK signaling was not required for Hippo pathway impairment, we also knocked down bsk expression in scrib mutant cells using a bskRNAi transgene." . _:b473777083 . _:b473777084 . _:b473777085 . _:b473777039 . _:b473777086 . _:b473777087 . _:b473777072 . _:b473777073 . _:b473777038 . _:b473777074 . _:b12702234 . _:b473777075 . _:b473777000 . _:b473777076 . _:b473777111 . . _:b473777033 . _:b473777077 . . _:b473777078 . _:b473777079 . _:b473777064 . _:b473777065 . _:b473777032 . _:b473776988 . _:b473777066 . _:b473777067 . _:b473777068 . _:b473777069 . _:b473777035 . _:b473777070 . . _:b473777071 . . _:b12702254 "Like the Hippo pathway, mammalian homologues of the Drosophila neoplastic tumor suppressors, as well as aPKC, are increasingly implicated as important players in human cancers [reviewed in [>>28<<]]." . _:b473777056 . _:b473777057 . _:b473777034 . _:b473777058 . _:b473777059 . _:b473777060 . . _:b473777061 . _:b473777045 . _:b473776832 . _:b473777062 . _:b473777063 . _:b473777062 . _:b473777044 . _:b473776838 . _:b473777047 . _:b473777046 . . _:b473776892 . _:b473776996 . _:b473777041 . _:b473776868 . . _:b473777040 . _:b473776859 . _:b473776931 . _:b473777043 . _:b473777042 . _:b473777053 . _:b473777052 . _:b473777030 . _:b12702169 "Upstream regulation of Hippo signaling occurs through at least two transmembrane proteins, Ft and Crb [reviewed in [>>44<<]]. Crb acts through Ex [32-34], and Ft acts through both Ex [14,15,19] and the unconventional myosin Dachs (D) [12]. The overexpression of ex can promote ectopic Hippo signaling, and when expressed in otherwise wild type clones can" . _:b473777055 . _:b473777036 . _:b473777054 . _:b473777049 . _:b473776993 . _:b473777048 . . . . _:b473777051 . _:b12702157 . _:b473777050 . _:b473776835 . _:b473776977 . _:b473777061 . _:b473777106 . _:b473776925 . _:b473777060 . _:b473776803 . _:b473777039 . . _:b473776849 . _:b473777063 . _:b473776817 . _:b473777083 . _:b473777062 . _:b473777057 . . . . _:b473776955 . _:b473777056 . _:b473777059 . _:b12702263 "In contrast to lgl, reduced levels of Scrib in the eye disc to levels where apico-basal cell polarity is only mildly affected does not impair Hippo signaling [>>35<<], although prior studies with null alleles have demonstrated both aPKC-dependent proliferation as well as ectopic JNK signaling in scrib mutant eye disc clones [38]." . _:b473777058 . _:b473777069 . _:b473776791 "13"^^ . _:b473777068 . _:b473777071 . _:b12702253 . _:b12702250 "genes that regulate cell polarity, scrib, discs large (dlg) and lethal giant larvae (lgl), as well as mutants within the endocytic pathway including avalanche (avl), tumor suppressor protein 101 (TSG101) and Rab5 [reviewed in [>>22<<]]. The loss of apico-basal cell polarity and overgrowth phenotypes of a number of these mutants, including scrib, lgl, avl and TSG101 are dependent upon atypical protein kinase C (aPKC) activity, since mutant phenotypes can be rescued by" . . _:b473777070 . _:b473776787 "17"^^ . _:b473777065 . _:b473777073 . _:b473776788 "17"^^ . _:b12702163 . _:b12702172 "Crb acts through Ex [32-34], and Ft acts through both Ex [14,>>15<<,19] and the unconventional myosin Dachs (D) [12]." . _:b473776789 "15"^^ . _:b12702158 "Loss of scrib also promotes tumorigenesis in cooperation with oncogenic Ras signaling [reviewed in [>>28<<]]. In a \"two hit\" Drosophila tumorigenesis model we have previously shown that although scrib mutant eye clones die via JNK-mediated apoptosis, if RasACT or its downstream effector, Rafgof, is expressed in the mutant clones, cell death is" . _:b473777064 . _:b12702261 . _:b12702225 "As the cell morphology defects in scrib mutant eye disc clones are aPKC-dependent [>>38<<], it will be important to determine what role aPKC signaling plays in the scrib mutant wing disc phenotypes." . _:b473776790 "14"^^ . _:b473777067 . _:b473777066 . _:b12702263 . . _:b473777077 . _:b473776798 "9"^^ . . _:b12702243 "Drosophila has long been recognized as an important model organism for elucidating oncogenic and tumor suppressor pathways [reviewed in [>>1<<]]. Traditionally two distinct classes of tumor suppressor mutants have been described, the loss of which cause either hyperplastic or neoplastic overgrowth [reviewed in [2]]." . _:b473776856 . _:b473776799 "8"^^ . _:b473777076 . _:b473776857 . _:b473776858 . _:b473776859 . _:b473777005 . _:b473776860 . . _:b473777079 . _:b473776861 . _:b473776862 . _:b473776796 "10"^^ . _:b473776863 . _:b473776848 . _:b473776849 . _:b12702259 . _:b473777078 . _:b473776797 "9"^^ . _:b473776850 . . . _:b473776851 . _:b473776951 . _:b473776795 "11"^^ . _:b473776852 . . _:b473776853 . _:b473777073 . _:b473776854 . . _:b12702259 . _:b473776855 . _:b473776840 . _:b12702247 . _:b12702170 . _:b473776794 "12"^^ . _:b12702247 . _:b473777072 . _:b473776841 . _:b473776842 . _:b473776843 . _:b473776888 . _:b473776844 . _:b473776793 "12"^^ . _:b12702163 "Consistent with the scrib mutant defects in Hippo signaling being aPKC-dependent, ectopic activation of aPKC has been shown to be sufficient to impair Hippo signaling [>>35<<,37]. This has been linked to a capacity for aPKC to activate JNK [37], however, we have previously shown that although the expression of a truncated activated allele of aPKC (aPKC\u0394N) in eye disc clones induces JNK-dependent cell death, if" . _:b473777075 . _:b473776845 . _:b473776846 . _:b12702167 "aPKC-dependent impairment to Hippo signaling, we next wanted to determine if aPKC signaling was also important for promoting tissue overgrowth in core Hippo pathway mutants such as wts that are known to upregulate aPKC protein levels [>>30<<,31]. However, although wtsRNAi-expressing clones ectopically express CycE and DIAP1 and overgrow (Figure 8A, B), similar to scrib mutant clones, coexpression of aPKCCAAXDN in wtsRNAi clones neither prevented the ectopic expression of CycE" . _:b473776847 . _:b473776832 . _:b12702251 . _:b473776833 . _:b473777074 . _:b473776792 "13"^^ . _:b473776834 . . . _:b473776835 . _:b12702198 . _:b473776806 "7"^^ . _:b473776836 . _:b473776837 . _:b473777085 . _:b473776838 . _:b473776839 . _:b473776807 "7"^^ . _:b473776888 . . _:b473776889 . _:b12702265 . _:b473777084 . _:b473777040 . _:b473776890 . _:b12702193 . _:b473776891 . _:b473776804 "7"^^ . . _:b473776892 . . _:b473777087 . _:b473776980 . _:b473776893 . _:b473776870 . _:b473776894 . _:b12702153 "Indeed, as in the eye disc [>>38<<,39], loss of scrib in the wing disc similarly led to the ectopic expression of the JNK pathway reporter msn-lacZ (Figure 4C)." . _:b473776895 . _:b473776805 "7"^^ . _:b12702247 . _:b473776880 . _:b473776881 . _:b473777086 . . _:b473776882 . _:b473776883 . _:b12702215 "However, this interpretation is complicated by Ex's capacity to directly bind to, and sequester Yki activity [>>46<<]. In fact, even ex overexpression was not sufficient to block ectopic CycE expression in scrib mutant clones, and epistasis experiments confirm that the deregulated Hippo signaling in scrib mutants is at least partially epistatic to both" . _:b473776802 "7"^^ . _:b473776884 . . _:b12702164 . _:b473777081 . _:b473776885 . _:b473776886 . _:b473776887 . _:b473776803 "7"^^ . _:b12702141 . _:b473776872 . . _:b12702165 . _:b473777080 . _:b473776873 . _:b473776874 . . _:b473776875 . _:b473776876 . _:b473776800 "8"^^ . _:b473777083 . _:b473776877 . _:b473776878 . _:b473776879 . _:b473776864 . _:b473776801 "8"^^ . _:b473777082 . _:b473776865 . . _:b473776866 . _:b473776814 "6"^^ . _:b473776867 . _:b473776921 . _:b473776868 . _:b473776869 . _:b12702152 . _:b473777093 . . _:b473777098 . _:b473776870 . _:b473776815 "6"^^ . _:b473776871 . _:b473776792 . _:b473776793 . _:b473777092 . _:b473777017 . _:b473776794 . _:b473776812 "6"^^ . _:b473776795 . _:b473776796 . _:b473776797 . _:b12702245 "Over recent years, a large number of hyperplastic tumor suppressor mutants have been united into a single pathway, the Hippo pathway [reviewed in [>>3<<]]. Core components of the Hippo pathway include the serine-threonine kinases Hippo (Hpo) and Warts (Wts), and their adaptor proteins, Salvador (Sav) and Mob-As-Tumor-Suppressor (Mats)." . _:b473777095 . _:b473776798 . _:b473776813 "6"^^ . _:b473776799 . _:b473777094 . _:b473776810 "6"^^ . _:b473776787 . _:b12702188 _:b12702208 . _:b473777102 . _:b473776788 . _:b12702188 _:b12702209 . _:b473777089 . _:b473776789 . _:b12702188 _:b12702210 . _:b473776790 . . _:b473776811 "6"^^ . _:b473776791 . _:b12702188 _:b12702211 . . _:b12702182 . _:b473777088 . _:b473776943 . _:b473776808 "7"^^ . . _:b473777091 . . _:b473776809 "6"^^ . _:b12702170 "Crb acts through Ex [>>32<<-34], and Ft acts through both Ex [14,15,19] and the unconventional myosin Dachs (D) [12]." . _:b473777090 . _:b473777101 . _:b473776821 "5"^^ . . _:b473776824 . _:b473776825 . _:b473777100 . _:b473776820 "5"^^ . _:b473776826 . _:b473776827 . _:b12702175 "The overexpression of ex can promote ectopic Hippo signaling, and when expressed in otherwise wild type clones can restrain clonal growth in a Hpo and Wts-dependent manner [>>13<<,17,41]. Significantly, the overexpression of ex in scrib mutant eye disc clones also restrained clonal growth, and this was the case even if JNK-mediated apoptosis of the clonal tissue was prevented (Figure 9A-F). Indeed, scrib clones" . _:b473776828 . _:b473776829 . _:b12702146 . _:b473777103 . _:b473776823 "5"^^ . _:b473776830 . _:b473776930 . _:b473776831 . _:b473776816 . _:b12702140 . _:b473776817 . _:b12702219 . _:b473777102 . _:b473776822 "5"^^ . _:b473776818 . _:b473776819 . _:b473776820 . _:b12702141 . . _:b12702218 . _:b473777097 . _:b473776821 . _:b473776817 "5"^^ . _:b473776822 . _:b473776823 . _:b12702156 . . _:b12702142 . _:b473776808 . . _:b473776809 . _:b473777096 . _:b473776816 "5"^^ . _:b473776810 . _:b473776811 . _:b473776812 . _:b12702143 . _:b473777080 . _:b473776813 . _:b473777099 . _:b473776819 "5"^^ . _:b473777084 . _:b473776814 . _:b473776815 . _:b473776800 . . _:b473777098 . _:b473776801 . _:b473776818 "5"^^ . _:b473776802 . _:b473776803 . _:b473776804 . _:b473776805 . _:b473777109 . _:b473776829 "5"^^ . _:b473776806 . _:b473776877 . _:b473776807 . _:b473776938 . _:b473777108 . _:b473776828 "5"^^ . _:b12702155 . _:b473777009 . _:b473776831 "4"^^ . _:b473777111 . _:b12702139 . _:b12702148 . _:b473776830 "4"^^ . _:b473777110 . _:b473776810 . _:b473776935 . _:b12702149 . _:b473776821 . _:b473777105 . _:b473776825 "5"^^ . _:b12702150 . _:b473776962 . _:b473777104 . _:b473776824 "5"^^ . . _:b473776809 . _:b12702151 . _:b473777107 . _:b473776827 "5"^^ . _:b12702144 . _:b473777106 . _:b473776826 "5"^^ . _:b12702145 . _:b473776837 "4"^^ . . _:b12702146 . _:b473776830 . _:b473776836 "4"^^ . _:b473777116 . _:b12702147 . _:b473776839 "4"^^ . _:b12702156 . _:b473776838 "4"^^ . . _:b473776895 . _:b12702194 . _:b473777001 . _:b12702157 . _:b473776833 "4"^^ . _:b473777113 . _:b473776999 . _:b12702246 . . _:b12702158 . _:b473776832 "4"^^ . _:b473777112 . _:b473777100 . _:b12702159 . _:b473776835 "4"^^ . _:b473777115 . _:b473777096 . _:b473776910 . _:b12702152 . _:b473776834 "4"^^ . _:b473777114 . . . _:b473777029 . _:b12702153 . _:b473776845 "4"^^ . _:b473776948 . _:b473776997 . _:b12702154 . _:b473776844 "4"^^ . _:b12702155 . _:b473776847 "4"^^ . _:b12702164 . _:b473776846 "4"^^ . _:b12702224 . _:b12702165 . _:b473776841 "4"^^ . . _:b473777104 . _:b12702166 . _:b473776840 "4"^^ . _:b12702162 "in scrib mutant clones could both be rescued by the expression of a membrane-tethered, kinase-dead (dominant negative) form of aPKC (aPKCCAAXDN), although this was not sufficient to rescue the mutant cells from JNK-dependent cell death [>>38<<]. Therefore to determine if the scrib mutant defects in Hippo pathway activity were aPKC-dependent, we examined ex-lacZ and fj-lacZ in scrib mutant clones expressing aPKCCAAXDN, that were also protected from apoptosis by the coexpression" . _:b12702167 . _:b473776843 "4"^^ . _:b12702160 . _:b473776842 "4"^^ . _:b473776865 . _:b12702199 . _:b473776853 "3"^^ . _:b12702161 . _:b473776793 . . _:b473777010 . . . _:b12702162 . _:b473776852 "4"^^ . _:b473776855 "3"^^ . _:b12702163 . _:b473777115 . _:b12702154 "Indeed, as in the eye disc [38,>>39<<], loss of scrib in the wing disc similarly led to the ectopic expression of the JNK pathway reporter msn-lacZ (Figure 4C)." . _:b473776854 "3"^^ . _:b12702172 . . . _:b12702184 . _:b12702173 . _:b473776849 "4"^^ . _:b12702180 . _:b12702179 . . . _:b12702174 . _:b473776848 "4"^^ . _:b473777093 . . _:b12702175 . _:b473776851 "4"^^ . _:b12702168 . _:b473776850 "4"^^ . _:b12702174 . . . _:b12702169 . _:b473776861 "3"^^ . _:b473776871 . _:b473776860 "3"^^ . _:b12702170 . _:b473776864 . _:b473776863 "3"^^ . _:b12702171 . . . _:b473776862 "3"^^ . _:b12702180 . _:b473776857 "3"^^ . . _:b12702181 . . _:b473776856 "3"^^ . _:b12702264 "cell polarity is only mildly affected does not impair Hippo signaling [35], although prior studies with null alleles have demonstrated both aPKC-dependent proliferation as well as ectopic JNK signaling in scrib mutant eye disc clones [>>38<<]. Furthermore, whilst homozygous scrib mutant wing disc overgrowth is reduced in response to limiting Yki levels [35], it has not been determined if Hippo signaling is impaired in this tissue and whether the genetic interaction with yki" . _:b12702182 . . _:b473777037 . _:b473776859 "3"^^ . _:b12702183 . _:b473776858 "3"^^ . _:b12702209 "[26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12 [75]; UAS-scrib19.2 [38]; FRT82B, scrib1 [76]; scrib3 [77]; UAS-scribRNAi (VDRC #27424); UAS-sdRNAi (NIG #8544R-2); wtsX1 [78]; UAS-wtsRNAi (NIG #12072R-1); ykiB5 [>>7<<]; UAS-ykiRNAi [5]." . _:b12702176 . _:b473777072 . _:b473776869 "3"^^ . _:b12702177 . "PMC0" . _:b473776868 "3"^^ . _:b12702178 . _:b473776952 . _:b12702237 "cell polarity pathways is not confined to traditional apico-basal epithelial polarity regulators such as Scrib and aPKC, since the Hippo pathway components ft, fj and ds also participate in planar cell polarity pathways [reviewed in [>>56<<]], as do scrib [57], lgl [58] and aPKC [59,60]." . _:b473776871 "3"^^ . _:b12702179 . _:b473776870 "3"^^ . _:b12702188 . _:b473776865 "3"^^ . _:b12702189 . _:b473776864 "3"^^ . _:b12702190 . _:b473776808 . . _:b12702171 "Crb acts through Ex [32-34], and Ft acts through both Ex [>>14<<,15,19] and the unconventional myosin Dachs (D) [12]." . _:b12702254 . _:b473776867 "3"^^ . _:b12702191 . . _:b473776866 "3"^^ . _:b12702184 . _:b473776877 "3"^^ . _:b12702185 . _:b473776934 . _:b473776876 "3"^^ . _:b12702186 . _:b473776960 . _:b473777081 . _:b473776879 "3"^^ . _:b12702187 . _:b473776882 . _:b473776804 . _:b473776796 . _:b473776878 "3"^^ . _:b12702196 . _:b473776873 "3"^^ . _:b12702197 . _:b473777068 . _:b12702158 . . _:b473776872 "3"^^ . _:b12702198 . _:b473776946 . . _:b473776797 . _:b473776875 "3"^^ . _:b12702199 . _:b473776939 . _:b473776874 "3"^^ . _:b12702192 . . _:b473776885 "3"^^ . _:b12702193 . _:b473776959 . _:b473776967 . _:b473776884 "3"^^ . _:b12702194 . _:b473776954 . _:b473776887 "3"^^ . _:b12702195 . _:b473776886 "3"^^ . _:b12702204 . . _:b12702160 "To further confirm the importance of impaired Hippo signaling in scrib- + Rafgof tumorigenesis, we also halved the gene dosage of yki in the tumor background, and knocked down yki function within the tumor using a ykiRNAi transgene [>>5<<]. Consistent with the effects of sdRNAi, both methods of limiting yki function were unable to restore pupation or prevent tumor overgrowth throughout an extended larval phase of development, however, they significantly reduced tumor size" . _:b473776881 "3"^^ . _:b12702205 . . _:b473776880 "3"^^ . _:b12702206 . . . _:b473776883 "3"^^ . _:b12702207 . . . _:b473776882 "3"^^ . _:b12702200 . _:b473777075 . _:b12702140 "We have previously reported that scrib mutant clones of tissue in the eye disc exhibit ectopic CycE expression and excessive cell proliferation, although this is restrained through JNK-dependent apoptosis [>>39<<]. If JNK signaling is blocked in scrib mutant clones by expressing a dominant negative version of Drosophila JNK, bsk (bskDN), apoptosis is prevented and mutant cells are observed to ectopically proliferate posterior to the morphogenetic" . _:b473776893 "3"^^ . _:b12702201 . _:b473777007 . _:b12702197 . . _:b12702252 "antagonism between the junctional tumor suppressors and aPKC has been demonstrated by the ability of aPKC to associate with and phosphorylate Lgl, thereby releasing Lgl from the cell cortex and thus potentially inhibiting Lgl function [>>26<<], and the ability of Lgl to inhibit aPKC-dependent phosphorylation of other key targets [27]." . . _:b473776892 "3"^^ . _:b12702202 . . . _:b473777033 . _:b473776895 "3"^^ . _:b12702203 . _:b473777050 . _:b473776894 "3"^^ . _:b473776889 "3"^^ . _:b12702211 "Clonal analysis utilized either MARCM (mosaic analysis with repressible cell marker) [>>79<<] with FRT82B and ey-FLP1 to induce clones and mCD8-GFP expression to mark mutant tissue, or for negatively marked scrib1 clones, hs-FLP with FRT82B, Ubi-GFP." . _:b473776888 "3"^^ . _:b473776891 "3"^^ . _:b473776998 . . . _:b473776890 "3"^^ . . _:b473776901 "3"^^ . . _:b473776900 "3"^^ . _:b473776945 . _:b473776903 "3"^^ . . . _:b473776854 . . _:b473776989 . _:b12702165 "This has been linked to a capacity for aPKC to activate JNK [>>37<<], however, we have previously shown that although the expression of a truncated activated allele of aPKC (aPKC\u0394N) in eye disc clones induces JNK-dependent cell death, if JNK signaling is blocked, ectopic cell proliferation still ensues" . _:b473776902 "3"^^ . . _:b473776904 . _:b12702193 "were used: ey-FLP1, UAS-mCD8-GFP;;Tub-GAL4, FRT82B, Tub-GAL80 [64]; y, w, hs-FLP; FRT82B, Ubi-GFP; en-GAL4; UAS-DaPKC\u0394N [26]; UAS-DaPKCCAAXDN [65]; UAS-aPKCRNAi (VDRC #2907); bsk2 [66]; UAS-bskDN [67]; UAS-bskRNAi (NIG #5680R-1); d1 [>>68<<]; dGC13 [12]; exe1 [69]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74];" . _:b473776897 "3"^^ . _:b473776896 "3"^^ . _:b473776899 "3"^^ . _:b473776862 . . . _:b473776898 "3"^^ . _:b12702191 . _:b473776909 "3"^^ . _:b473776909 . _:b473776908 "3"^^ . _:b473776911 "3"^^ . . _:b473776910 "3"^^ . _:b473776984 . _:b473776836 . . _:b12702227 . _:b473776905 "3"^^ . _:b12702259 "Crb itself acts to regulate Hippo signaling by binding to Ex [32], and either excessive Crb activity or loss of Crb results in deregulation of Ex and an impairment to Hippo signaling resulting in tissue overgrowth [>>32<<-35]. The neoplastic tumor suppressors scrib, dlg and lgl also interact with the Hippo pathway. dlg, lgl or scrib mutant follicle cells surrounding the female ovary have elevated levels of Yki targets Cyclin E and DIAP1, and exhibit strong" . _:b473776904 "3"^^ . _:b12702236 "Crb, aPKC [30,31], and F-actin [48], although, interestingly, despite the potential for increased Crb, aPKC and F-actin to promote tissue hyperplasia, the overgrowth in Hippo pathway mutants is independent of the apical hypertrophy [30,>>31<<]. This is consistent with our own work demonstrating that wts and ft mutant tissue overgrowth was independent of aPKC signaling." . . _:b473776907 "3"^^ . _:b473777014 . _:b12702221 . _:b473776906 "3"^^ . _:b473777011 . _:b473776917 "3"^^ . _:b473776916 "3"^^ . _:b473776919 "3"^^ . . _:b12702238 . . _:b473776918 "3"^^ . _:b473776787 . _:b473776913 "3"^^ . . _:b473776912 "3"^^ . _:b12702183 . . _:b473777045 . _:b473776915 "3"^^ . _:b12702150 "In contrast, Sd is largely dispensable for normal eye disc growth and proliferation and specifically mediates Hippo pathway mutant overgrowth [4,>>5<<]. Therefore, to determine if the ectopic cell proliferation and altered cell morphology of scrib mutant cells were due to loss of Hippo pathway signaling we utilized RNAi-mediated knockdown of sd function in scrib mutant eye disc clones" . . . _:b473776914 "3"^^ . _:b473776925 "2"^^ . _:b12702223 . . . . _:b473776924 "2"^^ . _:b473776790 . _:b473776927 "2"^^ . _:b12702198 "[26]; UAS-DaPKCCAAXDN [65]; UAS-aPKCRNAi (VDRC #2907); bsk2 [66]; UAS-bskDN [67]; UAS-bskRNAi (NIG #5680R-1); d1 [68]; dGC13 [12]; exe1 [69]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [>>71<<]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12 [75]; UAS-scrib19.2 [38]; FRT82B, scrib1 [76]; scrib3 [77]; UAS-scribRNAi (VDRC #27424); UAS-sdRNAi (NIG" . _:b473776926 "2"^^ . _:b473776921 "2"^^ . . _:b473776920 "2"^^ . _:b473777069 . . _:b473776923 "2"^^ . . _:b12702223 "Certainly loss of scrib is notable for eliciting much stronger cell morphology defects in the eye disc than loss of lgl [>>39<<,50]. As the cell morphology defects in scrib mutant eye disc clones are aPKC-dependent [38], it will be important to determine what role aPKC signaling plays in the scrib mutant wing disc phenotypes." . _:b473776968 . _:b473776922 "2"^^ . . . . _:b12702241 . _:b473776933 "2"^^ . . _:b473776932 "2"^^ . _:b473776935 "2"^^ . _:b473776934 "2"^^ . _:b473777090 . _:b473776929 "2"^^ . . . _:b12702258 . . _:b473776928 "2"^^ . _:b12702245 . _:b12702259 . . _:b473776931 "2"^^ . . _:b473776858 . _:b473777043 . _:b12702196 "Ubi-GFP; en-GAL4; UAS-DaPKC\u0394N [26]; UAS-DaPKCCAAXDN [65]; UAS-aPKCRNAi (VDRC #2907); bsk2 [66]; UAS-bskDN [67]; UAS-bskRNAi (NIG #5680R-1); d1 [68]; dGC13 [12]; exe1 [69]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [>>69<<]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12 [75]; UAS-scrib19.2 [38]; FRT82B, scrib1 [76]; scrib3 [77]; UAS-scribRNAi (VDRC" . . _:b473776930 "2"^^ . . _:b473776941 "2"^^ . _:b473776940 "2"^^ . . _:b473776866 . . _:b473776943 "2"^^ . . . _:b473776942 "2"^^ . _:b473776937 "2"^^ . _:b473776936 "2"^^ . _:b473776940 . . _:b473776964 . _:b473776939 "2"^^ . _:b12702259 . . _:b12702177 "The overexpression of ex can promote ectopic Hippo signaling, and when expressed in otherwise wild type clones can restrain clonal growth in a Hpo and Wts-dependent manner [13,17,>>41<<]. Significantly, the overexpression of ex in scrib mutant eye disc clones also restrained clonal growth, and this was the case even if JNK-mediated apoptosis of the clonal tissue was prevented (Figure 9A-F). Indeed, scrib clones" . _:b473776938 "2"^^ . . _:b473776949 "2"^^ . . _:b12702240 . _:b473776908 . _:b473776948 "2"^^ . _:b473777052 . . _:b473776798 . . _:b473776951 "2"^^ . _:b473776911 . . _:b473776950 "2"^^ . _:b473776945 "2"^^ . . _:b473776944 "2"^^ . _:b473776947 "2"^^ . . _:b473776946 "2"^^ . . _:b473776957 "2"^^ . _:b473776956 "2"^^ . _:b473777056 . _:b12702205 "in all figures except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [73]; Ras85De1b [74]; UAS-dRas1V12 [75]; UAS-scrib19.2 [>>38<<]; FRT82B, scrib1 [76]; scrib3 [77]; UAS-scribRNAi (VDRC #27424); UAS-sdRNAi (NIG #8544R-2); wtsX1 [78]; UAS-wtsRNAi (NIG #12072R-1); ykiB5 [7]; UAS-ykiRNAi [5]." . _:b473776959 "2"^^ . _:b473776958 "2"^^ . _:b12702143 "Targets examined included protein levels of DIAP1 [40], and expression of the enhancer traps for four-jointed (fj-lacZ) and ex (ex-lacZ), both of which are known to function as readouts of impaired Hippo signaling [>>17<<,41]." . _:b473776953 "2"^^ . _:b12702170 . _:b12702202 "(NIG #5680R-1); d1 [68]; dGC13 [12]; exe1 [69]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A [26]; UAS-phlgof (UAS-Rafgof) [>>73<<]; Ras85De1b [74]; UAS-dRas1V12 [75]; UAS-scrib19.2 [38]; FRT82B, scrib1 [76]; scrib3 [77]; UAS-scribRNAi (VDRC #27424); UAS-sdRNAi (NIG #8544R-2); wtsX1 [78]; UAS-wtsRNAi (NIG #12072R-1); ykiB5 [7]; UAS-ykiRNAi [5]." . _:b473776952 "2"^^ . . . . _:b473776937 . . _:b473776955 "2"^^ . _:b473776954 "2"^^ . _:b473776965 "2"^^ . _:b473776964 "2"^^ . _:b12702253 "of aPKC to associate with and phosphorylate Lgl, thereby releasing Lgl from the cell cortex and thus potentially inhibiting Lgl function [26], and the ability of Lgl to inhibit aPKC-dependent phosphorylation of other key targets [>>27<<]. Like the Hippo pathway, mammalian homologues of the Drosophila neoplastic tumor suppressors, as well as aPKC, are increasingly implicated as important players in human cancers [reviewed in [28]]." . _:b473776967 "2"^^ . _:b473776802 . _:b473776966 "2"^^ . _:b473776961 "2"^^ . . _:b473776960 "2"^^ . _:b473776974 . _:b473776963 "2"^^ . . . _:b473776962 "2"^^ . _:b473777088 . _:b473776973 "2"^^ . _:b12702180 "In the wing disc, loss of scrib is also associated with JNK activation, and although JNK signaling has been shown to be sufficient to downregulate the Hippo pathway in the wing disc [>>37<<], Hippo pathway signaling remains impaired in scrib mutants even when JNK is blocked." . . _:b473776972 "2"^^ . _:b473777003 . _:b473776975 "2"^^ . . _:b473776974 "2"^^ . _:b12702248 . _:b473776969 "2"^^ . _:b473776968 "2"^^ . _:b473776971 "2"^^ . _:b473776906 . _:b473777067 . _:b473776970 "2"^^ . . _:b473776981 "2"^^ . _:b12702202 . _:b473776980 "2"^^ . . _:b473776953 . _:b473776941 . _:b473776983 "2"^^ . _:b473776933 . _:b473776942 . _:b473776982 "2"^^ . _:b12702228 "Indeed, the role of JNK in neoplasia is proving to be complex, since in some contexts it functions as an oncogene to promote neoplasia, whilst in different contexts it acts as a tumor suppressor through the induction of apoptosis [38,39,>>51<<-53]. Its effects upon Hippo pathway regulation may therefore also be context dependent." . _:b473776977 "2"^^ . _:b473776976 "2"^^ . _:b12702234 "Loss of wts is capable of eliciting neoplastic overgrowth [55], and Hippo pathway mutants induce apical hypertrophy with increased levels of apical cell determinants such as Crb, aPKC [30,31], and F-actin [>>48<<], although, interestingly, despite the potential for increased Crb, aPKC and F-actin to promote tissue hyperplasia, the overgrowth in Hippo pathway mutants is independent of the apical hypertrophy [30,31]." . _:b12702212 "discussion" . _:b473776979 "2"^^ . _:b473776978 "2"^^ . _:b12702244 . _:b473776989 "2"^^ . _:b473777085 . _:b473776988 "2"^^ . _:b473776991 "2"^^ . . _:b12702141 "clones by expressing a dominant negative version of Drosophila JNK, bsk (bskDN), apoptosis is prevented and mutant cells are observed to ectopically proliferate posterior to the morphogenetic furrow (MF) resulting in clonal overgrowth [>>38<<]. The ectopic cell proliferation in scrib mutant clones expressing bskDN is similar to mutants in the Hippo pathway, and therefore to determine if the Hippo pathway is impaired by the loss of scrib, we examined known targets of" . _:b473776990 "2"^^ . . _:b473776820 . _:b473776985 "2"^^ . _:b473776984 "2"^^ . . . _:b473776987 "2"^^ . _:b473776986 "2"^^ . . . _:b473776997 "2"^^ . _:b473776887 . . _:b12702265 "Furthermore, whilst homozygous scrib mutant wing disc overgrowth is reduced in response to limiting Yki levels [>>35<<], it has not been determined if Hippo signaling is impaired in this tissue and whether the genetic interaction with yki reflects a general sensitivity of scrib mutant tissue to limiting levels of survival/proliferation functions." . _:b473776996 "2"^^ . _:b473776999 "2"^^ . . _:b12702212 . _:b473776998 "2"^^ . _:b12702213 . _:b473776788 . _:b473776993 "2"^^ . _:b12702214 . _:b473776899 . _:b473776827 . _:b473776992 "2"^^ . _:b12702215 . . _:b12702235 "as Crb, aPKC [30,31], and F-actin [48], although, interestingly, despite the potential for increased Crb, aPKC and F-actin to promote tissue hyperplasia, the overgrowth in Hippo pathway mutants is independent of the apical hypertrophy [>>30<<,31]. This is consistent with our own work demonstrating that wts and ft mutant tissue overgrowth was independent of aPKC signaling." . _:b473776995 "2"^^ . _:b12702208 . _:b12702148 . _:b473776994 "2"^^ . _:b12702209 . . _:b473777005 "2"^^ . _:b12702224 "Certainly loss of scrib is notable for eliciting much stronger cell morphology defects in the eye disc than loss of lgl [39,>>50<<]. As the cell morphology defects in scrib mutant eye disc clones are aPKC-dependent [38], it will be important to determine what role aPKC signaling plays in the scrib mutant wing disc phenotypes." . _:b12702210 . _:b12702231 . _:b473777070 . . _:b12702211 . _:b473777004 "2"^^ . . _:b473777007 "2"^^ . _:b12702220 . _:b12702215 . _:b12702226 "Indeed, the role of JNK in neoplasia is proving to be complex, since in some contexts it functions as an oncogene to promote neoplasia, whilst in different contexts it acts as a tumor suppressor through the induction of apoptosis [>>38<<,39,51-53]. Its effects upon Hippo pathway regulation may therefore also be context dependent." . _:b473777006 "2"^^ . _:b12702221 . . . _:b473777001 "2"^^ . _:b12702222 . _:b473777000 "2"^^ . _:b12702223 . _:b473777003 "2"^^ . _:b12702216 . _:b473777002 "2"^^ . _:b12702217 . . . _:b473777013 "2"^^ . _:b12702218 . _:b12702190 . _:b473776869 . _:b473777012 "2"^^ . _:b12702219 . _:b12702248 "It is proposed that the primary function of the Hippo pathway is to incorporate positional cues within an epithelial field to dictate the ultimate size of organ development [>>20<<]. The pathway is highly conserved and also functions to restrain organ size in mammals. Furthermore, increasing evidence links Hippo pathway deregulation to tumorigenesis [reviewed in [21]]." . _:b473777015 "2"^^ . _:b12702228 . _:b12702203 . . _:b12702229 . _:b473777014 "2"^^ . _:b473776917 . . _:b12702230 . _:b473777009 "2"^^ . . . _:b473777008 "2"^^ . _:b12702231 . _:b12702190 "The following Drosophila stocks were used: ey-FLP1, UAS-mCD8-GFP;;Tub-GAL4, FRT82B, Tub-GAL80 [64]; y, w, hs-FLP; FRT82B, Ubi-GFP; en-GAL4; UAS-DaPKC\u0394N [>>26<<]; UAS-DaPKCCAAXDN [65]; UAS-aPKCRNAi (VDRC #2907); bsk2 [66]; UAS-bskDN [67]; UAS-bskRNAi (NIG #5680R-1); d1 [68]; dGC13 [12]; exe1 [69]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [71];" . _:b473777011 "2"^^ . _:b12702224 . _:b473777010 "2"^^ . _:b12702225 . _:b12702181 "conclusions" . . _:b473777004 . _:b12702226 . _:b473777021 "2"^^ . _:b473776789 . _:b473777020 "2"^^ . _:b12702227 . _:b473777048 . _:b473776788 . _:b473777023 "2"^^ . _:b12702236 . _:b473776791 . _:b473777022 "2"^^ . _:b12702237 . _:b473776885 . _:b473776863 . _:b473776790 . . . _:b12702238 . _:b473777017 "2"^^ . . _:b12702239 . _:b473777016 "2"^^ . _:b12702148 "Removing Yki function can rescue Hippo pathway mutant overgrowth, however, Yki is also required for normal cell proliferation in the eye disc [>>7<<]. In contrast, Sd is largely dispensable for normal eye disc growth and proliferation and specifically mediates Hippo pathway mutant overgrowth [4,5]. Therefore, to determine if the ectopic cell proliferation and altered cell morphology" . _:b473777019 "2"^^ . _:b12702232 . _:b473776787 . _:b473777018 "2"^^ . _:b12702233 . _:b473777029 "2"^^ . _:b12702234 . _:b473776797 . _:b473777028 "2"^^ . _:b12702235 . _:b12702152 "Loss of lgl in the wing disc also impairs Hippo signaling, and this has been shown to depend upon JNK signaling [>>37<<]. Indeed, as in the eye disc [38,39], loss of scrib in the wing disc similarly led to the ectopic expression of the JNK pathway reporter msn-lacZ (Figure 4C)." . _:b473776796 . _:b473777031 "2"^^ . _:b12702244 . _:b473776799 . _:b473777030 "2"^^ . _:b12702245 . _:b473776932 . _:b473776798 . _:b473777025 "2"^^ . _:b12702246 . . _:b473776793 . _:b12702246 . _:b473776944 . _:b473776903 . _:b12702247 . _:b12702201 . _:b473777024 "2"^^ . _:b473776792 . . _:b473777027 "2"^^ . _:b12702240 . _:b12702200 . _:b473777020 . _:b473776795 . _:b473777026 "2"^^ . _:b12702241 . _:b473777023 . _:b473776794 . _:b473777037 "2"^^ . _:b12702242 . _:b473776805 . _:b473777038 . _:b473777036 "2"^^ . _:b12702243 . _:b473776804 . _:b473777039 "2"^^ . _:b12702252 . _:b473776807 . _:b473777038 "2"^^ . _:b12702253 . _:b473776890 . _:b473776806 . _:b12702164 "Consistent with the scrib mutant defects in Hippo signaling being aPKC-dependent, ectopic activation of aPKC has been shown to be sufficient to impair Hippo signaling [35,>>37<<]. This has been linked to a capacity for aPKC to activate JNK [37], however, we have previously shown that although the expression of a truncated activated allele of aPKC (aPKC\u0394N) in eye disc clones induces JNK-dependent cell death, if" . _:b473777033 "2"^^ . _:b12702254 . _:b12702246 "the nucleus where it binds to its DNA binding partner, Scalloped (Sd), and promotes expression of proteins involved in cell proliferation (Cyclin E; CycE), cell growth (Myc) and cell survival (Drosophila Inhibitor of Apoptosis 1; DIAP1) [>>4<<-11]. It is the dual role of the Hippo pathway in regulating both cell proliferation and survival functions that makes its loss such a potent driver of tissue overgrowth." . _:b473776801 . _:b473777032 "2"^^ . _:b12702255 . . _:b473776800 . . . . _:b473777035 "2"^^ . _:b12702248 . _:b473776803 . _:b473777034 "2"^^ . _:b12702249 . . _:b473776802 . . _:b12702161 "Despite the reduction in tumor overgrowth, however, knockdown of yki was unable to prevent tumor cells from adopting an invasive morphology and appearing to move between the brain lobes, as has been previously described [>>38<<], indicating that the tumor cells retained invasive capabilities (see additional file 6)." . _:b473777077 . _:b473777045 "2"^^ . _:b12702250 . _:b473776813 . . _:b12702251 . _:b12702146 "However, a previous report has highlighted the role that JNK signaling can play in promoting Hippo pathway impairment [>>37<<], and therefore we also examined the expression of the reporters in scrib mutant clones in which JNK signaling was blocked by the expression of bskDN." . _:b473777044 "2"^^ . . _:b473776812 . _:b473776845 . _:b12702260 . _:b473777047 "2"^^ . _:b473776815 . _:b473777046 "2"^^ . _:b12702261 . _:b473776814 . _:b473777041 "2"^^ . _:b12702262 . . _:b473776809 . _:b473777040 "2"^^ . _:b12702263 . . _:b473776808 . . _:b12702256 . _:b473777043 "2"^^ . _:b473776811 . _:b12702157 "Consistent with previous reports [>>35<<], although giant larvae were still formed throughout an extended larval phase of development, wing disc overgrowth was dramatically reduced, resulting in significantly smaller wing discs with disorganized morphology (Figure 4F)." . _:b473777019 . _:b12702257 . _:b473777042 "2"^^ . _:b473776810 . _:b473777053 "2"^^ . _:b12702258 . _:b473776821 . _:b12702143 . _:b473777052 "2"^^ . _:b12702259 . _:b473776820 . _:b12702192 . _:b473777055 "2"^^ . _:b473776823 . . _:b12702186 . _:b473777054 "2"^^ . . _:b473776822 . . _:b473777049 "2"^^ . _:b473777114 . . _:b473776817 . . _:b473777048 "2"^^ . . _:b473776816 . _:b473776883 . _:b12702264 . _:b12702191 "The following Drosophila stocks were used: ey-FLP1, UAS-mCD8-GFP;;Tub-GAL4, FRT82B, Tub-GAL80 [64]; y, w, hs-FLP; FRT82B, Ubi-GFP; en-GAL4; UAS-DaPKC\u0394N [26]; UAS-DaPKCCAAXDN [65]; UAS-aPKCRNAi (VDRC #2907); bsk2 [>>66<<]; UAS-bskDN [67]; UAS-bskRNAi (NIG #5680R-1); d1 [68]; dGC13 [12]; exe1 [69]; ex697 (ex-lacZ in all figures except Figure 9J which is exe1) [69]; UAS-ex [70]; ftfd [71]; UAS-ftRNAi (VDRC #9396); fj-lacZ [72]; UAS-lgl5.1 [26]; UAS-lgl3A" . _:b473777051 "2"^^ . . _:b473776794 . _:b473776819 . . _:b12702265 . _:b473777050 "2"^^ . _:b473776818 . . . _:b473777061 "2"^^ . . _:b473776889 . _:b473776829 . _:b473777060 "2"^^ . _:b473776828 . _:b473776876 . _:b12702144 "Targets examined included protein levels of DIAP1 [40], and expression of the enhancer traps for four-jointed (fj-lacZ) and ex (ex-lacZ), both of which are known to function as readouts of impaired Hippo signaling [17,>>41<<]." . _:b473777063 "2"^^ . _:b473776905 . _:b473776850 . . _:b473776831 . . _:b473777062 "2"^^ . _:b473776830 . _:b473777057 "2"^^ . _:b12702204 . _:b473776825 . _:b473776992 . _:b473776828 . _:b473776879 . _:b12702255 . _:b473777056 "2"^^ . _:b473776824 . . _:b473777059 "2"^^ . . _:b12702168 "impairment to Hippo signaling, we next wanted to determine if aPKC signaling was also important for promoting tissue overgrowth in core Hippo pathway mutants such as wts that are known to upregulate aPKC protein levels [30,>>31<<]. However, although wtsRNAi-expressing clones ectopically express CycE and DIAP1 and overgrow (Figure 8A, B), similar to scrib mutant clones, coexpression of aPKCCAAXDN in wtsRNAi clones neither prevented the ectopic expression of CycE or" . _:b473776827 . _:b12702228 . _:b473777058 "2"^^ . _:b473776826 . _:b473776872 . _:b473777069 "2"^^ . _:b473776837 . _:b473776829 . _:b473777068 "2"^^ . _:b473776836 . _:b12702226 . . _:b12702247 . _:b12702247 . _:b473777071 "2"^^ . _:b473776839 . _:b12702225 . _:b473777070 "2"^^ . _:b12702260 . _:b473776838 . _:b473777028 . _:b12702250 . _:b473777065 "2"^^ . _:b12702142 . _:b473776833 . _:b473777064 "2"^^ . _:b473776832 . . _:b473777067 "2"^^ . _:b473776835 . _:b473777066 "2"^^ . _:b473776834 . _:b473777077 "2"^^ . _:b473777065 . _:b473776845 . _:b473777076 "2"^^ . . _:b473776844 . _:b473777079 "2"^^ . _:b473777110 . _:b473776898 . _:b473776847 . . _:b473777078 "2"^^ . _:b473776965 . _:b12702142 "Targets examined included protein levels of DIAP1 [>>40<<], and expression of the enhancer traps for four-jointed (fj-lacZ) and ex (ex-lacZ), both of which are known to function as readouts of impaired Hippo signaling [17,41]." . _:b473776846 . _:b473777073 "2"^^ . _:b473777035 . _:b473776846 . _:b473776841 . . _:b12702242 _:b12702252 . _:b12702242 _:b12702253 . _:b473777072 "2"^^ . _:b12702242 _:b12702254 . _:b12702242 _:b12702255 . _:b473776840 . . _:b12702167 . _:b12702242 _:b12702248 . _:b12702242 _:b12702249 . _:b473777075 "2"^^ . _:b12702242 _:b12702250 . _:b12702242 _:b12702251 . _:b473776843 . _:b12702242 _:b12702244 . . _:b473777074 "2"^^ . _:b12702242 _:b12702245 . _:b12702242 _:b12702246 . _:b12702242 _:b12702247 . _:b473776842 . _:b473777085 "2"^^ . . _:b473776853 . _:b12702242 _:b12702243 . _:b473776986 . _:b473777084 "2"^^ . _:b473776852 . _:b12702258 "Crb itself acts to regulate Hippo signaling by binding to Ex [>>32<<], and either excessive Crb activity or loss of Crb results in deregulation of Ex and an impairment to Hippo signaling resulting in tissue overgrowth [32-35]." . _:b12702182 "Loss of Hippo pathway signaling or ectopic activation of the Yki or Sd homologues, (YAP and TEAD family proteins respectively), is emerging as a powerful oncogenic force [reviewed in [>>21<<]]. Similarly, the mammalian Scrib module is increasingly implicated in tumorigenesis [reviewed in [28]], and mammalian Scrib can restrain tissue transformation by oncogenic Ras [61], and Myc [62]. Mammalian Scrib also functions within" . _:b12702242 _:b12702264 . _:b12702242 _:b12702265 . _:b473777087 "2"^^ . . . _:b473776855 . _:b12702242 _:b12702260 . . _:b473777086 "2"^^ . _:b12702242 _:b12702261 . _:b12702242 _:b12702262 . _:b12702168 . _:b12702242 _:b12702263 . _:b473776854 . _:b12702242 _:b12702256 . _:b12702176 . _:b12702154 . . _:b12702242 _:b12702257 . _:b473777081 "2"^^ . _:b12702242 _:b12702258 . _:b12702242 _:b12702259 . _:b473776849 . . _:b473777080 "2"^^ . . _:b473776848 . _:b473776994 . _:b473777083 "2"^^ . . _:b12702184 "Similarly, the mammalian Scrib module is increasingly implicated in tumorigenesis [reviewed in [28]], and mammalian Scrib can restrain tissue transformation by oncogenic Ras [>>61<<], and Myc [62]." . _:b473776851 . . _:b12702159 . _:b473776801 . _:b473777082 "2"^^ . _:b473776850 . _:b473777093 "2"^^ . _:b473776861 . . _:b12702246 . _:b473777092 "2"^^ . . . _:b473776860 . _:b473777095 "2"^^ . _:b473776863 . _:b473777079 . _:b473777094 "2"^^ . . _:b12702264 . _:b473776862 . _:b473777116 . _:b473777089 "2"^^ . _:b473776857 . _:b473777088 "2"^^ . _:b473776929 . _:b473776856 . _:b473777091 "2"^^ . _:b473776859 . _:b473777090 "2"^^ .