Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila

Autophagy is a catabolic process that is negatively regulated by growth and has been implicated in cell death. We find that autophagy is induced following growth arrest and precedes developmental autophagic cell death of Drosophila salivary glands. Maintaining growth by expression of either activated Ras or positive regulators of the class I phosphoinositide 3-kinase (PI3K) pathway inhibits autophagy and blocks salivary gland cell degradation. Developmental degradation of salivary glands is also inhibited in autophagy gene (atg) mutants. Caspases are active in PI3K-expressing and atg mutant salivary glands, and combined inhibition of both autophagy and caspases increases suppression of gland degradation. Further, induction of autophagy is sufficient to induce premature cell death in a caspase-independent manner. Our results provide in vivo evidence that growth arrest, autophagy, and atg genes are required for physiological autophagic cell death and that multiple degradation pathways cooperate in the efficient clearance of cells during development.     

Steroid regulation of autophagic programmed cell death during development

Apoptosis and autophagy are morphologically distinct forms of programmed cell death. While autophagy occurs during the development of diverse organisms and has been implicated in tumorigenesis, little is known about the molecular mechanisms that regulate this type of cell death. Here we show that steroid-activated programmed cell death of Drosophila salivary glands occurs by autophagy. Expression of p35 prevents DNA fragmentation and partially inhibits changes in the cytosol and plasma membranes of dying salivary glands, suggesting that caspases are involved in autophagy. The steroid-regulated BR-C, E74A and E93 genes are required for salivary gland cell death. BR-C and E74A mutant salivary glands exhibit vacuole and plasma membrane breakdown, but E93 mutant salivary glands fail to exhibit these changes, indicating that E93 regulates early autophagic events. Expression of E93 in embryos is sufficient to induce cell death with many characteristics of apoptosis, but requires the H99 genetic interval that contains the rpr, hid and grim proapoptotic genes to induce nuclear changes diagnostic of apoptosis. In contrast, E93 expression is sufficient to induce the removal of cells by phagocytes in the absence of the H99 genes. These studies indicate that apoptosis and autophagy utilize some common regulatory mechanisms.     

Autophagy functions in programmed cell death

Autophagic cell death is a prominent morphological form of cell death that occurs in diverse animals. Autophagosomes are abundant during autophagic cell death, yet the functional role of autophagy in cell death has been enigmatic. We find that autophagy and the Atg genes are required for autophagic cell death of Drosophila salivary glands. Although caspases are present in dying salivary glands, autophagy is required for complete cell degradation. Further, induction of high levels of autophagy results in caspase-independent autophagic cell death. Our results provide the first in vivo evidence that autophagy and the Atg genes are required for autophagic cell death and confirm that autophagic cell death is a physiological death program that occurs during development.

Addendum to: Berry DL, Baehrecke EH. Growth arrest and autophagy are required for programmed salivary gland cell degradation in Drosophila. Cell 2007; 131:1137-48.     

The engulfment receptor Draper is required for autophagy during cell death

Autophagy is a process to degrade and recycle cytoplasmic contents. Autophagy is required for survival in response to starvation, but has also been associated with cell death. How autophagy functions during cell survival in some contexts and cell death in others is unknown. Drosophila larval salivary glands undergo programmed cell death requiring autophagy genes, and are cleared in the absence of known phagocytosis. Recently, we demonstrated that Draper (Drpr), the Drosophila homolog of C. elegans engulfment receptor CED-1, is required for autophagy induction

during cell death, but not during cell survival. drpr mutants fail to clear salivary glands. drpr knockdown in salivary glands prevents the induction of autophagy, and Atg1 misexpression in drpr null mutants suppresses salivary gland persistence. Surprisingly, drpr knockdown cell-autonomously prevents autophagy induction in dying salivary gland cells, but not in larval fat body cells following starvation. This is the first engulfment factor shown to function in cellular self-clearance, and the first report of a cell-death-specific autophagy regulator.     

The engulfment receptor Draper is required for autophagy during cell death

Autophagy is a process to degrade and recycle cytoplasmic contents. Autophagy is required for survival in response to starvation, but has also been associated with cell death. How autophagy functions during cell survival in some contexts and cell death in others is unknown. Drosophila larval salivary glands undergo programmed cell death requiring autophagy genes, and are cleared in the absence of known phagocytosis. Recently, we demonstrated that Draper (Drpr), the Drosophila homolog of C. elegans engulfment receptor CED-1, is required for autophagy induction during cell death, but not during cell survival. drpr mutants fail to clear salivary glands. drpr knockdown in salivary glands prevents the induction of autophagy, and Atg1 misexpression in drpr null mutants suppresses salivary gland persistence. Surprisingly, drpr knockdown cell-autonomously prevents autophagy induction in dying salivary gland cells, but not in larval fat body cells following starvation. This is the first engulfment factor shown to function in cellular self-clearance, and the first report of a cell-death-specific autophagy regulator.Key words: autophagy, Draper, programmed cell death, engulfment, developmentProgrammed cell death is required for animal development and tissue homeostasis. Improper cell death leads to pathologies including autoimmunity and cancer. Several morphological forms of cell death occur during animal development, including apoptosis and autophagic cell death. Autophagic cell death is characterized by the presence of autophagosomes in dying cells that are not known to be engulfed by phagocytes. Autophagic cell death is observed during several types of mammalian developmental cell death, including regression of the corpus luteum and involution of mammary and prostate glands.During macroautophagy (autophagy), cytoplasmic components are sequestered by autophagosomes and delivered to the lysosome for degradation. Autophagy is a cellular response to stress required for survival in response to starvation. Whereas autophagy has been associated with cell death, it is unknown how autophagy is distinguished during cell death and cell survival. Autophagy is induced in Drosophila in response to starvation in the fat body where it promotes cell survival, while autophagy is induced by the steroid hormone ecdysone in salivary glands where it promotes cell death. This allows studies of autophagy in different cell types and in response to different stimuli.Drosophila larval salivary glands die with autophagic cell death morphology and autophagy is required for their degradation. Expression of the caspase inhibitor p35 enhances salivary gland persistence in Atg mutants, suggesting that caspases and autophagy function in parallel during salivary gland degradation. Either activation of caspases or Atg1 misexpression is sufficient to induce ectopic salivary gland clearance. We queried genome-wide microarray data from purified dying salivary glands and noted the induction of engulfment genes, those required for a phagocyte to consume and degrade a dying cell. We also noted few detectable changes in engulfment genes in Drosophila larvae during starvation.We found that Drpr, the Drosophila orthologue of C. elegans engulfment receptor CED-1, is enriched in dying salivary glands, and drpr null mutants have persistent salivary glands. Interestingly, whereas knockdown of drpr in phagocytic blood cells fails to influence salivary gland clearance, expression of drpr-RNAi in salivary glands prevents gland clearance. Drosophila drpr is alternatively spliced to produce three isoforms. We found that drpr-I-specific knockdown prevents salivary gland degradation and Drpr-I expression in salivary glands of drpr null mutants rescues salivary gland persistence. Therefore, drpr is autonomously required for salivary gland clearance. However, how Drpr is induced or activated during hormone-regulated cell death remains to be determined.drpr knockdown fails to influence caspase activation, and caspase inhibitor p35 expression in drpr null mutants enhances salivary gland persistence, suggesting that Drpr functions downstream or parallel to caspases in dying salivary glands. Interestingly, we found that drpr knockdown in salivary glands prevents the formation of GFP-LC3 puncta. Further, Atg1 misexpression in salivary glands of drpr null mutants suppresses salivary gland persistence. drpr is therefore required for autophagy induction in salivary glands, and Atg1 functions downstream of Drpr in this tissue. We found that several other engulfment genes are required for salivary gland degradation. However, the Drpr signaling mechanism leading to autophagy induction in salivary glands remains to be elucidated.We tested whether drpr is a general regulator of autophagy. The Drosophila fat body is a nutrient storage and mobilization organ akin to the mammalian liver, and is a well-established model to study starvation-induced autophagy. We found that drpr-RNAi expression in fat body clone cells fails to prevent GFP-Atg8 puncta formation in response to starvation. Similarly, drpr null fat body clone cells form Cherry-Atg8 puncta after starvation. Strikingly, drpr-RNAi expression in salivary gland clone cells inhibits the formation of GFP-Atg8 puncta. Therefore, drpr is cell-autonomously required for autophagy induction in dying salivary gland cells, but not for autophagy induction in fat body cells after starvation. These findings suggest that distinct signaling mechanisms regulate autophagy in response to nutrient deprivation compared to steroid hormone induction. Little is known about what distinguishes autophagy function in cell survival versus death. It is possible that varying levels of autophagy are induced during specific cell contexts and that high levels of autophagy could overwhelm a cell—leading to cell death. Autophagic degradation of specific cargo, such as cell death inhibitors, could also contribute to cell death.Given recent interest in manipulation of autophagy for therapies, it is possible that factors such as Drpr could be used as biomarkers to distinguish autophagy leading to cell death versus cell survival. While it is generally accepted that augmentation of protein clearance by autophagy during neurodegeneration would be beneficial, the role of autophagy in tumor progression is less clear. For example, monoallelic loss of the human Atg6 homolog beclin 1 is prevalent in human cancers, suggesting that autophagy is a tumorsuppressive mechanism. Thus, autophagy enhancers have been proposed for cancer prevention. However, autophagy occurs in tumor cells as a survival mechanism, and autophagy inhibitors have been proposed for anti-cancer therapies. Understanding how autophagy is regulated in different contexts is critical for appropriate therapeutic strategies.     

The Drosophila Mst ortholog,hippo, restricts growth and cell proliferation and promotes apoptosis

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 (wts), encoding a serine/threonine kinase, and salvador (sav), encoding a WW domain containing Wts-interacting protein. However, the mechanisms by which sav and wts regulate growth and apoptosis are not well understood. Here, we describe mutations in hippo (hpo), 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.     

Autophagy functions in programmed cell death

Autophagic cell death is a prominent morphological form of cell death that occurs in diverse animals. Autophagosomes are abundant during autophagic cell death, yet the functional role of autophagy in cell death has been enigmatic. We find that autophagy and the Atg genes are required for autophagic cell death of Drosophila salivary glands. Although caspases are present in dying salivary glands, autophagy is required for complete cell degradation. Further, induction of high levels of autophagy results in caspase-independent autophagic cell death. Our results provide the first in vivo evidence that autophagy and the Atg genes are required for autophagic cell death and confirm that autophagic cell death is a physiological death program that occurs during development.     

AP-1, but not NF-kappa B, is required for efficient steroid-triggered cell death in Drosophila

Extensive studies in vertebrate cells have assigned a central role to Rel/NF-kappa B and AP-1 family members in the control of apoptosis. We ask here whether parallel pathways might function in Drosophila by determining if Rel/NF-kappa B or AP-1 family members contribute to the steroid-triggered death of larval salivary glands during Drosophila metamorphosis. We show that two of the three Drosophila Rel/NF-kappa B genes are expressed in doomed salivary glands and that one family member, Dif, is induced in a stage-specific manner immediately before the onset of programmed cell death. Similarly, Djun is expressed for many hours before salivary gland cell death while Dfos is induced in a stage-specific manner, immediately before this tissue is destroyed. We show that null mutations in the three Drosophila Rel/NF-kappa B family members, either alone or in combination, have no apparent effect on this death response. In contrast, Dfos is required for the proper timing of larval salivary gland cell death as well as the proper induction of key death genes. This study demonstrates a role for AP-1 in the stage-specific steroid-triggered programmed cell death of larval tissues during Drosophila metamorphosis.     

Hid can induce, but is not required for autophagy in polyploid larval Drosophila tissues

The major cell death pathways are apoptosis and autophagy-type cell death in Drosophila. Overexpression of proapoptotic genes in developing imaginal tissues leads to the activation of caspases and apoptosis, but most of them show no effect on the polytenic cells of the fat body during the last larval stage. Surprisingly, overexpression of Hid induces caspase-independent autophagy in the fat body, as well as in most other larval tissues tested. Hid mutation results in inhibition of salivary gland cell death, but the disintegration of the larval midgut is not affected. Electron microscopy shows that autophagy is normally induced in fat body, midgut and salivary gland cells of homozygous mutant larvae, suggesting that Hid is not required for autophagy itself. Constitutive expression of the caspase inhibitor p35 produces identical phenotypes. Our results show that the large, post-mitotic larval cells do not react or activate autophagy in response to the same strong apoptotic stimuli that trigger apoptosis in small, mitotically active imaginal disc cells.     

Drosophila Casein Kinase 2 (CK2) Promotes Warts Protein to Suppress Yorkie Protein Activity for Growth Control

Drosophila Hippo signaling regulates Wts activity to phosphorylate and inhibit Yki in order to control tissue growth. CK2 is widely expressed and involved in a variety of signaling pathways. In this study we report that Drosophila CK2 promotes Wts activity to phosphorylate and inhibit Yki activity, which is independent of Hpo-induced Wts promotion. In vivo, CK2 overexpression suppresses hpo mutant-induced expanded (Ex) up-regulation and overgrowth phenotype, whereas it cannot affect wts mutant. Consistent with this, knockdown of CK2 up-regulates Hpo pathway target expression. We also found that Drosophila CK2 is essential for tissue growth as a cell death inhibitor as knockdown of CK2 in the developing disc induces severe growth defects as well as caspase3 signals. Taken together, our results uncover a dual role of CK2; although its major role is promoting cell survive, it may potentially be a growth inhibitor as well.