Imaginal discs from third-instar larvae were probed with either an antisense hpo probe. hpo is expressed in an unpatterned fashion throughout all imaginal discs including the eye imaginal disc (Harvey, 2003).
Proliferation and apoptosis must be precisely regulated to form organs with appropriate cell numbers and to avoid tumor growth. Hippo (Hpo), the Drosophila homolog of the mammalian Ste20-like kinases (Dan, 2001), MST1/2, promotes proper termination of cell proliferation and stimulates apoptosis during development. hpo mutant tissues are larger than normal because mutant cells continue to proliferate beyond normal tissue size and are resistant to apoptotic stimuli that usually eliminate extra cells. Hpo negatively regulates expression of Cyclin E to restrict cell proliferation, downregulates the Drosophila inhibitor of apoptosis protein DIAP1, and induces the proapoptotic gene head involution defective (hid) to promote apoptosis. The mutant phenotypes of hpo are similar to those of warts (wts), which encodes a serine/threonine kinase of the myotonic dystrophy protein kinase family, and salvador (sav), which encodes a WW domain protein that binds to Wts. Sav binds to a regulatory domain of Hpo that is essential for its function, indicating that Hpo acts together with Sav and Wts in a signalling module that coordinately regulates cell proliferation and apoptosis (Udan, 2003).
To identify genes that regulate tissue growth, a mutagenesis screen was performed by using the eyFLP system and three complementation groups were isolated that showed similar overgrowth phenotypes. These were sav (also known as shar-pei), wts and a previously uncharacterized locus that has been named 'hippo' (hpo) because of the dark, folded and overgrown cuticle of the mutant heads. Four alleles of hpo (hpoKC202, hpoKC203, hpoKC221 and hpoKC249) were isolated that all show similar overgrowth phenotypes. Adult mutant heads are enlarged, with larger eyes and expanded and folded cuticle on head and antennae. hpoKC202 mutant cells show overgrowths on head cuticle, halteres, thorax, wings and legs. Notably, the positions of pattern elements such as ommatidia, bristles and ocelli are relatively normal, and overgrown structures differentiated proper cell types. It is therefore concluded that Hpo is a general growth regulator required to restrict cell number and tissue size in adult structures. Analysis of hpoKC202 mutant clones in adult eyes by thin sections show that there is a considerable increase in spacing between photoreceptor clusters in mutant regions as compared with wild-type regions. This is due to a large increase in numbers of interommatidial cells, which are evident in hpoKC202 mutant mid-pupal retinae. These extra cells eventually differentiate into extra bristles and pigment cells. In contrast to the interommatidial cells, most hpoKC202 mutant ommatidia have normal numbers of photoreceptor, cone and primary pigment cells. Notably, the sizes of all cell types are normal in mutant eyes, although the morphology of photoreceptors are often abnormal. Both the expression of Elav, a marker for differentiating photoreceptor cells, and the R8 marker Senseless (Sens) were also analyzed to test whether hpo affects pattern formation during development. The initial spacing of R8 cells and the number of photoreceptor cells per ommatidium are normal in hpoKC202 mutant clones. Thus, mutations in hpo primarily affect tissue size but not patterning or differentiation (Udan, 2003).
The effects of loss of Hpo function on cell proliferation were tested by analysing the pattern of bromodeoxyuridine (BrdU) incorporation, which marks cells in S phase. In wild-type eye discs, BrdU-incorporating cells are randomly distributed anterior to the morphogenetic furrow, arrested in G1 in the furrow, and start either to differentiate into photoreceptor cells or to undergo an additional round of cell division referred to as the second mitotic wave posterior to the furrow. After the second mitotic wave, cells cease proliferation and differentiate into the remaining photoreceptor, cone, pigment and bristle cells. Mutant cells in eye discs that were almost completely mutant for hpoKC202 (subsequently referred to as eyFLP hpoKC202 eye discs because the clones were induced by eyFLP) properly synchronize their cell cycles in the furrow and progress through the second mitotic wave. In contrast to wild-type, however, cells in eyFLP hpoKC202 eye discs show ectopic incorporation of BrdU after the second mitotic wave. DNA synthesis was followed by cell division, as assessed by the ectopic expression of phosphorylated histone H3 (PH3), which marks mitotic chromosomes. Thus, Hpo mutant cells fail to arrest in G1 and continue to proliferate. This phenotype is cell autonomous and ectopic proliferation is restricted to uncommitted precursor cells and not observed in differentiating photoreceptor cells. Furthermore, Cyclin E, which is limiting for S-phase initiation in imaginal disc cells, is cell autonomously upregulated in hpoKC202 mutant clones. Likewise, Cyclin E messenger RNA is upregulated in and around the second mitotic wave in eyFLP hpoKC202 eye discs. Thus, the transcriptional repression of Cyclin E expression is probably an important downstream effect of Hpo to promote proliferation arrest of uncommitted precursor cells (Udan, 2003).
Because extra interommatidial cells are normally removed by developmentally induced apoptosis, the phenotype of hpoKC202 mutant retinae indicates that Hpo is required for apoptosis in addition to cell proliferation arrest. Indeed, in contrast to wild-type, clones of hpoKC202 mutant cells lack apoptotic cells, showing that hpo is required for developmentally regulated apoptosis in the retina. Whether hpo mutant cells are protected from ectopically induced cell death was tested. Overexpression of Hid in the developing eye induces apoptosis and results in loss of eye structures. hpoKC202 loss-of-function partially rescues cell death in eye discs that ectopically express Hid (Udan, 2003).
The proapoptotic genes hid, reaper, grim, sickle (skl) and Jafrac2 induce apoptosis by activating caspase cascades through direct targeting of DIAP1 for auto-ubiquitination and degradation, which in turn liberates caspases from DIAP1-mediated inhibition. hpoKC202 mutant clones show an increase in DIAP1 protein and mRNA. Regulation of DIAP1 might include transcriptional mechanisms because DIAP1 mRNA is upregulated just behind the furrow. The increase in DIAP1 might protect cells from apoptosis induced both in normal development and by ectopic expression of Hid. Notably, sav and wts mutants show similar defects in proliferation arrest and apoptosis (Udan, 2003).
Overexpression of several kinases including members of the Ste20 family has been used successfully in yeast, cell culture and Drosophila to activate signalling pathways (Dan, 2001). Thus, overexpression of human MST1/2 in cell culture activates caspases and triggers apoptosis. Ectopic expression of wild-type Hpo during imaginal disc development results in loss of eye, head and wing tissues through the arrest of cell proliferation and the induction of apoptosis. By contrast, overexpression of a kinase-dead variant of Hpo, HpoK71R, does not result in smaller structures, but partially rescues the phenotypes caused by overexpressing wild-type Hpo. Overexpression of Hpoauto, a hyperactive form of Hpo, produces phenotypes similar to those caused by overexpressing wild-type Hpo. The Hpoauto construct carries a deletion that is analogous to a deletion in human MST1 that causes an increase in kinase activity in vitro. Thus, kinase activity is required for Hpo action and HpoK71R is acting as a dominant-negative form of Hpo, suggesting that overexpression of Hpo activates its pathway. GMR-driven Hpo overexpression in the eye disc caused large amounts of apoptosis, similar to GMR-driven Hid expression. Hpo and Hid expression induce DNA fragmentation, activate Drice caspase, downregulate DIAP1 and induce nuclear condensation, which are all hallmarks of apoptosis. The small head and wing phenotypes are partially suppressed by coexpression of DIAP1 and of p35, a general caspase inhibitor. Expression is observed of the proapoptotic gene hid, which is required for developmentally regulated apoptosis observed during wild-type eye development (Udan, 2003).
GMR-driven Hpo expression upregulates hid mRNA and protein but does not effect grim or reaper mRNA. The small wing and eye phenotypes caused by Hpo overexpression are partially rescued by heterozygosity for hid, indicating that Hid is crucial for Hpo to exert its effects. Similarly, heterozygosity for wts suppresses the ectopic Hpo phenotype, suggesting that Hpo requires wts for its action. However, heterozygosity for Tsc1, a gene that functions in the target of rapamycin (TOR) signalling pathway that regulates cell growth, does not affect the Hpo overexpression phenotype. Hpo thus seems to regulate apoptosis at several levels. In one case, Hpo negatively regulates DIAP1 to promote apoptosis. The increase in DIAP1 in hpo mutant clones protect cells from Hid-induced apoptosis, indicating that Hpo effects cell death downstream of Hid. In addition, Hpo seems to act upstream of Hid, because overexpression of Hpo upregulates Hid expression and causes Hid-dependent cell death. In naturally occurring cell death pathways that require Hpo activity, such as the killing of interommatidial cells, a death-inducing signal may activate Hpo, which in turn may induce expression and activation of Hid, as well as downregulation of DIAP1 to trigger apoptosis. It has been shown that the activity of Hid is regulated at the transcriptional and posttranslational level and that hid is required for apoptosis in the retina. MAPK, for example, promotes cell survival by downregulating Hid activity through direct phosphorylation and through transcriptional mechanisms that depend on the downstream transcription factor Pointed. Although no significant changes of MAPK phosphorylation has been detected either in Hpo mutant clones or by Hpo overexpression, subtle effects of Hpo on MAPK activity may contribute to the effect of Hpo on Hid activity and apoptosis. Determining whether hid induction by Hpo is a direct effect or a secondary consequence of Hpo-induced cell death, however, requires further investigation (Udan, 2003).
Because Hpo is expressed ubiquitously, its activity is probably regulated by posttranscriptional mechanisms such as phosphorylation. Indeed, the activities of the mammalian homologs of Hpo, MST1/2, are regulated by phosphorylation during stress-induced cell death. The Hpo/Sav/Wts signalling pathway is unique in that it regulates apoptosis and proliferation. In fact, the hpo mutant overgrowth phenotypes are the outcome of deregulated cell proliferation, which generates extra cells, along with a lack of apoptosis, which allows extra cells to survive and to contribute to adult structures. Hpo, Wts and Sav mutants specifically affect cell numbers but not cell size, in contrast to other growth regulators such as InR signalling, Ras and Myc, which typically affect both cell size and cell number. In addition, the function of Hpo/Sav/Wts signalling is distinct from mechanisms directing cell-cycle exit during terminal differentiation because Hpo, Wts and Sav are required for cell proliferation arrest of uncommitted precursor cells but not differentiating cells. Because of this specificity for precursor cells, only the last cell types to differentiate in the retina, namely the interommatidial pigment and bristle cells, are produced in excess, whereas the number of photoreceptor and cone cells is normal in mutant ommatidia. Wts negatively regulates the activity of Cdc2/Cyclin A33, and Sav, Wts and Hpo negatively regulate Cyclin E. Thus, excessive activity of Cdc2/Cyclin A and Cyclin E may contribute to drive cell proliferation of mutant cells. The promotion of cell-cycle progression alone, however, cannot account for the excess growth (mass accumulation) of mutant tissues. Thus, Hpo/Sav/Wts signalling must regulate additional targets to restrict cell growth and cell proliferation (Udan, 2003).
Hpo, Wts and Sav are highly conserved in vertebrates. As in Drosophila, the mammalian homologs of Hpo, MST1/2, have been implicated in regulating apoptosis. LATS acts as a tumor suppressor gene in vertebrates and the human ortholog of Sav (hWW45) is mutated in cancer cell lines. It will be interesting to determine whether MST1/2 signalling is downregulated in cancer cells (Udan, 2003).
Establishing and maintaining homeostasis is critical to the well-being of an organism and is determined by the balance of cell proliferation and death. Two genes that function together to regulate growth, proliferation, and apoptosis in Drosophila are warts, encoding a serine/threonine kinase, and salvador, encoding a WW domain containing Wts-interacting protein. However, the mechanisms by which sav and wts regulate growth and apoptosis are not well understood. Mutations are described in hippo, which encodes a protein kinase most related to mammalian Mst1 and Mst2. Like wts and sav, hpo mutations result in increased tissue growth and impaired apoptosis characterized by elevated levels of the cell cycle regulator cyclin E and apoptosis inhibitor DIAP1. Hpo, Sav, and Wts interact physically and functionally, and regulate DIAP1 levels, likely by Hpo-mediated phosphorylation and subsequent degradation. Thus, Hpo links Sav and Wts to a key regulator of apoptosis (Harvey, 2003).
Three alleles of hippo are lethal either when homozygous or in trans to another allele. Eyes containing hpo mutant clones and wild-type clones have an overrepresentation of mutant tissue when compared to eyes containing clones of the wild-type parental chromosome suggesting that the mutant tissue may have a relative growth advantage. Mutant ommatidial facets are slightly larger than wild-type facets and sometimes contain extra interommatidial bristles. When homozygous clones of hpo were generated in other imaginal discs using hsFLP, outgrowths of tissue were observed and portions of wings containing large hpo clones were larger than the corresponding portion of a wild-type wing, indicating a role for hpo in regulating organ size in tissues other than the eye. Retinal sections of adult eyes containing hpo clones reveal that mutant ommatidia appear to have the normal complement and arrangement of photoreceptor cells. However, hpo mutant ommatidia appear to have significantly more tissue between adjacent ommatidia. Cell outlines are visualized more readily in the pupal retina. In contrast to the single layer of interommatidial cells observed in wild-type retinas, mutant ommatidia have several additional interommatidial cells. These phenotypic abnormalities are very similar to those observed in sav or wts mutant clones (Harvey, 2003).
sav or wts mutant cells in the eye imaginal disc fail to exit from the cell cycle at the appropriate time. The additional rounds of cell division generate an excess of interommatidial cells. Elevated levels of cyclin E protein detected in mutant cells may underlie the delayed cell cycle exit (Harvey, 2003).
In a disc mosaic for the wild-type parent chromosome, BrdU-incorporation was evident in the anterior portion of the disc and in a narrow stripe, the second mitotic wave (SMW), but not in the morphogenetic furrow (MF) or posterior to the SMW where cells arrest in the G1 phase of the cell cycle. In hpo mosaic eye discs, the pattern of S phases was normal in the anterior portion of the disc and in the SMW but in mutant portions of the disc, BrdU-incorporating nuclei were observed posterior to the SMW and also in the MF. Thus, hpo cells continue to cycle when surrounding wild-type cells are arrested in G1, indicating that hpo function is essential for timely cell cycle exit. In discs containing clones of the wild-type parental chromosome, mitoses, visualized with anti-phospho histone H3, were observed in the anterior portion of the eye disc and in several rows of developing ommatidia immediately posterior to the SMW. In discs mosaic for hpo however, extra mitoses were seen in mutant clones many ommatidial rows posterior to the SMW, indicating that at least a subset of hpo mutant cells continue to divide when wild-type cells are mitotically quiescent. These abnormalities are similar to, though less severe than, those found in sav and wts imaginal discs (Harvey, 2003).
Elevated levels of cyclin E were found in hpo mutant clones immediately anterior to the MF, in the SMW, and posterior to the SMW. Cyclin E expression appeared normal in hpo clones in the most anterior portions of the third instar larval eye-antennal disc. In contrast, cyclins A, B, and D were expressed at normal levels throughout the disc. In wild-type discs cyclin E RNA is expressed in a narrow stripe corresponding to the SMW. In discs mosaic for hpo, the level of cyclin E RNA was elevated and the expression domain of cyclin E was broader. Additionally, an increase in cyclin E mRNA was detected by semiquantitative RT-PCR performed on eye imaginal discs composed almost entirely of hpo mutant tissue. This indicates that, at least in part, hpo regulates cyclin E at the level of transcription or RNA stability but additional posttranscriptional regulation is also possible. In experiments where hpo function was reduced in S2 cells by RNAi, the levels of cyclin E protein were increased without an obvious change in RNA levels as assessed by Northern blotting. Thus, hpo may be capable of regulating cyclin E levels at both transcriptional and posttranscriptional levels (Harvey, 2003).
The cycling properties of hpo mutant cells were analyzed by generating clones of mutant cells in third instar larval wing discs. Larvae were heat shocked 48 hr after egg deposition (AED) and wing discs were dissected 120 hr AED. Following dissociation with trypsin and staining with Hoechst, cells were subjected to flow cytometry. Cell size, as gauged by forward scatter, and DNA content were measured and found to be almost indistinguishable between wild-type and hpo mutant cells (Harvey, 2003).
The number of cells in hpo mutant clones is consistently larger than in wild-type sister clones. The median population doubling time in hpo clones was 13.1 hr, which is significantly shorter than that of the GFP-bearing tester chromosome, which was 14.7 hr. By comparison, clones derived from the parent FRT42D chromosome had a population doubling time that was not significantly different from the same tester chromosome. It is unlikely that the increased cell numbers in hpo clones can be explained by a block in apoptosis, since overexpression of the caspase inhibitor p35 in wing disc cells at this stage of development does not appreciably alter the population doubling time. Thus, cells appear to divide faster in hpo clones. Since cell size is essentially unchanged, this would imply that hpo cells have an increased rate of growth (mass accumulation) and a commensurate increase in the rate of cell division (Harvey, 2003).
In wild-type pupal retinas, excess interommatidial cells are eliminated in a wave of apoptosis during the midpupal stage. There is a defect in apoptosis in sav and wts mutant tissue. As a result, the additional cells generated by excess cell division in sav and wts tissue are not eliminated and account for the increased number of interommatidial cells (Harvey, 2003).
In a genetic screen for mutations that restrict cell growth and organ size, a new tumor suppressor gene, dMST (CG11228), was identified that encodes the Drosophila homolog of the mammalian Ste20 kinase family members MST1 and MST2. Loss-of-function mutations in dMST result in overgrown tissues containing more cells of normal size. dMST mutant cells exhibit elevated levels of Cyclin E and DIAP1, increased cell growth and proliferation, and impaired apoptosis. dMST forms a complex with Salvador (Sav) and Warts (Wts)/Large tumors (Lats), a WW domain-containing protein and a Ser/Thr kinase resepectively, two tumor suppressors also implicated in regulating both cell proliferation and apoptosis, suggesting that they act in common pathways (Jia, 2003).
To identify novel tumor suppressor genes, the Drosophila genome was systematically screened for mutations that cause tissue overgrowth phenotypes. From this screen, three alleles of a gene named dMST (Drosophila homolog of MST1 and MST2) were identified. dMSTBF33 was used for most analyses described in this study; the molecular nature of dMSTBF33 suggests that it is likely to be a null allele (Jia, 2003).
Compared with wild-type eyes, dMST mosaic eyes are significantly larger and often protrude out in folds. Tumorous outgrowths are also observed when dMST clones are induced in other places, including the thorax, wing, and haltere. Hence, dMST is generally required for restricting tissue growth and organ size (Jia, 2003).
To determine how dMST controls organ size, labeled dMST clones were generated in wing discs and cell size and clone size were compared between mutant clones and twin spots. dMST mutant cells do not exhibit discernible changes in cell size. Moreover, dMST mutant cells differentiate into wing margin bristles of normal size. However, dMST clones occupy significantly larger areas and contain more cells than do wild-type twin spots. Since mutant clones and twin spots are derived from mitotic sister cells born at the same developmental stages, the increase in cell numbers and tissue mass of dMST mutant clones over twin spots suggests that dMST mutant cells grow and proliferate faster than do wild-type cells. Hence, the increase in size of dMST mosaic organs is caused by an increase in cell number but not cell size (Jia, 2003).
To further explore cell proliferation defects caused by dMST mutations, focus was placed on eye development. In wild-type eye discs, a single stripe of cells, referred to as the second mitotic wave (SMW), enters S phase synchronously posterior to the morphogenetic furrow(MF), and little bromodeoxyuridine (BrdU) labeling is present posterior to the SMW. In contrast, dMST mosaic discs exhibit extensive BrdU incorporation posterior to the SMW. To determine if extra mitosis also occurs in dMST mutant eye discs, the anti-phosphohistone H3 (pH3) antibody was used to label cells in M phase. In wild-type eye discs, few cells posterior to the SMW exhibit pH3 staining. In contrast, dMST mutant discs contain increased number of cells in M phase posterior to the SMW, suggesting that dMST mutations increase cell proliferation (Jia, 2003).
Cyclin E is an important regulator of S-phase initiation and progression in imaginal disc development. In wild-type discs, Cyclin E is up-regulated at the SMW. dMST mutant clones accumulate high levels of Cyclin E in a cell-autonomous fashion, which is consistent with the increased cell proliferation in dMST mutant discs (Jia, 2003).
In wild-type eyes, excessive cells between differentiated ommatidias are eliminated by a wave of apoptosis at early pupal stage so that a single layer of cells exists between two adjacent ommatidias. In contrast, the dMST mutant disc contains multiple layers of interommatidial cells. The persistence of excessive interommatidial cells in dMST mutant discs implies that apoptosis could be compromised. To test this, cell death was examined in wild-type and dMST mosaic pupal retina 38 h after pupa formation (APF); apoptosis was found to be diminished in dMST mutant cells. Hence, dMST is required for apoptosis during development (Jia, 2003).
In Drosophila, apoptosis is triggered by death inducers, including head involution defective (hid). Expressing GMR-hid induces precocious cell death in larval eye discs, which is blocked in dMST mutant cells. As a consequence, adult eyes derived from GMR-hid-expressing discs that also contain dMST mutant clones are larger than those derived from wild-type discs expressing GMR-hid. In Drosophila, death promoters induce apoptosis in part by down-regulating the levels of the death inhibitor DIAP1. dMST mutant cells exhibit higher levels of DIAP1 than do wild-type cells, suggesting that dMST promotes cell death at least in part by down-regulating the levels of DIAP1. The expression of a diap1-lacZ reporter gene is elevated in dMST mutant cells, indicating that dMST inhibits the transcription of diap1 (Jia, 2003).
The dMST mutations were mapped by high-resolution meiotic recombination. Several candidate genes were sequences for molecular lesions present in mutagenized chromosomes containing dMST alleles. Both dMSTJM1 and dMSTBF33 introduce point mutations in the open reading frame (ORF) of annotated gene CG11228, which encodes the Drosophila homolog of mammalian Ste20 kinase family members MST1 and MST2. A full-length cDNA corresponding to CG11228 was placed under the control of a tubulinalpha1 promoter to generate tub-dMSTf. Both dMSTJM1 and dMSTBF33 homozygotes expressing one copy of tub-dMSTf are viable, morphologically normal, and fertile, demonstrating that CG11228 is dMST. dMSTJM1 introduced a single amino acid substitution at a conserved residue (G181E), whereas dMSTBF33 introduced a stop codon at amino acid 174, suggesting that dMSTBF33 is likely to be a null allele (Jia, 2003).
When overexpressed in larval eye discs by using the GMR-gal4 driver, dMSTn, but not its kinase dead form (dMSTnK>R), induced ectopic apoptosis, leading to the formation of small rough eyes. Although expressing GMR-Wts alone does not cause any significant defect, coexpressing GMR-Wts with dMSTn enhances the defects caused by expressing dMSTn alone. For example, although flies expressing GMR-Gal4/dMSTn are viable, coexpressing GMR-Wts with GMR-gal4/dMSTn causes flies to die as pharate adults that have smaller eyes than those of flies expressing GMR-gal4/dMSTn alone. Similar phenotypic enhancement was obtained when two copies of UAS-dMSTn were expressed under GMR-gal4. In contrast, the defects caused by dMSTn are not enhanced by GMR-Sav. These genetic interactions are consistent with the findings that dMSTn binds Wts but not Sav (Jia, 2003).
To determine the genetic epistasis, dMSTn was overexpressed with GMR-gal4 in eye discs that contain wts or sav mutant clones. dMSTn induces ectopic Drice activation in sav mutant cells, as in the case of wild-type cells. In contrast, the ectopic Drice activation induced by dMSTn is diminished in wts mutant cells. Consistently, dMSTn blocks the up-regulation of DIAP1 in sav, but not in wts, mutant cells. In addition, dMSTn blocks the up-regulation of Cyclin E in sav but not in wts mutant cells. These results suggest that dMST acts downstream of sav but upstream of or in parallel with wts to restrict cell proliferation and promote apoptosis (Jia, 2003).
The MST subfamily of Ser/Thr kinases is classified as putative mitogen-activated protein kinase kinase kinase kinase (MAP4K). Although numerous studies have been carried out to address their biochemical properties and regulations in cultured cells, their physiological roles remain elusive. dMST is shown to play a pivotal role in controlling cell number and organ size in Drosophila development. dMST fulfills such a role both by restricting cell growth and proliferation and by promoting cell death. The cell-autonomous elevation of Cyclin E and DIAP1 levels in dMST mutant clones suggest that dMST regulates cell proliferation and cell death cell autonomously. Biochemical evidence is provided that dMST, Sav, and Wts form a complex. Genetic epitasis study suggests sav acts upstream of dMST whereas wts acts downstream of or in parallel with dMST. Sav could regulate the formation or activity of dMST/Wts kinase complex. Alternatively, Sav could act to bridge the dMST/Wts kinase complex to its substrates, a function that can be bypassed by excess amounts of dMSTn (Jia, 2003).
It should be noted that sav, dMST, and wts may not simply act in a linear pathway, because wts mutant phenotypes appear to be more severe than those caused by sav or dMST mutations. In addition, sav wts double-mutant phenotypes are stronger than sav or wts single-mutant phenotypes. Hence, although the results suggest that dMST, Sav, and Wts act in common pathways, they may exist in multiple complexes and have independent functions (Jia, 2003).
It has been shown that human Sav is mutated in several cancer cell lines, and mice lacking a homolog of wts/lats develop hyperplasia and tumors in several tissues, raising the possibility that mammalian MST1 and MST2 may also function as tumor suppressors. In support of this notion, MST1 and MST2 have been implicated in promoting cell death in cultured mammalian cells. It remains to be determined whether MST1 and MST2 also regulate cell proliferation in mammals. Given the functional conservation between many insect and mammalian pathways, it is speculated that the mammalian MST/Sav/Lats pathway may also participate in restricting cell number and organ size during normal development, and its malfunction may lead to cancers (Jia, 2003).
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).
Dendritic fields are important determinants of neuronal function. However, how neurons establish and then maintain their dendritic fields is not well understood. Polycomb group (PcG) genes are required for maintenance of complete and nonoverlapping dendritic coverage of the larval body wall by Drosophila class IV dendrite arborization (da) neurons. In esc, Su(z)12, or Pc mutants, dendritic fields are established normally, but class IV neurons display a gradual loss of dendritic coverage, while axons remain normal in appearance, demonstrating that PcG genes are specifically required for dendrite maintenance. Both multiprotein Polycomb repressor complexes (PRCs) involved in transcriptional silencing are implicated in regulation of dendrite arborization in class IV da neurons, likely through regulation of homeobox (Hox) transcription factors. Genetic interactions and association between PcG proteins and the tumor suppressor kinase Warts (Wts) is demonstrated, providing evidence for their cooperation in multiple developmental processes including dendrite maintenance (Parrish, 2007).
Dendrite arborization patterns are a hallmark of neuronal type; yet how dendritic arbors are maintained after they initially cover their receptive field is an important question that has received relatively little attention. The Drosophila PNS contains different classes of sensory neurons, each of which has a characteristic dendrite arborization pattern, providing a system for analysis of signals required to achieve specific dendrite arborization patterns. Class IV neurons are notable among sensory neurons because they are the only neurons whose dendrites provide a complete, nonredundant coverage of the body wall. This study found tha the function of Polycomb group genes is required specifically in class IV da neurons to regulate dendrite development. In the absence of PcG gene function, class IV dendrites initially cover the proper receptive field but subsequently fail to maintain their coverage of the field. Time-lapse analysis of dendrite development in esc or Pc mutants suggests that a combination of reduced terminal dendrite growth and increased dendrite retraction likely accounts for the gradual loss of dendritic coverage in these mutants. Maintenance of axonal terminals in class IV da neurons is apparently unaffected by loss of PcG gene function, suggesting that PcG genes function as part of a program that specifically regulates dendrite stability (Parrish, 2007).
Establishment of dendritic territories in class IV neurons is regulated by homotypic repulsion, and this process proceeds normally in the absence of PcG function. In PcG mutants, class IV neurons tile the body wall by 48 h AEL, similar to wild-type controls. However, beginning at 48 h AEL, likely as a result of reduced dendritic growth and increased terminal dendrite retraction, class IV neurons of PcG mutants gradually lose their dendritic coverage. In contrast, the axon projections and terminal axonal arbors of PcG mutants show no obvious defects. Although an early role for PcG genes in regulating axon development cannot be ruled out, MARCM studies showed that PcG genes are required for the maintenance of dendrites but not axons in late larval development. Thus, different genetic programs appear to be responsible for the establishment and maintenance of dendritic fields, and for the maintenance of axons and dendrites (Parrish, 2007).
It is well established that PcG genes participate in regulating several important developmental processes including expression of Hox genes for the specification of segmental identity. In comparison, much less is known about the function of PcG genes in neuronal development. Studies of the expression patterns of PcG genes and the consequences of overexpression of PcG genes suggest that PcG genes may affect the patterning of the vertebrate CNS along the anterior-posterior (AP) axis, analogous to their functions in specifying the body plan. A recent study demonstrates that the PcG gene Polyhomeotic regulates aspects of neuronal diversity in the Drosophila CNS. The current study now links the function of PcG genes to maintenance of dendritic coverage of class IV sensory neurons. Thus it will be interesting to determine whether PcG genes play a conserved role in the regulation of dendrite maintenance (Parrish, 2007).
Since Hox genes function in late aspects of neuronal specification and axon morphogenesis, it seems possible that regulation of Hox genes by PcG genes may be important for aspects of post-mitotic neuronal morphogenesis, including dendrite development. The PcG genes esc and E(z) are required for proper down-regulation of BX-C Hox gene expression in class IV neurons. The timing of this change in BX-C expression corresponds to the time frame during which PcG genes are required for dendritic maintenance. Furthermore, post-mitotic overexpression of BX-C genes in class IV da neurons, but not other classes of da neurons, is sufficient to cause defects in dendrite arborization, thus phenocopying the mutant effects of PcG genes. Finally, it was found that Hox genes are required cell-autonomously for dendrite development in class IV neurons, and loss of Hox gene function causes defects in terminal dendrite dynamics that are opposite to the defects caused by loss of PcG genes. Therefore, it seems likely that PcG genes regulate dendrite maintenance in part by temporally regulating BX-C Hox gene expression (Parrish, 2007).
Several recent studies have focused on the identification of direct targets of PcG-mediated silencing, demonstrating that PcG genes regulate expression of distinct classes of genes in different cellular contexts. During Drosophila development, PRC proteins likely associate with >100 distinct loci, and the chromosome-associate profile of PRC proteins appears dynamic. Therefore, identifying the targets of PcG-mediated silencing in a given developmental process has proven difficult. Thus far, alleles of >20 predicted targets of PcG-mediated silencing have been analyzed for roles in establishment or maintenance of dendritic tiling and a potential role has been found for only Hox genes. Future studies will be required to identify additional targets of PcG-mediated silencing in regulation of dendrite maintenance (Parrish, 2007).
PcG genes are broadly expressed, so it seems likely that interactions with other factors or post-translational mechanisms may be responsible for the cell type-specific activity of PcG genes. Indeed, PcG genes genetically interact with components of the Wts signaling pathway to regulate dendrite development specifically in class IV neurons. Based on the observations that wts mutants also show derepression of Ubx in class IV neurons and that Wts can physically associate with PcG components, it seems likely that Wts may directly or indirectly influence the activity of PcG components. In proliferating cells, Wts phosphorylates the transcriptional coactivator Yorkie to regulate cell cycle progression and apoptosis, demonstrating that Wts can directly influence the activity of transcription factors. In support of a possible role for Wts directly modulating PcG function, several recent reports have documented roles for phosphorylation in regulating PcG function both in Drosophila and in vertebrates. Thus, it is possible that some of the components involved in PcG-mediated silencing are regulated by Wts phosphorylation. Alternatively, association of Wts with PcG proteins may facilitate Wts-mediated phosphorylation of chromatin substrates (Parrish, 2007).
The tumor suppressor kinase Hpo regulates both establishment and maintenance of dendritic tiling in class IV neurons through its interactions with Trc and Wts, respectively, but how Hpo coordinately regulates these downstream signaling pathways is currently unknown. Similar to mutations in wts, mutations in PcG genes interact with mutations in hpo to regulate dendrite maintenance but show no obvious interaction with trc, consistent with the observation that PcG gene function is dispensable for establishment of dendritic tiling. Although it is possible that different upstream signals control Hpo-mediated regulation of establishment and maintenance of dendritic tiling, the nature of such signals remain to be determined. Another possibility is that the activity of the Wts/PcG pathway could be antagonized by additional unknown factors that promote establishment of dendritic tiling (Parrish, 2007).
In addition to their interaction in regulating dendrite maintenance, PcG genes and wts interact to regulate expression of the Hox gene Scr during leg development. This finding suggests that the Hpo/Wts pathway may play a general role in contributing to PcG-mediated regulation of Hox gene expression. The presence of ectopic sex combs provides a very simple and sensitive readout of wts/PcG gene interactions and should form the basis for conducting large-scale genetic screens to identify other genes that interact with wts or PcG genes and participate in this genetic pathway (Parrish, 2007).
In Drosophila, the body axes are specified during oogenesis through interactions between the germline and the overlying somatic follicle cells. A Gurken/TGF-alpha signal from the oocyte to the adjacent follicle cells assigns them a posterior identity. These posterior cells then signal back to the oocyte, thereby inducing the repolarization of the microtubule cytoskeleton, the migration of the oocyte nucleus, and the localization of the axis specifying mRNAs. However, little is known about the signaling pathways within or from the follicle cells responsible for these patterning events. It study shows that the Salvador Warts Hippo (SWH) tumor-suppressor pathway is required in the follicle cells in order to induce their Gurken- and Notch-dependent differentiation and to limit their proliferation. The SWH pathway is also required in the follicle cells to induce axis specification in the oocyte, by inducing the migration of the oocyte nucleus, the reorganization of the cytoskeleton, and the localization of the mRNAs that specify the anterior-posterior and dorsal-ventral axes of the embryo. This work highlights a novel connection between cell proliferation, cell growth, and axis specification in egg chambers (Meignin, 2007).
Multicellular organisms develop through an orchestrated temporal and spatial pattern of cell behavior, which is controlled by cell-to-cell signaling. In Drosophila melanogaster, the establishment of the embryonic axes occurs in the oocyte and depends on a sequence of signals between the germline and the somatic cells. First, Gurken (Grk) signals from the oocyte to the adjacent follicle cells (FCs), in which Torpedo (Top, EGFR) is activated, and this signal instructs them to adopt a posterior identity. The posterior FCs (PFCs) then send an unidentified signal back to the oocyte, leading to the movement of the nucleus from the posterior to the dorsoanterior (DA) corner and the repolarization of the microtubule (MT) cytoskeleton, with the minus ends at the anterior and lateral cortex and the plus ends at the posterior. This repolarization results in the localization of the mRNAs that encode key patterning factors. grk mRNA is next to the nucleus at the DA corner of the oocyte. At this corner, Grk instructs the overlying FCs to adopt dorsal fates. In contrast, oskar (osk) and bicoid (bcd) mRNAs are localized at the posterior and anterior pole, respectively, thus defining the anterior posterior (AP) embryonic axis and the germ cells. Although several genes are required in the FCs to control these events, little is known about the signaling pathways within and from the FCs (Meignin, 2007).
One of the genes required for axis formation during oogenesis is the tumor suppressor merlin (mer). However, it is not known whether Mer influences axis specification directly or what signaling pathways lie downstream of Mer. In other tissues, Mer is known to activate the Salvador Warts Hippo (SWH) pathway, which is a tumor-suppressor pathway. Inhibition of the SWH pathway leads to a characteristic overgrowth phenotype in adult organs because of an overproliferation of cells, increased cell growth, and defects in apoptosis. To test whether the SWH pathway is required in the function of Mer in axis formation, the localization of grk, bcd, and osk mRNA was examined in egg chambers with warts (wts) and hippo (hpo) mutant FCs. wts and hpo encode two serine/threonine kinases that are core components of this pathway. In both cases, grk mRNA is mislocalized at the posterior, osk mRNA is mislocalized at the center, and bcd mRNA is mislocalized at the posterior and anterior poles. The mislocalization of these mRNAs could be due to failure of the MTs to repolarize, as has been previously shown in grk/EGFR and mer mutants. In wild-type oocytes, the MTs are organized in an AP gradient. In contrast, in egg chambers with hpo mutant FCs, the MTs are distributed diffusely all over the oocyte cytoplasm. Considering these results, together with previous characterizations of similar phenotypes, it is concluded that the oocyte cytoskeleton in mutant egg chambers for the SWH pathway is disorganized with the MT plus ends at the center and the minus ends at the anterior and posterior poles. These defects resemble those described in oocytes lacking the Grk signal. In wts mutants, however, Grk protein is detected at the posterior pole, where grk mRNA is mislocalized. This demonstrates that the axis-specification defects in wts mutant egg chambers are not a consequence of the absence of Grk protein (Meignin, 2007).
It was shown that mer is required in the FCs for the repolarizing signal back to the germline and consequently for the migration of the oocyte nucleus from the posterior to the DA corner. Similarly, when mutant FC clones were generated for wts, hpo, and expanded (ex), an activator of the SWH pathway, the oocyte nucleus fails to migrate to the anterior. Another protein that is upstream of the SWH pathway is the giant atypical cadherin fat (ft). However, egg chambers with ft mutant FCs show no defects in oocyte polarity, and both the nucleus and Staufen (Stau) [a marker for osk mRNA] are always properly localized. In other epithelia, hpo and wts are required to repress the activity of Yorkie (Yki) and overexpression of yki phenocopies loss-of-function mutations of hpo and wts. Similarly, it was found that overexpression of yki in the FCs also causes the mislocalization of Stau and the oocyte nucleus. These results indicate that the SWH pathway, with the exception of Ft, might be required for the repolarizing signal back from the FCs to the oocyte (Meignin, 2007).
Because this signal is sent by the PFCs, whether the SWH pathway is required only in these cells was analyzed. In egg chambers with wild-type PFCs within an otherwise hpo or wts mutant epithelium, as well as in hpo, wts, and ex germline clones, the oocyte polarity is unaffected. However, in egg chambers with hpo mutant PFCs in an otherwise wild-type epithelium, the oocyte nucleus is mislocalized. It was also observed that when only a few cells at the posterior are mutant, Stau localizes in the region of the oocyte that faces the posterior wild-type cells. The SWH pathway is not required in the polar cells for axis determination because egg chambers with hpo or wts mutant PFCs and wild-type polar cells show oocyte polarity defects. It is concluded that the SWH pathway is required only in the PFCs to induce axis specification in the oocyte (Meignin, 2007).
In contrast to the monolayered wild-type epithelium, anterior and posterior, but not lateral, hpo and wts mutant cells form a bilayered, and occasionally a multilayered, epithelium. Given that the SWH pathway is required to control proliferation in epithelia of imaginal discs, whether the bilayered epithelium is a result of overproliferation was analyzed. At stage 6 of oogenesis, wild-type FCs undergo a Notch-dependent switch from a mitotic cell cycle to an endocycle. For this reason, phosphohistone 3 (PH3), a marker for mitotic cells, is detected only until that stage and never later. In contrast, hpo mutant anterior and posterior FCs are often positive for PH3 at stage 7-10B, indicating that these cells are still dividing. Similar results are obtained in yki overexpressing FCs. Taken together, these findings show that the SWH pathway is required for the control of proliferation at the anterior and posterior FCs (Meignin, 2007).
The formation of a multilayered epithelium was also observed in stage 3-5 mutant FCs, although the number of dividing cells is similar to that of the wild-type. It has been recently shown that the aberrant orientation of the mitotic spindle in the FCs results in the formation of a multilayered epithelium. Therefore the orientation of the mitotic spindle was analyzed in wild-type and hpo mutant cells. It was observed that, contrary to wild-type cells, the mitotic spindle in mutant FCs is often at an angle or perpendicular to the membrane. This aberrant orientation disrupts the remaining daughter cells within the same plane, thereby resulting in a bilayered epithelium (Meignin, 2007).
Often, tumor suppressors are important for the polarity of the epithelia. To determine whether this is the case for the SWH pathway, the atypical (novel) Protein Kinase C (nPKC), an apical marker, and Disc large (Dlg), a lateral marker, were examined in the FCs. In wild-type cells, as well as in hpo mutant FCs that maintain a monolayer epithelium, nPKC and Dlg localize at the apical and lateral membrane, respectively. However, when the mutant epithelium forms several layers of cells, nPKC and Dlg are often mislocalized, with a reduction of the nPKC staining and an expansion of the Dlg-positive membrane. Nevertheless, a certain degree of the polarity in these cells is maintained because nPKC is always apical in the cells that are in contact with the oocyte (Meignin, 2007).
Because SWH pathway mutant cells do not exit mitosis and keep dividing, it is possible that their differentiation is impaired. To address this question, the expression of Fasciclin III (FasIII) and eyes absent (eya) were analyzed in wild-type and wts and hpo mutant FCs. FasIII and Eya are downregulated in a Notch-dependent manner in the main-body FCs after stage 6 of oogenesis. However, the levels of FasIII in hpo mutant PFCs and Eya in wts mutant PFCs remain high after stage 6. To further assess the effect of the SWH pathway on the Notch-dependent maturation of the FCs, the expression of Hindsight (Hnt), a transcription factor that is upregulated by Notch signaling in all FCs was examined.. In hpo posterior FC clones, this Hnt upregulation is blocked. Contrary to notch clones, however, hpo lateral and anterior clones do not show defects in FasIII, Eya, or Hnt expression. Furthermore, border, centripetal, and stretched cells that are mutant for hpo migrate normally. Considering all these results together, it is concluded that the SWH pathway is essential for the PFCs to fully differentiate (Meignin, 2007).
The findings described above, together with the proliferation defects in hpo and wts mutant cells, suggest that the SWH pathway is required for Notch signaling. To test whether this is the case, the expression of universal Notch transcriptional reporters was analyzed in wild-type and hpo mutant FCs. In wild-type egg chambers, the Notch reporter E(spl)mß7-lacZ is expressed in all FCs upon Notch activation at stage 6 of oogenesis. In contrast, it was found that in hpo mutant cells, the levels of E(spl)mß7-lacZ are weakly reduced in 53% of the clones and normally expressed in the rest. It has been shown that in wing imaginal discs, mer and ex are required to control Notch localization in the cell and consequently its activity. Similarly, the subcellular distribution of Notch is affected in hpo mutant FCs. Contrary to the wild-type, in which Notch accumulates in the apical membrane, Notch expands to other membranes and is often detected in clusters in hpo clones. The results point out that hpo is essential in the PFCs for the Notch-dependent expression of several differentiation markers, such as FasIII, Eya, and Hnt, and for Notch subcellular localization. These observations and the weak defects on the Notch reporters support a function of the SWH pathway in modulating Notch signaling (Meignin, 2007).
Because the SWH pathway is required for the polarization of the oocyte, as well as for the differentiation of the PFCs, whether the mutant cells are competent to respond to Grk and indeed adopt a posterior fate was analyzed. Dystroglycan (DG) is expressed in all FCs at early stages of oogenesis, but upon Grk signaling, DG forms an AP gradient with lower levels at the PFCs. The fact that this Grk-dependent gradient of DG is also observed in the hpo mutant epithelia suggests that the mutant cells are responsive to the Grk signaling. Similarly, when hpo clones affect only a portion of the PFCs, the posterior fate marker pointed is expressed as in the wild-type in 40% of the cases. However, in 60% of the egg chambers with partial hpo posterior clones, and in all cases when all the PFCs are mutant, the expression of pointed is abolished. These results illustrate that hpo is required to fully process the Grk/EGFR signal in the PFCs. Conversely, in grk mutant egg chambers, the Hpo-dependent expression of Hnt is not affected, suggesting that the EGFR pathway is not required for the activation of the SWH pathway in the PFCs (Meignin, 2007).
Considering all these results together, it is concluded that the SWH pathway is involved in the Notch- and Gurken-dependent maturation of the PFCs. Whether the SWH pathway modulates this maturation directly or indirectly, for example by affecting membrane properties, needs to be further investigated (Meignin, 2007).
To study whether the oocyte polarity defects in egg chambers with FCs mutants for the SWH pathway are a consequence of the FCs proliferation and differentiation defects, egg chambers with ex and ft mutant PFCs were analyzed. Egg chambers with ft PFCs occasionally form a bilayer, although they never have defects in oocyte polarity, suggesting that the morphological disruption of the epithelia in itself does not block the repolarizing signal. Egg chambers with ex PFCs show weak defects in the epithelium, with a bilayer rarely formed and restricted to only a few mutant cells, but Stau is never properly localized. However, Hnt is not properly expressed in stage 7 ex mutant FCs, suggesting that the mislocalization of Stau is a consequence of the ex mutant cells being undifferentiated at the stage when the repolarizing signal is sent to the oocyte. These results suggest that the defects in oocyte polarity are probably due to a lack of proper differentiation of FCs in SWH mutant egg chambers (Meignin, 2007).
This study has analyzed the requirement of the SWH pathway during oogenesis. Several of the components of this pathway, but not ft, are required in the PFCs to induce the axis specification in the germline. The defects in oocyte polarity, however, are probably due to a lack of proper differentiation of the PFCs in SWH mutant egg chambers. In addition, the pathway is required in the terminal cells to control their proliferation. It has already been shown that terminal follicle cells are different from lateral follicle cells. The distinct spatial requirement of the SWH pathway for differentiation and proliferation is another feature that distinguishes the terminal from the lateral FCs, and the posterior from the anterior FCs. These results point out that this dual function of the SWH pathway might be achieved by modulation of the Notch and EGFR signals. In conclusion, the SWH pathway lies at the intersection of two signaling pathways and is permissive for the signal that is sent from the follicle cells to repolarize the oocyte (Meignin, 2007).
The Salvador Warts Hippo (SWH) network limits tissue size in Drosophila and vertebrates. Decreased SWH pathway activity gives rise to excess proliferation and reduced apoptosis. The core of the SWH network is composed of two serine/threonine kinases Hippo (Hpo) and Warts (Wts), the scaffold proteins Salvador (Sav) and Mats, and the transcriptional coactivator Yorkie (Yki). Two band 4.1 related proteins, Merlin (Mer) and Expanded (Ex), have been proposed to act upstream of Hpo, which in turn activates Wts. Wts phosphorylates and inhibits Yki, repressing the expression of Yki target genes. Recently, several planar cell polarity (PCP) genes have been implicated in the SWH network in growth control. This study shows that, during oogenesis, the core components of the SWH network are required in posterior follicle cells (PFCs) competent to receive the Gurken (Grk)/TGFβ signal emitted by the oocyte to control body axis formation. These results suggest that the SWH network controls the expression of Hindsight, the downstream effector of Notch, required for follicle cell mitotic cycle-endocycle switch. The PCP members of the SWH network are not involved in this process, indicating that signaling upstream of Hpo varies according to developmental context (Polesello, 2007).
Body axis formation is a critical stage of development in most multicellular organisms. In Drosophila melanogaster, the anteroposterior (AP) body axis is determined by the polarization of the developing oocyte. The egg chamber is composed of 16 germ cells (15 nurse cells plus the oocyte) and the follicular epithelium. Specification of the AP axis requires active transport of several mRNAs along the microtubule network, thereby resulting in asymmetric mRNA and protein localization inside the oocyte. For example, bicoid (bcd) and oskar (osk) mRNAs localize to and control the formation of the anterior and posterior poles, respectively. This process is initiated through bidirectional signaling between the oocyte and the adjacent follicle cells. In midoogenesis egg chambers, grk mRNA is localized between the oocyte nucleus and the plasma membrane at the presumptive posterior pole and targets the Grk signal to the posterior follicle cells (PFCs) only. Grk is believed to be the ligand for the Torpedo/DER (EGFR) signaling pathway, which controls PFC identity. Once they are specified, the PFCs send an unknown signal back to the oocyte; this signal is required to establish oocyte posterior polarity (Polesello, 2007).
Mer, which has recently been proposed to be part of the SWH network in tissue-size control, has been suggested to play a role in signal back. Therefore whether other members of this network could play a role in body axis formation was addressed. It was tested whether hpo, like mer, is required in PFCs to control oocyte polarity by generating FLP/FRT mitotic clones of mutant cells in the egg chamber was tested with either a kinase-dead (hpoJM1) or a truncating (hpoBF33) allele of hpo. These two alleles behave similarly in all subsequent experiments (Polesello, 2007).
In wild-type egg chambers, the RNA-binding proteins Staufen (Stau) and Osk are localized in a crescent at the posterior pole of the oocyte. When the PFCs were mutant for hpo (visualized by the lack of GFP), both Osk and Stau are mislocalized. If all PFCs were mutant, both Stau and Osk were found in the middle of the oocyte or were absent in some cases for Osk. When hpo clones affected only a portion of the PFCs, Stau was mislocalized almost exclusively in the mutant part, showing the importance of the crosstalk between PFCs and the oocyte (Polesello, 2007).
In hpo germline clones, Stau localization is unaffected if the PFCs are wild-type, suggesting that Hpo is not required for secretion of the Grk signal by the oocyte. Similarly, hpo activity in polar cells is not sufficient to rescue hpo PFC phenotypes because chambers with mutant PFCs and wild-type (GFP-positive) polar cells show disrupted Stau localization. Together, these data suggest that hpo is required in the PFCs to control oocyte polarity (Polesello, 2007).
By using Stau localization as a readout, it was found that like mer and hpo, ex, sav, mats, wts, and yki are playing a role in PFCs to control oocyte polarity, suggesting that 'canonical' Hpo signaling is responsible for the observed phenotype. In contrast, fat (ft) and discs overgrown (dco) are not required in PFCs to control oocyte polarity. This suggests that the core components of the SWH network but not the SWH-associated PCP genes are required for anteroposterior axis formation (Polesello, 2007).
The microtubule cytoskeleton plays an active role in the correct localization of posterior determinants such as Osk mRNA and Stau. Therefore, whether the microtubules are normally organized when the PFCs were mutant for hpo was tested. The oocyte nucleus is initially positioned at the posterior pole (up to stage 6) and migrates to an anterodorsal localization in a microtubule-dependent manner after the signal back from the PFCs (stages 7-14). The oocyte nucleus fails to migrate to an anterodorsal position in 50% of egg chambers with PFC hpo clones. The expression of a tubulin-GFP fusion protein was drived in the germline to visualize the microtubule network. In control oocytes, tubulin-GFP forms a regular network of filaments with a stronger accumulation at the anterior pole corresponding to the nucleation site. Egg chambers with hpo mutant PFCs present ectopic Tubulin-GFP accumulation at the posterior pole of the oocyte. Apart from this defect, the general aspect of the microtubule network is normal in egg chambers with hpo PFC clones, even when the oocyte nucleus has failed to migrate to the anterior end. Finally, microtubule polarity was examined by using both Nod-βGalactosidase (Nod-βGal, minus end marker-anterior) and Kinesin-βGalactosidase (Kin-βGal, plus end marker-posterior) fusion proteins. When the PFCs were mutants for hpo, Nod-βGal was present at both poles or only at the posterior of the oocyte when the nucleus failed to migrate. When all PFCs were hpo mutant, Kin-βGal localization was in a diffuse cloud in the middle of the ooplasm. As for Stau, only half of the Kin-βGal was normally localized when only part of the PFCs were hpo mutant. Together these data support the idea that core components of the SWH pathway are required in the PFCs to build oocyte polarity, controlling microtubule-network orientation (Polesello, 2007).
Because the SWH network is known to control cell number, a phosphorylated Histone 3 (PH3) antibody was used to follow cell division in the follicle cells. During egg-chamber development, follicle cells undergo normal mitotic divisions up to stage 6, thereby giving rise to ~650 follicle cells surrounding the germ cells. Follicle cells then switch from mitotic cycles to three rounds of endoreplication cycles (endocycles) during stages 7-10A. Thus, follicle cells normally stop proliferating after stage 6, as assayed by the absence of PH3-positive cells. hpo PFC clones still contained PH3-positive cells until stage 10B. This excess proliferation observed in hpo mutant cells gives rise to both a reduction of the size of follicle cell nuclei (reduced endocycling) and formation of double layers of cells at the posterior of the egg chamber. Formation of extra layers in the follicular epithelium has been reported to result from misorientation of the mitotic spindle. Normally, the mitotic spindle is parallel to the surface of the germline cells but appears randomly oriented in hpo mutant PFCs because both parallel and perpendicularly oriented spindles were observed. This defect in the mitotic-spindle orientation is probably responsible for the double-layer formation. The proliferation defect specifically affects PFCs because reduced nuclei, ectopic PH3 foci or double layers were not obvious elsewhere. Finally, it was found that loss of the core components of the SWH network, but not of ex for which the proliferation defect is weaker, produced a double cell layer (Polesello, 2007).
In imaginal discs, loss of SWH pathway genes leads to increased expression of Yki target genes. Whether this is also the case in PFCs was tested. As expected, disruption of SWH activity in PFCs gave rise to an increase in ex expression, although no changes were detected in DIAP1 or cycE expression. ex upregulation was restricted to the PFCs in both wts mutant cells and yki gain-of-function experiments. These results suggest that core components of the SWH network specifically control proliferation of a particular subset of follicle cells required for body axis establishment (Polesello, 2007).
Because hpo mutant PFCs were still dividing after stage 6, whether hpo loss of function could affect PFC polarity was assessed. Armadillo (Arm) and Discs large (Dlg) normally label the adherens junctions and the lateral region of the cell, respectively. In hpo mutant PFCs, these were found all around the cells. In addition, the level of Arm, atypical Protein Kinase C (aPKC), and phosphorylated Moesin (P-Moe) were increased. Nevertheless, some aspects of the polarity in these cells were preserved because aPKC was still localized in the apical domain facing the oocyte (Polesello, 2007).
Grk signals via the EGF receptor Torpedo (Top) and activates the Ras signaling pathway, specifying the PFC identity. The PFC fate can be followed by the expression of the Ras target pointed (pnt-LacZ). In the absence of hpo, pnt-LacZ expression was disrupted in most but not all PFC clones. Nevertheless, hpo mutant PFCs were still able to activate the Jak/STAT pathway in response to a signal emerging from the polar cells, (monitored with a STAT reporter suggesting that the polarity defect observed in hpo mutant PFCs does not affect their ability to receive secreted signals in general. wts mutant PFCs were negative for the dpp-LacZ reporter, a specific marker of the anterior follicle cell fate (stretch and centripetal cells), suggesting that when the SWH pathway is compromised, the PFCs are not merely transformed into anterior cells. In addition, it was found that hpo mutant PFCs present characteristics of immature cells such as maintenance of Fasciclin III (FASIII) and eyes absent (eya) expression. Normally, the level of these two genes is downregulated when the follicle cells switch from mitotic cycles to endocycles. It is noted that, when hpo mutant PFCs were FASIII positive, they did not express pnt-LacZ and vice versa. In addition, it was found that pnt-LacZ-positive hpo mutant PFCs have normal Stau localization. This suggests that the primary defect in hpo mutant cells is the failure to mature. In the rare cases where hpo mutant PFCs mature properly, they are competent to transduce the Grk signal, and oocyte polarity is normal (Polesello, 2007).
Notch (N) is required in the follicle cells for the mitotic-endocycle switch that occurs at stage 6 and for controlling follicle cell identity. N mutant follicle cells, like hpo mutant PFCs, keep proliferating because they are stuck in an immature state and continue to express undifferentiated markers such as FASIII. Recently, members of the SWH network were reported to modulate N activity by affecting its subcellular localization. N protein, which localizes to the apical part of the follicle cells, is downregulated at midoogenesis. This downregulation is delayed in wts and hpo mutant PFCs, possibly causing a defect in N signaling. Hindsight (Hnt), a target of N, which starts to be expressed in all follicle cells at stage 7 after N activation, was examined. Expression of Hnt in hpo mutant PFCs is compromised. In addition, it was found that the expression of Cut, which is normally inhibited by Hnt at stage 7, was maintained in hpo and wts clones up to stage 10. Finally, whether the modulation of N activity by the SWH network was direct was tested by looking at the expression of direct N reporters. no obvious reduction of the m7-LacZ reporter was found in hpo PFC clones. However, because of the perdurance of the β-galactosidase protein, this type of reporter is more suitable to follow increases rather than decreases in signaling. It therefore cannot be entirely rule out that the SWH network might directly affect Notch activity. Nevertheless, together these data show that inactivation of the SWH network compromises the regulation of downstream targets of Notch such as Hnt and Cut. As is the case for FASIII, misregulation of these genes is restricted to the PFCs in a SWH mutant background (Polesello, 2007).
Because of this spatial restriction of SWH activity to PFCs, whether the SWH network could be part of the Torpedo/Ras pathway acting downstream of the Grk signal was tested. ras, wts double loss-of-function clones were examined. ras, wts clones present characteristics of both ras and wts single-mutant clones, namely upregulation of Dystroglycan (DG), as observed in ras clones, and maintenance of FASIII protein, as observed in wts clones. In addition, grk mutant egg chambers present only DG upregulation but no FASIII modification and no substantial change in ex expression. It is therefore concludes that the SWH network and EGFR/Ras signaling are likely to act in parallel to control respectively PFC maturation and identity and that Grk is not the ligand that controls the SWH network activation (Polesello, 2007).
A last concern was to test whether the SWH network is involved in the PFC signal back that controls oocyte polarity. To tackle this point, attempts were made to uncouple the possible signal back to the oocyte from the PFC maturation phenotypes. ex loss of function, which affects Stau localization but presents a very reduced proliferation rate and double-layer formation compared to other SWH members, was examined. Unfortunately, ex loss of function still affected Arm, FASIII, and Cut protein levels in the PFCs, in particular at midoogenesis, when both the N and Grk signals act. Therefore mer, cut double mutants were generated. In theory, this should force the cells to differentiate (lack of cut) and still affect SWH activity (lack of mer). As expected, whereas mer loss of function alone elicited both Cut upregulation and Stau mislocalization, mer/cut PFC clones were able to induce normal oocyte polarity, manifested by correct Stau localization. It is concluded that the activity of the SWH network is required to control PFC maturation, but this pathway is probably not involved in the signal-back process (Polesello, 2007).
In conclusion, this study has shown that the core components of the SWH network are required specifically to allow the maturation of the PFCs receiving the Grk signal, thus controlling AP body axis formation. The PFC defect is due to a lack of Hnt expression in response to Notch signaling. Because the function of the SWH network is restricted to the PFCs, one interesting speculation is that it is an added layer of Notch regulation specific to PFCs, which, given their crucial role in initiating body axis formation, need robust control of signaling. Placing this regulatory element in complement and in parallel to the signal that initiates PFC specification (Grk) would ensure, in cooperation with the Unpaired signal (Jak/STAT pathway) from the polar cells, a tight and robust boundary between the PFCs and the rest of the follicle cells (Polesello, 2007).
Finally the results make a clear distinction between the core components of the SWH network (hpo, sav, wts, mats, and yki) and mer, ex on one hand and the PCP genes (ft and dco) on the other. It is speculated that the core components are used in a variety of contexts during development, whereas the PCP genes are restricted to organ-size specification (Polesello, 2007).
The conserved Hippo kinase pathway plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. Whereas the function of the core kinase cascade, consisting of the serine/threonine kinases Hippo and Warts, in phosphorylating and thereby inactivating the transcriptional coactivator Yorkie is well established, much less is known about the upstream events that regulate Hippo signaling activity. The FERM domain proteins Expanded and Merlin appear to represent two different signaling branches that feed into the Hippo pathway. Signaling by the atypical cadherin Fat may act via Expanded, but how Merlin is regulated has remained elusive. This study shows that the WW domain protein Kibra is a Hippo signaling component upstream of Hippo and Merlin. Kibra acts synergistically with Expanded, and it physically interacts with Merlin. Thus, Kibra predominantly acts in the Merlin branch upstream of the core kinase cascade to regulate Hippo signaling (Baumgartner, 2010).
Overexpression of Drosophila Kibra in the developing eye has been shown to decrease the size of the adult organ. Four different loss-of-function alleles of Kibra were generated to define its function in growth control. Deletion of the first exon (harboring the translational start site) by imprecise excision of a P element resulted in the alleles Kibra1 and Kibra2. Kibra3, a mutation in the initiating ATG, was generated by means of an EMS reversion mutagenesis of the EP-mediated Kibra overexpression phenotype. Finally, the entire Kibra locus was removed by the hybrid element insertion (HEI) technique. All alleles were lethal when homozygous and failed to complement each other but were complemented by the precise P element excision used as a control throughout this study. All mutants displayed the same growth phenotypes, and homozygous mutant animals died as first-instar larvae. It is concluded that all Kibra alleles are genetically null (Baumgartner, 2010).
Kibra mutant heads were enlarged in comparison to controls. Similarly, wings containing posterior compartments largely mutant for Kibra were larger than control wings. The presence of a UAS-Kibra overexpression construct, without any Gal4 driver, rescued the lethality of Kibra homozygous mutant flies as well as the size defects of Kibra mutant organs, proving that the growth alterations are caused by the loss of Kibra function. Thus, Kibra is a general regulator of growth that is required to restrict organ size (Baumgartner, 2010).
To determine the cause of the Kibra mutant overgrowth phenotypes, a clonal analysis in wing imaginal discs was performed. Clones of Kibra mutant cells were larger than their corresponding wild-type sister clones. The number of cells per clone was increased in Kibra mutant clones compared to wild-type clones but not to the same extent as the clone size. However, FACS analysis revealed that cell size was unchanged in Kibra mutant cells, suggesting a change in cellular architecture in cells devoid of Kibra function. It is concluded that Kibra mutant clones in the wing imaginal disc were enlarged because Kibra mutant cells exhibit a proliferative advantage over wild-type cells (Baumgartner, 2010).
Tangential sections were analyzed of mosaic compound eyes consisting of Kibra mutant cells surrounded by heterozygous cells. The mutant ommatidia were normally structured and the different cell types properly differentiated, but the interommatidial regions were enlarged compared to the control. The increased distance between mutant ommatidia was due to more cells, because clones of Kibra mutant cells in the pupal retina displayed an increase in the number of interommatidial cells. Supernumerary interommatidial cells are a hallmark of inactivation of the Hippo pathway. Whereas a complete loss of Hippo signaling causes a pronounced excess of interommatidial cells, a mild extra interommatidial cell phenotype is observed in mutants that reduce but do not abrogate Hippo signaling, such as ex or Mer (Baumgartner, 2010).
A reduction in Hippo signaling activity results in extra interommatidial cells because the developmental apoptosis in pupal retinae is largely eliminated. Conversely, overexpression of hpo or ex induces apoptosis in third instar eye discs. Overexpression of Kibra in clones in the wing imaginal disc reduced clone size. Kibra-overexpressing clones contained fewer cells than control clones. To investigate whether overexpression of Kibra induces apoptosis, Kibra overexpression clones were generated in the third instar eye disc by using the Gene-Switch system. Indeed, the Kibra-overexpressing clones located anterior to the morphogenetic furrow (MF) showed an increase in programmed cell death as judged by staining for cleaved Caspase-3 and TUNEL staining, suggesting that overexpression of Kibra induces inappropriate apoptosis of proliferating cells. Consistently, co-overexpression of Diap1, a direct Yorkie transcriptional target, partially rescued the small eye phenotype associated with Kibra overexpression. Co-overexpression of CycE, another target of the Hippo pathway, also resulted in a partial rescue of the small eye. The size of Kibra-overexpressing eyes was further restored by concomitant overexpression of Diap1 and CycE. These results suggest that the effects elicited by Kibra overexpression are at least partly due to a reduction in the expression of the Hippo pathway target genes Diap1 and CycE (Baumgartner, 2010).
The striking similarities of the Kibra, ex, and Mer phenotypes prompted a genetic test of whether Kibra restricts tissue size via Hippo signaling. Interaction studies were started at the level of the transcriptional coactivator yki, which induces target genes promoting cell proliferation and cell survival and is inactivated by Hippo signaling. Three lines of evidence suggest that Kibra acts via inactivation of Yki. First, the coexpression of Kibra and yki during eye development suppressed the eye size reduction caused by Kibra and resulted in the same overgrowth phenotype as observed in eyes overexpressing yki alone. Second, the growth advantage of Kibra mutant cells was completely abolished by the concomitant loss of yki function. Third, a pupal lethal hypomorphic combination of Kibra alleles was rescued to viability by removal of a single copy of yki (Baumgartner, 2010).
To determine whether (and at which level) Kibra acts in the Hippo pathway to inactivate Yki, a series of epistasis tests were performed. It was found that the loss-of-function phenotypes of hpo, sav, and wts were epistatic to the Kibra overexpression phenotype, indicating that Kibra acts upstream of Hpo (Baumgartner, 2010).
Next, interaction with the upstream components Ex and Mer was tested. Overexpression of ex in a Kibra mutant background resulted in an intermediate phenotype. Vice versa, overexpression of Kibra also yielded an additive effect in an ex mutant head. Conversely, Kibra overexpression failed to reduce organ size in a Mer mutant head, indicating that Kibra requires Mer to exert its function. The eyFlp/FRT recombination system (without cell lethal) was used to generate mosaic animals with heads largely homozygous for ex and Mer mutations, as well as ex Kibra and Mer Kibra double mutations, respectively. Both ex and Mer mosaic heads showed only mild overgrowth. Strikingly, pupae with mosaic heads doubly mutant for ex and Kibra did not eclose, and normal head structures were displaced by overgrown tissue. In contrast, flies with Mer Kibra mosaic heads were viable. However, Mer Kibra double mutant clones showed stronger overgrowth than Mer clones. Reducing ex function during eye development by the expression of a hairpin RNAi construct did not alter the wild-type eye size but resulted in a severe enhancement of the Kibra loss-of-function phenotype, and the resulting eyes resembled those of hpo mutants. Reducing Mer function caused subtle overgrowth but enhanced the Kibra mutant phenotype much less (Baumgartner, 2010).
Whereas single mutants for ex and Mer cause a mild overgrowth phenotype, ex Mer double mutants display strong synergistic effects, suggesting that the two FERM domain proteins act in separate branches to activate Hippo signaling. These findings suggest that Kibra acts primarily upstream of Mer. However, since Mer Kibra double mutant clones show stronger overgrowth than Mer mutant clones and a reduction of Mer function enhances the Kibra loss-of-function phenotype, Kibra also contributes to Mer-independent regulation of Yki activity (Baumgartner, 2010).
To confirm that Kibra acts via Hippo signaling, whether Kibra mutant clones upregulated the expression of a Diap1 enhancer element (diap1-GFP4.3) that had been published to be a minimal Hippo responsive element (HRE) was tested. A pronounced upregulation of diap1-GFP4.3 was evident in clones of hpo mutant cells posterior and, to a weaker extent, anterior to the MF in eye imaginal discs. Cells lacking Kibra function also upregulated diap1-GFP4.3 expression, although to a lesser degree and with restriction to the differentiating tissue posterior to the MF. Clones of ex mutant cells, in resemblance to hpo clones, upregulated diap1-GFP4.3 strongly behind and somewhat weaker before the MF, whereas Mer mutant cells, like Kibra mutant cells, upregulated diap1-GFP4.3 expression weakly and solely posterior to the MF. Thus, the loss of Kibra results in an upregulation of a Hippo signaling reporter gene. The similar response of diap1-GFP4.3 to loss of Kibra or Mer suggests that Kibra and Mer act in the same way on Hippo signaling to regulate the HRE (Baumgartner, 2010).
This study provides genetic and biochemical evidence that the WW domain protein Kibra is a Hippo signaling component. Several lines of evidence indicate that Kibra acts predominantly in the Mer branch. First, the mild overgrowth phenotype caused by loss of Kibra function is akin to the Mer phenotype. Second, genetic epistasis experiments place Kibra upstream of Mer. Third, the effects of Kibra and Mer loss-of-function on a reporter for Hippo signaling activity are very similar. Fourth, Kibra and Mer synergise with ex in a similar fashion. Fifth, Kibra physically interacts with Mer. However, since the genetic analysis of Kibra also revealed a synergism with Mer, Kibra also acts on Yki activity in a Mer-independent manner (Baumgartner, 2010).
FERM domain proteins, such as Mer, have been suggested to connect membrane proteins with the underlying cortical cytoskeleton in order to integrate signals from the membrane and initiate intracellular signaling cascades. Thus, it is conceivable that Mer, together with as yet unknown proteins, assembles downstream cytoplasmic components of the Hippo pathway at the membrane and that controlled assembly and stabilization of such multiprotein complexes regulates the activity of the Hippo kinase cascade. In such a scenario, adaptor proteins providing multiple protein-protein interaction domains are of special interest (Baumgartner, 2010).
The WW domain protein Kibra binds Mer and could enable signaling events at the membrane/cytoskeleton interface that activate the Hpo kinase cascade. Since a truncated Kibra protein lacking the WW domains interacts more fiercely with Mer, it is likely that the physical association of Kibra and Mer is modulated by binding of other factors to the WW domains of Kibra (Baumgartner, 2010).
Interestingly, the effects caused by the concomitant loss of ex and Kibra functions are more severe than those elicited by mutated Hippo signaling core components. In addition to massively overgrowing, clones of ex Kibra double mutant cells round up, a behavior that was never observed in clones of hpo mutant cells. Furthermore, the diap1-GFP4.3 reporter indicates higher Yki activity in proliferating ex Kibra mutant eye imaginal disc cells as compared to hpo mutant cells. It thus appears that Yki activity is unleashed in cells lacking both ex and Kibra functions. Since Ex has been shown to directly bind Yki, it is tempting to speculate that Kibra participates in a distinct (Mer-independent) mechanism to prevent nuclear Yki localization (Baumgartner, 2010).
The Salvador-Warts-Hippo (SWH) pathway contains multiple growth-inhibitory proteins that control organ size during development by limiting activity of the Yorkie oncoprotein. Increasing evidence indicates that these growth inhibitors act in a complex network upstream of Yorkie. This complexity is emphasised by the distinct phenotypes of tissue lacking different SWH pathway genes. For example, eye tissue lacking the core SWH pathway components salvador, warts or hippo is highly overgrown and resistant to developmental apoptosis, whereas tissue lacking fat or expanded is not. This study explores the relative contribution of SWH pathway proteins to organ size control by determining their temporal activity profile throughout Drosophila eye development. Eye tissue lacking fat, expanded or discs overgrown displays elevated Yorkie activity during the larval growth phase of development, but not in the pupal eye when apoptosis ensues. Fat and Expanded do possess Yorkie-repressive activity in the pupal eye, but loss of fat or expanded at this stage of development can be compensated for by Merlin. Fat appears to repress Yorkie independently of Dachs in the pupal eye, which would contrast with the mode of action of Fat during larval development. Fat is more likely to restrict Yorkie activity in the pupal eye together with Expanded, given that pupal eye tissue lacking both these genes resembles that of tissue lacking either gene. This study highlights the complexity employed by different SWH pathway proteins to control organ size at different stages of development (Milton, 2010).
The SWH pathway controls Drosophila eye size by limiting growth during the larval stage of development and by restricting proliferation and promoting apoptosis during pupal development. Eyes lacking core SWH pathway components (e.g. sav, wts or hpo) are significantly larger than eyes lacking the non-core components ft, ex, dco or Mer. Owing to this disparity, it has been hypothesized that ft and ex only partially affect SWH pathway activity, whereas sav, wts and hpo have stronger effects, or, alternatively, that non-core components affect pathway activity in a temporally restricted fashion. Analysis of tissue recessive for ft, ex or dco3 revealed that Yki activity was elevated during larval eye development when tissues are actively growing and proliferating, but not during pupal development when apoptosis ensues, supporting the idea that Ft, Ex and Dco influence SWH pathway activity in a temporally restricted fashion. However, when tissue lacking both Mer and ft, or Mer and ex, was analysed, Yki activity was found to be elevated during both larval and pupal development, similar to the Yki activity profile observed in tissue lacking core SWH pathway proteins. This is consistent with previous reports showing that Mer acts in parallel to both Ft and Ex, and that these proteins can compensate for each other to control SWH pathway activity. Therefore, Ft and Ex do contribute to SWH pathway regulation in the pupal eye to ensure appropriate exit from the cell cycle and developmental apoptosis, but these functions can be executed by Mer in their absence, suggesting a degree of plasticity in the regulation of Yki activity by non-core SWH pathway proteins. The ability of Mer to compensate for Ft or Ex cannot simply be explained by compensatory increases in Mer protein in pupal eye tissues lacking ft or ex, since Mer expression levels were found to be unaltered in these tissues (Milton, 2010).
Previous analyses of tissue lacking both ft and ex showed that these proteins function, at least in part, in parallel to control growth of larval imaginal discs. The current analysis of ft,ex double-mutant tissue suggests that these proteins are likely to function together to control Yki activity in the pupal eye. Yki activity was not elevated in tissue lacking ft, ex or both genes, showing that these genes cannot compensate for each other in the pupal eye. This is consistent with the notion that Ft influences the activity of downstream SWH pathway proteins by multiple mechanisms, an idea that is supported by THE analysis of the requirement of the atypical myosin, Dachs, for Ft signalling in the pupal eye. During larval imaginal disc development, Ft can influence Yki activity by repressing Dachs activity, which in turn can repress the core SWH pathway protein Wts. Analysis of pupal eye tissue that lacks both Mer and ft, or Mer, ft and dachs, showed that Yki activity was elevated in each scenario. This shows that in the pupal eye, the ability of Ft to compensate for Mer is not reliant on Dachs, and implies that Ft can employ different modes of signal transduction throughout eye development. However, because Ft and Mer can compensate for each other it is not possible to formally conclude that normal signal transduction by Ft in the pupal eye occurs independently of Dachs (Milton, 2010).
Expression of Ex is tightly controlled in response to alterations in SWH pathway activity at both the transcriptional and post-transcriptional levels. Interestingly, it was also found that Ex expression is controlled in a temporal fashion throughout eye development; Ex is expressed at relatively high levels in the larval eye, but at very low levels in the pupal eye. Despite the fact that Ex expression is very low in the pupal eye, it clearly retains function at this stage of development because it can compensate for loss of Mer to restrict Yki activity. The dynamic expression profile of Ex suggests that factors that influence its expression play an important role in defining overall eye size in Drosophila. At present, only two transcriptional regulatory proteins have been shown to influence the expression of ex: Yki and Sd. There are conflicting reports on whether Yki and Sd control basal expression of ex in larval imaginal discs. It is clear, however, that Yki and Sd collaborate to drive ex expression when the activity of the SWH pathway is suppressed, presumably as part of a negative-feedback loop. Despite the fact that basal ex expression is low in the pupal eye, the ex promoter is still responsive to Yki, as Ex expression is substantially elevated in pupal eye clones lacking hpo or Mer and ex. Future investigation of the ex promoter will help to clarify understanding of the complex fashion by which expression of the ex gene is controlled, and should aid understanding of eye size specification in Drosophila (Milton, 2010).
This study emphasises the complexity of the means by which the activity of core SWH pathway proteins is regulated by non-core proteins such as Ft, Ex, Mer and Dco. The signalling mechanisms employed by non-core proteins appear to differ at discrete stages of development in order to achieve appropriate organ size during the larval growth period of eye development, and to subsequently sculpt the eye by regulating apoptosis during pupal development (Milton, 2010).
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date revised: 15 August 2011
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