A candidate for a Salvador-interacting protein is encoded by the warts (wts; also known as LATS) gene that encodes a serine-threonine kinase. Clones of wts tissue generate outgrowths that resemble tumors. Nine alleles of wts were identified in the screen, and the phenotype of sav3 is similar to that elicited by hypomorphic mutations in wts. Null alleles of wts display a more severe phenotype. Like sav, wts clones in the pupal retina have additional interommatidial cells. Larval imaginal discs containing large wts clones are enlarged and convoluted. Larval eye discs that contain eyFLP-induced wts clones are composed mostly of mutant tissue with small regions of wild-type tissue. Many additional BrdU-incorporating nuclei are observed in mutant clones posterior to the SMW. As observed with sav, the stripe of cyclin E RNA expression is also broadened in these discs. Moreover, the normal cell death that occurs in the pupal retina is almost completely abolished in wts mutant clones. Thus, as for sav, wts mutations generate additional interommatidial cells resulting from both increased cell proliferation posterior to the SMW as well as reduced apoptosis in the pupal retina. In addition, Drice activation induced by GMR-hid is markedly diminished in wts clones (Tapon, 2002).
Overexpression of sav alone using the GMR promoter has no effect, and overexpression of wts generates subtle irregularities in ommatidial architecture. However, combined overexpression of sav and wts results in a smaller eye where the ommatidial pattern is highly irregular. This effect appears to reflect a synergistic increase in cell death in the eye discs of flies that express both transgenes as well as a minor effect on reducing cell proliferation assoiated with the SMW (Tapon, 2002).
Thus, Sav and Wts may function in the same pathway and may bind to each other. Indeed, the Sav protein has a Group I WW domain that is predicted to interact with the PPXY (PY) motif, five of which are found in the Wts protein. To test whether Drosophila Sav and Wts proteins could physically interact, a GST pull-down assay was employed. The region containing the two potential WW domains of Sav was fused to GST and incubated with cell lysates that expressed Myc-tagged Wts protein. Using this assay, Wts was found to interact specifically with the region of Sav that contained the WW domain. Furthermore, a 15 amino acid peptide, designed to mimic one of the PY motifs of Wts, was found to inhibit the interaction between the WW domain region of Sav and Wts. An identical peptide where the tyrosine residue that is required for interaction with type I WW domains had been replaced by an alanine did not prevent this interaction. Thus, at least under the conditions of this experiment, Sav and Wts interact in a WW domain- and PY motif-dependent fashion, suggesting that an analogous interaction could occur in vivo (Tapon, 2002).
Discs containing clones of the wts null allele, wtslatsX1, are much larger than discs containing sav3 clones. If all sav functions were wts dependent, the double mutant phenotype should not be more severe than the wts phenotype. When mutant clones were generated with eyFLP, average disc sizes were 39,669 pixels for sav3, wtslatsX1 double mutant discs and 31,360 pixels for wtslatsX1 discs. Thus, the double mutant discs were significantly larger than the wtslatsX1 discs. Thus, while sav and wts appear to function together in certain ways, they are also likely to have functions that are independent of each other (Tapon, 2002).
Tissue growth during animal development is tightly controlled so that the organism can develop harmoniously. The salvador (sav) gene, which encodes a scaffold protein, restricts cell number by coordinating cell-cycle exit and apoptosis during Drosophila development. Hippo (Hpo), the Drosophila ortholog of the mammalian MST1 and MST2 serine/threonine kinases, is a partner of Sav. Hippo was described in five publications that appeared simutaneously: Pantalacci (2003) identified Hippo in a yeast two-hybrid screen in a search for Salvador interacting proteins, Udan (2003) identifed and positionally cloned hippo in a mutagenesis screen for genes that regulate tissue growth, and Harvey (2003), Jia (2003) and Wu (2003) identified hippo in screens for genes that restrict growth and cell number. Loss of hpo function leads to sav-like phenotypes, whereas gain of hpo function results in the opposite phenotype. Whereas Sav and Hpo normally restrict cellular quantities of the Drosophila inhibitor of apoptosis protein DIAP1 (Thread), overexpression of Hpo destabilizes DIAP1 in cell culture. DIAP1 is phosphorylated in a Hpo-dependent manner in S2 cells and Hpo can phosphorylate DIAP1 in vitro. Thus, Hpo may promote apoptosis by reducing cellular amounts of DIAP1. In addition, Sav is an unstable protein that is stabilized by Hpo. It is proposed that Hpo and Sav function together to restrict tissue growth in vivo (Pantalacci, 2003; Harvey, 2003; Jia, 2003; Udan, 2003 and Wu, 2003).
The dMST mutant phenotypes closely resemble those caused by sav or wts mutations. It has been shown that Sav and Wts physically and genetically interact, suggesting that they may act in common pathways. To determine if dMST could act in the same pathways, coimmunoprecipitation assays with Sav and Wts were performed. S2 cells were transfected with DNA constructs expressing HA-tagged Sav and Flag-tagged full-length dMST (dMSTf), its N-terminal fragment containing the kinase domain (dMSTn), or its C-terminal fragment containing the regulatory domains (dMSTc). Both dMSTf and dMSTc, but not dMSTn, coimmunoprecipitate with Sav, suggesting that dMST binds Sav through its C-terminal regulatory region. The dimerization domain at the C terminus of dMST appears to be essential for interaction as deletion of this domain from dMSTc abolishes its ability to bind Sav (Jia, 2003).
To define the domain in Sav that binds dMST, a series of truncated forms of Sav were generated. Both C-terminal fragments, SavC1 and SavC2, bind dMST. In contrast, all the C-terminally truncated fragments, including SavDeltaC1, SavDeltaC2, and SavDeltaC3, fail to bind dMST, suggesting that dMST binds the C-terminal region of Sav and the coiled-coil domain of Sav is essential (Jia, 2003).
dMST also binds Wts. S2 cells were transfected with DNA constructs expressing Myc-tagged Wts and Flag-tagged dMSTf, dMSTn, or dMSTc. Wts binds dMSTf and dMSTn, but not dMSTc, suggesting that Wts interacts with the N-terminal region of dMST. The interaction between Wts and dMST is not affected by Sav, since coexpression of Sav does not increase the amount of dMSTf coimmunoprecipitated with Wts. However, it remains possible that Sav might regulate dMST/Wts interaction in vivo at physiological concentration. Taken together, these results suggest that dMST, Wts, and Sav form a complex in which Wts and Sav bind the kinase and regulatory domains of dMST, respectively (Jia, 2003).
Sav quantities increase markedly in the presence of Hpo. Furthermore, Sav mobility on acrylamide gels shifts toward higher molecular weights. Sav immunoprecipitated from lysates containing Hpo was treated with phosphatases and it was found that this band shift disappeared, confirming that Sav becomes phosphorylated in the presence of Hpo. Sav is also stabilized by treatment with the proteasome inhibitor LLnL, suggesting that Sav is normally targeted for degradation by the proteasome. Unexpectedly, kinase-dead Hpo also induces stabilization and a mobility shift of Sav, whereas Hpo lacking the Sav-binding domain has little effect (Pantalacci, 2003).
Correct organ size is determined by the balance between cell death and proliferation. Perturbation of this delicate balance leads to cancer formation. Hippo (Hpo), the Drosophila ortholog of MST1 and MST2 (Mammalian Sterile 20-like 1 and 2) is a key regulator of a signaling pathway that controls both cell death and proliferation. This pathway is so far composed of two Band 4.1 proteins, Expanded (Ex) and Merlin (Mer), two serine/threonine kinases, Hpo and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki). It has been proposed that Ex and Mer act upstream of Hpo, which in turn phosphorylates and activates Wts. Wts phosphorylates Yki and thus inhibits its activity and reduces expression of Yki target genes such as the caspase inhibitor DIAP1 and the micro RNA bantam. However, the mechanisms leading to Hpo activation are still poorly understood. In mammalian cells, members of the Ras association family (RASSF) of tumor suppressors have been shown to bind to MST1 and modulate its activity. In this study it is shown that the Drosophila RASSF ortholog (dRASSF) restricts Hpo activity by competing with Sav for binding to Hpo. In addition, dRASSF also possesses a tumor-suppressor function (Polesello, 2006).
The mammalian RASSF family comprises six different loci encoding a variety of splice variants. Most transcripts encode proteins that contain a Ras association domain (RA), an N-terminal C1-type zinc finger, and a C-terminal SARAH (Sav RASSF Hippo) domain. RASSF family members, most notably RASSF1A, are frequently silenced in a variety of solid tumors. Thus, it has been proposed that RASSF genes act as tumor suppressors (Polesello, 2006).
The biological function of these genes is not well understood. RASSF1A and Nore1A have both been shown to interact with MST1 via its SARAH domain. Overexpression of RASSF1A or Nore1A inhibits MST1 activation, but coexpression of these RASSF proteins with Ras enhanced MST1 activity. RASSF1A knockout mice have mildly increased tumor susceptibility, confirming that RASSF genes can act as tumor suppressors. The weakness of the mouse phenotype, which is at odds with the frequency of RASSF1A inactivation in human tumors, can be ascribed to redundancy with other family members (Polesello, 2006).
By contrast, Drosophila melanogaster has a single RASSF family member, which is encoded by the CG4656 gene and which will be referred to as dRASSF. Like its vertebrate counterparts, dRASSF encodes a protein bearing an RA and SARAH domain at its C terminus. It also possesses a LIM domain that shares some similarities with C1 zinc fingers at its N terminus (Polesello, 2006).
Mutant alleles of dRASSF were generated by imprecise excision of two nearby transposons, GE23517 and EY2800. Multiple alleles, which delete up to the fourth intron, including the initiating ATG, were obtained. Some transcript was still detected in dRASSFX16, dRASSFX36, but a strong reduction was found in dRASSF44.2, which lacks the transcription start. However, antibodies raised against the C terminus (amino acids 792–806) and a nonconserved region (amino acids 294–308) of dRASSF showed that full-length dRASSF is absent in lysates from all mutant lines, suggesting the dRASSF mutants are indeed loss-of-function mutations for the locus. All of these alleles were viable and behaved identically in subsequent assays. In addition, dRASSF staining was severely reduced in FLP/FRT-generated dRASSF mutant clones in the eye-imaginal disc, the larval precursor to the adult eye (Polesello, 2006).
Although the dRASSF mutant flies are viable, they present a clear growth defect in comparison to wild-type animals when reared in carefully controlled conditions. dRASSF mutant flies were 15% lighter than their wild-type counterparts, a phenotype which was significantly rescued by introduction of a single copy of a dRASSF rescue construct, although wild-type levels of dRASSF were not fully restored. dRASSF mutant flies were fully fertile and normally proportioned but sensitive to γ-irradiation. Wing surface area was reduced by 8% in dRASSF mutant flies, whereas wing hair density was unaffected. This suggests that the growth defect of dRASSF mutant flies is due to a reduction in cell number and not a defect in cell size (Polesello, 2006).
In mammals, members of the RASSF family are known to interact with MST1 and thus to modulate its pro-apoptotic activity. Therefore whether dRASSF can interact with Hpo was tested. Coimmunoprecipitation (Co-IP) experiments were performed in Drosophila Kc cells with dRASSF antibodies to immunoprecipitate endogenous protein. As expected, dRASSF robustly coimmunoprecipitated with Hpo. The association between Hpo and Sav is mediated by these proteins' shared SARAH domains. Likewise, Hpo's SARAH domain is required for its association with dRASSF, as shown by the fact that a truncated form of Hpo (HpoΔC) lacking this domain fails to bring down dRASSF. Thus, the Hpo SARAH domain can associate with both Sav and dRASSF (Polesello, 2006).
Sav is stabilized by the presence of Hpo. Therefore whether dRASSF levels are modulated by Hpo was tested. dRASSF immunostaining was reduced in clones mutant for a hpo allele that lacks the SARAH domain. In addition, RNAi-mediated depletion of Hpo from Drosophila Kc cells resulted in a reduction of endogenous dRASSF expression, whereas dRASSF transcripts were unaffected. By contrast, dRASSF levels were unaffected in clones mutant for other Hpo-pathway members, such as ex, sav, and wts. These results suggest that direct binding to Hpo through its SARAH domain, rather than signaling through the Hpo pathway, is necessary for dRASSF stability. This is analogous to the situation for Sav, which is also stabilized by a kinase-dead form of Hpo (Polesello, 2006).
Because Hpo, Sav, and dRASSF all contain a SARAH domain, it was speculated that dRASSF might also bind Sav. To test this, whether dRASSF interacts with Sav was investigated by co-IP but no such an interaction was detected. Because the possibility of a ternary complex had been raised by another study, whether the three proteins could be found in the same complex was tested. Hpo, Sav, and dRASSF were co-expressed in cultured Kc cells. As expected, Hpo was able to bind Sav and dRASSF. However, Sav immunoprecipitates only contained Hpo and not dRASSF, and dRASSF immunoprecipitates contained Hpo but not Sav. Identical results were obtained with endogenous IPs by using dRASSF and Sav antibodies. These data support the notion that Sav and dRASSF are not present in the same complex but are in two different Hpo complexes (Polesello, 2006).
Sav has been shown to be a positive regulator of the Hpo pathway, whereas genetic results suggest that dRASSF might antagonize Hpo function. It was therefore of interest to determining whether complexing with Sav or dRASSF might influence Hpo activity. Immunoprecipitates were probed with an phospho-MST1 antibody that recognizes phosphorylated (active) Hpo. Interestingly, although Hpo that was coimmunoprecipitated with dRASSF showed barely detectable levels of phosphorylation, the Sav-associated fraction was highly phosphorylated. Thus, Hpo can exist as two pools, a highly active Sav-associated pool and an inactive dRASSF-associated pool. This correlates with data showing that Nore1 can repress MST1 activity in mammalian cells. This also suggests that Sav can promote Hpo activation and provides the first direct evidence of a function for the Hpo/Sav interaction (Polesello, 2006).
Next, the prediction that dRASSF depletion would promote Hpo activation was tested. Like that of Hpo's mammalian counterparts, phosphorylation of endogenous Hpo can be potently stimulated by the drug Staurosporine (STS) in Kc cells. Although RNAi depletion of dRASSF alone was not able to induce Hpo phosphorylation, dRASSF depletion markedly potentiated STS-induced Hpo activation. Thus, dRASSF restricts Hpo activation in cultured cells (Polesello, 2006).
Given their opposing effects on Hpo activation, the relationship between Sav and dRASSF was investigated. Depletion of dRASSF in Kc cells gives rise to an increase in Sav protein levels. Although dRASSF levels were unaltered in sav mutant clones, overexpression of Sav in the wing disc results in a robust decrease of dRASSF staining. Whether dRASSF and Sav compete to bind Hpo was tested. To address this question, because Sav and dRASSF repress each other's expression and dRASSF has reduced affinity for phosphorylated Hpo, separate Kc cell lysates expressing a kinase-dead form of Hpo (HpoKD-Flag), Sav-HA, and HA-dRASSF were mixed and IPs were performed after the proteins were allowed to bind overnight. Both Sav and dRASSF were able to interact with Hpo. In these conditions, increasing the amount of Sav was able to displace the dRASSF fraction bound to Hpo, showing that Sav and dRASSF are competing to bind Hpo. The outcome of the competition probably determines the stability of Sav and dRASSF; both proteins are downregulated when Hpo is depleted by RNAi. Thus, it is suggested that interplay between the inhibitor dRASSF and the activator Sav determines the level of Hpo activation and therefore affects body size (Polesello, 2006).
This model was tested by performing genetic-interaction experiments. A mutant allele of hpo was crossed into the dRASSF mutant background and the adult body mass was measured. The body-mass reduction of dRASSF mutant flies (15% reduction) was substantially rescued by removal of just one copy of Hpo (8% reduction). Flies overexpressing Sav showed a reduction of 10% in weight and 5% in wing area, mimicking dRASSF loss of function. This wing defect was significantly increased in a dRASSF mutant background. In addition, misexpression of dRASSF was able to robustly rescue the rough-eye phenotype elicited by coexpression of Sav and Wts. These data support the notion that dRASSF can antagonize Sav-mediated Hpo activation in vivo (Polesello, 2006).
Though the results are consistent with biochemical data on mammalian RASSF family members, they are at odds with the fact that RASSF genes are commonly silenced in tumor cells. It has been proposed that one RASSF protein, Nore1, possesses a tumor-suppressor function that is independent of MST1 and MST2. Two lines of evidence to support this notion were found. First, in vivo clones were made in the head (by using the eyeless FLP system) that were mutant for two hpo hypomorphic alleles, hpo42–48 and hpoKC203, that remove the SARAH domain in a dRASSF mutant background. Interestingly, the overgrowth phenotype elicited by these hpo alleles was strongly enhanced by loss of dRASSF. By contrast, a hpo allele (hpo42–47) bearing an inactivating deletion in the kinase domain but an intact SARAH domain was barely if at all enhanced by dRASSF loss of function. This suggests that dRASSF may possess a tumor-suppressor function, which may be uncovered when the Hpo function is compromised (Polesello, 2006).
In addition, the relationship between Ras1 and dRASSF was examined because the mammalian RASSF proteins have all been shown to bind Ras proteins. In Drosophila imaginal tissues, Ras1 mutant clones grow poorly and are eliminated by apoptosis. When double-mutant clones for Ras1 and dRASSF were made in the developing eye, a substantial rescue was observed of the growth defect observed in clones mutant for Ras1 alone. This rescue of Ras loss of function was the result of both increased proliferation quantified with phosphorylated Histone 3 staining and a reduction of apoptosis visualized with a cleaved-Caspase 3 antibody. Thus, dRASSF appears to antagonize Ras1 signaling in growth control, which is again suggestive of a “tumour-suppressing” effect distinct from its “oncogenic” role in opposing the Hpo pathway. However, it has been suggest that NORE1 may also have both Ras- and MST-independent functions. Future experiments will therefore be aimed at gaining a better understanding of the RASSFs' growth-restricting functions. The fact that the dRASSF mutations are viable might therefore reflect the facts that its ability to regulate the Hpo pathway may be redundant with other modes of regulation and that loss of dRASSF's tumor-suppressive activity is balanced by loss of its growth-promoting activity. It has been proposed that MST2 may be inactivated by binding to Raf-1. It will be interesting to determine whether this mode of regulation is redundant with RASSF (Polesello, 2006).
In summary, mutant alleles of the sole Drosophila ortholog of the RASSF family of tumor suppressors were generated. Surprisingly, dRASSF mutant flies are smaller than control flies. This growth defect can probably be ascribed in part to dRASSF's ability to antagonize Hpo signaling by competing with Sav for binding to Hpo. In addition, dRASSF also possesses a tumor-suppressor activity, which is uncovered when hpo or Ras1 function is compromised. It will be interesting to investigate whether some mammalian RASSF proteins share these properties (Polesello, 2006).
In the eye disc, sav is expressed in a stripe in the MF, and expression decreases in the region of the second mitotic wave (SMW). Expression increases once again posterior to the SMW. Thus, to a first approximation, sav expression coincides with regions of temporary or permanent cell cycle arrest and supports the notion that sav functions in promoting exit from the cell cycle (Tapon, 2002).
In sav1 clones in the adult retina, almost all the ommatidia contain the normal complement of eight photoreceptor cells. However, there is increased spacing between adjacent ommatidia. In contrast to wild-type retinas from late pupae that contain a single layer of interommatidial cells, mutant clones contain many additional interommatidial cells. Generation of sav1 mutant clones in a white+ background indicates that most of these additional interommatidial cells contain pigment. Thus, these cells can undergo terminal differentiation. The more disorganized retinas of the sav3 allele display all of these phenotypic abnormalities. In addition, almost half of the ommatidia in sav3 clones lack one or more photoreceptor cells (Tapon, 2002).
In wild-type imaginal discs, S phases, as visualized by BrdU incorporation, are observed anterior to the morphogenetic furrow (MF) and as a single stripe of incorporation posterior to the furrow referred to as the second mitotic wave (SMW). In sav clones, many BrdU-incorporating nuclei are observed posterior to the SMW. Clones spanning the MF have some BrdU-incorporating nuclei in the anterior half of the MF, a region that is normally composed of cells arrested in G1. Using the anti-phosphohistone H3 antibody, additional cells in mitosis are also visualized in sav mutant clones posterior to the MF, suggesting that at least some of these cells are completing additional cell cycles. BrdU incorporation persists in mutant clones during the first 12 hr after puparium formation (APF) but has ceased by 24 hr APF. Thus, sav mutant cells continue to proliferate for 12–24 hr after wild-type cells stop dividing but are eventually able to exit from the cell cycle and undergo terminal differentiation (Tapon, 2002).
In cycling cells in the anterior portion of the eye imaginal disc, the distribution of mutant cells in the cell cycle, as assessed by flow cytometry, is extremely similar to that of wild-type cells. The mutant cells are very slightly smaller than their wild-type counterparts. Posterior to the MF, mutant populations have an increased proportion of cells in S and G2, indicating that mutant cells continue to cycle in this portion of the disc. Mutant cells are of normal size. The population doubling times of clones of mutant cells and wild-type cells generated in the wing imaginal disc during the proliferative phase of development did not differ significantly. Thus, when they are proliferating, mutant cells behave like wild-type cells. However, exit from the cell cycle is delayed in sav cells (Tapon, 2002).
Elevated levels of Cyclin E protein are found in the basal nuclei of sav clones posterior to the MF. These are the nuclei of the undifferentiated cells that continue to proliferate in sav clones. Such discs were examined for levels of cyclin E RNA. When sav clones are generated using eyFLP, a large proportion of cells in third instar discs are mutant, and these discs contain large patches of mutant tissue. In wild-type discs, cyclin E RNA is expressed in a narrow stripe immediately posterior to the morphogenetic furrow. In discs containing sav clones, the stripe of expression is broader and more intense, indicating that cyclin E RNA levels are elevated in these discs. Thus, the increased level of Cyclin E protein is likely to result, at least in part, from an increase in cyclin E RNA levels (Tapon, 2002).
In wild-type eyes, excessive interommatidial cells are eliminated by a wave of apoptosis that is evident in 38 hr pupal retinas. Even in sav mutant clones, cell proliferation, as assessed by BrdU incorporation, has ceased within 24 hr APF. When mosaic retinas were examined 38 hr APF, cell death is mostly confined to the wild-type portions of the retina. Thus, the apoptotic cell deaths that are part of normal retinal development appear to require sav function (Tapon, 2002).
Apoptosis in the pupal retina requires hid function, since hid mutants display additional interommatidial cells. Hid is thought to induce caspase activation by binding to the DIAP1/Thread protein and preventing it from inhibiting caspase function. Overexpression of hid using the eye-specific GMR promoter generates a small eye. The induction of cell death by hid is severely impaired in sav mutant clones. As a consequence, eyes derived from GMR-hid-expressing discs that contain sav mutant clones are larger than those derived from wild-type discs that express GMR-hid. Since sav function is required for hid-induced cell death, sav is likely to function either downstream of hid or in a parallel pathway (Tapon, 2002).
Several studies have shown that Hid and Rpr activate caspases by another mechanism in which they induce the autoubiquitination of DIAP1 and target it for degradation by the proteasome. DIAP1 levels are markedly elevated in sav clones in the larval eye disc and remain elevated in the interommatidial cells in mutant clones in the pupal eye disc. Thus, increased levels of DIAP1 in sav cells may be able to overcome the effect of many proapoptotic signals (Tapon, 2002).
To examine DIAP1 RNA levels, in situ hybridization was used to examine 20 wild-type discs and 20 mutant discs. The presence of sav (GFP-) clones in the mutant discs was confirmed by examining the discs by fluorescence microscopy prior to hybridization. There is a modest level of DIAP1 RNA expression posterior to the furrow in both populations of discs and no evidence of increased DIAP1 RNA in the discs containing sav clones. Thus, at least at this level of detection, the increased DIAP1 expression in sav cells does not appear to result from increased transcription (Tapon, 2002).
In wild-type eye discs, DIAP1 protein is expressed at higher levels posterior to the morphogenetic furrow. DIAP1 protein levels are downregulated by GMR-rpr or, to a lesser extent, by GMR-hid expression. In sav mutant clones expressing GMR-rpr, DIAP1 protein levels remain elevated. Similar results are observed with GMR-hid. Thus, neither GMR-rpr nor GMR-hid appears capable of downregulating the elevated levels of DIAP1 sufficiently in sav clones to activate caspases (Tapon, 2002).
Expression of hid or reaper (rpr) in the eye imaginal disc results in activation of the effector caspase Drice. An antibody that recognizes the cleaved (activated) form of Drice was used to stain eye discs expressing GMR-hid or GMR-rpr. In wild-type cells, Drice is activated by GMR-hid or GMR-rpr. However, in clones of sav tissue, Drice activation by either GMR-hid or GMR-rpr is almost completely blocked. These experiments indicate that sav blocks activation of Drice by both rpr and hid (Tapon, 2002).
A mutant form of Hid (Hid-Ala5) is resistant to inactivation by MAP kinase phosphorylation. GMR-hid-Ala5 is a more potent inducer of cell death than is GMR-hid, as assessed by the extent of Drice activation in the eye disc. Cell death induced by GMR-hid-Ala5 is only partially blocked in sav clones, indicating that the increased potency of Hid-Ala-5 may be able to overcome increased DIAP1 levels (Tapon, 2002).
The mutations in sav1, sav2, and sav4 result in stop codons in positions 289, 231, and 160, respectively, that would truncate the protein N-terminal to the WW domains. As expected, the more N-terminally located sav4 mutation has a more severe phenotype than sav1 or sav2. Surprisingly, the sav3 mutation, which elicits the most severe phenotype, maps 3' to those found in sav1 and sav2. The sav3 mutation causes a frameshift and generates a protein consisting of 406 sav-encoded amino acids and a C-terminal portion of 84 amino acids derived from the use of an alternate open reading frame that has no sequence similarity to any protein in the database. It is possible that sav1, sav2, and sav4 proteins may have some residual activity despite the absence of the WW domains and that sav3 is a null allele. The sav3 allele may have a more severe phenotype because the novel C-terminal sequences may further impair its stability or function. Alternatively, the novel C terminus of the sav3 protein may confer some neomorphic properties. Any such properties, if present, are not apparent in the presence of the wild-type protein, since sav3/+ flies display no overt phenotypic abnormalities. In different transheterozygous combinations, sav3 is similar in strength to a deletion. In four independent experiments, sav1/sav3 animals and sav1/Df(3R)EB6 animals have hatching rates of 85.5% (SD 2.5%) and 83.3% (SD 3.2%), respectively, and 90%–95% of the animals of each genotype subsequently failed to grow and died as first instar larvae. Thus, at least by this criterion, sav3 behaves like a null mutation. Importantly, the abnormalities in cell proliferation and apoptosis were analyzed using at least two different sav alleles and only quantitative differences were observed between sav3 and the weaker alleles (Tapon, 2002).
During animal development, organ size is determined primarily by the amount of cell proliferation, which must be tightly regulated to ensure the generation of properly proportioned organs. However, little is known about the molecular pathways that direct cells to stop proliferating when an organ has attained its proper size. Mutations have been identified in a novel gene, shar-pei, that is required for proper termination of cell proliferation during Drosophila imaginal disc development. Clones of shar-pei mutant cells in imaginal discs produce enlarged tissues containing more cells of normal size. This phenotype is the result of both increased cell proliferation and reduced apoptosis. Hence, shar-pei restricts cell proliferation and promotes apoptosis. By contrast, shar-pei is not required for cell differentiation and pattern formation of adult tissue. Shar-pei is also not required for cell cycle exit during terminal differentiation, indicating that the mechanisms directing cell proliferation arrest during organ growth are distinct from those directing cell cycle exit during terminal differentiation. shar-pei, identified by Nolan (2002) and termed salvador in that study, encodes a WW-domain-containing protein that has homologs in worms, mice and humans, suggesting that mechanisms of organ growth control are evolutionarily conserved (Kango-Singh, 2002).
To identify novel components of growth control pathways, a genetic screen was performed in adult Drosophila to isolate mutants in which tissue size but not tissue patterning is affected. Because genes involved in growth control may have ubiquitous functions, it was anticipated that animals homozygous for mutations in these genes might die during embryogenesis. Therefore randomly mutagenized chromosome arms that were made homozygous only in the head using an eyeless enhancer driven Flipase transgene (eyFLP) were screened. Mutations in several genes were isolated that resulted in enlarged heads but did not affect patterning. These include mutations in the Drosophila homologs of PTEN and TSC1/2 tumor suppressor genes, which act in cell growth control pathways that affect cell number as well as cell size, mutations in warts/lats, a previously described tumor suppressor gene encoding a Ser/Thr kinase that affect cell number but not cell size and mutations in a previously undescribed gene. This gene was named 'shar-pei' because of its folded cuticle phenotype in the head, which resembles the folded skin of Shar-pei dogs (Kango-Singh, 2002).
Mutations in shrp were isolated on chromosome arm 3R using chemical mutagenesis. Complementation tests showed that six mutations (shrp1-6) that caused a head overgrowth phenotype fail to complement each other. All mutations showed a very similar phenotype and caused early larval lethality. Given the nature of the molecular lesions it is likely that they are either null alleles or very severe loss-of-function alleles. All experiments involving cell clones were performed with at least three independent alleles (Kango-Singh, 2002).
The heads of flies in which over 90% of cells are homozygous mutant for shrp1 are proportionally larger than other structures but have a normal overall pattern, including bristles, ocelli and ommatidia. All mutant fly heads have folded head cuticle and eye tissue and over 15% of flies are severely affected. Smaller clones generated by heat-shock induced Flipase expression do not exhibit this folding phenotype. Folding may therefore be a secondary consequence of limited space within the pupal case, which does not allow overgrown tissue to fully expand. In addition to producing structures that are too big, shrp mutant cells appear to out-compete wild-type cells. The phenotype suggests that shrp mutant cells proliferate more rapidly than wild-type cells (Kango-Singh, 2002).
To test whether shrp affects cell proliferation in tissues other than the head, random clones were generated by heat-shock induced Flipase expression. Such mutant clones resulted in overgrowths on thorax, wings, halteres and legs. As observed for the eye and head, these structures differentiated the correct tissue-specific cell types. It is concluded that shrp is generally required to restrict the size of imaginal disc-derived adult structures, whereas tissue-specific cell-type specification and differentiation remain unaffected in shrp mutant cells (Kango-Singh, 2002).
To define the developmental basis for the enlarged tissue phenotypes, a focus was placed on patterning and cell proliferation in the developing eye because the eye has a precise pattern of cell types and highly regulated cell proliferation. The pattern of differentiated photoreceptor cells was analyzed in adult shrp mutant clones in 1 µm sections. Eight photoreceptors per ommatidium with a normal trapezoidal arrangement are observed, indicating that this aspect of pattern formation is not affected. However, spacing between individual photoreceptor clusters is significantly increased in shrp5 clones when compared with wild-type areas. To test whether the increased space is due to an excess of interommatidial cells, wild-type and mutant midpupal retinas were stained with an antibody against Discs-large (Dlg), a protein that localizes to apical junctions and hence reveals cell outlines. It was found that shrp4 mutant clones exhibit a dramatic increase in the numbers of interommatidial cells when compared with wild type. These extra interommatidial cells differentiate into pigment cells that produce normal pigmentation when clones are induced in a w+ background. These data indicate that Shrp regulates cell number but not differentiation in the retina (Kango-Singh, 2002).
The extra interommatidial cells could be due to excess cell proliferation, increased spacing of photoreceptor clusters during patterning, lack of apoptosis or a combination thereof. In wild-type flies, interommatidial cells are initially produced in excess but the extra cells are later eliminated by apoptosis during pupal development in a process that requires cell-cell signaling. This system generates a very precise retinal lattice. To determine whether shrp mutant cells initiate the apoptotic program, shrp mosaic pupal retinas were stained with an antibody that detects the activated form of Drice, a caspase that triggers the apoptotic program and specifically marks cells undergoing apoptosis. Many Drice-positive cells were detected in wild-type retinal tissue, but none were found in shrp3 mutant territories. Importantly, all Drice-positive cells were wild type. This suggests that the apoptotic pathway is blocked in shrp mutant cells and that this block occurs upstream of Drice activation. It is concluded that shrp mutant cells do not receive or are resistant to signals that induce apoptosis (Kango-Singh, 2002).
To test directly whether lack of apoptosis is sufficient to produce the shrp mutant phenotype, the phenotype of shrp mutant retinas was compared with that of wild-type retinas in which apoptosis was blocked by expressing the apoptosis inhibitor p35. Ectopic expression of p35 eliminates most, if not all, normally occurring cell death in the retina and results in extra interommatidial cells. However, the number of additional cells is significantly less than that observed in shrp4 mutant clones. Therefore, while lack of apoptosis allows additional cells to survive, it is not sufficient to explain the amount of extra interommatidial cells generated in shrp mutants (Kango-Singh, 2002).
To test directly whether shrp affects cell proliferation, the distribution of cell cycle progression was monitored in mutant third larval eye discs by bromodeoxyuridine (BrdU) incorporation. In wild-type discs, BrdU-incorporating cells are randomly distributed in front of the morphogenetic furrow. In the furrow, cells synchronously arrest in G1 and do not incorporate BrdU. Posterior to the furrow, cells go through a synchronous S phase in the second mitotic wave, revealed as a band of cells incorporating BrdU. Few BrdU-positive cells are found posterior to the second mitotic wave. shrp1 mutant cells also synchronize their cell cycles in the furrow and progress normally through the second mitotic wave. However, in contrast to wild-type cells, shrp1 mutant cells still display BrdU incorporation after the second mitotic wave. The extra DNA synthesis is followed by cell division, as revealed by ectopic expression of phosphorylated histone H3 (PH3), which marks chromosomes during mitosis. This phenotype of shrp is cell autonomous, because only mutant cells undergo extra rounds of cell proliferation, and all territories of mutant cells show the excess interommatidial cell phenotype in pupal retinas, whereas non-mutant tissue appears wild type. Extra cell proliferation continues into the pupal stage but ceases by 24 hours after pupariation. Double labeling with BrdU and antibodies against Elav to detect differentiating photoreceptor cells revealed that only Elav-negative cells incorporate BrdU. Therefore, shrp is required to arrest cell proliferation in developmentally uncommitted cells after the second mitotic wave, but is not required for cell cycle arrest of differentiating photoreceptor cells. The ectopic proliferation produces extra interommatidial cells, which together with the lack of apoptosis, are sufficient to explain the overgrowth phenotypes observed in pupal and adult retinas (Kango-Singh, 2002).
Cyclin E is limiting for S-phase initiation and progression during imaginal disc development and several tumor suppressor genes negatively regulate its activity or levels. Cyclin E levels are upregulated in shrp1 mutant cells in the second mitotic wave and posterior to it. Elevated levels were also observed just anterior to the second mitotic wave, although this effect was not as pronounced. The effect on Cyclin E is cell autonomous and observed in most or all mutant cells, even though only a fraction of them are actively progressing through S phase. Thus, the effect of Shrp on cell proliferation arrest may be mediated by regulating the levels of Cyclin E (Kango-Singh, 2002).
The data show that although shrp mutant cells are able to exit the cell cycle during cell differentiation, they are delayed in arresting cell proliferation at the end of eye imaginal disc growth. To determine whether shrp has a function in uncommitted cells anterior to the morphogenetic furrow, it was necessary to measure whether mutant eye discs are already larger than wild-type before ommatidial clusters are specified. Because initial spacing of photoreceptor clusters is normal in shrp mutant eye discs, the final number of ommatidia provides a measure of the number of cells present in mutant eye discs before R8 cells are specified in the morphogenetic furrow. The numbers of ommatidia were determined in wild-type and mutant retinas by counting clusters of photoreceptor cells revealed by Elav-Gal4 driven GFP expression. Mutant retinas contained an average of 913 ommatidial clusters, whereas wild-type retinas had an average of 776 photoreceptor clusters. It is concluded that shrp mutant eye discs are already larger than normal at the time when the positions of ommatidia are specified in the morphogenetic furrow. Shrp thus functions in uncommitted cells anterior to the morphogenetic furrow (Kango-Singh, 2002).
To test whether shrp affects the rate of cell proliferation during the growth phase of imaginal discs, cell numbers were compared in mutant clones and their associated twin clones in third instar wing discs. To reduce variability in the proliferation rate of wild-type twin clones, isogenized FRT 82B ubi-GFP chromosomes were used to generate mitotic clones. Cell numbers in shrp3 mutant clones were almost always larger than their twin clones, and the difference in cell numbers was significant when assessed using a t-test. The same experiment with a second allele, shrp4, showed similar differences. By contrast, cell numbers in clones of the isogenized wild-type chromosome on which the shrp mutations were induced during the mutagenesis screen were similar and not significantly different from the corresponding ubi-GFP/ubi-GFP twin clones. Based on these cell counts and assuming exponential proliferation, the cell division rate of shrp mutant cells is 1.10 times faster than that of wild-type cells. These data thus indicate that shrp mutant cells proliferated more. This phenotype is also manifest in mosaic adult eyes, where shrp1 mutant cells out-compete wild-type cells. Determination of the distribution of cell cycle phases in third instar wing discs using FACS analysis shows that the population of shrp4 mutant cells has the same distribution of cell cycle phases as wild-type cells. Thus, shrp mutant cells do not accelerate a particular step in the cell cycle. Rather, mutant cells show an even acceleration of cell cycle progression (Kango-Singh, 2002).
Manipulating the activity of cell growth regulators such as components of the insulin receptor signaling pathway results in larger organs because of more and larger cells. To determine whether shrp also affects cell size, mosaic wing discs were stained with antibodies against Dlg to detect apical cell outlines. Cells in shrp3 mutant clones show normal cell sizes, as judged by cell outlines and have normal height as judged by the thickness of the wing disc epithelium in the mutant clones. In addition, rhabdomeres of mutant photoreceptor cells were of normal diameter, and shrp mutant cone and pigment cells are of normal size at the pupal stage. Furthermore, forward light scatter (FSC) data, a measurement of cell size collected by FACS analysis confirms that mutant cells have normal size. Therefore, Shrp does not regulate cell size. Rather, extra proliferation of shrp mutant cells is induced by stimulation of cell growth and cell cycle progression, resulting in balanced growth and extra cells that are of normal size (Kango-Singh, 2002).
It is proposed that the arrest of cell proliferation during imaginal disc development is controlled by several genetically separable mechanisms. (1) Cells stop proliferating when imaginal discs have reached their correct sizes. This process requires Shrp function. (2) Cells permanently exit the cell cycle during terminal differentiation. Because terminal cell cycle exit is part of cell differentiation, it is directly regulated by patterning mechanisms that determine the identity and position of each individual cell. This regulation is governed by tissue-and cell-type specific enhancers of cell cycle regulators such as dacapo, cyclin E and string, which all have complex cis- regulatory regions. Similarly, patterned regulation of cell cycle progression may occur before terminal differentiation, as is observed in the second mitotic wave in the eye and in the zone of non-proliferation along the presumptive wing margin in the wing disc. None of these processes are affected in shrp mutants. Thus, the direct control of cell cycle progression by patterning mechanisms acts epistatically to the control of cell proliferation observed during organ growth and can impose cell cycle arrest on cells that otherwise may continue to proliferate. Therefore, shrp mutations do not deregulate cell proliferation of terminally arrested cells, and cells differentiate normally. In summary, Shrp identifies a molecular mechanism that is required to arrest cell proliferation during organ growth and that appears to be distinct from the ones used to arrest cells during terminal cell differentiation (Kango-Singh, 2002).
What are the downstream effectors of Shrp that are deregulated in shrp mutants and induce cell proliferation? shrp mutant clones behind the second mitotic wave in eye discs show elevated levels of Cyclin E. Notably, Cyclin E was elevated in all developmentally uncommitted cells of the clones, apparently irrespective of the phase of the cell cycle. The effect on Cyclin E levels may thus be direct and not just a reflection of the ectopic cell proliferation observed in mutant clones. Precise regulation of Cyclin E expression and activity is crucial because ectopic expression of Cyclin E induces entry into S phase and limited cell proliferation in imaginal discs and embryos. Several other negative regulators of cell proliferation directly regulate the levels of Cyclin E activity. Dap directly inhibits Cyclin E/Cdk2 complexes, and Archipelago is required for degradation of Cyclin E. The regulation of Cyclin E is thus likely to be an important downstream effect of Shrp function (Kango-Singh, 2002).
However, ectopic expression of Cyclin E alone is not sufficient to generate the number of extra cells observed in shrp mutant tissues. Artificial acceleration of the cell cycle by ectopic expression of specific cell cycle regulators such as E2F accelerates cell division, but does not stimulate cell growth rates, and cells divide when they are smaller. This results in an increase in cell number and a concomitant decrease in cell size, yet does not affect the overall tissue size. Thus, cell cycle progression is not sufficient to drive cell and organ growth. Conversely, stimulating cell growth alone produces larger organs, but also affects cell size. For example, artificially stimulating the activities of Ras, Myc or insulin receptor signaling produces more and bigger cells and thus bigger but otherwise normal flies. Thus, cell proliferation during organ growth requires coordinate stimulation of cell cycle progression and cell growth to produce normal sized cells. Because shrp mutant cells maintain normal size, Shrp appears to be required to regulate cell growth and cell cycle progression coordinately. Thus, Shrp probably regulates other targets driving cell cycle and cell growth in addition to Cyclin E (Kango-Singh, 2002 and references therein).
Several other mutations have been described that fail to arrest imaginal disc growth and were thus classified as tumor suppressor genes. These include discs-large (dlg), lethal giant larva (lgl) and scribble (scrib), encoding proteins that form an architectural complex localized to septate junctions. Mutations in these genes disrupt septate junctions and apical-basal polarity of epithelial cells and result in neoplastic overgrowth of imaginal discs. Mutations in a second group of Drosophila tumor suppressor genes cause hyperplastic overgrowth of imaginal discs that retain their single layered epithelial structure. These include warts/lats, which encodes a kinase that regulates the activity of Cdc2; fat, a large Cadherin; hyperplastic discs, a E3 ubiquitin ligase, and discs overgrown, a Drosophila homolog of Casein kinase IDelta/Epsilon. The imaginal disc overgrowth in mutants of both groups occurs during an extended larval period, and embryonic requirements for these genes appear to be provided by maternal contributions. By contrast, homozygous shrp mutant animals die as first/second instar larvae, which do not show gross morphological defects. Thus, zygotic expression of shrp is required for early larval viability, whereas that of other tumor suppressor genes is not. Cells homozygous mutant for neoplastic or hyperplastic tumor suppressor genes generally differentiate abnormally and show defects in cell morphology and/or pattern formation. These phenotypes are thus different from those of shrp mutant cell clones, which overproliferate but differentiate with normal cell morphology and patterning. In addition to these differences, clones of cells homozygous mutant for shrp proliferate more rapidly and have reduced apoptosis, while cells mutant for most other tumor suppressor genes have reduced viability and a decreased rate of cell proliferation. Only fat and warts/lats mutant cell clones proliferate faster, similar to shrp mutant cells. However, the phenotypes of shrp, fat and warts/lats differ, because fat and warts/lats affect the morphology and pattern of adult tissues in addition to cell number. Therefore, shrp affects cell number more specifically than these other mutants, and future work will have to establish whether and how Shrp interacts with other tumor suppressor gene products to control tissue size (Kango-Singh, 2002).
In summary, these studies provide evidence that Shrp functions in the decision of imaginal disc cells to terminate proliferation and to exit the cell cycle once the correct disc size is attained. The determination of the effectors of Shrp action should reveal mechanisms by which cell growth and cell cycle progression are coordinately regulated during organ growth and how cells arrest proliferation once organs have reached their correct size. The presence of Shrp homologs in mouse and human suggest the existence of a conserved organ size control mechanism in mammals. The characterization of Shrp function should therefore provide valuable insights into the mechanisms that underlie tissue size regulation and cause disproportionate growth and tumorigenesis when defective (Kango-Singh, 2002).
Color vision in Drosophila relies on the comparison between two color-sensitive photoreceptors, R7 and R8. Two types of ommatidia in which R7 and R8 contain different rhodopsins are distributed stochastically in the retina and appear to discriminate short (p-subset) or long wavelengths (y-subset). The choice between p and y fates is made in R7, which then instructs R8 to follow the corresponding fate, thus leading to a tight coupling between rhodopsins expressed in R7 and R8. warts, encoding large tumor suppressor (Lats) and melted encoding a PH-domain protein, play opposite roles in defining the yR8 or pR8 fates. By interacting antagonistically at the transcriptional level, they form a bistable loop that insures a robust commitment of R8 to a single fate, without allowing ambiguity. This represents an unexpected postmitotic role for genes controlling cell proliferation (warts and its partner hippo and salvador) and cell growth (melted) (Mikeladze-Dvali, 2005).
The fly eye provides a powerful system to study cell-fate decisions: it develops from a flat epithelium into a complex three-dimensional structure of multiple cell types in less than a week. The adult eye allows the fly to perform various visual tasks, ranging from motion detection and the discrimination of colors to measuring the orientation of polarized light for navigation (Mikeladze-Dvali, 2005).
In the fly compound eye, each of the 800 ommatidia is a single optical unit that contains 8 photoreceptor cells (PRs). The 8 PRs form widely expanded membrane structures, the rhabdomeres, which contain the photosensitive Rhodopsins (Rh). The rhabdomeres of the six outer PRs (R1-R6) form a trapezoid. R1-R6 all express the broad spectrum rhodopsin1 (rh1 or ninaE) and are morphologically and functionally invariant in all ~800 ommatidia (Mikeladze-Dvali, 2005).
The center of the trapezoid is occupied by the two inner PRs, R7 and R8. The rhabdomeres of R7 are positioned on top of R8, so that they share the same optic path. Inner PRs are involved in color vision and can be viewed as equivalent to vertebrate cones. Each R7 and R8 expresses only one of the four rhodopsins, rh3, rh4, rh5, or rh6 in a highly regulated manner, defining three different subtypes of ommatidia: 'yellow' (y), 'pale' (p) (for their appearance under UV illumination), and the 'dorsal rim area' (DRA). Ommatidia in the DRA express rh3 in both R7 and R8 and are specified in a very restricted region by the gene homothorax. They are believed to function as polarized light detectors (Mikeladze-Dvali, 2005).
In contrast, color vision depends on the y and p ommatidial subtypes that are randomly distributed through the main part of the retina, with a bias of y (~70%) over p subtype (~30%). In the p subtype, R7 expresses the UV-sensitive Rh3 and R8 the blue-sensitive Rh5. In the y subtype, R7 expresses a distinct UV-sensitive Rh4 while R8 expresses the green-sensitive Rh6. As in many other sensory systems, expression of a given Rhodopsin excludes all others to prevent sensory overlap. While the p subtype is better suited to discriminate among shorter wavelengths, the y subtype should discriminate amongst longer wavelengths (Mikeladze-Dvali, 2005).
The choice between the p and y fate is first made in R7: once an R7 commits to the p fate and expresses rh3, it sends an instructive signal to the underlying R8, which then also commits to the p fate and expresses rh5. In the absence of the R7 signal (i.e., when R7 expresses rh4 or in a sevenless mutant), R8 commits to the y fate and expresses rh6. The stochastic choice appears to be made by each R7 independently of its neighbors, resulting in the biased random distribution of p and y ommatidia throughout the main part of the retina (for review see Mikeladze-Dvali, 2005).
Four genes required in R8 cells for ensuring the correct choice of y versus p cell fate have been identified. The warts (wts) gene, which encodes the Drosophila large tumor suppressor (also known as lats) and melted (melt) play a critical role in the specification of p and y R8 cells, without affecting the R7 choice. wts encodes a Ser/Thr kinase, while melt encodes a Pleckstrin Homology (PH) domain protein. wts is necessary and sufficient for R8 to adopt the y fate, while melt plays the opposite role and specifically induces the p fate in R8. wts and melt are expressed in a complementary manner in the yR8 and pR8 subsets, respectively. Evidence is presented that the two genes repress each other's transcription to form a bistable loop. melt seems to respond to the R7 signal, while wts appears to regulate the output of the loop. The tumor-suppressor genes hippo (hpo) and salvador (sav), which encode the two molecular partners of Wts/Lats, have phenotypes identical to wts. Interestingly, melt has been reported to regulate growth and fat metabolism in Drosophila. Thus, genes known to regulate both cell growth (melt) and proliferation (wts, sav, hpo) interact antagonistically during retinal patterning (Mikeladze-Dvali, 2005).
Precise patterning of dendritic fields is essential for neuronal circuit formation and function, but how neurons establish and maintain their dendritic fields during development is poorly understood. In Drosophila class IV dendritic arborization neurons, dendritic tiling, which allows for the complete but non-overlapping coverage of the dendritic fields, is established through a 'like-repels-like' behaviour of dendrites mediated by Tricornered (Trc), one of two NDR (nuclear Dbf2-related) family kinases in Drosophila. The other NDR family kinase, the tumour suppressor Warts/Lats (Wts), regulates the maintenance of dendrites; in wts mutants, dendrites initially tile the body wall normally, but progressively lose branches at later larval stages, whereas the axon shows no obvious defects. Biochemical and genetic evidence is provided for the tumour suppressor kinase Hippo (Hpo) as an upstream regulator of Wts and Trc for dendrite maintenance and tiling, respectively, thereby revealing important functions of tumour suppressor genes of the Hpo signalling pathway in dendrite morphogenesis (Emoto, 2006).
Dendritic arborization patterns are critical to a neuron's ability to receive and process impinging signals. Whereas neurons normally maintain the gross morphology of their dendrites, cortical neurons of Down's syndrome patients gradually lose dendritic branches after initially forming normal dendritic fields. Thus, neurons appear to have separate mechanisms for establishment and maintenance of their dendritic fields (Emoto, 2006).
Dendritic tiling is an evolutionarily conserved mechanism for neurons of the same type to ensure complete but non-redundant coverage of dendritic fields. In the mammalian visual system, for instance, dendrites of each retinal ganglion cell type cover the entire retina with little overlap, like tiles on a floor. In Drosophila, the dendritic arborization sensory neurons can be divided into four classes (I–IV) based on their dendrite morphology, and the dendritic field of class IV dendritic arborization neurons is shaped, in part, through a like-repels-like tiling behaviour of dendrite terminals. The NDR family kinase Trc and its activator Furry (Fry) has been identified as essential regulators of dendritic tiling and branching of class IV dendritic arborization neurons. These proteins are evolutionarily conserved and probably serve similar functions in neurons of different organisms (Emoto, 2006).
In addition to Trc, Drosophila has one other NDR family kinase, Wts, which is a tumour suppressor protein that functions in the coordination of cell proliferation and cell death in flies. To uncover the cell-autonomous functions of Wts in neurons, MARCM (mosaic analysis with a repressive cell marker) was ised to generate mCD8–GFP-labelled wts clones in a heterozygous background. Wild-type class IV neurons elaborate highly branched dendrites that cover essentially the entire body wall. Compared to wild-type ddaC (dorsal dendrite arborization neuron C) neurons, wts clones showed a severe and highly penetrant simplification of dendritic trees, with significantly reduced number (wild type, 575.1; wts, 255.6) and length (wild type, 1,457.0; wts, 590.4) of dendritic branches, and hence a greatly reduced dendritic field (Emoto, 2006).
In contrast to the severe dendritic defects caused by loss of Wts function, wts mutant ddaC axons entered the ventral nerve cord at the appropriate position and showed arborization patterns very similar to wild-type controls, with their axons terminating on the innermost fascicle and sending ipsilateral branches anteriorly and posteriorly and sometimes also a collateral branch towards the midline. Thus, Wts seems to have a crucial role in dendrite-specific morphogenesis in post-mitotic neurons (Emoto, 2006).
In proliferating cells, Wts is part of a signalling complex for tumour suppression that includes the adaptor protein Salvador (Sav) and the serine/threonine kinase Hpo. sav mutant ddaC MARCM clones were examined and dendritic defects were observed similar to wts MARCM clones. In severely affected clones (3 of 15 clones), most of the high-order branches were missing, whereas moderately affected clones (12 of 15 clones) exhibited a partial loss of their fine branches and major branches (Emoto, 2006).
To confirm that Wts and Sav function in the same pathway, genetic interaction between wts and sav in regulating dendrite morphogenesis was tested. Whereas heterozygous wts or sav mutants had no obvious dendritic phenotype, trans-heterozygous combinations of wts and sav alleles resulted in simplified dendrites similar to moderately affected sav clones. Furthermore, sav wts double mutant clones showed a severe dendrite defect comparable to wts mutant clones. Thus, Wts and Sav most probably function together in class IV neurons to regulate dendrite morphogenesis (Emoto, 2006).
The dendritic phenotypes of wts mutants and sav mutants might result from defects in branch formation and/or elongation, or loss of normally formed dendrites. Therefore ddaC dendrites were examined at different time points of larval development using the pickpocket-EGFP reporter, which is specifically expressed in class IV dendritic arborization neurons. Wild-type ddaC neurons elaborated primary and secondary dendritic branches by 24–28 h after egg laying, but large regions of the body wall were not yet covered by dendrites. By 48–52 h after egg laying, the major branches reached the dorsal midline, and the open spaces between major branches were filled with fine branches, resulting in complete dendritic coverage of the body wall. This tiling of dendrites persisted throughout the rest of larval development. In wts and sav mutants, ddaC dendrites were indistinguishable from those of wild-type controls at 24–28 h after egg laying. By 48–52 h after egg laying, wts and sav dendrites tiled the body wall as in wild type. During the next 24 h, however, dendrites of wts and sav mutants no longer tiled the body wall. Therefore, wts and sav seem to be required for maintenance of the already established tiling of dendrites (Emoto, 2006).
The loss of dendrites was further documented in live mutant larvae imaged for 30 h starting in early second instar larvae (48–50 h after egg laying). In wild-type larvae, ddaC dendrites grew steadily; the number of terminal branches increased by 23.0 over this time period. By contrast, dendrites of wts and sav mutants gradually lost their fine branches (decrease of 27.5 and 31.5, respectively) as well as some of the major branches by 78–80 h after egg laying (Emoto, 2006).
Class-IV-neuron-specific expression of wts and sav largely rescued the dendritic phenotype of wts and sav mutants, respectively, confirming that Wts and Sav act cell autonomously in class IV neurons. Furthermore, no detectable defect in patterning of the epidermis (anti-Armadillo antibody) or muscle (Tropomyosin::GFP reporter) was observed in wts or sav mutant third instar larvae. Taken together, these results indicate that the Wts/Sav signalling pathway functions in class IV neurons to maintain dendritic arborizations (Emoto, 2006).
Wts kinase activity is regulated, at least in part, by the Ste20-like serine/threonine kinase Hpo. Indeed, ddaC clones mutant for hpo exhibited simplified dendritic trees in third instar larvae, similar to wts or sav mutant clones, but showed more extensive dendritic arborizations in earlier larval stages (second to early third instar), consistent with the involvement of Hpo in the maintenance of dendrites. Notably, in hpo mutant clones at earlier developmental stages, dendritic branches were often found to overlap. Both the dendritic tiling and maintenance phenotypes were rescued by hpo expression in MARCM clones, consistent with the cell-autonomous function of Hpo in class IV neurons. Because this tiling defect in hpo mutant clones was similar to the tiling defects of trc mutant clones, whether hpo could genetically interact with trc to regulate dendritic tiling was tested. Compared with wild-type controls, trans-heterozygous combinations of trc and hpo exhibited obvious iso-neuronal as well as hetero-neuronal tiling defects, whereas wts and hpo trans-heterozygotes displayed simplified dendrites similar to wts mutants. These dendritic defects were consistently observed in multiple allelic combinations between hpo and trc or wts. In contrast, trans-heterozygous combinations of trc and wts showed no significant dendritic phenotypes. Furthermore, overexpression of wild-type Trc, but not Wts, in hpo MARCM clones partially rescued the dendritic tiling defects in class IV neurons. Thus, Hpo acts through Trc and Wts to regulate dendritic tiling and maintenance, respectively (Emoto, 2006).
Not only did Hpo interact genetically with Trc and Wts, its physical association with these NDR kinases could be detected in vivo. When Flag-tagged Trc was expressed using a nervous-system-specific Gal4 driver, anti-Flag antibodies immunoprecipitated Trc together with Hpo. Similarly, Myc-tagged Wts co-immunoprecipitated with Hpo expressed in embryonic nervous systems. Hpo co-immunoprecipitation appeared to be specific, because Misshapen, another Ste20-like kinase protein present in neurons, was not co-immunoprecipitated by anti-Flag or anti-Myc antibodies in similar experiments. These results suggest that Hpo associates with Trc and Wts in the Drosophila nervous system (Emoto, 2006).
To examine further the physical interaction between Trc and Hpo, analogous experiments were carried out in Drosophila S2 cells co-transfected with a haemagglutinin (HA)-tagged Trc construct and a Flag-tagged Hpo construct containing the full open reading frame, an amino-terminal fragment containing the kinase domain, or a carboxy-terminal fragment containing the regulatory domain. Full-length Hpo and the C-terminal portion of Hpo, but not the N-terminal fragment, were co-immunoprecipitated with Trc, suggesting that the C-terminal domain of Hpo is sufficient for Trc–Hpo complex formation (Emoto, 2006).
Hpo physically interacts with Wts and promotes Wts phosphorylation at multiple serine/threonine sites, including two sites, S920 and T1083 of Drosophila Wts, that appear to be necessary for Wts kinase activation. Indeed, Wts protein with mutations in the S920 and T1083 residues was unable to rescue the wts mutant dendritic phenotypes. Given that the corresponding phosphorylation sites in Trc are critical for Trc activation as well as control of dendritic tiling and branching, it was of interest to know whether Hpo may promote Trc phosphorylation at the critical serine and/or threonine residue. Wild-type Hpo, but not catalytically inactive Hpo or the Misshapen kinase, led to substantial incorporation of 32P-labelled phosphate into recombinant Trc or Trc with a mutation at the S292 site (S292A), but not the T449A mutant form of Trc. Analogous results were obtained with Wts. These results support a model in which Hpo associates with and phosphorylates Trc and Wts at a critical threonine residue to regulate dendritic tiling and maintenance, respectively (Emoto, 2006).
Both genetic and biochemical evidence reveals that Hpo regulates complementary aspects of dendrite development through two distinct downstream signalling pathways: the Trc kinase pathway for tiling and the Wts kinase pathway for maintenance. These studies of class IV dendritic arborization neurons, together with the recent report that Wts signalling is required for cell fate specification of photoreceptor cells in Drosophila retina, demonstrate that the Wts signalling pathway is important for post-mitotic neurons. In proliferating cells, Wts phosphorylates Yorkie (Yki), a transcriptional co-activator, to regulate cell cycle and apoptosis in growing cells. However, Yki is dispensable for Hpo/Wts-mediated dendrite maintenance. Hpo probably functions as an upstream kinase for Trc, as well as Wts, in neurons by phosphorylating a functionally essential threonine, which may also be regulated by MST3, a Ste20-like kinase closely related to Hpo. Given the evolutionary conservation of known components in the Trc and Wts signalling pathways, it will be important to identify their relevant downstream targets and explore mechanisms that coordinate the establishment and maintenance of dendritic fields, and to determine the role of Trc and Wts signalling in the mammalian nervous system (Emoto, 2006).
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date revised: 25 July 2007
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