Temporal dynamics of apoptosis-induced proliferation in pupal wing development: implications for regenerative ability.

BACKGROUND: The ability of animals to regenerate damaged tissue is a complex process that involves various cellular mechanisms. As animals age, they lose their regenerative abilities, making it essential to understand the underlying mechanisms that limit regenerative ability during aging. Drosophila melanogaster wing imaginal discs are epithelial structures that can regenerate after tissue injury. While significant research has focused on investigating regenerative responses during larval stages our comprehension of the regenerative potential of pupal wings and the underlying mechanisms contributing to the decline of regenerative responses remains limited. RESULTS: Here, we explore the temporal dynamics during pupal development of the proliferative response triggered by the induction of cell death, a typical regenerative response. Our results indicate that the apoptosis-induced proliferative response can continue until 34 h after puparium formation (APF), beyond this point cell death alone is not sufficient to induce a regenerative response. Under normal circumstances, cell proliferation ceases around 24 h APF. Interestingly, the failure of reinitiating the cell cycle beyond this time point is not attributed to an incapacity to activate the JNK pathway. Instead, our results suggest that the function of the ecdysone-responsive transcription factor E93 is involved in limiting the apoptosis-induced proliferative response during pupal development. CONCLUSIONS: Our study shows that apoptosis can prolong the proliferative period of cells in the wing during pupal development as late as 34 h APF, at least 10 h longer than during normal development. After this time point, the regenerative response is diminished, a process mediated in part by the ecdysone-responsive transcription factor E93.

Cell proliferation and Notch signaling coordinate the formation of epithelial folds in the Drosophila leg.

The formation of complex three-dimensional organs during development requires precise coordination between patterning networks and mechanical forces. In particular, tissue folding is a crucial process that relies on a combination of local and tissue-wide mechanical forces. Here, we investigate the contribution of cell proliferation to epithelial morphogenesis using the Drosophila leg tarsal folds as a model. We reveal that tissue-wide compression forces generated by cell proliferation, in coordination with the Notch signaling pathway, are essential for the formation of epithelial folds in precise locations along the proximo-distal axis of the leg. As cell numbers increase, compressive stresses arise, promoting the folding of the epithelium and reinforcing the apical constriction of invaginating cells. Additionally, the Notch target dysfusion plays a key function specifying the location of the folds, through the apical accumulation of F-actin and the apico-basal shortening of invaginating cells. These findings provide new insights into the intricate mechanisms involved in epithelial morphogenesis, highlighting the crucial role of tissue-wide forces in shaping a three-dimensional organ in a reproducible manner.

Regulation and coordination of the different DNA damage responses in Drosophila.

Cells have evolved mechanisms that allow them to respond to DNA damage to preserve genomic integrity and maintain tissue homeostasis. These responses include the activation of the cell cycle checkpoints and the repair mechanisms or the induction of apoptosis that eventually will eliminate damaged cells. These "life" vs. "death" decisions differ depending on the cell type, stages of development, and the proliferation status of the cell. The apoptotic response after DNA damage is of special interest as defects in its induction could contribute to tumorigenesis or the resistance of cancer cells to therapeutic agents such as radiotherapy. Multiples studies have elucidated the molecular mechanisms that mediate the activation of the DNA damage response pathway (DDR) and specifically the role of p53. However, much less is known about how the different cellular responses such as cell proliferation control and apoptosis are coordinated to maintain tissue homeostasis. Another interesting question is how the differential apoptotic response to DNA damage is regulated in distinct cell types. The use of Drosophila melanogaster as a model organism has been fundamental to understand the molecular and cellular mechanisms triggered by genotoxic stress. Here, we review the current knowledge regarding the cellular responses to ionizing radiation as the cause of DNA damage with special attention to apoptosis in Drosophila: how these responses are regulated and coordinated in different cellular contexts and in different tissues. The existence of intrinsic mechanisms that might attenuate the apoptotic pathway in response to this sort of DNA damage may well be informative for the differences in the clinical responsiveness of tumor cells after radiation therapy.

Coordination between cell proliferation and apoptosis after DNA damage in Drosophila.

Exposure to genotoxic stress promotes cell cycle arrest and DNA repair or apoptosis. These "life" or "death" cell fate decisions often rely on the activity of the tumor suppressor gene p53. Therefore, the precise regulation of p53 is essential to maintain tissue homeostasis and to prevent cancer development. However, how cell cycle progression has an impact on p53 cell fate decision-making is mostly unknown. In this work, we demonstrate that Drosophila p53 proapoptotic activity can be impacted by the G2/M kinase Cdk1. We find that cell cycle arrested or endocycle-induced cells are refractory to ionizing radiation-induced apoptosis. We show that p53 binding to the regulatory elements of the proapoptotic genes and its ability to activate their expression is compromised in experimentally arrested cells. Our results indicate that p53 genetically and physically interacts with Cdk1 and that p53 proapoptotic role is regulated by the cell cycle status of the cell. We propose a model in which cell cycle progression and p53 proapoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage.

Dpp and Hedgehog promote the glial response to neuronal apoptosis in the developing Drosophila visual system.

Damage in the nervous system induces a stereotypical response that is mediated by glial cells. Here, we use the eye disc of Drosophila melanogaster as a model to explore the mechanisms involved in promoting glial cell response after neuronal cell death induction. We demonstrate that these cells rapidly respond to neuronal apoptosis by increasing in number and undergoing morphological changes, which will ultimately grant them phagocytic abilities. We found that this glial response is controlled by the activity of Decapentaplegic (Dpp) and Hedgehog (Hh) signalling pathways. These pathways are activated after cell death induction, and their functions are necessary to induce glial cell proliferation and migration to the eye discs. The latter of these 2 processes depend on the function of the c-Jun N-terminal kinase (JNK) pathway, which is activated by Dpp signalling. We also present evidence that a similar mechanism controls glial response upon apoptosis induction in the leg discs, suggesting that our results uncover a mechanism that might be involved in controlling glial cells response to neuronal cell death in different regions of the peripheral nervous system (PNS).

JNK and JAK/STAT signalling are required for inducing loss of cell fate specification during imaginal wing discs regeneration in Drosophila melanogaster.

The regenerative process after tissue damage relies on a variety of cellular responses that includes compensatory cell proliferation and cell fate re-specification. The identification of the signalling networks regulating these cellular events is a central question in regenerative biology. Tissue regeneration models in Drosophila have shown that two of the signals that play a fundamental role during the early stages of regeneration are the c-Jun N-terminal kinase (JNK) and JAK/STAT signalling pathways. These pathways have been shown to be required for controlling regenerative proliferation, however their contribution to the processes of cellular reprogramming and cell fate re-specification that take place during regeneration are largely unknown. Here, we present evidence for a previously unrecognised function of the cooperative activities of JNK and JAK/STAT signalling pathways in inducing loss of cell fate specification in imaginal discs. We show that co-activation of these signalling pathways induces both the cell fate changes in injured areas, as well as in adjacent cells. We have also found that this function relies on the activity of the Caspase initiator encoded by the gene dronc.

Drosophila as a Model System to Study Cell Signaling in Organ Regeneration.

Regeneration is a fascinating phenomenon that allows organisms to replace or repair damaged organs or tissues. This ability occurs to varying extents among metazoans. The rebuilding of the damaged structure depends on regenerative proliferation that must be accompanied by proper cell fate respecification and patterning. These cellular processes are regulated by the action of different signaling pathways that are activated in response to the damage. The imaginal discs of Drosophila melanogaster have the ability to regenerate and have been extensively used as a model system to study regeneration. Drosophila provides an opportunity to use powerful genetic tools to address fundamental problems about the genetic mechanisms involved in organ regeneration. Different studies in Drosophila have helped to elucidate the genes and signaling pathways that initiate regeneration, promote regenerative growth, and induce cell fate respecification. Here we review the signaling networks involved in regulating the variety of cellular responses that are required for discs regeneration.

Analysis of the Function of Apoptosis during Imaginal Wing Disc Regeneration in Drosophila melanogaster.

Regeneration is the ability that allows organisms to replace missing organs or lost tissue after injuries. This ability requires the coordinated activity of different cellular processes, including programmed cell death. Apoptosis plays a key role as a source of signals necessary for regeneration in different organisms. The imaginal discs of Drosophila melanogaster provide a particularly well-characterised model system for studying the cellular and molecular mechanisms underlying regeneration. Although it has been shown that signals produced by apoptotic cells are needed for homeostasis and regeneration of some tissues of this organism, such as the adult midgut, the contribution of apoptosis to disc regeneration remains unclear. Using a new method for studying disc regeneration in physiological conditions, we have defined the pattern of cell death in regenerating discs. Our data indicate that during disc regeneration, cell death increases first at the wound edge, but as regeneration progresses dead cells can be observed in regions far away from the site of damage. This result indicates that apoptotic signals initiated in the wound spread throughout the disc. We also present results which suggest that the partial inhibition of apoptosis does not have a major effect on disc regeneration. Finally, our results suggest that during disc regeneration distinct apoptotic signals might be acting simultaneously.

The bHLH factors extramacrochaetae and daughterless control cell cycle in Drosophila imaginal discs through the transcriptional regulation of the Cdc25 phosphatase string.

One of the major issues in developmental biology is about having a better understanding of the mechanisms that regulate organ growth. Identifying these mechanisms is essential to understand the development processes that occur both in physiological and pathological conditions, such as cancer. The E protein family of basic helix-loop helix (bHLH) transcription factors, and their inhibitors the Id proteins, regulate cell proliferation in metazoans. This notion is further supported because the activity of these factors is frequently deregulated in cancerous cells. The E protein orthologue Daughterless (Da) and the Id orthologue Extramacrochaetae (Emc) are the only members of these classes of bHLH proteins in Drosophila. Although these factors are involved in controlling proliferation, the mechanism underlying this regulatory activity is poorly understood. Through a genetic analysis, we show that during the development of epithelial cells in the imaginal discs, the G2/M transition, and hence cell proliferation, is controlled by Emc via Da. In eukaryotic cells, the main activator of this transition is the Cdc25 phosphatase, string. Our genetic analyses reveal that the ectopic expression of string in cells with reduced levels of Emc or high levels of Da is sufficient to rescue the proliferative defects seen in these mutant cells. Moreover, we present evidence demonstrating a role of Da as a transcriptional repressor of string. Taken together, these findings define a mechanism through which Emc controls cell proliferation by regulating the activity of Da, which transcriptionally represses string.

Pattern reorganization occurs independently of cell division during Drosophila wing disc regeneration in situ.

One of the most intriguing problems in developmental biology is how an organism can replace missing organs or portions of its body after injury. This capacity, known as regeneration, is conserved across different phyla. The imaginal discs of Drosophila melanogaster provide a particularly well-characterized model for analyzing regeneration. We have developed a unique method to study organ regeneration under physiological conditions using the imaginal discs of Drosophila. Using this method, we revisited different aspects of organ regeneration. The results presented in this report suggest that during the initial stages of regeneration, different processes occur, including wound healing, a temporary loss of markers of cell-fate commitment, and pattern reorganization. We present evidence indicating that all of these processes occur even when cell division has been arrested. Our data also suggested that Wingless is not required during the early stages of disc regeneration.

The bHLH factors Dpn and members of the E(spl) complex mediate the function of Notch signalling regulating cell proliferation during wing disc development.

The Notch signalling pathway plays an essential role in the intricate control of cell proliferation and pattern formation in many organs during animal development. In addition, mutations in most members of this pathway are well characterized and frequently lead to tumour formation. The Drosophila imaginal wing discs have provided a suitable model system for the genetic and molecular analysis of the different pathway functions. During disc development, Notch signalling at the presumptive wing margin is necessary for the restricted activation of genes required for pattern formation control and disc proliferation. Interestingly, in different cellular contexts within the wing disc, Notch can either promote cell proliferation or can block the G1-S transition by negatively regulating the expression of dmyc and bantam micro RNA. The target genes of Notch signalling that are required for these functions have not been identified. Here, we show that the Hes vertebrate homolog, deadpan (dpn), and the Enhancer-of-split complex (E(spl)C) genes act redundantly and cooperatively to mediate the Notch signalling function regulating cell proliferation during wing disc development.

The bHLH factor deadpan is a direct target of Notch signaling and regulates neuroblast self-renewal in Drosophila.

A defining feature of stem cells is their capacity to renew themselves at each division while producing differentiated progeny. How these cells balance self-renewal versus differentiation is a fundamental issue in developmental and cancer biology. The Notch signaling pathway has long been known to influence cell fate decisions during development. Indeed, there is a great deal of evidence correlating its function with the regulation of neuroblast (NB) self-renewal during larval brain development in Drosophila. However, little is known about the transcription factors regulated by this pathway during this process. Here we show that deadpan (dpn), a gene encoding a bHLH transcription factor, is a direct target of the Notch signaling pathway during type II NB development. Type II NBs undergo repeated asymmetric divisions to self-renew and to produce immature intermediate neural progenitors. These cells mature into intermediate neural progenitors (INPs) that have the capacity to undergo multiple rounds of asymmetric division to self-renew and to generate GMCs and neurons. Our results indicate that the expression of dpn at least in INPs cells depends on Notch signaling. The ectopic expression of dpn in immature INP cells can transform these cells into NBs-like cells that divide uncontrollably causing tumor over-growth. We show that in addition to dpn, Notch signaling must be regulating other genes during this process that act redundantly with dpn.

Upregulation of glypicans in Hippo mutants alters the coordinated activity of morphogens.

The function of the conserved Drosophila Hippo signaling pathway has been shown to be required to limit cell proliferation. Several studies have identified different target genes of this pathway that could modulate this function. However, the ectopic expression of these genes cannot account for all of the hyperplasic and pattern defects displayed by Hippo signaling mutants. We have recently identified two new targets of the Hippo pathway, the heparan sulfate proteoglycans (HSPGs) encoded by division abnormally delayed (dally) and dally-like protein (dlp). The function of these glypicans is required to modulate the activity of different signaling pathways triggered by diffusible ligands. Thus, our results link the function of the Hippo pathway with the control of the activity of several signaling pathways required for the definition of the size and pattern of an organ.

The tumor suppressor genes dachsous and fat modulate different signalling pathways by regulating dally and dally-like.

The activity of different signaling pathways must be precisely regulated during development to define the final size and pattern of an organ. The Drosophila tumor suppressor genes dachsous (ds) and fat (ft) modulate organ size and pattern formation during imaginal disc development. Recent studies have proposed that Fat acts through the conserved Hippo signaling pathway to repress the expression of cycE, bantam, and diap-1. However, the combined ectopic expression of all of these target genes does not account for the hyperplasic phenotypes and patterning defects displayed by Hippo pathway mutants. Here, we identify the glypicans dally and dally-like as two target genes for both ft and ds acting via the Hippo pathway. Dally and Dally-like modulate organ growth and patterning by regulating the diffusion and efficiency of signaling of several morphogens such as Decapentaplegic, Hedgehog, and Wingless. Our findings therefore provide significant insights into the mechanisms by which mutations in the Hippo pathway genes can simultaneously alter the activity of several signaling pathways, compromising the control of growth and pattern formation.

Epithelial cell adhesion in the developing Drosophila retina is regulated by Atonal and the EGF receptor pathway.

In the Drosophila retina, photoreceptor differentiation is preceded by significant cell shape rearrangements within and immediately behind the morphogenetic furrow. Groups of cells become clustered into arcs and rosettes in the plane of the epithelium, from which the neurons subsequently emerge. These cell clusters also have differential adhesive properties: adherens junction components are upregulated relative to surrounding cells. Little is known about how these morphological changes are orchestrated and what their relevance is for subsequent neuronal differentiation. Here, we report that the transcription factor Atonal and the canonical EGF receptor signalling cascade are both required for this clustering and for the accompanying changes in cellular adhesion. In the absence of either component, no arcs are formed behind the furrow, and all cells show low Armadillo and DE-cadherin levels, although in the case of EGFR pathway mutants, single, presumptive R8 cells with high levels of adherens junction components can be seen. Atonal regulates DE-cadherin transcriptionally, whereas the EGFR pathway, acting through the transcription factor Pointed, exerts its effects on adherens junctions indirectly, at a post-transcriptional level. These observations define a new function for EGFR signalling in eye development and illustrate a mechanism for the control of epithelial morphology by developmental signals.

Atrophin contributes to the negative regulation of epidermal growth factor receptor signaling in Drosophila.

Dentato-rubral and pallido-luysian atrophy (DRPLA) is a dominant, progressive neurodegenerative disease caused by the expansion of polyglutamine repeats within the human Atrophin-1 protein. Drosophila Atrophin and its human orthologue are thought to function as transcriptional co-repressors. Here, we report that Drosophila Atrophin participates in the negative regulation of Epidermal Growth Factor Receptor (EGFR) signaling both in the wing and the eye imaginal discs. In the wing pouch, Atrophin loss of function clones induces cell autonomous expression of the EGFR target gene Delta, and the formation of extra vein tissue, while overexpression of Atrophin inhibits EGFR-dependent vein formation. In the eye, Atrophin cooperates with other negative regulators of the EGFR signaling to prevent the differentiation of surplus photoreceptor cells and to repress Delta expression. Overexpression of Atrophin in the eye reduces the EGFR-dependent recruitment of cone cells. In both the eye and wing, epistasis tests show that Atrophin acts downstream or in parallel to the MAP kinase rolled to modulate EGFR signaling outputs. We show that Atrophin genetically cooperates with the nuclear repressor Yan to inhibit the EGFR signaling activity. Finally, we have found that expression of pathogenic or normal forms of human Atrophin-1 in the wing promotes wing vein differentiation and acts as dominant negative proteins inhibiting endogenous fly Atrophin activity.

The orientation of cell divisions determines the shape of Drosophila organs.

Organ shape depends on the coordination between cell proliferation and the spatial arrangement of cells during development. Much is known about the mechanisms that regulate cell proliferation, but the processes by which the cells are orderly distributed remain unknown. This can be accomplished either by random division of cells that later migrate locally to new positions (cell allocation) or through polarized cell division (oriented cell division; OCD). Recent data suggest that the OCD is involved in some morphogenetic processes such as vertebrate gastrulation, neural tube closure, and growth of shoot apex in plants; however, little is known about the contribution of OCD during organogenesis. We have analyzed the orientation patterns of cell division throughout the development of wild-type and mutant imaginal discs of Drosophila. Our results show a causal relationship between the orientation of cell divisions in the imaginal disc and the adult morphology of the corresponding organs, indicating a key role of OCD in organ-shape definition. In addition, we find that a subset of planar cell polarity genes is required for the proper orientation of cell division during organ development.