Ligand-independent activation of the Hedgehog pathway displays non-cell autonomous proliferation during eye development in Drosophila

Deregulation of the Hedgehog (Hh) signaling pathway is associated with the development of human cancer including medullobastoma and basal cell carcinoma. Loss of Patched or activation of Smoothened in mouse models increases the occurrence of tumors. Likewise, in a Drosophila eye model, deregulated Hedgehog signaling causes overgrowth of eye and head tissues. Surprisingly, we show that cells with deregulated Hh signaling do not or only little contribute to the tissue overgrowth. Instead, they become more sensitive to apoptosis and may eventually be eliminated. Nevertheless, these mutant cells increase proliferation in the adjacent wild-type tissue, i.e., in a non-cell autonomous manner. This non-cell autonomous effect is position-dependent and restricted to mutant cells in the anterior portion of the eye. We also observe precocious non-cell autonomous differentiation in genetic mosaics with deregulated Hh signaling. Together, these non-cell autonomous growth and differentiation phenotypes in the Drosophila eye model reveal another strategy by which oncogenes may generate a supportive micro-environment for tumor growth.     

Notch signaling activates Yorkie non-cell autonomously in Drosophila

In Drosophila imaginal epithelia, cells mutant for the endocytic neoplastic tumor suppressor gene vps25 stimulate nearby untransformed cells to express Drosophila Inhibitor-of-Apoptosis-Protein-1 (DIAP-1), conferring resistance to apoptosis non-cell autonomously. Here, we show that the non-cell autonomous induction of DIAP-1 is mediated by Yorkie, the conserved downstream effector of Hippo signaling. The non-cell autonomous induction of Yorkie is due to Notch signaling from vps25 mutant cells. Moreover, activated Notch in normal cells is sufficient to induce non-cell autonomous Yorkie activity in wing imaginal discs. Our data identify a novel mechanism by which Notch promotes cell survival non-cell autonomously and by which neoplastic tumor cells generate a supportive microenvironment for tumor growth.     

The Kto-Skd Complex Can Regulate ptc Expression by Interacting with Cubitus interruptus (Ci) in the Hedgehog Signaling Pathway

The hedgehog (Hh) signaling pathway plays a very important role in metazoan development by controlling pattern formation. Drosophila imaginal discs are subdivided into anterior and posterior compartments that derive from adjacent cell populations. The anterior/posterior (A/P) boundaries, which are critical to maintaining the position of organizers, are established by a complex mechanism involving Hh signaling. Here, we uncover the regulation of ptc in the Hh signaling pathway by two subunits of mediator complex, Kto and Skd, which can also regulate boundary location. Collectively, we provide further evidence that Kto-Skd affects the A/P-axial development of the whole wing disc. Kto can interact with Cubitus interruptus (Ci), bind to the Ci-binding region on ptc promoter, which are both regulated by Hh signals to down-regulate ptc expression.     

The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development

Cell proliferation and programmed cell death are closely controlled during animal development. Proliferative stimuli generally also induce apoptosis, and anti-apoptotic factors are required to allow net cell proliferation. Genetic studies in Drosophila have led to identification of a number of genes that control both processes, providing new insights into the mechanisms that coordinate cell growth, proliferation, and death during development and that fail to do so in diseases of cell proliferation. We present evidence that the Drosophila Sterile-20 kinase Slik promotes cell proliferation and controls cell survival. At normal levels, Slik provides survival cues that prevent apoptosis. Cells deprived of Slik activity can grow, divide, and differentiate, but have an intrinsic survival defect and undergo apoptosis even under conditions in which they are not competing with normal cells for survival cues. Like some oncogenes, excess Slik activity stimulates cell proliferation, but this is compensated for by increased cell death. Tumor-like tissue overgrowth results when apoptosis is prevented. We present evidence that Slik acts via Raf, but not via the canonical ERK pathway. Activation of Raf can compensate for the lack of Slik and support cell survival, but activation of ERK cannot. We suggest that Slik mediates growth and survival cues to promote cell proliferation and control cell survival during Drosophila development.     

Vsx2/Chx10 ensures the correct timing and magnitude of Hedgehog signaling in the mouse retina

Vertebrate retinal progenitor cells (RPCs) undergo a robust proliferative expansion to produce enough cells for the retina to form appropriately. Vsx2 (formerly Chx10), a homeodomain protein expressed in RPCs, is required for sufficient proliferation to occur. Sonic Hedgehog protein (SHH), secreted by retinal ganglion cells (RGCs), activates Hedgehog (Hh) signaling in RPCs and is also required for sufficient proliferation to occur. Therefore, we sought to determine if reduced Hh signaling is a contributing factor to the proliferation changes that occur in the absence of Vsx2. To do this, we examined Shh expression and Hh signaling activity in the homozygous ocular retardation J (orJ) mouse, which harbors a recessive null allele in the Vsx2 gene. We found that Shh expression and Hh signaling activity are delayed during early retinal development in orJ mice and this correlates with a delay in the onset of RGC differentiation. At birth, reduced expression of genes regulated by Hh signaling was observed despite the production of SHH ligand. orJ RPCs respond to pre-processed recombinant SHH ligand (SHH-N) in explant culture as evidenced by increased proliferation and expression of Hh target genes. Interestingly, proliferation in the orJ retina is further inhibited by cyclopamine, an antagonist of Hh signaling. Our results suggest that reduced Hh signaling contributes to the reduced level of RPC proliferation in the orJ retina, thereby revealing a role for Vsx2 in mediating mitogen signaling.     

Wengen,the Sole Tumour Necrosis Factor Receptor in Drosophila,Collaborates with Moesin to Control Photoreceptor Axon Targeting during Development

Photoreceptor neurons (R cells) in the Drosophila eye define a map of visual space by connecting to targets in distinct layers of the optic lobe, with R1-6 cells connecting to the lamina (the first optic ganglion) and R7 and R8 cells connecting to the medulla (the second optic ganglion). Here, we show that Wengen (Wgn) directly binds Moesin (Moe) through a cytosolic membrane proximal domain and this interaction is important for mediating two distinct aspects of axonal targeting. First, we show that loss of wgn or moe function disrupts cell autonomous R8 axon targeting. Second, we report that wgn or moe mutants show defects in R2–R5 targeting that result from disruption of non-cell autonomous effects, which are secondary to the cell autonomous R8 phenotype. Thus, these studies reveal that the Wgn-Moe signaling cascade plays a key role in photoreceptor target field innervations through cell autonomous and non-cell autonomous mechanisms.     

Regulation of apoptosis of rbf mutant cells during Drosophila development

Inactivation of the retinoblastoma gene Rb leads to defects in cell proliferation, differentiation, or apoptosis, depending on specific cell or tissue types. To gain insights into the genes that can modulate the consequences of Rb inactivation, we carried out a genetic screen in Drosophila to identify mutations that affected apoptosis induced by inactivation of the Retinoblastoma-family protein (rbf) and identified a mutation that blocked apoptosis induced by rbf. We found this mutation to be a new allele of head involution defective (hid) and showed that hid expression is deregulated in rbf mutant cells in larval imaginal discs. We identified an enhancer that regulates hid expression in response to developmental cues as well as to radiation and demonstrated that this hid enhancer is directly repressed by RBF through an E2F binding site. These observations indicate that apoptosis of rbf mutant cells is mediated by an upregulation of hid. Finally, we showed that bantam, a miRNA that regulates hid translation, is expressed in the interommatidial cells in the larval eye discs and modulates the survival of rbf mutant cells.     

All Mammalian Hedgehog Proteins Interact with Cell Adhesion Molecule,Down-regulated by Oncogenes (CDO) and Brother of CDO (BOC) in a Conserved Manner

Hedgehog (Hh) signaling proteins stimulate cell proliferation, differentiation, and tissue patterning at multiple points in animal development. A single Hh homolog is present in Drosophila, but three Hh homologs, Sonic Hh, Indian Hh, and Desert Hh, are present in mammals. Distribution, movement, and reception of Hh signals are tightly regulated, and abnormal Hh signaling is associated with developmental defects and cancer. In addition to the integral membrane proteins Patched and Smoothened, members of the Drosophila Ihog family of adhesion-like molecules have recently been shown to bind Hh proteins with micromolar affinity and positively regulate Hh signaling. Cell adhesion molecule-related, down-regulated by oncogenes (CDO) and Brother of CDO (BOC) are the closest mammalian relatives of Drosophila Ihog, and CDO binds Sonic Hh with micromolar affinity and positively regulates Hh signaling. Despite these similarities, structural and biochemical studies have shown that Ihog and CDO utilize nonorthologous domains and completely different binding modes to interact with cognate Hh proteins. We report here biochemical and x-ray structural studies of Sonic, Indian, and Desert Hh proteins both alone and complexed with active domains of CDO and BOC. These results show that all mammalian Hh proteins bind CDO and BOC in the same manner. We also show that interactions between Hh proteins and CDO are weakened at low pH. Formation of Hh-mediated Hh oligomers is thought to be an important feature of normal Hh signaling, but no conserved self-interaction between Hh proteins is apparent from inspection of 14 independent Hh-containing crystal lattices.     

The Drosophila Sterile-20 Kinase Slik Controls Cell Proliferation and Apoptosis during Imaginal Disc Development

Cell proliferation and programmed cell death are closely controlled during animal development. Proliferative stimuli generally also induce apoptosis, and anti-apoptotic factors are required to allow net cell proliferation. Genetic studies in Drosophila have led to identification of a number of genes that control both processes, providing new insights into the mechanisms that coordinate cell growth, proliferation, and death during development and that fail to do so in diseases of cell proliferation. We present evidence that the Drosophila Sterile-20 kinase Slik promotes cell proliferation and controls cell survival. At normal levels, Slik provides survival cues that prevent apoptosis. Cells deprived of Slik activity can grow, divide, and differentiate, but have an intrinsic survival defect and undergo apoptosis even under conditions in which they are not competing with normal cells for survival cues. Like some oncogenes, excess Slik activity stimulates cell proliferation, but this is compensated for by increased cell death. Tumor-like tissue overgrowth results when apoptosis is prevented. We present evidence that Slik acts via Raf, but not via the canonical ERK pathway. Activation of Raf can compensate for the lack of Slik and support cell survival, but activation of ERK cannot. We suggest that Slik mediates growth and survival cues to promote cell proliferation and control cell survival during Drosophila development.     

Neuronal necrosis and spreading death in a Drosophila genetic model

Brain ischemia often results in neuronal necrosis, which may spread death to neighboring cells. However, the molecular events of neuronal necrosis and the mechanisms of this spreading death are poorly understood due to the limited genetic tools available for deciphering complicated responses in mammalian brains. Here, we engineered a Drosophila model of necrosis in a sub-population of neurons by expressing a leaky cation channel in the Drosophila eye. Expression of this channel caused necrosis in defined neurons as well as extensive spreading of cell death. Jun N-terminal kinase (JNK)-mediated, caspase-independent apoptosis was the primary mechanism of cell death in neurons, while caspase-dependent apoptosis was primarily involved in non-neuronal cell death. Furthermore, the JNK activation in surrounding neurons was triggered by reactive oxygen species (ROS) and Eiger (Drosophila tumor necrosis factor α (TNFα)) released from necrotic neurons. Because the Eiger/ROS/JNK signaling was also required for cell death induced by hypoxia and oxidative stress, our fly model of spreading death may be similar to brain ischemia in mammals. We performed large-scale genetic screens to search for novel genes functioning in necrosis and/or spreading death, from which we identified several classes of genes. Among them, Rho-associated kinase (ROCK) had been reported as a promising drug target for stroke treatment with undefined mechanisms. Our data indicate that ROCK and the related trafficking pathway genes regulate neuronal necrosis. We propose the suppression of the function of the trafficking system, ROS and cytokines, such as TNFα, as translational applications targeting necrosis and spreading death.     

The Drosophila homolog of methionine sulfoxide reductase A extends lifespan and increases nuclear localization of FOXO

Methionine sulfoxide reductase A (msrA) was previously found to increase resistance to oxidative stress and longevity in animals. We identified Drosophila msrA (dmsrA), a Drosophila homolog of human msrA, as a downstream effector of forkhead box O (FOXO) signaling in Drosophila, which enhances resistance to oxidative stress and increases survival under stressed conditions. Additionally, overexpression of dmsrA in neurons extended the lifespan of flies. Moreover, overexpression of dmsrA in fat body cells caused FOXO to translocate to the nucleus, implying that this possible positive feedback loop between dmsrA and FOXO could potentiate the antioxidant activity of dmsrA and increase the lifespan in Drosophila.     

Ubr3, a Novel Modulator of Hh Signaling Affects the Degradation of Costal-2 and Kif7 through Poly-ubiquitination

Hedgehog (Hh) signaling regulates multiple aspects of metazoan development and tissue homeostasis, and is constitutively active in numerous cancers. We identified Ubr3, an E3 ubiquitin ligase, as a novel, positive regulator of Hh signaling in Drosophila and vertebrates. Hh signaling regulates the Ubr3-mediated poly-ubiquitination and degradation of Cos2, a central component of Hh signaling. In developing Drosophila eye discs, loss of ubr3 leads to a delayed differentiation of photoreceptors and a reduction in Hh signaling. In zebrafish, loss of Ubr3 causes a decrease in Shh signaling in the developing eyes, somites, and sensory neurons. However, not all tissues that require Hh signaling are affected in zebrafish. Mouse UBR3 poly-ubiquitinates Kif7, the mammalian homologue of Cos2. Finally, loss of UBR3 up-regulates Kif7 protein levels and decreases Hh signaling in cultured cells. In summary, our work identifies Ubr3 as a novel, evolutionarily conserved modulator of Hh signaling that boosts Hh in some tissues.     

POSH promotes cell survival in Drosophila and in human RASF cells

In Drosophila, Eiger, a tumor necrosis factor α (TNFα) superfamily ligand, induces cell death by activating the c-Jun N-terminal kinase (JNK) pathway. Here, we report that overexpression of Plenty of SH3s (POSH) suppresses Eiger-induced cell death and produces highly deformed tissues. These results imply that high levels of POSH protect tissues from cell death. In humans, rheumatoid arthritis synovial fibroblasts (RASF) are generally resistant to apoptosis. We show that POSH is expressed at relatively high levels in RASF, and its reduction by RNAi sensitizes these cells to Fas-mediated apoptosis. Thus, we demonstrate that POSH promotes cell survival in Drosophila and in human RASF.     

A novel Drosophila Girdin-like protein is involved in Akt pathway control of cell size

The Akt signaling pathway is well known to regulate cell proliferation and growth. Girdin, a novel substrate of Akt, plays a crucial role in organization of the actin cytoskeleton and cell motility under the control of Akt. We here identified a novel Girdin-like protein in Drosophila (dGirdin), which has two isoforms, dGirdin PA and dGirdin PB. dGirdin shows high homology with human Girdin in the N-terminal and coiled-coil domains, while diverging at the C-terminal domain. On establishment of transgenic fly lines, featuring knockdown or overexpression of dGirdin in vivo, overexpression in the wing disc cells induced ectopic apoptosis, implying a role in directing apoptosis. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size. Furthermore, this was enhanced by half reduction of the Akt gene dose, suggesting that Akt positively regulates dGirdin. In the wing disc, cells in which dGirdin was knocked down exhibited disruption of actin filaments. From these in vivo analyses, we conclude that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway.