When dying is not the end: Apoptotic caspases as drivers of proliferation.

Caspases are well known for their role as executioners of apoptosis. However, recent studies have revealed that these lethal enzymes also have important mitogenic functions. Caspases can promote proliferation through autonomous regulation of the cell cycle, as well as by induction of secreted signals, which have a profound impact in neighboring tissues. Here, I review the proliferative role of caspases during development and homeostasis, in addition to their key regenerative function during tissue repair upon injury. Furthermore, the emerging properties of apoptotic caspases as drivers of carcinogenesis are discussed, as well as their involvement in other diseases. Finally, I examine further effects of caspases regulating death and survival in a non-autonomous manner.

Spreading the word: non-autonomous effects of apoptosis during development, regeneration and disease.

Apoptosis, in contrast to other forms of cell death such as necrosis, was originally regarded as a 'silent' mechanism of cell elimination designed to degrade the contents of doomed cells. However, during the past decade it has become clear that apoptotic cells can produce diverse signals that have a profound impact on neighboring cells and tissues. For example, apoptotic cells can release factors that influence the proliferation and survival of adjacent tissues. Apoptosis can also affect tissue movement and morphogenesis by modifying tissue tension in surrounding cells. As we review here, these findings reveal unexpected roles for apoptosis in tissue remodeling during development, as well as in regeneration and cancer.

The benefits of aging: cellular senescence in normal development.

Senescence is a form of cellular aging that limits the proliferative capacity of cells. Senescence can be triggered by different stress stimuli, such as DNA damage or oncogene activation. Two recent articles published in Cell have uncovered an unexpected role for cellular senescence during development, as a process that contributes to remodeling and patterning of the embryo. These findings are exciting and have important implications for the understanding of normal developmental and the evolutionary origin of senescence.

Apoptotic cells can induce non-autonomous apoptosis through the TNF pathway.

Apoptotic cells can produce signals to instruct cells in their local environment, including ones that stimulate engulfment and proliferation. We identified a novel mode of communication by which apoptotic cells induce additional apoptosis in the same tissue. Strong induction of apoptosis in one compartment of the Drosophila wing disc causes apoptosis of cells in the other compartment, indicating that dying cells can release long-range death factors. We identified Eiger, the Drosophila tumor necrosis factor (TNF) homolog, as the signal responsible for apoptosis-induced apoptosis (AiA). Eiger is produced in apoptotic cells and, through activation of the c-Jun N-terminal kinase (JNK) pathway, is able to propagate the initial apoptotic stimulus. We also show that during coordinated cell death of hair follicle cells in mice, TNF-α is expressed in apoptotic cells and is required for normal cell death. AiA provides a mechanism to explain cohort behavior of dying cells that is seen both in normal development and under pathological conditions. DOI:http://dx.doi.org/10.7554/eLife.01004.001.

Mitogenic signaling from apoptotic cells in Drosophila.

Apoptotic cells of Drosophila not only activate caspases, but also are able to secrete developmental signals like Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) before dying. Since Dpp and Wg are secreted in growing tissues and behave as growth factors, it was proposed that they play a role in compensatory proliferation, the process by which a growing blastema can restore normal size after massive apoptosis. We discuss recent results showing that there is normal compensatory proliferation in the absence of Dpp/Wg signaling, thus indicating it has no significant role in the process. Furthermore, we argue that Dpp/Wg signaling is not a resident feature of apoptotic cells, but a side effect of the necessary activation of the JNK pathway. Nevertheless, the ectopic JNK/Dpp/Wg signaling may have an important role in tissue regeneration. Recent work in other organisms suggests that paracrine signaling from apoptotic cells may be of general significance in wound healing and tissue regeneration in metazoans.

A tumor-suppressing mechanism in Drosophila involving cell competition and the Hippo pathway.

Mutant larvae for the Drosophila gene lethal giant larva (lgl) develop neoplastic tumors in imaginal discs. However, lgl mutant clones do not form tumors when surrounded by wild-type tissue, suggesting the existence of a tumor-suppressing mechanism. We have investigated the tumorigenic potential of lgl mutant cells by generating wing compartments that are entirely mutant for lgl and also inducing clones of various genetic combinations of lgl(-) cells. We find that lgl(-) compartments can grow indefinitely but lgl(-) clones are eliminated by cell competition. lgl mutant cells may form tumors if they acquire constitutive activity of the Ras pathway (lgl(-) UAS-ras(V12)), which confers proliferation advantage through inhibition of the Hippo pathway. Yet, the majority of lgl(-) UAS-ras(V12) clones are eliminated in spite of their high proliferation rate. The formation of a tumor requires in addition the formation of a microenvironment that allows mutant cells to evade cell competition.

Apoptosis in Drosophila: compensatory proliferation and undead cells.

Apoptosis (programmed cell death) is a conserved process in all animals, used to eliminate damaged or unwanted cells after stress events or during normal development to sculpt larval or adult structures. In Drosophila, it is known that stress events such as irradiation or heat shock give rise to high apoptotic levels which remove more than 50% of cells in imaginal discs. However, the surviving cells are able to restore normal size and pattern, indicating that they undergo additional proliferation. This compensatory proliferation is still poorly understood. One widely used method to study the properties of apoptotic cells is to keep them alive by expressing in them the baculoviral protein P35, which blocks the activity of the effector caspases. These "undead" cells acquire special features, such as the emission of the growth signals Dpp and Wg, changes in cellular morphology and induction of proliferation in neighbouring cells. Here, we review the various methods used in Drosophila to block apoptosis and its consequences, and focus on the generation and properties of undead cells in the wing imaginal disc. We describe their effects in epithelial architecture and growth in some detail, and discuss the possible relationship between undead cells and compensatory proliferation.

The role of Dpp and Wg in compensatory proliferation and in the formation of hyperplastic overgrowths caused by apoptotic cells in the Drosophila wing disc.

Non-lethal stress treatments (X-radiation or heat shock) administered to Drosophila imaginal discs induce massive apoptosis, which may eliminate more that 50% of the cells. Yet the discs are able to recover to form final structures of normal size and pattern. Thus, the surviving cells have to undergo additional proliferation to compensate for the cell loss. The finding that apoptotic cells ectopically express dpp and wg suggested that ectopic Dpp/Wg signalling might be responsible for compensatory proliferation. We have tested this hypothesis by analysing the response to irradiation-induced apoptosis of disc compartments that are mutant for dpp, for wg, or for both. We find that there is compensatory proliferation in these compartments, indicating that the ectopic Dpp/Wg signalling generated by apoptotic cells is not involved. However, we demonstrate that this ectopic Dpp/Wg signalling is responsible for the hyperplastic overgrowths that appear when apoptotic ('undead') cells are kept alive with the caspase inhibitor P35. We also show that the ectopic Dpp/Wg signalling and the overgrowths caused by undead cells are due to a non-apoptotic function of the JNK pathway. We propose that the compensatory growth is simply a homeostatic response of wing compartments, which resume growth after massive cellular loss until they reach the final correct size. The ectopic Dpp/Wg signalling associated with apoptosis is inconsequential in compartments with normal apoptotic cells, which die soon after the stress event. In compartments containing undead cells, the adventitious Dpp/Wg signalling results in hyperplastic overgrowths.

Dpp of posterior origin patterns the proximal region of the wing.

The decapentaplegic (dpp) gene encodes a long-range morphogen that plays a key role in the patterning of the wing imaginal disc of Drosophila (Nellen, D., Burke, R., Struhl, G. and Basler, K. 1996. Direct and long-range action of a DPP morphogen gradient. Cell 85, 357-368.). The current view is that dpp is transcriptionally active in a narrow band of anterior compartment cells close to the anterio-posterior (A/P) compartment border. Once the Dpp protein is synthesised, it travels across the A/P border and diffuses forming concentration gradients in the two compartments (reviewed in Lawrence, P.A., Struhl, G. 1996. Morphogens, compartments, and pattern: lessons from drosophila? Cell 85, 951-961.). We have found a new site of dpp expression in the posterior wing compartment which appears during the third larval period. This source of Dpp signal generates a local gradient of Dpp pathway activity, which is independent of that originating in the anterior compartment. We show that this posterior tier of Dpp activity is functionally required for normal wing development: the elimination of dpp expression in the posterior compartment results in defective adult wings in which pattern elements such as the alula and much of the axillary cord are not formed. Moreover, these structures develop normally in the absence of anterior dpp expression. Thus the normal wing pattern requires distinct Dpp organizer activities in the anterior and posterior compartments. We further show that, unlike the anterior dpp expression domain, the posterior one is not dependent on Hedgehog activity but is dependant on the activity of the IRO complex gene mirror. Since there is a similar expression in the haltere disc, we suggest that this late appearing posterior Dpp activity may be an attribute of dorsal thoracic discs.

Dpp signaling and the induction of neoplastic tumors by caspase-inhibited apoptotic cells in Drosophila.

In Drosophila, stresses such as x-irradiation or severe heat shock can cause most epidermal cells to die by apoptosis. Yet, the remaining cells recover from such assaults and form normal adult structures, indicating that they undergo extra growth to replace the lost cells. Recent studies of cells in which the cell death pathway is blocked by expression of the caspase inhibitor P35 have raised the possibility that dying cells normally regulate this compensatory growth by serving as transient sources of mitogenic signals. Caspase-inhibited cells that initiate apoptosis do not die. Instead, they persist in an "undead" state in which they ectopically express the signaling genes decapentaplegic (dpp) and wingless (wg) and induce abnormal growth and proliferation of surrounding tissue. Here, using mutations to abolish Dpp and/or Wg signaling by such undead cells, we show that Dpp and Wg constitute opposing stimulatory and inhibitory signals that regulate this excess growth and proliferation. Strikingly, we also found that, when Wg signaling is blocked, unfettered Dpp signaling by undead cells transforms their neighbors into neoplastic tumors, provided that caspase activity is also blocked in the responding cells. This phenomenon may provide a paradigm for the formation of neoplastic tumors in mammalian tissues that are defective in executing the cell death pathway. Specifically, we suggest that stress events (exposure to chemical mutagens, viral infection, or irradiation) that initiate apoptosis in such tissues generate undead cells, and that imbalances in growth regulatory signals sent by these cells can induce the oncogenic transformation of neighboring cells.

Caspase inhibition during apoptosis causes abnormal signalling and developmental aberrations in Drosophila.

Programmed cell death or apoptosis plays an important role in the development of multicellular organisms and can also be induced by various stress events. In the Drosophila wing imaginal disc there is little apoptosis in normal development but X-rays can induce high apoptotic levels, which eliminate a large fraction of the disc cells. Nevertheless, irradiated discs form adult patterns of normal size, indicating the existence of compensatory mechanisms. We have characterised the apoptotic response of the wing disc to X-rays and heat shock and also the developmental consequences of compromising apoptosis. We have used the caspase inhibitor P35 to prevent the death of apoptotic cells and found that it causes increased non-autonomous cell proliferation, invasion of compartments and persistent misexpression of the wingless (wg) and decapentaplegic (dpp) signalling genes. We propose that a feature of cells undergoing apoptosis is to activate wg and dpp, probably as part of the mechanism to compensate for cell loss. If apoptotic cells are not eliminated, they continuously emit Wg and Dpp signals, which results in developmental aberrations. We suggest that a similar process of uncoupling apoptosis initiation and cell death may occur during tumour formation in mammalian cells.

The brinker gradient controls wing growth in Drosophila.

The Decapentaplegic (Dpp) morphogen gradient controls growth and patterning in the Drosophila appendages. There is recent evidence indicating that the Dpp gradient is converted into an inverse gradient of activity of the gene brinker (brk), which encodes a transcriptional repressor and is negatively regulated by the Dpp pathway. We have studied how alterations in the Brk gradient affect the growth of the wing disc. We find that there is a negative correlation between brk activity and growth of the disc: high levels of brk prevent or reduce growth, whereas loss of brk activity results in excessive growth. This effect is concentration dependent: different amounts of Brk produce distinct rates of growth. Furthermore, our results demonstrate that although brk is able to induce apoptosis where there is a sharp difference in Brk levels, its role as a growth repressor is not achieved by inducing apoptosis but by reducing cell proliferation. Brk appears to downregulate the activity of genes that control cell proliferation, such as bantam.