To examine the role of programmed cell death in differentiation of the embryonic central nervous system midline, a reaper-deficiency mutant strain has been used Df(3R)H99 (or H99) in conjunction with strains containing cell-type-specific markers. Midline cell death has been identified both by the presence of excess midline cells in H99 mutants and by the engulfment of dying midline cells by macrophages in wild-type embryos. These developmental deaths are lineage-specific: prominent midline glial death was observed, while little if any death was detected among the ventral unpaired median neurons. Examination of H99 mutants indicates that cell death is not required for the formation of macrophage precursors, or for their subsequent migration throughout the embryo; however, in the absence of dying cells, macrophage precursors do not exhibit morphological differentiation or phagocytosis. In both wild-type and H99 mutant embryos, a subset of macrophages migrate along the ventral midline. This midline migration is not observed in single-minded mutants, in which ventral midline cells fail to develop. Programmed cell death plays a crucial role in the development of the central nervous system midline, and dying midline cells are rapidly eliminated by phagocytic macrophages. It seems that the generation of engulfment signals in cells undergoing programmed cell death is downstream of reaper gene function, and that central nervous system midline and/or ventral epidermal cells provide directional cues for migrating macrophages (Zhou, 1995).

An analysis has been carried out of the correlation between the pattern of expression of reaper and morphogenetic movements affecting head development. The defects in head development resulting from the absence of apoptosis in embryos deficient for rpr have also been investigated. In the head, domains of high incidence of cell death as marked by expression of rpr correlate with regions where most morphogenetic movements occur; these regions are involved in formation of mouth structures, the internalization of neural progenitors, and head involution. Cellular events driving these movements are delamination, invagination, and intercalation, as well as disruption and reformation of contacts among epithelial cells. At the late blastoderm stage (stage 5/6), a transient low level expression of rpr is seen in stripes delimiting the anterior and posterior trunk. This diffuse expression subsides by the onset of gastrulation (stage 7) and is replaced by multiple strongly expressed foci in the head, as well as a few in the tail region. Patchy expression of rpr is seen in the anterior endoderm and head mesoderm during stages 7-10. These tissues give rise to the anterior midgut and hemocytes, respectively. Nassif (1998) provides detailed descriptions of six expression domains in the head, as follows:

  1. Gnathocephalon: A high level and complex pattern of rpr expression is observed in the three gnathal segments (mandibular, maxillary, and labial). These large, interconnected domains can be distinguished on the basis of time of onset and peak expression of rpr. The dorsal gnathal domain is located most dorsally, bordering the optic lobe; it shows expression of rpr first, during stages 10-11. During stage 11, the dorsal portions of the gnathal segments are dramatically reduced in size and fuse into a single lobe-like structure, the dorsal ridge. Later during stage 11, rpr expression is activated in three domains, shaped like inverted horseshoes, that outline the mandibular, maxillary, and labial lobes. During stage 12, a third domains of rpr expression, the ventral gnathal domain, is observed in the mid-ventral portion of the gnathal segments. Within this region, rpr expression is strongest in the maxillary lobe, and in a labial stripe that flanks the salivary placode, which is itself free of rpr expression (Nassif, 1998).
  2. The ventral procephalon: Anterior to the gnathocephalon, in the ventral procephalon, lie the antennal and intercalary segment, the antennal segment being found just dorsal to the intercalary segment. These two segments give rise to the antennal and hypopharyngeal lobes, respectively. A large focus of rpr expression, the ventral antennal domain, appears during stage 11 in the anterior-ventral antennal region. During late stage 11, this focus becomes prominent, appearing as an array of three highly expressing cell clusters arranged in a crescent. During stage 12, a second focus of rpr expression, the dorsal antennal domain, appears in the dorsal part of the antennal domain, adjacent to the gnathocephalon and coincident with the region where fusion between antennal lobe and gnathocephalon will occur. During stage 11, a stripe-like focus, appears that marks the boundary between the intercalary and antennal segments (Nassif, 1998).
  3. Stomatogastric nervous system: During early stage 11, a reaper focus appears transiently in the dorsal portion of the esophagus in a placode that will give rise to the stomatogastric nervous system. Expression in this focus fades and then later reappears, during stages 13 and 14, in the three SNS vesicles that have invaginated from the placode (Nassif, 1998).
  4. Clypeolabrum: Apart from the strongly expressing optic lobe focus in the posterior procephalon, the clypeolabrum is the most prominent domain of rpr expression in the early embryo. Expression within the clypeolabrum, typical of most strongly expressing foci, is mottled, with single or small groups of strongly expressing cells surrounded by cells with weaker expression. While expression throughout most of the clypeolabrum declines during stage 11, expression in a small domain remains until stage 13. Later, during stages 13 and 14, a more ventral portion of the labrum that will form the pharynx roof (epipharynx) shows a prominent focus of rpr expression. Cell death in the midline of the clypeolabrum contributes to the medial shift of the labral sensilla, which in wild type arise on either side of the clypeolabrum (Nassif, 1998).
  5. Medial procephalon: During stage 13, rpr expression begins in the dorsomedial procephalon. It is from here that neural progenitors segregate from the surface ectoderm in a "mass-delamination" movement that is distinct from the individual delamination movements of the majority of brain neuroblasts that occur at an earlier stage. Later, during stages 14-15, scattered and relatively weak rpr expression can be seen in the medial procephalon as it folds into the dorsal pouch. In rpr mutants, the number of vesicles associated with the dorsal surface of the brain is significantly increased (Nassif, 1998).
  6. Posterior procephalon: A large focus containing 30-50 rpr-expressing cells, designated optic lobe 1, is seen at the boundary between dorsal procephalon (future brain and optic lobe) and amnioserosa. Expression in OL1 is high during stages 7-10, a period of approximately 2 hours. During stage 11, while expression in OL1 declines, rpr expression is activated in two to three small groups of cells (which togethar are designated OL2) located slightly more anterior and medial to OL1. These OL2 cells lie at the border between dorsomedial brain and optic lobe. Finally, during stage 12, rpr is expressed in a large focus (OL3) that borders the invaginating optic lobe. Cell death is shown to play three morphogenetic functions in the development of the optic lobe: (1) reducing the number of cells, (2) facilitating the ventral shift of the optic lobe primordium, which normally occurs during early embryogenesis (and presumably involves major horizontal intercalation of cells in the optic lobe primordium), and (3) enabling the optic lobe primordium to separate from the surface epithelium following invagination (Nassif, 1998).

In all domains expressing rpr, each involving apoptosis, profound morphogenetic movements take place during embryogenesis. These include the following major processes:

The analysis of rpr-deficient embryos demonstrates that despite the widespread occurrence of apoptosis during normal head morphogenesis, many aspects of this process proceed in an apparently unperturbed manner even when cell death is blocked. In particular, movements that happen early during embryonic development and that are evolutionarily more ancient (e.g., formation of the dorsal ridge and the pharynx) take place almost normally in rpr-deficient embryos. Later events which are mostly associated with head involution (e.g., retraction of the clypeolabrum, formation of the dorsal pouch, fusion of lateral gnathal lobes) are evolutionarily more recent and fail to occur normally in rpr-deficient embryos (Nassif, 1998).

Egfr signaling is required in a narrow medial domain of the head ectoderm (here called ‘head midline’) that includes the anlagen of the medial brain (including the dorsomedial and ventral medial domain of the brain, termed DMD and VMD respectively), the visual system (optic lobe, larval eye) and the stomatogastric nervous system (SNS). These head midline cells differ profoundly from their lateral neighbors in the way they develop. Three differences are noteworthy: (1) Like their counterparts in the mesectoderm, the head midline cells do not give rise to typical neuroblasts by delamination, but stay integrated in the surface ectoderm for an extended period of time. (2) The proneural gene l’sc, which transiently (for approximately 30 minutes) comes on in all parts of the procephalic neurectoderm while neuroblasts delaminate, is expressed continuously in the head midline cells for several hours. (3) Head midline cells, similar to ventral midline cells of the trunk, require the Egfr pathway. In embryos carrying loss-of-function mutations in Egfr, spi, rho, S and pnt, most of the optic lobe, larval eye, SNS and dorsomedial brain are absent. This phenotype arises by a failure of many neurectodermal cells to segregate (i.e., invaginate) from the ectoderm; in addition, around the time when segregation should take place, there is an increased amount of apoptotic cell death, accompanied by reaper expression, which removes many head midline cells. In embryos where Egfr signaling is activated ectopically by inducing rho, or by argos (aos) or yan loss-of-function, head midline structures are variably enlarged. A typical phenotype resulting from the overactivity of Egfr signaling is a ‘cyclops’ like malformation of the visual system, in which the primordia of the visual system stay fused in the dorsal midline. The early expression of cell fate markers, such as sine oculis in Spitz-group mutants, is unaltered (Dumstrei, 1998).

The deficiency of the reaper (rpr), grim and hid genes [Df(3L)H99] blocks apoptosis in Drosophila. Overexpression of any of these three genes results in ectopic apoptosis in embryos. It was therefore of interest to see whether Dakt1 mutants result in apoptosis through the mis-expression of rpr, grim or hid. This was found not to be the case, since Dakt1 mutant embryos do not show overexpression of these genes. Loss of rpr, grim and hid in H99 do not suppress the phenotype of Dakt1 GLC embryos. These results suggest that Dakt1 and H99 modulate apoptosis via distinct mechanisms. To test the involvement of caspases in Dakt1-mediated apoptosis, the baculoviral caspase-inhibitory protein p35 was expressed in Dakt1 embryos. Ectopic p35 has been shown to block caspase activity and suppress apoptosis in Dakt1 embryos, and hs-p35 effectively blocks apoptosis in Dakt1 embryos, demonstrating the requirement for caspase activity in this process (Staveley, 1998).

These epistasis tests suggest that Dakt1 does not function upstream of the rpr, grim and hid gene functions in the embryo. It is possible, though, that Dakt1 might be regulated by the rpr, grim and hid genes (at the H99 locus) and in fact act downstream of these genes. This presents two possibilities: (1) Dakt1 and the H99 locus represent independent pathways; (2) the H99 locus might repress Dakt1 function. This study thus provides the first genetic evidence implicating PKB as an anti-apoptotic factor (Staveley, 1998).

Larval and Pupal

Endogenous E2F falls from high to very low levels as cells initiate DNA synthesis during a developmentally regulated G1-S-transition in the eye disc. Ectopic E2F expression drives many otherwise quiescent cells to enter S phase. Subsequently, cells throughout the discs express reaper and then die. Ectopic E2F expression during S phase in normally cycling cells blocks their re-entry into S phase in the following cell cycle. Thus an elevation in the level of E2F is sufficient to induce imaginal disc cells to enter S phase, and the downregulation of E2F upon entry into S phase may be essential to prevent the induction of apoptosis (Asano, 1996).

To understand the role apoptosis plays in nervous system development and to gain insight into the mechanisms by which steroid hormones regulate neuronal apoptosis, the death of a set of peptidergic neurons was investigated in the CNS of Drosophila. Typically, apoptosis in Drosophila is induced by the expression of the genes reaper, grim, or head involution defective (hid). Genetic evidence is provided that the death of these neurons requires reaper and grim gene function. Consistent with this genetic analysis, these doomed neurons accumulate reaper and grim transcripts prior to the onset of apoptosis. These neurons also accumulate low levels of hid, although the genetic analysis suggests that hid may not play a major role in the induction of apoptosis in these neurons. The death of these neurons is dependent on the fall in the titer of the steroid hormone 20-hydroxyecdysone that occurs at the end of metamorphosis: the accumulation of both reaper and grim transcripts is inhibited by this steroid hormone. These observations support the notion that 20E controls apoptosis by regulating the expression of genes that induce apoptosis (Draizen, 1999).

Drosophila metamorphosis is characterized by diverse developmental phenomena, including cellular proliferation, tissue remodeling, cell migration, and programmed cell death. Cells undergo one or more of these processes in response to the hormone 20-hydroxyecdysone (ecdysone), which initiates metamorphosis at the end of the third larval instar and before puparium formation (PF) via a transcriptional hierarchy. Additional pulses of ecdysone further coordinate these processes during the prepupal and pupal phases of metamorphosis. Larval tissues such as the gut, salivary glands, and larval-specific muscles undergo programmed cell death and subsequent histolysis. The imaginal discs undergo physical restructuring and differentiation to form rudimentary adult appendages such as wings, legs, eyes, and antennae. Ecdysone also triggers neuronal remodeling in the central nervous system (White, 1999).

Wild-type patterns of gene expression in D. melanogaster during early metamorphosis were examined by assaying whole animals at stages that span two pulses of ecdysone. Microarrays were constructed containing 6240 elements that included more than 4500 unique cDNA expressed sequence tag (EST) clones along with a number of ecdysone-regulated control genes having predictable expression patterns. These ESTs represent approximately 30% to 40% of the total estimated number of genes in the Drosophila genome. In order to gauge expression levels, microarrays were hybridized with fluorescent probes derived from polyA+ RNA isolated from developmentally staged animals. The time points examined are relative to PF, which last approximately 15 to 30 min, during which time the larvae cease to move and evert their anterior spiracles. Nineteen arrays were examined representing six time points relative to PF: one time point before the late larval ecdysone pulse; one time point just after the initiation of this pulse (4 hours BPF), and time points at 3, 6, 9, and 12 hours after PF (APF). The prepupal pulse of ecdysone occurs 9 to 12 hours APF (White, 1999).

In order to manage, analyze, and disseminate the large amount of data, a searchable database was constructed that includes the average expression differential at each time point. The analysis set consists of all elements that reproducibly fluctuate in expression threefold or more at any time point relative to PF, leaving 534 elements containing sequences represented by 465 ESTs and control genes. More than 10% of the genes represented by the ESTs display threefold or more differential expression during early metamorphosis. This may be a conservative estimate of the percentage of Drosophila genes that change in expression level during early metamorphosis, because of the stringent criteria used for their selection (White, 1999).

To interpret these data, genes were grouped according to similarity of expression patterns by two methods. The first relied on pairwise correlation statistics, and the second relied on the use of self-organizing maps (SOMs). Differentially expressed genes fall into two main categories. The first category contains genes that are expressed at >18 hours BFP (before the late larval ecdysone pulse) but then fall to low or undetectable levels during this pulse. These genes are potentially repressed by ecdysone and make up 44% of the 465 ESTs identified in this set. The second category consists of genes expressed at low or undetectable levels before the late larval ecdysone pulse but then are induced during this pulse. These genes are potentially induced by ecdysone and make up 31% of the 465 ESTs. Consequently, 75% of genes that changed in expression by threefold or more do so during the late larval ecdysone pulse that marks the initial transition from larva to prepupa. This result is consistent with the extreme morphological changes that are about to occur in these animals. There are clearly discrete subdivisions of gene expression within these categories (White, 1999).

Larval-specific tissues such as larval muscles, the midgut, and the salivary glands undergo programmed cell death during metamorphosis. Genes involved in programmed cell death were identified in these experiments. The apoptosis-activating reaper gene has previously been shown to be ecdysone-inducible, and this is reflected in the data. Expression of the Drosophila caspase-1 gene is also observed during the prepupal ecdysone pulse but not during the late larval pulse. This gene is also an activator of apoptosis, and mutants display melanotic tumors and larval lethality. Induction of a cell death inhibitor gene, thread (also known as Diap1), is observed during the late larval pulse but not the prepupal phase. The DIAP1 protein includes inhibitor-of-apoptosis (IAP) domains and has been identified as a factor that can block reaper activity. Because different tissues begin apoptosis at different stages of development, changes in the expression of inhibitors and activators of apoptosis are expected to be tissue-specific. For example, the expression profiles observed for the caspase-1 activator and the Diap1 inhibitor are those expected in tissues such as the larval salivary glands. Tissue-specific information on the induction of these genes will be important to an understanding of the coordination of apoptosis during metamorphosis (White, 1999).

Postembryonic neuroblasts are stem cell-like precursors that generate most neurons of the adult Drosophila central nervous system (CNS). Their capacity to divide is modulated along the anterior-posterior body axis, but the mechanism underlying this is unclear. Clonal analysis of identified precursors in the abdomen shows that neuron production stops because the cell death program is activated in the neuroblast, while it is still engaged in the cell cycle. A burst of expression of the Hox protein Abdominal-A (AbdA) specifies the time at which apoptosis occurs, thereby determining the final number of progeny that each neuroblast generates. These studies identify a mechanism linking the Hox axial patterning system to neural proliferation, and this involves temporal regulation of precursor cell death rather than the cell cycle (Bello, 2003).

An embryonic period of neuroblast divisions produces neurons that will form the functional CNS of the larva. Following this, there is a larval and pupal phase of neurogenesis that accounts for over 90% of the neurons present in the adult CNS. The precursors responsible for this, called postembryonic neuroblasts (pNBs), share a lineage with their embryonic counterparts and most probably are the same cells. Although each hemisegment of the early embryo contains an invariant number of 30 neuroblasts, in the larva this is no longer the case. For example, in the thorax, each larval hemisegment retains about 23 of the initial 30 neuroblasts, while in the central abdomen only three remain. The dramatic reduction in the number of abdominal neuroblasts occurs late in embryogenesis and depends on cell death mediated by the proapoptotic gene reaper. As a consequence, the surviving abdominal precursors that will contribute progeny to the adult CNS are well separated and can be readily identified as either the ventromedial (vm), ventrolateral (vl), or dorsolateral (dl) pNB (Bello, 2003 and references therein).

The Drosophila compound eye is formed by selective recruitment of undifferentiated cells into clusters called ommatidia during late larval and early pupal development. Ommatidia at the edge of the eye often lack the full complement of photoreceptors and support cells, and undergo apoptosis during mid-pupation. This cell death is triggered by the secreted glycoprotein Wingless, which activates its own expression in peripheral ommatidia via a positive feedback loop. Wingless signaling elevates the expression of the pro-apoptotic factors head involution defective, grim and reaper, which are required for ommatidial elimination. It is estimated that approximately 6%-8% of the total photoreceptor pool in each eye is removed by this mechanism. In addition, the retinal apoptosis previously reported in apc1 mutants occurs at the same time as the peripheral ommatidial cell death and also depends on head involution defective, grim and reaper. The implications of these findings for eye development and function in Drosophila and other organisms is considered (Lin, 2004).

Compensatory proliferation in Drosophila imaginal discs requires Dronc-dependent p53 activity

The p53 transcription factor directs a transcriptional program that determines whether a cell lives or dies after DNA damage. Animal survival after extensive cellular damage often requires that lost tissue be replaced through compensatory growth or regeneration. In Drosophila, damaged imaginal disc cells can induce the proliferation of neighboring viable cells, but how this is controlled is not clear. This paper provides evidence that Drosophila p53 has a previously unidentified role in coordinating the compensatory growth response to tissue damage. The sole p53 ortholog in Drosophila, is required for each component of the response to cellular damage, including two separate cell-cycle arrests, changes in patterning gene expression, cell proliferation, and growth. These processes are regulated by p53 in a manner that is independent of DNA-damage sensing but that requires the initiator caspase Dronc. These results indicate that once induced, p53 amplifies and sustains the response through a positive feedback loop with Dronc and the apoptosis-inducing factors Hid and Reaper. How cell death and cell proliferation are coordinated during development and after stress is a fundamental question that is critical for an understanding of growth regulation. These data suggest that p53 may carry out an ancestral function that promotes animal survival through the coordination of responses leading to compensatory growth after tissue damage (Wells, 2006; full text of article).

The repair of tissue after cellular damage can be critical to the survival of the animal. Previous studies demonstrated that undead cells stimulate the proliferation of neighboring cells, providing a model for how damaged and dying cells contribute to the replacement of lost tissue. With this model, it was found that the wing imaginal disc responds to this damage as a whole by deploying a multi-step process that ends with compensatory growth. p53 functions in a dronc-dependent manner at each step of the tissue-replacement process. Furthermore, p53 and the initiator caspase dronc may be generally required for tissue recovery in imaginal discs, because it was found that blastema formation was significantly impaired during regeneration induced in either p53 or dronc mutant leg discs (Wells, 2006).

The data suggest that p53 is induced and becomes functional in undead cells by a mechanism that does not require DNA-damage sensing or activation of the stress kinase AMPK. Rather, Dronc, an initiator caspase homologous to caspase-9, is necessary and sufficient to induce all aspects of the growth regulation by p53. It is not known how Dronc activity results in p53 expression and activity in these cells, but many caspase substrates are not directly involved in apoptosis. As an example, one of the first caspase substrates identified was the cytokine IL-1β, which regulates many aspects of the inflammatory response. Induction of p53 mRNA in undead cells is prevented in dronc mutant discs, and thus it is possible that a regulator of p53 is cleaved by Dronc, leading to its expression and ultimately to its ability to regulate the compensatory growth response in the imaginal discs. Regardless of the molecular mechanism, the data argue for direct communication between Dronc and p53 in response to tissue damage (Wells, 2006).

Collectively, these experiments imply that p53 serves as a master coordinator of tissue repair in imaginal discs, regulating both cell-autonomous and non-cell-autonomous cell-cycle arrests, the expression of the pattern-regulating genes wg and dpp, and compensatory cell proliferation and growth. Based on these results, it is suggested that cellular damage activates Dronc, which in a nonapoptotic role causes the induction of p53 mRNA and leads to p53 activity. It is proposed that p53 then acts as an overall damage monitor, in a role that includes its conserved functions in apoptosis (here, induction of hid and rpr expression) and growth arrest (by repression of stg/cdc25), but also allows for induction of signals that promote compensatory growth of the disc. The results suggest that p53 monitors tissue damage through a feed-forward loop with Dronc and the pro-apoptotic genes hid and rpr, which both amplifies and sustains the growth-regulating signal (Wells, 2006).

An intriguing puzzle left unanswered by these results is why the growth response to undead cells occurs only several days after they are generated: both HhGal4 and EnGal4 drive expression of Hid or Rpr from early embryonic stages, yet even with careful observation no growth phenotype was detected until the middle part of the third instar. Caspases are active in cells expressing Hid or Rpr + P35 at early time points, indicating that these cells are not immune to the apoptotic response early in development. The genes involved in the apoptotic response are subject to many levels of control, including that by micro-RNAs (miRNAs). Hid protein expression, for example, is suppressed by Bantam, a miRNA highly expressed early in imaginal disc development, but declining as development progresses. It is likely that rpr is also regulated by miRNA gene silencing. Hence, the delay of the growth response in discs with undead cells may reflect a requirement for threshold levels of these factors to fully activate the feedback loop. At the very least it emphasizes that the regulation of growth and cell death during wing disc development is complex and has multiple inputs, many of which are poorly understood (Wells, 2006).

Activity thresholds appear to play an important role in the processes induced by undead cells. Dronc, for instance, is haploinsufficient for its effect in compensatory proliferation. It is possible that the apoptotic functions of Dronc require a relatively low activity level, but that high Dronc activity allows activation of the p53-dependent tissue-damage response. Regulation of Dronc by critical activity thresholds could provide the animal some regenerative capacity and increase its chances for survival when conditions are appropriate for tissue repair (Wells, 2006).

As expected given its role in coordinating many cellular behaviors, p53 modulates the activity or expression of myriad effectors. Regulatory effectors of Drosophila p53 are only beginning to be identified, and these data add stg/cdc25 to the list. One of the first detectable disc responses to undead cells is G2 arrest, mediated by loss of stg mRNA. Cdc25 is also regulated by vertebrate p53 but is inhibited post-transcriptionally by p53-dependent 14-3-3 activity (Levine, 2006). Experiments with irradiated p53 mutant animals have not revealed a cell-cycle arrest role. However, recent work indicates that dp53 also regulates a G1 checkpoint under conditions of metabolic stress; thus, like vertebrate p53, Drosophila p53 can activate both a G1 and a G2 checkpoint in response to tissue stress. Other effectors and targets involved in the compensatory proliferation process remain unknown, although expression profiling experiments from irradiated p53 mutants identified several potential targets, several of which do not have obvious roles in cell death or DNA repair (Wells, 2006).

How does Drosophila p53 control the signaling that leads to compensatory proliferation? The events observed — G2 arrests in two different cell populations, ectopic expression of wg, and compensatory growth — are all regulated by p53. It is possible that p53 directly and coordinately controls each of these processes by regulating the expression of specific effectors. However, because the response is both cell autonomous and non-cell autonomous, the idea is favored that these processes are interdependent, but sequentially activated. It is envisioned that as a result of Dronc activation in undead cells, p53 induces loss of stg, leading to G2 arrest, and hid and rpr expression, initiating the feedback loop. It is postulate that cells then synthesize factors that stimulate their survival and proliferation. The non-cell-autonomous arrest in the anterior compartment may be a secondary effect of undead cells in the posterior. High levels of TUNEL activity was observed in the anterior cells of these discs, which could feasibly activate p53 in those cells. However, no p53 mRNA was detected in anterior cells. One possibility is that the DNA fragmentation resulting from dying anterior cells could activate ATM and Chk2 in those cells. Consistent with this, although loss of either of these kinases did not affect undead cell induction of Wg expression or compensatory growth, the cell-cycle arrest in anterior cells was reduced in a fraction of atm and chk2 mutants (Wells, 2006).

What is the growth-stimulating signal induced by undead cells? While its identity is still unclear, both Wg and Dpp have been implicated in this role. This makes sense, because Wg and Dpp are the major pattern organizers of all imaginal discs and are also involved in regulating their growth, and furthermore they are known to be induced in disc regeneration. However, although wg and dpp are ectopically expressed in undead cells, it was found that targets of both are sharply downregulated, specifically in the undead cells. These data also show that undead cells are able to proliferate and contribute to the compensatory growth. Thus, although the nonautonomous stimulation of growth (anterior cells near the A/P boundary) could be due to increased Dpp signaling, it is suspected that the autonomous growth stimulation is due to other, unidentified factors (Wells, 2006).

This study identified a growth-regulatory role for p53 that seems counter to its role as a tumor suppressor in vertebrates. However, it is speculated that the ability of p53 to sense and respond to tissue damage and promote compensatory proliferation and regeneration in Drosophila reflects an ancestral function, aspects of which have been appropriated for developmental processes and distributed among p53, p63, and p73 during vertebrate evolution. Although p63 and p73 initially were proposed to have evolved as duplications of p53, reanalysis of the phylogenetic relationship between the three family members has suggested that p63 may be the ancestral gene. p63 and p73 are structurally similar to p53 but contain an additional SAM domain. p53 is the sole member of the family encoded in the Drosophila genome, and although dp53 does not contain a SAM domain, based on the sequence of the DNA binding domain, the most highly conserved region of p53, it is more related to vertebrate p63 than to p53. After irradiation, cell-cycle arrest is not p53 dependent in either Drosophila or the nematode C. elegans, and therefore it has been proposed that the ancestral p53 function is apoptosis, rather than a “repair, then death” response when damage cannot be repaired. The experiments argue that as in vertebrates, p53 plays a role in cell-cycle arrest after tissue damage. The additional functions of p53 in promoting cell proliferation may have been conserved in p63, which regulates progenitor cell renewal in the epidermis. Other processes that require cell renewal may also be regulated by p53. For example, p53 mutants are reported to have fertility defects, so it is tempting to speculate that stem cell renewal in the gonad requires this previously unappreciated role of Drosophila p53 (Wells, 2006).

Role of mitochondrial remodeling in programmed cell death in Drosophila melanogaster

The role of mitochondria in Drosophila programmed cell death remains unclear, although certain gene products that regulate cell death seem to be evolutionarily conserved. This study found that developmental programmed cell death stimuli in vivo and multiple apoptotic stimuli ex vivo induce dramatic mitochondrial fragmentation upstream of effector caspase activation, phosphatidylserine exposure, and nuclear condensation in Drosophila cells. Unlike genotoxic stress, a lipid cell death mediator induces an increase in mitochondrial contiguity prior to fragmentation of the mitochondria. Dynamin related protein 1 (Drp1), is important for mitochondrial disruption. Using genetic mutants and RNAi-mediated knockdown of drp-1, it was found that Drp1 not only regulates mitochondrial fission in normal cells, but mediates mitochondrial fragmentation during programmed cell death. Mitochondria in drp-1 mutants fail to fragment, resulting in hyperplasia of tissues in vivo and protection of cells from multiple apoptotic stimuli ex vivo. Thus, mitochondrial remodeling is capable of modifying the propensity of cells to undergo death in Drosophila (Goyal, 2007).

Programmed cell death (PCD) plays an important role in sculpting tissues during animal development. The molecular regulators that are central to this process seem to be evolutionarily conserved from worms to mammals and include autocatalytic initiator caspases, trans-activable effector caspases, cytosolic activating factors (APAF-1), and multidomain Bcl-2 proteins. The proapoptotic Bcl-2-family proteins oligomerize and permeabilize mitochondria, releasing intermembrane space components such as cytochrome-C and Smac/DIABLO into the cytosol, where they activate initiator caspases by an ATP-dependent mechanism. Initiator caspases trans-activate effector caspases that cleave multiple cellular substrates, resulting in DNA degradation, nuclear condensation, and loss of cell integrity (Goyal, 2007 and references therein).

Mitochondrial outer-membrane permeabilization has been proposed to depend on the mitochondrial fission and fusion machinery. Consistent with this, mitochondria undergo dramatic fragmentation very close in time to cytochrome-C release during mammalian cell death. Furthermore, an increase in mitochondrial fragmentation and a block in mitochondrial fusion are essential for cell death progression. In normal cells, the balance in the rates of mitochondrial fission and fusion regulated by Dynamin-related protein-1 (Drp-1), Fis-1 and endophilin (fission), or Mitofusins and Opa-1 (fusion) maintains the dynamic, interconnected mitochondrial tubules. An increase in recruitment of Drp-1 to the mitochondria accentuates staurosporine, lipid, and free oxygen radical stress-induced mitochondrial outer-membrane permeabilization. Moreover, multiple apoptotic stimuli induce mitochondrial recruitment of the proapoptotic Bcl-2-family protein, Bax, to Drp-1 and Mitofusin-2-positive putative mitochondrial fragmentation sites in a Fis-1-dependent manner, consistent with a role for mitochondrial fission and fusion machinery in cell death (Goyal, 2007).

In Drosophila, RHG-family proteins (Reaper, Hid and Grim), genotoxic stresses, and protein synthesis inhibitors antagonize Drosophila inhibitor of apoptosis protein-1 (DIAP-1)-mediated inhibition of the activation of the apical caspase Dronc in an ARK- (Drosophila APAF-1) and ATP-dependent manner, leading to effector caspase activation and cell death. The role of mitochondria in this process is unclear. Cytochrome-C has been shown to be differentially displayed from the mitochondria during cell death. Knockdown of Drosophila cytochrome-C did not affect cell death triggered by genotoxic stress in vitro and ex vivo or developmental stimuli in vivo, although certain nonapoptotic caspase activation pathways utilized during sperm individualization were affected. Furthermore, mitochondrial morphology during Drosophila PCD has not been previously reported (Goyal, 2007 and references therein).

This study shows that multiple apoptotic stimuli result in mitochondrial fragmentation upstream of caspase activation, phosphatidylserine exposure, and nuclear condensation in Drosophila cells. While etoposide induced mitochondrial fragmentation, C6-ceramide resulted in an increase in mitochondrial contiguity prior to its fragmentation. drp-1 mutant or RNAi-treated S2R+ cells are considerably protected from multiple apoptotic stimuli, consistent with reduced mitochondrial fragmentation. Thus, mitochondrial remodeling plays an important role in modifying the propensity of cells to undergo PCD in Drosophila (Goyal, 2007).

Precisely timed ecdysone pulses induce Reaper and Hid expression in the Drosophila larval midgut (0 hr after puparium formation [APF]) or the salivary gland (10 hr APF) and trigger developmental PCD. Mitochondria, visualized by using matrix-targeted GFP (Mito-GFP) in acridine orange-positive, dying prepupal midgut cells (1 hr APF) and salivary glands (minus 4 hr APF), are remarkably fragmented, unlike third-instar larval (-4 hr APF) mitochondria. Quantification revealed a dramatic decrease in the prepupal mitochondrial cross-sectional area (CSA; midgut and salivary gland and a significant increase in the number of mitochondria per cell. Moreover, ecdysone-induced mitochondrial fragmentation is mimicked ex vivo on third-instar larval wing discs by using 1 mM ecdysone for 2 hr. In addition, overexpression of Hid resulted in mitochondrial fragmentation in acridine orange-positive eye disc cells. Thus, mitochondria in Drosophila tissues fragment during PCD, as has been reported in C. elegans and mammalian cells (Goyal, 2007).

To assess the role of mitochondrial remodeling in PCD, mitochondrial morphology was temporally characterized in etoposide-, actinomycin-D-, cycloheximide-, or C6-ceramide (a lipid cell death mediator)-treated larval hemocytes and the S2R+ cell line. A 3- to 4-fold increase in nuclear condensation (6 hr) was preceded by effector caspase activation (5 hr) and phosphatidylserine (PS) exposure in propidium iodide (PI)-negative hemocytes (6 hr). These cells subsequently (10 hr) became characteristically blebbed and PI permeable. The number of etoposide-treated apoptotic hemocytes increased with time. Interestingly, mitochondrial fragmentation, as confirmed by quantifying functionally isolated mitochondria at 3 hr, preceded the onset of PS exposure or nuclear damage. Quantification showed an increase in the number of mitochondria and the contribution of fragmented mitochondria to the mitochondrial cross-sectional area (CSA). Mitochondrial fragmentation was also observed in cycloheximide- or actinomycin-D-treated, Mito-YFP-transfected S2R+ cells (Goyal, 2007).

Surprisingly, mitochondria in C6-ceramide-treated (30-60 min) hemocytes that had normal nuclei were highly contiguous. Quantifying functionally isolated mitochondrial CSA per cell showed a significant increase in the contribution of tubular or extensively tubular mitochondria in these cells when compared with untreated cells. However, by 4 hr, these extensively tubular mitochondria underwent fragmentation in FITC-Annexin V (AnV)-negative hemocytes that had normal nuclei , similar to what was observed with genotoxic stress (Goyal, 2007).

Therefore, genotoxic stresses trigger mitochondrial fragmentation, while the lipid cell death mediator induces increased mitochondrial contiguity and subsequent fragmentation prior to phosphatidylserine exposure, nuclear condensation, and finally plasma membrane permeability during Drosophila cell death (Goyal, 2007).

In hemocytes incubated with an apoptotic stimulus, mitochondrial fragmentation (3-4 hr) preceded any detectable effector caspase activation. Furthermore, inhibiting caspases with zVAD-fmk or by overexpressing DIAP-1 (DIAP-1+) did not affect mitochondrial fragmentation, although hemocyte death was inhibited, as revealed by a lack of apoptotic markers. In addition, overexpression of Dcp-1, a Drosophila effector caspase, did not affect mitochondrial morphology. Thus, mitochondrial fragmentation is upstream of effector caspase activation (Goyal, 2007).

The drp-1 mutants used to study the role of mitochondrial remodeling during Drosophila PCD are functional null alleles, drp-12 (Gly293Ser mutation), picked in a forward screen for genes affecting neurotransmission and drp-1[KG 03815], a P element insertion between the first two exons of drp-1 (13510 in this study) and a hypomorph, nrdD46 (Arg278Trp mutation; 3665 in this study). drp-12, 13510, and the deficiency Df Exel6008 were second-instar larval lethal; however, drp-12 yielded bang-sensitive escapers. The hypomorphic trans-allelic combination of 3665/13510 was third-instar larval lethal, although it yielded a few temperature-sensitive adults. A genomic duplication of drp-1 (Dp [2;1] JS13) completely rescued the lethality associated with drp-12, 13510, and 3665/13510 (Goyal, 2007).

Mitochondria in drp-12 and 3665/13510 hemocytes were extensively tubular when compared with wild-type mitochondria. Quantifying mitochondrial morphology revealed a 2-fold decrease in the number of mitochondria and a significant increase in the contribution of tubular and extensively tubular mitochondria to the total mitochondrial CSA in drp-1 mutant hemocytes when compared with wild-type cells. Interestingly, 13510/+ hemocytes or eye disc cells displayed a dominant mitochondrial fission defect that was completely rescued by a genomic duplication of drp-1. The mitochondrial fission defect in mutant cells could result from a reduced mitochondrial association of Drp-1 (Goyal, 2007).

An increase in mitochondrial contiguity due to a loss of Drp-1 function was also confirmed by measuring fluorescence recovery after photobleaching (FRAP) of Mito-YFP in drp-1 RNAi-treated S2R+ cells that had extensively tubular mitochondria. Relative FRAP of Mito-YFP in a defined mitochondrial region in drp-1 RNAi-treated cells was significantly higher than that observed in mock RNAi-treated cells (Goyal, 2007).

drp-1 mutant hemocytes were protected from etoposide-induced death up to at least 10 hr, as revealed by a lack of caspase activation, PS exposure, or PI permeability in the majority (~80%) of these cells. Furthermore, drp-1 mutant and dsRNA-treated S2R+ cells were significantly protected from cycloheximide-, actinomycin-D-, or UV-B-induced death. Consistent with increased protection, mitochondria in the majority (~98%) of etoposide-treated drp-12 hemocytes failed to fragment. Interestingly, mitochondria in etoposide-treated 3665/13510 hemocytes revealed a tubular, yet beaded and swollen intermediate in mitochondrial fragmentation by 4 hr that yielded some fragmented mitochondria in few (~25%) cells later. Therefore, reduced (drp-12) or delayed (3665/13510) mitochondrial fragmentation decreased effector caspase activation and protected cells from genotoxic stress. Moreover, an increase in expression of Drp-1 in hemocytes resulted in enhancement of etoposide-induced cell death (Goyal, 2007).

The majority (~70%) of the C6-ceramide-treated drp-12 hemocytes did not show effector caspase activation or PS exposure and displayed significant protection, similar to what was observed with etoposide, although hemocytes derived from the weaker allelic combination, 13510/3665, were apoptotic. Unlike 13510/3665 mitochondria, drp-12 mitochondria failed to fragment, consistent with an essential role for Drp-1-mediated mitochondrial fragmentation during apoptosis in Drosophila. Moreover, developmental PCD in drp-12 mutant larvae was considerably reduced, as revealed by the enlarged central nervous system and a prominently elongated ventral ganglion, similar to other PCD-defective mutants reported (Goyal, 2007).

During metamorphosis, the first ecdysone pulse triggers mitochondrial fragmentation in prepupal tissues, although it is after the second ecdysone pulse that salivary gland histolysis occurs. It is likely that DIAP-1 inhibits caspases in these cells that have fragmented mitochondria until it is downregulated at the transcriptional level or degraded after the second ecdysone pulse. Interestingly, this was mimicked ex vivo in etoposide-treated DIAP-1+ hemocytes (Goyal, 2007).

The data presented in this study show involvement of mitochondrial fragmentation for ARK-mediated Dronc activation during cell death. The RHG-family proteins that localize to the mitochondria might activate Drp-1-mediated mitochondrial fragmentation. This could result in exposure of cytochrome-C or release of Peanut, which antagonize DIAP-1-mediated suppression of Dronc. However, since Drosophila PCD is unaffected upon knockdown of cytochrome-C, mitochondrial fragmentation in Drosophila and mammalian cells would increase mitochondrial surface area and perhaps the concentration of bulky head group lipids on the outer mitochondrial membrane, facilitating recruitment of proapoptotic proteins. Drp-1 might organize sites for Drosophila Bcl-2-family protein Debcl function on mitochondria that are similar to mitochondrial sites of Bax recruitment in mammalian cells (Goyal, 2007 and references therein).

These results provide the first evidence that Drp-1-mediated mitochondrial fragmentation upstream of effector caspase activation modifies apoptotic sensitivity. Thus, mitochondrial fragmentation, like caspase activation, plays a conserved and unifying role in diverse cell death pathways from worms to mammals. Although the function of the highly contiguous mitochondria during lipid-induced cell death remains poorly understood, this study brings to the forefront a modulatory role for mitochondrial remodeling in determining the susceptibility of Drosophila cells to death.

Mitochondrial disruption in Drosophila apoptosis

Mitochondrial disruption is a conserved aspect of apoptosis, seen in many species from mammals to nematodes. Despite significant conservation of other elements of the apoptotic pathway in Drosophila, a broad role for mitochondrial changes in apoptosis in flies remains unconfirmed. This study shows that Drosophila mitochondria become permeable in response to the expression of Reaper and Hid, endogenous regulators of developmental apoptosis. Caspase activation in the absence of Reaper and Hid is not sufficient to permeabilize mitochondria, but caspases play a role in Reaper- and Hid-induced mitochondrial changes. Reaper and Hid rapidly localize to mitochondria, resulting in changes in mitochondrial ultrastructure. The dynamin-related protein, Dynamin related protein 1 (Drp1), is important for Reaper- and DNA-damage-induced mitochondrial disruption. Significantly, it was shows that inhibition of Reaper or Hid mitochondrial localization or inhibition of Drp1 significantly inhibits apoptosis, indicating a role for mitochondrial disruption in fly apoptosis (Abdelwahid, 2007).

A role for mitochondria in apoptosis appears to be conserved from mammals to nematodes to yeast. The lack of clear evidence that mitochondria play a role in Drosophila apoptosis has prompted discussion of whether flies represent an evolutionary outlier in this highly conserved process. The data strongly suggests that mitochondrial disruption also plays a role in Drosophila apoptosis (Abdelwahid, 2007).

The data show that mitochondria rapidly become permeable to Cyt c when Rpr or Hid are expressed, both in cultured cells and in vivo. This alteration in mitochondrial permeability was also seen during DNA-damage-induced apoptosis. Importantly, it was demonstrated that the mitochondrial permeabilization during DNA-damage-induced apoptosis is dependent on the genes in the H99 interval. Taken together, these data indicate that Rpr and Hid are both necessary and sufficient for mitochondrial permeabilization (Abdelwahid, 2007).

In contrast, apoptosis induced by Actinomycin D, UV, and DIAP1 RNAi does not result in mitochondrial permeabilization. This indicates that caspase activation alone is not sufficient to induce mitochondrial permeabilization and that the mitochondrial permeabilization seen on Rpr or Hid induction is not simply a general late event in apoptosis. The efficient cell killing by Actinomycin D, UV, and DIAP1 RNAi also implies that mitochondrial permeabilization is not important for all apoptosis in Drosophila cells. Rather, it suggests that the Rpr and Hid proteins have a specific activity on the mitochondria that results in mitochondrial permeabilization to execute apoptosis in a timely manner (Abdelwahid, 2007).

The effects of Rpr and Hid on mitochondria were not limited to permeabilization. It was found that mitochondrial morphology is dramatically altered within 90 min of Rpr or Hid expression, in both S2 cells and embryos. A variety of defects were found in mitochondrial ultrastructure ranging from a rounded appearance, to bulging (and occasional rupture) of the outer mitochondrial membrane, to swelling of the matrix and disruption of the cristae. This was rarely seen with other inducers of apoptosis. Rpr and Hid may directly cause altered mitochondrial morphology or could act indirectly through other proteins localized at the mitochondria (Abdelwahid, 2007).

The absence of mitochondrial permeabilization in cells treated with DIAP1 dsRNA indicates that the mitochondrial function of Rpr and Hid is independent of their ability to inhibit DIAP1. This is confirmed by data showing that expression of DeltaN-Rpr results in mitochondrial permeabilization despite the fact that this protein lacks the necessary motif to inhibit DIAP1 antiapoptotic activity. Taken together, these data demonstrate that Rpr and Hid have dual activities in the cell, both to inhibit DIAP1 and to permeabilize mitochondria. Data from other labs have suggested that Rpr is a multifunctional protein. The data confirm that Rpr has multiple proapoptotic activities in the fly (Abdelwahid, 2007).

The dual functionality of Rpr and Hid parallel the recently described role of C. elegans Egl-1 in mitochondrial damage. Egl-1 induces apoptosis by binding to Ced-9 to promote both the activation of the caspase Ced-3 and mitochondrial fragmentation. Similarly, Rpr and Hid bind to DIAP1, displacing active caspases and act on mitochondria to promote mitochondrial disruption. One difference between C. elegans and flies appears to be the requirement for caspase activity in the mitochondrial disruption. In C. elegans, Ced-3 is not required for fragmentation but is required for apoptosis in response to fragmentation. In Drosophila, caspase activity participates in the mitochondrial changes (Abdelwahid, 2007).

Two lines of evidence support a role for mitochondrial disruption in Drosophila apoptosis. First, Rpr and Hid must localize to mitochondria to elicit a full apoptotic response. Second, if mitochondrial disruption is blocked by inhibiting Dynamin related protein 1 (Drp1) expression, a decrease is seen in apoptosis. These data clearly indicate that mitochondrial localization of Rpr and Hid is required for a full apoptotic response in S2 cells. This agrees with previous data on Rpr and also with studies on a Grim mutant lacking a mitochondrial localization signal. Mitochondrial localization of Hid has been demonstrated in a heterologous system. In the Haining study, Hid killing was not compromised in the absence of mitochondrial localization, in contrast to the current observations in Drosophila cells. A role for mitochondrial localization is also supported by the finding that two mutant forms of Hid that lack mitochondrial localization in mammalian cells behave as weak loss-of-function alleles in the fly (Abdelwahid, 2007).

The mitochondrial fission protein Drp1 is implicated in mitochondrial disruption during apoptosis in yeast, nematodes, and mammals. The current data indicate a role for this protein in Rpr-induced and DNA-damage-induced mitochondrial disruption in S2 cells and in the embryo. Furthermore, the inhibition of mitochondrial disruption after Drp1 knockdown is correlated with a decrease in apoptosis, strongly suggesting that mitochondrial disruption contributes to the apoptotic response. It is interesting to note that Drp1 plays a conserved role in apoptosis in a wide variety of organisms but seems to function downstream of different pathways. In mammals, inhibition of Drp1 blocks apoptosis in response to activation of proapoptotic Bcl-2 family members. In C. elegans, Drp1 inhibition blocks endogenous death downstream of Egl1 and Ced9, also Bcl-2 family proteins. Even in yeast, the role of Drp1 in cell death can be modulated by Bcl-2 family proteins. Surprisingly, in flies, Drp1 appears to be acting downstream of a different family of apoptosis inducers, the RHG proteins. It remains to be seen whether a role for the fly Bcl-2 family proteins can be established in mitochondrial disruption (Abdelwahid, 2007).

Release of apoptogenic factors, most notably Cyt c, from the mitochondria is an essential step in most apoptosis in mammalian systems. However, the current work confirms the findings of others that Cyt c, although released from mitochondria by Rpr and Hid, is not important for Rpr or Hid killing. It should be noted that Cyt c has been shown to be important in some Drosophila developmental apoptosis. In these deaths, Hid is likely to act upstream of Cyt c release. If Cyt c release is required in some cells for Hid-mediated caspase activation, why not in S2 cells? It is possible that there are both Cyt c-dependent and -independent mechanisms for activating caspases, and these may be cell-type dependent. Recent data from mice carrying a nonapoptogenic form of Cyt c supports this model, since this study suggests that there is both Cyt c-dependent and -independent apoptosis during mouse development (Abdelwahid, 2007).

If release of Cyt c is not an essential step in apoptosis in most fly cells, is another apoptosis-inducing factor released during mitochondrial disruption? In mammalian cells, release of other mitochondrial proteins such as SMAC/Diablo, Omi/HTRA2, and AIF are proposed to contribute to apoptosis. There is some evidence that released mitochondrial factors do not contribute to caspase activation in the fly. Unlike in the mammalian system, mitochondrial lysates cannot activate caspases in fly cytoplasmic lysates. An alternative possibility is that mitochondrial disruption per se might contribute to apoptosis in the fly through inhibition of normal mitochondrial functions essential for cell viability. This might serve as a backup system, to maximize apoptosis in cells that express low levels of the RHG proteins. A similar role for mitochondrial disruption has been proposed in C. elegans (Abdelwahid, 2007).

In sum, it is concluded from these studies that Drosophila is not an outlier in evolution with regard to the involvement of mitochondria in the apoptotic process. Rather, the data indicate that mitochondrial changes contribute to Drosophila apoptosis. The findings suggest that the view of the role of mitochondria in cell death has to be broadened beyond the release of proapoptotic factors, to include the general disruption of mitochondria, ensuring that doomed cells have no chance of recovery. Such a model would fit not only the changes seen in mammalian mitochondria, but also those found in yeast, C. elegans, and flies as well (Abdelwahid, 2007).

Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper

Temporal patterning of neural progenitors is one of the core mechanisms generating neuronal diversity in the central nervous system. This study shows that, in the tips of the outer proliferation center (tOPC) of the developing Drosophila optic lobes, a unique temporal series of transcription factors not only governs the sequential production of distinct neuronal subtypes but also controls the mode of progenitor division, as well as the selective apoptosis of NotchOFF or NotchON neurons during binary cell fate decisions. Within a single lineage, intermediate precursors initially do not divide and generate only one neuron; subsequently, precursors divide, but their NotchON progeny systematically die through Reaper activity, whereas later, their NotchOFF progeny die through Hid activity. These mechanisms dictate how the tOPC produces neurons for three different optic ganglia. It is concluded that temporal patterning generates neuronal diversity by specifying both the identity and survival/death of each unique neuronal subtype (Bertet, 2014).

Although apoptosis is a common feature of neurogenesis in both vertebrates and Drosophila, the mechanisms controlling this process are still poorly understood. For instance, several studies in Drosophila have shown that, depending on the context, Notch can either induce neurons to die or allow them to survive during binary cell fate decisions. This is the case in the antennal lobes where Notch induces apoptosis in the antero-dorsal projecting neurons lineage (adpn), whereas it promotes survival in the ventral projecting neurons lineage (vPN). In both of these cases, the entire lineage makes the same decision whether the NotchON or NotchOFF cells survive or die. This suggests that, in this system, Notch integrates spatial signals to specify neuronal survival or apoptosis (Bertet, 2014).

This study shows that, during tOPC neurogenesis, neuronal survival is determined by the interplay between Notch and temporal patterning of progenitors. Indeed, within the same lineage, Notch signaling leads to two different fates: it first induces neurons to die, whereas later, it allows them to survive. This switch is due to the sequential expression of three highly conserved transcription factors-Dll/Dlx, Ey/Pax-6, and Slp/Fkh-in neural progenitors. These three factors have distinct functions, with Dll promoting survival of NotchOFF neurons, Ey inducing apoptosis of NotchOFF neurons, and Slp promoting survival of NotchON neurons. These data suggest that Ey induces death of NotchOFF neurons by activating the proapoptotic factor hid. Thus, Dll probably antagonizes Ey activity by preventing Ey from activating hid. The data also suggest that Notch signaling induces neuronal death by activating the proapoptotic gene rpr. Thus, Slp might promote survival of NotchON neurons by directly repressing rpr expression or by preventing Notch from activating it. In both cases, the interplay between Notch and Slp modifies the default fate of NotchON neurons, allowing them to survive. Further investigations will test these hypotheses and determine how Dll, Ey, Slp, and Notch differentially activate/repress hid and rpr (Bertet, 2014).

Although the tOPC and the main OPC have related temporal sequences, their neurogenesis is very different. This difference is in part due to the fact that newly specified tOPC neuroblasts express Dll, which controls neuronal survival, instead of Hth. Why do tOPC neuroblasts express Dll? The tOPC, which is defined by Wg expression in the neuroepithelium, is flanked by a region expressing Dpp. Previous studies have shown that high levels of Wg and Dpp activate Dll expression in the distal cells of the Drosophila leg disc. Wg and Dpp could therefore also activate Dll in the neuroepithelium and at the beginning of the temporal series in tOPC progenitors. Another difference between the main OPC and tOPC neurogenesis is that Ey and Slp have completely different functions in these regions. Indeed, unlike in the main OPC, Ey and Slp control the survival of tOPC neurons. This suggests that autonomous and/or nonautonomous signals interact with these temporal factors and modify their function in the tOPC (Bertet, 2014).

Finally, tOPC neuroblasts produce neurons for three different neuropils of the adult visual system, the medulla, the lobula, and the lobula plate. This ability could be due to the particular location of this region in the larval optic lobes. Indeed, the tOPC is very close to the two larval structures giving rise to the lobula and lobula plate neuropils-Dll-expressing neuroblasts are located next to the lobula plug, whereas D-expressing neuroblasts are close to the IPC. Interestingly, Dll and D neuroblasts specifically produce lobula plate neurons. This raises the possibility that these neuroblasts and/or the neurons produced by these neuroblasts receive signals from the lobula plug and the IPC, which instruct them to specifically produce lobula plate neurons. These nonautonomous signals could also modify the function of Ey and Slp in the tOPC (Bertet, 2014).

In summary, this study demonstrates that temporal patterning of progenitors, a well-conserved mechanism from Drosophila to vertebrates, generates neural cell diversity by controlling multiple aspects of neurogenesis, including neuronal identity, Notch-mediated cell survival decisions, and the mode of intermediate precursor division. In the tOPC temporal series, some factors control two of these aspects (Ey), whereas others have a specialized function (Dll, Slp, and D). This suggests that temporal patterning does not consist of a unique series of transcription factors controlling all aspects of neurogenesis but instead consists of multiple superimposed series, each with distinct functions (Bertet, 2014).


Polar cells are pairs of specific follicular cells present at each pole of Drosophila egg chambers. They are required at different stages of oogenesis for egg chamber formation and establishment of both the anteroposterior and planar polarities of the follicular epithelium. Definition of polar cell pairs is a progressive process since early stage egg chambers contain a cluster of several polar cell marker-expressing cells at each pole, while as of stage 5, they contain invariantly two pairs of such cells. Using cell lineage analysis, it has been demonstrated that these pre-polar cell clusters have a polyclonal origin and derive specifically from the polar cell lineage, rather than from that giving rise to follicular cells. In addition, selection of two polar cells from groups of pre-polar cells occurs via an apoptosis-dependent mechanism and is required for correct patterning of the anterior follicular epithelium of vitellogenic egg chambers. Prevention of pre-polar cell death and subsequent generation of supernumerary polar cells may lead to production of an excess of signaling molecules, such as Unpaired, and alteration of endogenous morphogen gradients which could explain why both squamous cells and border cells exhibit aberrant behavior when pre-polar cell death is blocked (Besse, 2003).

Thus, each pair of mature polar cells derives from a pool of precursor pre-polar cells within which supernumerary cells are eliminated via an apoptosis-dependent mechanism. This mechanism probably requires both caspase activity and the 'death' gene reaper, since death is inhibited by ectopic expression of the bacculoviral p35 protein and is associated with specific induction of reaper expression. However, whereas the self-death machinery appears to be evolutionary conserved, a wide range of distinct signaling mechanisms can be used to elicit apoptosis. Cellular interactions within or without the pre-polar cell cluster may also be crucial for regulation of the selective pre-polar cell loss. In the present study, no correlation could be made between pre-polar cell position and cell removal, at least for apoptosis events occurring after egg chamber budding. It would be interesting nonetheless to examine Notch signaling as a survival factor in this system. Indeed, induction of Notch loss-of-function clones in prefollicular cells is associated with absence of polar cells. Conversely, egg chambers with terminal clones expressing an activated form of Notch contain up to 6 polar cell marker-positive cells. Such phenotypes, interpreted as reflecting a role for Notch signaling in polar cell specification, could also correspond to a Notch-dependent control of apoptosis within the pre-polar cell lineage (Besse, 2003).

Apoptotic cells can induce compensatory cell proliferation through the JNK and the Wingless signaling pathways: DIAP1 antagonists reaper and hid can activate the JNK pathway which in turn is required for inducing wg and cell proliferation

In many metazoans, damaged and potentially dangerous cells are rapidly eliminated by apoptosis. In Drosophila, this is often compensated for by extraproliferation of neighboring cells, which allows the organism to tolerate considerable cell death without compromising development and body size. Despite its importance, the mechanistic basis of such compensatory proliferation remains poorly understood. Apoptotic cells are shown to express the secretory factors Wingless and Decapentaplegic. When cells undergoing apoptosis were kept alive with the caspase inhibitor p35, excessive nonautonomous cell proliferation was observed. Significantly, Wg signaling is necessary and, at least in some cells, also sufficient for mitogenesis under these conditions. Finally, evidence is provided that the DIAP1 antagonists reaper and hid can activate the JNK pathway and that this pathway is required for inducing wg and cell proliferation. These findings support a model where apoptotic cells activate signaling cascades for compensatory proliferation (Ryoo, 2004).

To investigate how the inhibition of diap1 may lead to mitogen expression, attention was focused on Dronc and the Jun N-terminal Kinase (JNK) pathway. Dronc has been implicated in compensatory proliferation, and its activity can be inhibited by the expression of droncDN. In addition, the JNK signaling pathway was considered as a candidate, since its activity is known to correlate with many forms of stress-provoked apoptosis, including disruption of morphogens, cell competition, and rpr expression. In Drosophila, the JNK pathway can be effectively blocked by the expression of puckered (puc), which encodes a phosphatase that negatively regulates JNK (Ryoo, 2004).

To induce patches of undead cells, wing imaginal discs were generated with mosaic clones expressing hid and p35. 48 hr after induction, these imaginal discs contained hid-expressing clones that autonomously induced wg. Using this experimental setup, it was asked whether additional expression of either droncDN or puc would block wg induction in undead cells. When droncDN was coexpressed, a subset of the hid-expressing population was still able to induce wg. In contrast, when puc was coexpressed, wg induction by hid was almost completely blocked. These results provide evidence that the JNK pathway is required for wg induction under these conditions but fail to uncover a similar requirement for Dronc (Ryoo, 2004).

To independently investigate the role of puc and droncDN in compensatory proliferation, the size of wing discs harboring undead cells was measured and they were compared with those of the sibling controls. Under the experimental conditions, wing discs harboring hid- and p35-expressing clones were on average 53% larger than their sibling controls. Coexpression of puc within these undead clones significantly limited growth, resulting in only a small increase in wing disc size that was not statistically significant. In contrast, coexpression of droncDN did not limit growth. Wing size measurements also correlated with the degree of wg induction. The larger size of discs harboring hid- and p35-expressing cells is not due simply to extra cell survival: (1) these undead cells are derived from the normal lineage; (2) the size of wing discs expressing hid, p35, and puc serves as a control. In this case, although a large number of undead cells were generated, no significant increase in disc size was observed, in stark contrast to the discs expressing hid and p35 only. It is concluded that the JNK pathway is required for the nonautonomous growth promoting activity of the undead cells (Ryoo, 2004).

To confirm a role of puc in imaginal disc growth, rpr and p35 werecoexpressed in wild-type and puc−/+ imaginal discs. Like hid, rpr is a DIAP1 antagonist, but with a weaker cell killing activity when overexpressed in imaginal disc cells. In a puc+/+ background, a small amount of ectopic wg expression was observed, indicative of rpr's weaker DIAP1 inhibiting activity. In contrast, ectopic wg expression was strongly enhanced in puc−/+ discs. Because the puc allele used, pucE69, also acts as a lacZ reporter, JNK pathway induction could be monitored simultaneously. wg induction in undead cells correlates very well with puc-lacZ expression, with a stronger induction at the center of the wing pouch. These results further support the role of JNK in the induction of wg (Ryoo, 2004).

Next to be tested was whether the reduction of puc had an effect on apoptosis-induced cell proliferation. Whereas puc−/+ discs expressing only p35 had BrdU incorporation similar to wild-type discs, coexpression of rpr and p35 in puc−/+ led to a significant increase in BrdU incorporation. Also, the size of these discs were on average 41% larger than those coexpressing rpr and p35 in a puc+/+ background. Taken together, these results show that diap1 inhibition leads to JNK activation and that JNK activity promotes wg induction and cell proliferation (Ryoo, 2004).

To directly test if JNK signaling can activate wg and dpp expression, hepCA, a constitutively active form of hemipterous (hep), the Drosophila JNK kinase was conditionally expressed. Expression of hepCA causes induction of wg-lacZ within 22 hr and to a lesser extent also dpp-lacZ. These ß-gal-expressing cells shifted basally and were apoptotic as assayed by anti-active caspase-3 antibody labeling. Hid protein levels were also elevated in these cells. Significantly, since p35 was not use to block apoptosis in this experiment, this demonstrates that wg and dpp can be induced not only in undead cells, but also in 'real' apoptotic cells (Ryoo, 2004).

This study provides evidence that the central apoptotic regulators can control the activity of mitogenic pathways. In particular, inhibition of DIAP1, either via expression of Reaper and Hid or by mutational inactivation, leads to the induction of the putative mitogens wg and dpp. When apoptosis was initiated through DIAP1 inhibition but cells were kept alive by blocking caspases, the resulting 'undead cells' exhibited strong mitogenic activity and stimulated tissue overgrowth. Inhibiting wg signaling with a conditional TCFDN blocked cell proliferation in imaginal discs, indicating that wg has an essential mitogenic function. Finally, evidence was provided that the JNK pathway mediates mitogen expression and imaginal disc overgrowth in response to rpr and hid. Based on these results, it is proposed that apoptotic cells actively signal to induce compensatory proliferation. DIAP1 inhibits both caspases as well as dTRAF1. According to this model, when DIAP1 is inhibited in response to cellular injury, the JNK pathway is activated and wg/dpp are induced in apoptotic cells. Secretion of these factors stimulates growth of proliferation-competent neighboring cells and leads to compensatory proliferation (Ryoo, 2004).

This study provides clear genetic evidence that diap1 is involved in compensatory proliferation. Overall, similar results were obtained with hypomorphic diap1 alleles (diap122-8s, diap133-1s), a null allele (diap1th5), and inactivation of diap1 by expression of Reaper and Hid. However, whereas expression of p35 effectively blocked apoptosis of diap122-8s/22-8s cells and in response to Reaper/Hid, it only partially suppressed the death of diap1th5/th5 cells. Consequently, the generation of undead cells was less efficient with the diap1th5 mutation. Moreover, these results suggest that the JNK pathway transduces the signal to activate mitogen expression and cell proliferation. Since IAPs have been shown to ubiquitylate TRAFs in both mammals and Drosophila and since no evidence was found for Dronc in growth promotion, it is attractive to speculate that JNK is regulated through direct DIAP1/TRAF1 interaction (Ryoo, 2004).

An important unresolved question is why compensatory proliferation is seen only in response to cellular injury, but not during normal developmental apoptosis. In particular, inactivation of DIAP1 by Reaper, Hid, and Grim is restricted not only to injury-provoked apoptosis, but also underlies most developmental cell deaths. One possible explanation is that activation of the JNK pathway is key to mitogenic signaling of apoptotic cells. Consistent with this idea, the JNK pathway is activated in response to tissue stress and injury, but not during developmental apoptosis. Furthermore, this study shows that JNK signaling can induce the expression of wg/dpp and nonautonomous cell proliferation. Therefore, it is possible that robust JNK activation and compensatory proliferation require the combined input of stress and apoptotic signals (Ryoo, 2004).

Effects of Mutation or Deletion

Virtually all programmed cell death that normally occurs during Drosophila embryogenesis is blocked in embryos homozygous for a small deletion that includes the reaper gene. Mutant embryos contained many extra cells and fail to hatch, but many other aspects of development appear quite normal. Deletions that include reaper also protect embryos from apoptosis caused by x-irradiation and developmental defects. However, high doses of X-rays induce some apoptosis in mutant embryos, and the resulting corpses are phagocytosed by macrophages. These data suggest that the basic cell death program is intact although it was not activated in mutant embryos. The DNA encompassed by the deletion has been cloned and the reaper gene has been identified on the basis of the ability of cloned DNA to restore apoptosis to cell death defective embryos in germ line transformation experiments. The reaper gene appears to encode a small peptide that shows no homology to known proteins, and Reaper messenger RNA is expressed in cells destined to undergo apoptosis (White, 1994).

Cell death was examined within lineages in the midline of Drosophila embryos. Approximately 50% of cells within the anterior, middle and posterior midline glial (MGA, MGM and MGP) lineages die by apoptosis after separation of the commissural axon tracts. Glial apoptosis is blocked in embryos deficient for reaper, where greater than wild-type numbers of midline glia (MG) are present after stage 12. Quantitative studies reveal that MG death follows a consistent temporal pattern during embryogenesis. Apoptotic MG are expelled from the central nervous system and were subsequently engulfed by phagocytic hemocytes. MGA and MGM survival is apparently dependent upon proper axonal contact (Sonnenfeld, 1995).

Mesectodermal cells (MEC) give rise to the first nervous system cells to become postmitotic and differentiate into identified cell types. Existing models of MEC lineage determination predict that there are between 2 and 6 midline glia (MG) precursors. A study was undertaken to clarify the origin of supernumerary MGs in embryos that lack reaper, head involution defective and grim, three closely linked proapoptotic genes. Drosophila embryos deficient for programmed cell death produce 9 midline glia (MG) in addition to the wild-type complement of 3.2 MG/segment. More than 3 of the supernumerary MG derive from the MGP (MG posterior) lineage and the remainder from the MGA/MGM (MG anterior and middle) lineage. There is one unidentified additional neuron in the mesectoderm of embryos deficient for apoptosis. The supernumerary MG are not diverted from other lineages nor do they arise from an altered pattern of mitosis. Instead, these MG appear to arise from a normally existing pool of 12 precursor cells, a number larger than anticipated by earlier studies. During normal development, MG survival is dependent upon signaling to the Drosophila EGF receptor. The persistence of supernumerary MG in embryos deficient for apoptosis does not alter the spatial pattern of Drosophila EGF receptor signaling. The number and position of MG that express genes dependent upon EGF receptor function, such as pointed or argos, are indistinguishable from wild type. Thus supernumary MG in H99 mutant embryos express EGF receptor but apparently receive insufficient receptor activation to express genes dependent on EGF receptor signaling. Genes of the spitz group are required for Drosophila EGF receptor function. Surviving MG in spitz group/H99 double mutants continue to express genes characteristic of the MG, but the cells fail to differentiate into ensheathing glia and are displaced from the nerve cord. It remains to be clarified how the MG progenitors are selected from the MEC population (Dong, 1997).

In Drosophila, the chromosomal region 75C1-2 contains at least three genes (reaper (rpr), head involution defective (hid), and grim) that have important functions in the activation of programmed cell death. To better understand how cells are killed by these genes, a well defined set of embryonic central nervous system midline cells have been used that normally exhibit a specific pattern of glial cell death. Most of the developing midline glia die and are quickly phagocytosed by migrating macrophages, whereas none of the ventral unpaired median neurons die during embryogenesis. Both rpr and hid are expressed in dying midline cells; the normal pattern of midline cell death requires the function of multiple genes in the 75C1-2 interval. The P[UAS]/P[Gal4] system was used to target expression of rpr and hid to midline cells. Targeted expression of rpr or hid alone is not sufficient to induce ectopic midline cell death. However, expression of both rpr and hid together rapidly induces ectopic midline cell death, resulting in axon scaffold defects characteristic of mutants with abnormal midline cell development. Midline-targeted expression of the baculovirus p35 protein, a caspase inhibitor, blocks both normal and ectopic rpr- and hid-induced cell death. Taken together, these results suggest that rpr and hid are expressed together and cooperate to induce programmed cell death during development of the central nervous system midline (Zhou, 1997).

To investigate whether rpr expression is sufficient to induce apoptosis, transgenic flies were generated that express rpr complementary DNA or the rpr open reading frame in cells that normally live. Transcription of rpr from a heat-inducible promoter rapidly causes wide-spread ectopic apoptosis and death of the fly. Ectopic overexpression of rpr in the developing retina results in eye ablation. The occurrence of cell death is highly sensitive to the dosage of the transgene. Because cell death induced by the protein encoded by rpr can be blocked by the baculovirus p35 protein, RPR appears to activate a death program mediated by a ced-3/ICE (interleukin-1 converting enzyme)-like protease (White, 1996).

The amnioserosa is an extraembryonic, epithelial tissue that covers the dorsal side of the Drosophila embryo. The initial development of the amnioserosa is controlled by the dorsoventral patterning genes. A group of genes, which is referred to as the U-shaped-group (ush-group), is required for maintenance of the amnioserosa tissue once it has differentiated. Using several molecular markers, amnioserosa development was examined in the ush-group mutants: u-shaped (ush), hindsight (hnt), serpent and tail-up (tup). The amnioserosa in these mutants is specified correctly and begins to differentiate as in wild type. However, following germ-band extension, there is a premature loss of the amnioserosa. This cell loss is a consequence of programmed cell death (apoptosis), carried out through the action of Reaper, in ush, hnt and srp, but not in tup mutants (Frank, 1996).

Nurse cells are cleared from the Drosophila egg chamber by apoptosis. DNA fragmentation begins in nurse cells at stage 12, following the completion of cytoplasm transfer from the nurse cells to the oocyte. During stage 13, nurse cells increasingly contain highly fragmented DNA and disappear from the egg chamber concomitantly with the formation of apoptotic vesicles containing highly fragmented nuclear material. In mutant egg chambers that fail to complete cytoplasm transport from the nurse cells (dumpless chambers), DNA fragmentation is markedly delayed and begins during stage 13, when the majority of cytoplasm is lost from the nurse cells. These data suggest the presence of cytoplasmic factors in nurse cells that inhibit the initiation of DNA fragmentation. The dumpless mutants studied include cheerio and kelch, which both have aberrant ring canal morphology that does not permit cytoplasm to pass easily from the nurse cells to the oocytes. The chickadee, singed and quail gene products are necessary for the proper formation of cytoplasmic actin filament bundles that form in nurse cells at stage 10B, just prior to the onset of cytoplasmic transport. reeper and hid are expressed in nurse cells beginning at stage 9 and continuing throughout stage 13. The grim transcript is not expressed as strongly as rpr or hid. The negative regulators DIAP1 and DIAP2 are also transcribed during oogenesis. However, germline clones homozygous for the deficiency Df(3)H99, which deletes rpr, hid and grim, undergo oogenesis in a manner morphologically indistinguishable from wild type, indicating that genes within this region are not necessary for apoptosis in nurse cells (Foley, 1998).

Ectopic death of retinal cells results from ectopic expression of rpr and grim in eye discs. Reduction of the level of Death related ced-3/Nedd2-like protein (Dredd) in Drosophila eyes reduces the level of ectopic cell death. Heterozygosity at the Dredd locus suppresses apoptosis in transgenic models of reaper- and grim-induced cell killing, demonstrating that levels of Dredd product can modulate signaling triggered by these death activators (Chen, 1998).

The Drosophila larva modulates its pattern of locomotion when exposed to light. Modulation of locomotion can be measured as a reduction in the distance traveled and by a sharp change of direction when the light is turned on. When the light is turned off this change of direction, albeit significantly smaller than when the light is turned on, is still significantly larger than in the absence of light transition. Mutations that disrupt adult phototransduction disrupt a subset of these responses. In larvae carrying these mutations the magnitude of change of direction when the light is turned on is reduced to levels indistinguishable from that recorded when the light is turned off, but it is still significantly higher than in the absence of any light transition. Similar results are obtained when these responses are measured in strains where the larval photoreceptor neurons have been ablated by mutations in the glass (gl) gene or by the targeted expression of the cell death gene head involution defective (hid). A mutation in the homeobox gene sine oculis (so) that ablates the larval visual system, or the targeted expression of the reaper (rpr) cell death gene, abolishes all responses to light detected as a change of direction. The existence of an extraocular light perception that does not use the same phototransduction cascade as the adult photoreceptors is proposed. The results indicate that this novel visual function depends on the blue-absorbing rhodopsin Rh1 and is specified by the so gene (Busto, 1999).

The role of Ras signaling was studied in the regulation of cell death during Drosophila eye development. Overexpression of Argos, a diffusible inhibitor of the EGF receptor and Ras signaling, causes excessive cell death in developing eyes at pupal stages. The Argos-induced cell death is suppressed by coexpression of the anti-apoptotic genes p35, diap1, or diap2 in the eye as well as by the Df(3L)H99 chromosomal deletion that lacks three apoptosis-inducing genes, reaper, head involution defective (hid) and grim. Transient misexpression of the activated Ras1 protein (Ras1V12) later in pupal development suppresses the Argos-induced cell death. Thus, Argos-induced cell death seems to have resulted from the suppression of the anti-apoptotic function of Ras. Conversely, cell death induced by overexpression of Hid is suppressed by gain-of-function mutations of the genes coding for MEK and ERK. These results support the idea that Ras signaling functions in two distinct processes during eye development, first triggering the recruitment of cells and later negatively regulating cell death (Sawamoto, 1998).

Fork head prevents apoptosis and promotes cell shape change during formation of the Drosophila salivary glands

The secretory tubes of the Drosophila salivary glands are formed by the regulated, sequential internalization of the primordia. Secretory cell invagination occurs by a change in cell shape, which includes basal nuclear migration and apical membrane constriction. In embryos mutant for fork head, the secretory primordia are not internalized and secretory tubes do not form. Secretory cells of fkh mutant embryos undergo extensive apoptotic cell death following the elevated expression of the apoptotic activator genes, reaper and head involution defective. The secretory cell death can be rescued in the fkh mutants and the rescued cells still do not invaginate. The rescued fkh secretory cells undergo basal nuclear migration in the same spatial and temporal pattern as in wild-type secretory cells, but do not constrict their apical surface membranes. These findings suggest at least two roles for fkh in formation of the embryonic salivary glands: an early role in promoting survival of the secretory cells, and a later role in secretory cell invagination, specifically in the constriction of the apical surface membrane (Myat, 2000).

The apoptotic cell death observed in the early secretory primordia of fkh mutants indicates that fkh is required for secretory cell survival. Thus, secretory cells may fail to invaginate in fkh mutants simply because the cells are dead or dying. Indeed, the ectopic expression of rpr and hid, but not grim, is effective in inducing early secretory cell death, which if extensive enough, prevents internalization. Alternatively, fkh may have two separate roles in the salivary gland, one to promote cell survival and another to control invagination of the primordia. To distinguish between these possibilities, the apoptotic secretory cell death was rescued in fkh mutants by generating embryos that were mutant for fkh and also carried Df(3L)H99, a small deficiency that deletes rpr, hid and grim (fkh H99). Normal salivary glands are formed in embryos homozygous for Df(3L)H99 (H99). In the fkh H99 embryos, dCREB-A staining is detected in the entire secretory placode at early stages This staining completely disappears by embryonic stage 13, suggesting that either fkh is required to maintain dCREB-A expression or that the fkh H99 secretory cells are still dying. To address this issue, the expression of another secretory marker protein, PS, whose expression is thought to be fkh independent, was analyzed. In wild-type embryos, PS is expressed at high levels in the salivary glands throughout embryogenesis. In fkh mutant embryos, PS is initially expressed in the entire secretory placode, and at reduced levels in the surviving ring of secretory cells. Importantly, PS is expressed to very high levels in all secretory cells throughout embryogenesis in the fkh H99 embryos. Nonetheless, the PS-expressing cells in the fkh H99 embryos are not internalized and remain at their site of origin on the ventral surface. Therefore, in addition to its early role in promoting secretory cell survival, Fkh is also required for the invagination of the secretory cells (Myat, 2000).

An essential role for the caspase dronc in developmentally programmed cell death in Drosophila

To examine genetic interactions between Nedd2-like caspase (Dronc) and other apoptotic pathway genes, two UAS-dronc transgenic lines (#23 and #80) were chosen that result in relatively low lethality when crossed to GMR-GAL4 and a recombinant second chromosome was generated for each of these transgenes with GMR-GAL4. When GMR-GAL4 UAS-dronc#80 was crossed to wild type w1118 flies at 25°C, adult flies that exhibited slightly rough and mottled eyes were observed. A similar phenotype has been observed in previous studies and has been shown to be due to ablation of the pigment and photoreceptor cells. Similar results were observed for GMR-GAL4, UAS-dronc#23. This phenotype became more severe when expression of dronc via GMR-GAL4 was increased by raising the temperature to 29°C. Because this eye phenotype can be modified by increasing the expression of dronc, it provided a dosage-sensitive system for examining genetic interactions between dronc and other genes of the apoptosis pathway. To test this further, whether co-expression of the baculovirus caspase inhibitor P35 from the GMR enhancer was able to suppress the eye phenotype of GMR-dronc at 29°C was examined. Co-expression of GMR-p35 dramatically improves the eye ablation phenotype of GMR-dronc. Thus, in this system, Dronc is sensitive to P35 in the Drosophila eye (Quinn, 2000).

Whether the GMR-dronc eye phenotype is sensitive to halving the dosage of the various Drosophila apoptosis-regulatory genes was tested. To assess whether the GMR-dronc eye phenotype is sensitive to the dosage of the H99 genes (reaper, hid, and grim), GMR-dronc flies were crossed to a deficiency removing the H99 genes, Df(3L)H99, at 29°C. The H99 deficiency dominantly suppressed the GMR-dronc eye phenotype. Thus, the cell death-inducing activity of dronc is sensitive to the dosage of the H99 genes. Furthermore, halving the dosage of dronc using a deficiency modifies the ablated eye phenotype of GMR-hid and GMR-rpr, suggesting that dronc is downstream of hid and rpr. To determine whether there was a genetic interaction with dronc and dark, whether decreasing the dosage of dark modified the eye phenotype of GMR-dronc at 29°C was examined. Three different P-element alleles of dark (darkCD4, darkCD8, and darkl(2)k11502) show suppression of the GMR-dronc eye phenotype, indicating that Dark plays a role in promoting Dronc-induced cell death in the eye. Halving the dosage of diap1 using deficiencies or the specific allele thread5 dominantly enhances the GMR-dronc eye phenotype at 25°C . In addition, these diap1 mutations dominantly enhance the lethality associated with GMR-dronc, resulting in at least 10-fold lower numbers of GMR-dronc/+; Df(diap1)/+ adult flies than expected. In contrast, a deficiency removing diap2 showed no effect on the GMR-dronc phenotype, and no lethal effects were observed. Thus diap1, but not a deficiency removing diap2, shows a dosage-sensitive interaction with dronc. By contrast, ectopic expression of diap1 or diap2 from the GMR promoter shows suppression of the GMR-dronc ablated eye phenotype, although GMR-diap2 results in much weaker suppression than GMR-diap1. Thus, both Diap1 and Diap2 are capable of directly or indirectly blocking Dronc-mediated cell death (Quinn, 2000).

The Drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by DIAP1, HID, and GRIM

Mutations that remove DRONC are not available. Therefore, to examine a possible role for DRONC as a cell death effector a form of DRONC, DRONCC318S, was generated in which the active site cysteine was altered to serine. Expression of similar forms of other caspases results in a suppression of caspase activity and caspase-dependent cell death. This may occur as a result of interaction of DRONCC318S with the Drosophila homolog of the caspase-activating protein Apaf-1, thus preventing the Drosophila Apaf-1 from binding to wild type DRONC and promoting its activation in a manner similar to that described for mammalian Apaf-1 and caspase-9. Transgenic Drosophila were generated in which DRONCC318S was expressed under the control of a promoter, known as GMR, that drives transgene expression specifically in the developing fly eye. The eyes of these flies, known as GMR-DRONCC318S flies, appear similar to those of wild type flies. To assay the ability of DRONCC318S to block cell death, GMR-DRONCC318S flies were crossed to flies overexpressing rpr (GMR-rpr), hid (GMR-hid), or grim (GMR-grim) under the control of the same promoter. GMR-driven expression of rpr, hid, or grim results in a small eye phenotype due to activation of caspase-dependent cell death. However, flies coexpressing GMR-DRONCC318S and one of the cell death activators showed a dramatic suppression of the small eye phenotype, indicating that cell death had been suppressed. The possibility cannot be ruled out that this suppression is a result of DRONCC318S forming nonproductive interactions with the Drosophila Apaf-1 that block its ability to activate other long prodomain caspases such as DCP-2/DREDD. However, these possibilities notwithstanding, these results suggest that DRONC activity is important for bringing about rpr-, hid-, and grim-dependent cell death (Hawkins, 2000).

reaper is required for neuroblast apoptosis during Drosophila development

Developmentally regulated apoptosis in Drosophila requires the activity of the reaper (rpr), grim and head involution defective (hid) genes. The expression of these genes is differentially regulated, suggesting that there are distinct requirements for their proapoptotic activity in response to diverse developmental and environmental inputs. To examine this hypothesis, a mutation that removes the rpr gene was generated. In flies that lack rpr function, most developmental apoptosis is unaffected. However, the central nervous systems of rpr null flies are very enlarged. This is due to the inappropriate survival of both larval neurons and neuroblasts. Importantly, neuroblasts rescued from apoptosis remain functional, continuing to proliferate and to generate many extra neurons. Males mutant for rpr exhibit behavioral defects resulting in sterility. Although both the ecdysone hormone receptor complex and p53 directly regulate rpr transcription, rpr was found to play a limited role in inducing apoptosis in response to either of these signals (Peterson, 2002).

A specific loss-of-function rpr mutation is essential to dissect the role of rpr in developmental apoptosis. The isolation of such a mutation has proved challenging; previous attempts to use chemical mutagens to create lethal or visible point mutations in the H99 region only resulted in the isolation of hid alleles, prompting the use of an alternative strategy. Males carrying a P element located in the non-stop gene, 225 kb proximal to rpr, were irradiated and candidate genomic deletions were identified. Loss of rpr genomic sequence was assayed by single embryo PCR. A single rpr deletion, XR38, was isolated. As assessed by in situ hybridization, homozygous XR38 embryos show no rpr expression, while no quantitative or qualitative changes in grim or hid mRNA expression were detected. The XR38 deletion is large, removing several genes, and XR38 homozygotes are lethal. However, flies of the genotype XR38/H99 are likely to be homozygously deleted for the rpr gene alone, since the proximal breakpoint of H99 lies only 15 kb from rpr, and no other predicted genes lie between rpr and this breakpoint. The distal breakpoint of the XR38 deletion lies between rpr and grim and was found to map more than 30 kb distal to rpr and more than 20 kb proximal to grim. There are no predicted genes between rpr and grim (Peterson, 2002).

The steroid hormone ecdysone regulates programmed cell death at metamorphosis and in the adult central nervous system. It is interesting to note that rising levels of ecdysone initiate degeneration in the larval midgut and salivary glands, while falling levels of the hormone are required for the death of the type II neurons in the newly eclosed adult. These different responses to ecdysone may be mediated by different isoforms of the receptor, because the doomed larval midgut and salivary gland cells express primarily the B1 isoform of the receptor, while the doomed neurons express the A isoform. Although these receptor isoforms share both ligand binding and DNA binding domains, they show functional differences (Peterson, 2002).

Expression of rpr is rapidly induced in the salivary glands after the prepupal pulse of ecdysone. A binding site for the ecdysone receptor complex is present in the rpr promoter, which is essential for rpr expression in the doomed salivary gland. Type II neurons also express rpr before they undergo apoptosis. Thus rpr is a strong candidate to be important in both of these deaths. It was found that salivary gland death is not affected in rpr mutant pupae, while the death of type II neurons is significantly inhibited. This disparity may be explained by the differences in the other genes expressed in these tissues. In the salivary glands, the induction of rpr expression is rapidly followed by hid expression. In this tissue, as in the embryo, it is likely that hid activity compensates for the absence of rpr. Expression of the caspase Dronc is also increased in response to ecdysone in these tissues. High levels of Dronc can induce apoptosis and may contribute to the histolysis of these tissues (Peterson, 2002).

In contrast to the findings in salivary gland and midgut, the ecdysone-regulated death of EcR-A-expressing neurons in the adult nervous system is inhibited in the absence of rpr. These cells express rpr and grim but not hid prior to their death. This expression pattern may be a common feature of neuronal tissue, since hid expression is not detectable in the embryonic central nervous system outside of the midline glia. In the adult nervous system, grim function is apparently not sufficient to induce apoptosis in many of the type II neurons. However, in the embryonic nervous system there is significant apoptosis in the absence of rpr. At this stage grim activity must be sufficient for most neural apoptosis, with the important exception of the death of the neuroblasts (Peterson, 2002).

In flies, as in mammalian tissues, cells undergo apoptosis in response to DNA damage, and this apoptosis requires the activity of the transcription factor p53. In flies, the expression of a dominant negative form of p53 largely inhibits X-ray-induced apoptosis. Drosophila p53, can directly bind to a radiation-inducible enhancer in the rpr promoter. These data strongly suggest that p53 induces apoptosis in response to DNA damage by activating rpr expression. Unexpectedly, no suppression of p53-induced apoptosis is detected in rpr null animals. However, X-ray-induced apoptosis is reduced in the absence of rpr. These data indicate that rpr is an important regulator of apoptosis induced by DNA damage, and that other apoptotic regulators are also involved. When p53 is strongly overexpressed in the eye, these other targets must be sufficient to overcome the absence of rpr. The functions of hid and/or grim are doubtless also involved in DNA damage-induced death, since X-ray-induced apoptosis is very strongly inhibited in H99 embryos (Peterson, 2002).

Two striking phenotypes are found in rpr mutants: the adult central nervous system in both males and females is enlarged, especially the abdominal part of the ventral nerve cord, and males are sterile. The hyperplasia of the CNS results in part from the abnormal persistence of some larval neurons in the adult ventral ganglia. More importantly, neuroblasts also survive inappropriately in rpr mutants. In the wild-type animal, most of the neuroblasts in the abdominal neuromeres die at the end of embryogenesis, while in the rpr mutant many of these neuroblasts survive and proliferate. The progeny of these ectopic neuroblast divisions differentiate into neurons that are integrated into the adult nervous system. Why are the neuroblasts particularly sensitive to the loss of rpr? One possibility is that rpr is the only apoptosis regulator expressed in these cells. hid is not expressed in the embryonic nervous system. Although widespread expression of grim is detected in the embryonic CNS, it is not known if neuroblasts express grim. A distinct expression of other apoptotic factors could also account for the specific requirement for rpr in neuroblast apoptosis (Peterson, 2002).

Mutations in the Drosophila Apaf1 homolog dark also result in enlargement of the larval CNS. This increased size results at least in part from the survival and proliferation of a few neuroblasts in the abdominal neuromeres. This implicates dark as being required for some rpr-dependent apoptosis. It is interesting to note that dark mutations, like rpr mutations, cause significant male sterility (Peterson, 2002).

The sterility of rpr mutant males appears to be behavioral, as they are unable to copulate, although other courtship behaviors appear normal. The cause of the male copulation defect is unknown, but it is interesting to speculate that the reduction in normal cell death in the abdominal neuromeres is in some way responsible for this behavioral deficit. Indeed, the focus of male copulatory behavior has been mapped to the abdominal nervous system by mosaic mapping techniques. The presence of additional neurons in the nervous system of rpr mutants might interfere with the organization of the appropriate neurons into a functional neural circuit required for copulation. Alternatively, the neural circuit in the CNS might be properly constructed but the presence of additional motorneurons might prevent coordinated movement of the abdomen during copulation (Peterson, 2002).

In C. elegans, the majority of developmental apoptosis occurs in the nervous system. In worms that are mutant for the genes ced-3 or ced-4, and thus lacking all apoptosis, there are extra neurons. However, ectopic cell proliferation has not been reported in these mutant animals. Neural hyperplasia is also seen in mice carrying engineered mutations in caspases 3 and 9 and in the Apaf1 caspase activator. A detailed analysis of brain development in caspase 3 knockout mice shows a marked increase in proliferating neuroblasts, similar to what is seen in rpr mutants. These mutants provide a graphic example of how normal development can be particularly disrupted when apoptosis of a stem cell population is inhibited, and these cells continue to proliferate. In the future, the rpr mutant flies will provide a unique model to explore the fate of ectopic neural stem cells and their progeny in the context of the nervous system (Peterson, 2002).

Lineage-specific cell death in postembryonic brain development of Drosophila

The Drosophila central brain is composed of thousands of neurons that derive from approximately 100 neuroblasts per hemisphere. Functional circuits in the brain require precise neuronal wiring and tight control of neuronal numbers. How this accurate control of neuronal numbers is achieved during neural development is largely unclear. Specifically, the role of programmed cell death in control of cell numbers has not been studied in the central brain neuroblast lineages. This study focusses on four postembryonic neuroblast lineages in the central brain identified on the basis that they express the homeobox gene engrailed (en). For each lineage, the total number of adult-specific neurons generated was determined, as well as number and pattern of en-expressing cells. Programmed cell death has a pronounced effect on the number of cells in the four lineages; approximately half of the immature adult-specific neurons in three of the four lineages are eliminated by cell death during postembryonic development. Moreover, programmed cell death selectively affects en-positive versus en-negative cells in a lineage-specific manner and, thus, controls the relative number of en-expressing neurons in each lineage. Furthermore, evidence is provided that Notch signaling is involved in the regulation of en expression. Based on these findings, it is concluded that lineage-specific programmed cell death plays a prominent role in the generation of neuronal number and lineage diversity in the Drosophila brain (Kumar, 2009).

In postembryonic CNS development of holometabolous insects such as flies, a combination of programmed cell death and neuronal process re-innervation allows the larval nervous system to reorganize and innervate new body structures. During metamorphosis many adult-specific neurons in the ventral ganglia are targeted by programmed cell death, particularly in abdominal segments. Furthermore, extensive cell death occurs during postembryonic development in the insect visual system, where cells are overproduced and those that do not make the appropriate targets are eliminated by apoptosis. By contrast, very little is currently known about the prevalence and functional roles of programmed cell death in development of the insect adult central brain (Kumar, 2009).

This report identifies four neuroblast lineages in the postembryonic central brain and finds that programmed cell death occurs in all four lineages, albeit to different extents. Whereas cell death plays only a minor role in the medial cluster MC1 lineage, it has dramatic effects in anterior cluster (AC), posterior cluster (PC) and medial cluster MC2 lineages, in which nearly half of the adult-specific neuronal progeny are programmed to die during larval development. It is noteworthy that the adult-specific neurons targeted by cell death are generated during larval development and are eliminated before their respective neuroblasts stop proliferating (12-24 hours after pupal formation). Because the cell death reported here occurs before neuronal differentiation, it is probably not involved in events of brain reorganization that take place during metamorphosis (Kumar, 2009).

Another central feature of the cell death events demonstrated here is that none of the four lineages is completely eliminated by cell death; all four neuroblasts and a significant number of their neuronal progeny survive at the end of larval development, and these neuronal progeny are largely present in the adult. In this sense, the programmed cell death reported here is likely to be functionally different from the cell death observed in the ventral ganglia, where the neuroblast itself undergoes apoptosis, regulating neuronal numbers in the abdominal segments (Kumar, 2009)

These experiments indicate that programmed cell death plays a prominent role in determining lineage-specific features; if cell death is blocked the total neuronal number increases in all four lineages and the number of en-expressing neurons increases in AC, PC and MC2. Furthermore, the axonal projection pattern of H99 mutant (deleting rpr, hid and grim) and Notch mutant en-expressing lineages was examined, comparing them to wild type. Both cell death defective H99 and Notch mutant PC lineages showed an additional projection that was not present in the wild type, whereas the other three H99 lineages did not appear to change drastically in their projection patterns. In conclusion, programmed cell death appears to contribute to the cellular diversity of neuronal lineages in the central brain (Kumar, 2009).

Studies on neuroblast lineages in the developing ventral ganglia indicate that proliferating neuroblasts generate a largely invariant clone of neural cells. In general, each neuroblast division produces a distinctly fated GMC, and each GMC division produces two sibling progeny of different fates. There is some evidence that the fate of these progeny is controlled by the parental GMC; the two siblings are restricted to a pair of different cell fates, with one sibling adopting an 'A' fate and the other adopting a 'B' fate. This, in turn, has led to a model in which a neuroblast lineage can be thought of as composed of two hemilineages, with one hemilineage comprising 'A'-fate cells and the other hemilineage comprising 'B'-fate cells. It is thought that an interaction between Notch and Numb is responsible for generating distinct neural fates of the two GMC daughter cells, with a loss of Notch or Numb resulting in reciprocal cell-fate duplication. However, Notch signaling does not appear to confer a particular fate; rather, it acts generically as a mechanism to enable two siblings to acquire different fates, and other developmental control genes that are inherited from the specific parental GMC are thought to be instrumental in determining the final identity of each progeny (Kumar, 2009).

Findings on lineage-specific cell death support a comparable model in which all four brain neuroblasts can generate one en-positive hemilineage and one en-negative hemilineage. In this model, programmed cell death is then targeted in a lineage-specific manner to either the en-negative hemilineage (AC, PC), or the en-positive hemilineage (MC2), or neither hemilineage (MC1). Alternatively, en-positive and en-negative neurons in the lineages could be generated in a temporal fashion and subsequently en-positive or en-negative neurons could be eliminated in a lineage-specific manner. However, the results suggest that this is unlikely. In particular, in the PC lineage, more than 80% of the two-cell clones examined were composed of one en-positive and one en-negative cell. If the above did occur, a significant number of two-cell en-positive clones should have been obtained along with two-cell clones comprising one en-positive and one en-negative neuron. Similar analysis of single and two cell clones in the other three en lineages is further required to confirm the occurrence of hemilineage-specific programmed cell death (Kumar, 2009).

Based on these experimental results, it was postulate that Notch signaling is an important generic mechanism underlying generation of the two different hemilineages, as in the absence of Notch signaling, cell-fate duplication of GMC siblings occurs. Indeed, analysis of Notch loss-of-function neuroblast clones suggests that in the absence of Notch signaling most of the neurons in the four lineages acquire an en-positive cell fate. Alternatively, in the four lineages examined, being positive for en may be the default state of the cells, and Notch induces secondary fate by repressing en in subsets of cells in each lineage. These en-positive neurons then appear to survive or undergo programmed cell death depending on the lineage-specific context. However, it remains to be seen whether Notch itself acts on the apoptotic machinery, independent of en (Kumar, 2009).

This study, used en as a molecular marker to identify four lineages in the postembryonic central brain. Might en itself be functionally involved in regulating programmed cell death in these lineages? For the PC lineage, there is some indication that the total clone size is reduced by approximately half in en loss-of-function mutants, compared with wild type. Although this suggests that en may be involved in promoting survival of en-positive neurons in PC (and probably AC), it does not explain the role of en in the MC2 lineage, where it would have to play an opposing role, as en-positive neurons die in this lineage. Thus, en could act either as a pro-apoptotic or an anti-apoptotic factor, depending on the lineage-specific context. Moreover, a direct genetic interaction between en and the apoptotic machinery remains to be investigated. As en is known to have multiple interactions with other proteins, a complex regulatory network involving target proteins of en may be responsible for regulating apoptosis in a lineage-specific manner. Further analysis of interactions with such target proteins is necessary to reveal the full regulatory network in more detail (Kumar, 2009).

The lineage-specific effects of cell death and of Notch signaling in AC and PC are distinctly different from those observed in MC1 or MC2 lineages. However, when compared with each other, many aspects of AC and PC lineages are similar. In wild type, both lineages consist of similar numbers of adult-specific neurons, and the majority (approximately 80%) of these neurons are positive for en, whereas neuroblasts and GMCs are negative for en in both lineages. Blocking cell death results in a substantial (approximately double) increase in total cell number in both lineages, and this increase is almost exclusively due to an increase in the number of surviving en-negative neurons in both lineages. Moreover, loss of Notch function causes a marked increase in the number of surviving en-positive neurons without affecting the number of en-negative neurons in both lineages. The only significant difference between AC and PC lineages observed in this study is that the AC lineage is located in the protocerebrum, whereas the PC lineage is located in the deutocerebrum (Kumar, 2009).

What might be responsible for these similarities in the AC and PC neuroblast lineages? There is some evidence for the existence of serially homologous neuroblasts in the fly brain and VNC. In the VNC, serially homologous neuroblasts, defined by comparable time of formation, similar positions in the neuromeric progenitor array and similar expression of developmental control genes, such as segment polarity genes, dorsoventral patterning genes and other molecular markers, can give rise to almost identical cell lineages. This suggests that similar regulatory interactions take place during development of serially homologous neuroblasts and their neural lineages. A comparison of molecular expression patterns in neuroblasts from different neuromeres of the brain and ventral ganglia suggests that several of them might be serial homologs of each other. For example, neuroblasts NB5-6 in the abdominal, thoracic and subesophageal ganglia have been proposed to be homologous to NBDd7 in the deutocerebrum and NBTd4 in the tritocerebrum (Kumar, 2009).

Given the remarkable similarities in AC and PC neuroblast lineages, it is possible that the protocerebral AC lineage and the deutocerebral PC lineages represent serial homologs. If this is the case, then investigations of the cellular and molecular mechanisms that control their lineage-specific development should be useful for understanding of how regionalized neural diversity in the brain evolves from a basic metameric ground state. However, as neither the combination of developmental control genes expressed in AC and PC neuroblasts nor the position of the two brain neuroblasts in their neuromeres of origin are currently known in sufficient detail, further experiments are needed before the issue of serial homology can be resolved for these brain neuroblast lineages (Kumar, 2009).

salvador promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines

So far, relatively few mechanisms have been shown to be capable of regulating both cell proliferation and cell death in a coordinated manner. In a screen for Drosophila mutations that result in tissue overgrowth, salvador (sav), a gene that promotes both cell cycle exit and cell death was identified. Elevated Cyclin E and DIAP1 levels are found in mutant cells, resulting in delayed cell cycle exit and impaired apoptosis. Salvador contains two WW domains and binds to the Warts protein kinase. The human ortholog of salvador (hWW45) is mutated in several cancer cell lines. Thus, salvador restricts cell numbers in vivo by functioning as a dual regulator of cell proliferation and apoptosis (Tapon, 2002).

In wild-type eyes, excessive interommatidial cells are eliminated by a wave of apoptosis that is evident in 38 hr pupal retinas. Even in sav mutant clones, cell proliferation, as assessed by BrdU incorporation, has ceased within 24 hr APF. When mosaic retinas were examined 38 hr APF, cell death is mostly confined to the wild-type portions of the retina. Thus, the apoptotic cell deaths that are part of normal retinal development appear to require sav function (Tapon, 2002).

Several studies have shown that Hid and Rpr activate caspases by another mechanism in which they induce the autoubiquitination of DIAP1 and target it for degradation by the proteasome. DIAP1 levels are markedly elevated in sav clones in the larval eye disc and remain elevated in the interommatidial cells in mutant clones in the pupal eye disc. Thus, increased levels of DIAP1 in sav cells may be able to overcome the effect of many proapoptotic signals (Tapon, 2002).

To examine DIAP1 RNA levels, in situ hybridization was used to examine 20 wild-type discs and 20 mutant discs. The presence of sav (GFP-) clones in the mutant discs was confirmed by examining the discs by fluorescence microscopy prior to hybridization. There is a modest level of DIAP1 RNA expression posterior to the furrow in both populations of discs and no evidence of increased DIAP1 RNA in the discs containing sav clones. Thus, at least at this level of detection, the increased DIAP1 expression in sav cells does not appear to result from increased transcription (Tapon, 2002).

In wild-type eye discs, DIAP1 protein is expressed at higher levels posterior to the morphogenetic furrow. DIAP1 protein levels are downregulated by GMR-rpr or, to a lesser extent, by GMR-hid expression. In sav mutant clones expressing GMR-rpr, DIAP1 protein levels remain elevated. Similar results are observed with GMR-hid. Thus, neither GMR-rpr nor GMR-hid appears capable of downregulating the elevated levels of DIAP1 sufficiently in sav clones to activate caspases (Tapon, 2002).

Expression of hid or reaper (rpr) in the eye imaginal disc results in activation of the effector caspase Drice (Ice). An antibody that recognizes the cleaved (activated) form of Drice was used to stain eye discs expressing GMR-hid or GMR-rpr. In wild-type cells, Drice is activated by GMR-hid or GMR-rpr. However, in clones of sav tissue, Drice activation by either GMR-hid or GMR-rpr is almost completely blocked. These experiments indicate that sav blocks activation of Drice by both rpr and hid (Tapon, 2002).

A mutant form of Hid (Hid-Ala5) is resistant to inactivation by MAP kinase phosphorylation. GMR-hid-Ala5 is a more potent inducer of cell death than is GMR-hid, as assessed by the extent of Drice activation in the eye disc. Cell death induced by GMR-hid-Ala5 is only partially blocked in sav clones, indicating that the increased potency of Hid-Ala-5 may be able to overcome increased DIAP1 levels (Tapon, 2002).

Elevated DIAP1 levels are likely to underlie the absence of the developmentally regulated apoptosis in sav clones in the pupal retina as well as the resistance to hid-induced and rpr-induced apoptosis in the larval imaginal disc. The elevated DIAP1 levels appear to result from alterations in posttranscriptional regulation of DIAP1 expression. Recent work has shown that both Rpr and Hid can downregulate DIAP1 levels either by promoting the autoubiquitination of DIAP1 or by causing a generalized inhibition of translation that especially impacts proteins with a short half-life such as DIAP1. Either of these mechanisms is likely to be less efficient in cells that already have elevated levels of DIAP1 (Tapon, 2002).

Drosophila nemo is an essential gene involved in the regulation of programmed cell death

Nemo-like kinases define a novel family of serine/threonine kinases that are involved in integrating multiple signaling pathways. They are conserved regulators of Wnt/Wingless pathways, which may coordinate Wnt with TGF-mediated signaling. Drosophila nemo was identified through its involvement in epithelial planar polarity, a process regulated by a non-canonical Wnt pathway. Ectopic expression of Nemo using the Gal4-UAS system results in embryonic lethality associated with defects in patterning and head development. An analyses of nemo phenotypes of germline clone-derived embryos is described. Lethality is observed associated with head defects and reduction of programmed cell death and it is concluded that nemo is an essential gene. Data is presented showing that nmo is involved in regulating apoptosis during eye development, based on both loss of function phenotypes and on genetic interactions with the pro-apoptotic gene reaper. Genetic data from the adult wing are presented that suggest the activity of ectopically expressed Nemo can be modulated by Jun N-terminal kinase (JNK) signaling. Such an observation supports the model that there is cross-talk between Wnt, TGFß and JNK signaling at multiple stages of development (Mirkovic, 2002).

It has also been determined that Nemo plays a role in apoptosis during retinal development, since nmo loss of function alleles contain additional secondary and tertiary pigment cells, which are normally removed through programmed cell death during retinal maturation. The ectopic expression of the pro-apoptotic gene reaper in the developing eye disc results in elevated levels of cell death as evidenced by a severely reduced and abnormal adult eye. Heterozygosity for several alleles of nmo can suppress the phenotype resulting in a larger adult eye. The ability of nmo to suppress the cell death caused by GMR-rpr expression supports the idea that both rpr and nmo are involved in promoting cell death and may act in parallel pathways that converge on regulation of the caspases. The data strongly implicate Nemo in the modulation of cell death within the retina and are consistent with observations in the embryo (Mirkovic, 2002).

The glial cell undergoes apoptosis in the microchaete lineage of Drosophila

Apoptosis plays a major role in vertebrate and invertebrate development. The adult Drosophila thoracic microchaete is a mechanosensory organ whose development has been extensively studied as a model of how cell division and cell determination intermingle. This sensory organ arises from a cell lineage that produces a glial cell and four other cells that form the organ. In this study, using an in vivo approach as well as fixed material, it has been shown that the glial cell undergoes nucleus fragmentation shortly after birth. Fragmentation was blocked after overexpression of the caspase inhibitor p35 or removal of the pro-apoptotic genes reaper, hid and grim, showing that the glial cell undergoes apoptosis. Moreover, it seems that fragments are eliminated from the epithelium by mobile macrophages. Forcing survival of the glial cells induces precocious axonal outgrowth but does not affect final axonal patterning and connectivity. However, under these conditions, glial cells do not fragment but leave the epithelium by a mechanism that is reminiscent of cell competition. Finally, evidence is presented showing that glial cells are committed to apoptosis independently of gcm and prospero expression. It is suggested that apoptosis is triggered by a cell autonomous mechanism (Fichelson, 2003).

Buffy, a Drosophila Bcl-2 protein, has anti-apoptotic and cell cycle inhibitory functions

Bcl-2 family proteins are key regulators of apoptosis. Both pro-apoptotic and anti-apoptotic members of this family are found in mammalian cells, but only the pro-apoptotic protein Debcl has been characterized in Drosophila. Buffy, the second Drosophila Bcl-2-like protein, is a pro-survival protein. Ablation of Buffy by RNA interference leads to ectopic apoptosis, whereas overexpression of buffy results in the inhibition of developmental programmed cell death and gamma irradiation-induced apoptosis. Buffy interacts genetically and physically with Debcl to suppress Debcl-induced cell death. Genetic interactions suggest that Buffy acts downstream of Reaper, Grim and Head involution defective, and upstream of the apical caspase Dronc. Furthermore, overexpression of buffy inhibits ectopic cell death in diap1 (th5) mutants. Taken together these data suggest that Buffy can act downstream of Rpr, Grim and Hid to block caspase-dependent cell death. Overexpression of Buffy in the embryo results in inhibition of the cell cycle, consistent with a G1/early-S phase arrest. These data suggest that Buffy is functionally similar to the mammalian pro-survival Bcl-2 family of proteins (Quinn, 2003).

To examine genetic interactions between Buffy and other apoptotic pathway genes, Glass multimer reporter (GMR)-GAL4 was used to drive the UAS-buffy transgene in the posterior region of the third instar eye imaginal disc. Recombinants of the UAS-buffy transgene with GMR-GAL4 on the second chromosome, when heterozygous (GMR-GAL4:UAS-buffy/+), produce flies with eyes of wild-type appearance. Similarly, ectopic expression of the Drosophila inhibitor of apoptosis, DIAP1, using the GMR driver results in normal-appearing adult eyes. However, expression of diap1 can inhibit apoptotic phenotypes generated by overexpression of caspases, rpr and hid. GMR-diap1 also suppresses the GMR-GAL4/+;UAS-debcl/+ ablated eye phenotype, consistent with the notion that Debcl induces apoptosis by functioning upstream of DIAP1-dependent caspase inhibition (Quinn, 2003).

A GH3-like domain in reaper is required for mitochondrial localization and induction of IAP degradation

Reaper is a potent pro-apoptotic protein originally identified in a screen for Drosophila mutants defective in apoptotic induction. Multiple functions have been ascribed to this protein, including inhibition of IAPs (inhibitors of apoptosis); induction of IAP degradation; inhibition of protein translation; and when expressed in vertebrate cells, induction of mitochondrial cytochrome c release. Structure/function analysis of Reaper has identified an extreme N-terminal motif that appears to be sufficient for inhibition of IAP function. This domain, although required for IAP destabilization, is not sufficient. Moreover, a small region of Reaper, similar to the GH3 domain of Grim, has been identified that is required for localization of Reaper to mitochondria, induction of IAP degradation, and potent cell killing. Although a mutant Reaper protein lacking the GH3 domain is deficient in these properties, these defects can be fully rectified by appending either the C-terminal mitochondrial targeting sequence from Bcl-xL or a homologous region from the pro-apoptotic protein HID. Together, these data strongly suggest that IAP destabilization by Reaper in intact cells requires Reaper localization to mitochondria and that induction of IAP instability by Reaper is important for the potent induction of apoptosis in Drosophila cells (Olson, 2003).

Integrin-dependent apposition of Drosophila extraembryonic membranes promotes morphogenesis and prevents JNK-pathway dependent, reaper independent, anoikis

Two extraembryonic tissues form early in Drosophila development. One, the amnioserosa, has been implicated in the morphogenetic processes of germ band retraction and dorsal closure. The developmental role of the other, the yolk sac, is obscure. By using live-imaging techniques, intimate interactions are reported between the amnioserosa and the yolk sac during germ band retraction and dorsal closure. These tissue interactions fail in a subset of myospheroid (mys: ßPS integrin) mutant embryos, leading to failure of germ band retraction and dorsal closure. The Drosophila homolog of mammalian basigin (EMMPRIN , CD147) -- an integrin-associated transmembrane glycoprotein -- is highly enriched in the extraembryonic tissues. Strong dominant genetic interactions between basigin and mys mutations cause severe defects in dorsal closure, consistent with basigin functioning together with ßPS integrin in extraembryonic membrane apposition. During normal development, JNK signaling is upregulated in the amnioserosa, as midgut closure disrupts contact with the yolk sac. Subsequently, the amnioserosal epithelium degenerates in a process that is independent of the reaper, hid, and grim cell death genes. In mys mutants that fail to establish contact between the extraembryonic membranes, the amnioserosa undergoes premature disintegration and death. It is concluded that intimate apposition of the amnioserosa and yolk sac prevents anoikis of the amnioserosa. Survival of the amnioserosa is essential for germ band retraction and dorsal closure. It is hypothesized that during normal development, loss of integrin-dependent contact between the extraembryonic tissues results in JNK-dependent amnioserosal disintegration and death, thus representing an example of developmentally programmed anoikis (Reed, 2004).

Physical interaction of the amnioserosa and yolk sac has been shown to play a crucial role in both germ band retraction and dorsal closure of the embryo. βPS integrin mediates extraembryonic membrane interactions that are required for survival of the amnioserosa. Anoikis of the amnioserosa occurs during normal development after closure of the midgut disrupts integrin-dependent apposition of the amnioserosa and yolk sac. In mys mutants, failure to establish apposition of extraembryonic membranes leads to premature anoikis of the amnioserosa. A possible role for JNK signaling and the reaper/hid/grim cell death genes in amnioserosal anoikis during normal development was investigated (Reed, 2004).

It is possible to visualize a subset of the amnioserosal cells as acridine orange positive either before they leave the tube or shortly thereafter. Both acridine orange staining and engulfment by hemocytes are hallmarks of dying cells. To determine whether death of amnioserosal cells might be reaper dependent, it was asked whether reaper expression could be visualized in the amnioserosal cells prior to or after extrusion. No reaper-expressing cells were detected. To further test whether amnioserosal cell death might be reaper dependent, the H99 deficiency [Df(3L)H99] was used; this deficiency removes the reaper, head involution defective (hid), and grim genes, and the amnioserosa with anti-HNT antibody was visualized. If amnioserosal death were reaper dependent, one would expect HNT-positive cells to persist in H99 mutants when compared with wild-type. Such persistence does not occur. While it is conceivable that HNT expression is downregulated in a persistent amnioserosa, the simplest interpretation of these data is that death of the amnioserosa is reaper independent. This conclusion is consistent with the recent suggestion that Drosophila embryos have a caspase-independent cell engulfment system, which is still operative in H99 mutants (Reed, 2004).

It has been shown that loss of integrin-dependent contact between cells and the extracellular matrix leads to cell death, a process referred to as anoikis. Anoikis is promoted by the Jun amino-terminal kinase (JNK) pathway. Previous analyses have shown that JNK signaling in the amnioserosa is downregulated prior to dorsal closure. In those analyses, puckered-lacZ expression was used as a read-out of JNK signaling, and it was shown that relocation of JUN and FOS proteins from the nucleus to the cytoplasm of amnioserosal cells correlates with downregulation of JNK signaling. While JNK signaling is downregulated in the amnioserosa prior to dorsal closure, JNK signaling is upregulated in this tissue as dorsal closure approaches completion. Thus, reactivation of JNK signaling in the amnioserosa follows loss of integrin-dependent apposition of the amnioserosa and yolk sac membrane and precedes amnioserosal disintegration and death. These data are consistent with the hypothesis that midgut closure disrupts integrin-dependent apposition of the amnioserosa and yolk sac, thus inducing JNK signaling in the amnioserosa and its subsequent anoikis (Reed, 2004).

It remains to be determined whether disintegration and death of the amnioserosa during normal development is caused solely by loss of contact with the yolk sac (i.e., is nonautonomously induced) versus whether signals from cell types other than the yolk -- or even an amnioserosa-autonomous program -- also play a role. For example, it is possible that upregulation of JNK signaling in the amnioserosa is independent of loss of contact with the yolk sac. Analysis of mutants lacking a midgut provide a test of this possibility: if disintegration and death of the amnioserosa occur even when apposition with the yolk sac is maintained, signals from other cell types or amnioserosa-autonomous processes would be implicated (Reed, 2004).

The specific role of JNK signaling in amnioserosal anoikis is difficult to assess because downregulation of JNK signaling in the amnioserosa and up-regulation of JNK signaling in the leading edge of the epidermis are required for dorsal closure. Thus JNK pathway mutants stall morphogenesis prior to dorsal closure, making it impossible to assess a possible later role. Expression of dominant-negative JNK specifically in the amnioserosa only later in development, when closure is almost complete, will be necessary to rigorously test the role of JNK activation in amnioserosal anoikis (Reed, 2004).

It is concluded that the extraembryonic tissues of Drosophila play a crucial role in directing embryonic morphogenesis. Close apposition of the yolk sac membrane and the basal cell membranes of the amnioserosa is dependent on βPS integrin. This intimate membrane association is required to promote survival and to prevent anoikis of the amnioserosa. The amnioserosa then directs germ band retraction and dorsal closure through physical contacts and/or signaling. Disintegration and death of the amnioserosa after closure of the epidermis and midgut correlates with upregulation of JNK signaling in the amnioserosa, is independent of reaper/hid/grim function, and is likely to represent the first example of developmentally programmed anoikis in Drosophila (Reed, 2004).

Ionizing radiation induces caspase-dependent but Chk2- and p53-independent cell death in Drosophila melanogaster

Ionizing radiation (IR) can induce apoptosis via p53, which is the most commonly mutated gene in human cancers. Loss of p53, however, can render cancer cells refractory to therapeutic effects of IR. Alternate p53-independent pathways exist but are not as well understood as p53-dependent apoptosis. Studies of how IR induces p53-independent cell death could benefit from the existence of a genetically tractable model. In Drosophila, IR induces apoptosis in the imaginal discs of larvae, typically assayed at 4-6 hr after exposure to a LD50 dose. In mutants of Drosophila Chk2 or p53 homologs, apoptosis is severely diminished in these assays, leading to the widely held belief that IR-induced apoptosis depends on these genes. This study shows that IR-induced apoptosis still occurs in the imaginal discs of chk2 and p53 mutant larvae, albeit with a delay. This phenomenon is a true apoptotic response because it requires caspase activity and the chromosomal locus that encodes the pro-apoptotic genes reaper, hid, and grim. Chk2- and p53-independent apoptosis is IR dose-dependent and is therefore probably triggered by a DNA damage signal. It is concluded that Drosophila has Chk2- and p53-independent pathways to activate caspases and induce apoptosis in response to IR. This work establishes Drosophila as a model for p53-independent apoptosis, which is of potential therapeutic importance for inducing cell death in p53-deficient cancer cells (Wichmann, 2006).

The Drosophila homologs of Chk2 and p53 are required, not for induction of apoptosis, but for timely induction of apoptosis in response to irradiation. Radiation-induced cell death still occurs in chk2 and p53 mutants, albeit with a delay. Four lines of evidence support the idea that this delayed cell death is apoptosis rather than necrosis: (1) it is detected by staining with AO, which has been shown to stain apoptotic but not necrotic cells; (2) it accompanies activation of caspases, a hallmark of apoptosis but not necrosis; (3) it requires caspase activity, which is required for apoptosis but not necrosis, and (4) it requires the chromosomal locus encoding the proapoptosis genes rpr, hid, and grim, whose protein products are required to inhibit DIAP1 and activate caspases. These results indicate that there is a Chk2-/p53-independent pathway that commits damaged cells to apoptosis and utilizes many of the same downstream components as the Chk2-/p53-dependent apoptosis pathway (Wichmann, 2006).

Two lines of evidence support the idea that DNA damage is the signal that induces Chk2-/p53-independent apoptosis after exposure to ionizing radiation. First, the amount of Chk2-/p53-independent apoptosis appears to increase with IR dose. This dose dependence suggests that the amount of DNA damage is what induces Chk2-/p53-independent apoptosis but does not rule out the contribution of other damages that result from IR. Second, higher levels of Chk2-/p53-independent apoptosis are observed when the ability to repair DNA is compromised, as in mei-41, p53 double mutants. Collectively, these data suggest that DNA damage caused by x-rays induces Chk2-/p53-independent apoptosis (Wichmann, 2006).

IR-induced apoptosis in chk2 and p53 mutants shows a temporal delay. IR-induced apoptosis is also delayed in H99 heterozygotes, possibly because H99 heterozygotes contain half the gene dose of the proapoptotic Smac/Diablo orthologs and it may take longer for the proapoptotic gene products to accumulate to the point of an apoptosis-stimulating threshold. IR induced increase in the transcripts of rpr and hid, two of the H99-encoded genes, still occurred in chk2 (rpr and hid) and p53 (hid) mutants, but to lower levels (for rpr) and after a delay. Therefore, apoptosis may be delayed in chk2 and p53 mutants because proapoptotic gene products take longer to accumulate to a threshold in the absence of Chk2 or p53 regulation. The data showing that IR-induced apoptosis is further delayed in a chk2, H99/+ double mutant, compared with a chk2 single mutant, support this claim. Furthermore, the results suggest the existence of at least another signaling pathway that does not operate through Chk2 or p53, but nonetheless links the same signal (DNA damage) to a similar outcome (accumulation of H99-encoded gene products) (Wichmann, 2006).

RT-PCR experiments revealed interesting differences in the identity and onset of induction of proapoptotic genes in chk2 and p53 mutants. rpr and hid are induced at 4 hr after irradiation in chk2 mutants, whereas hid and skl are induced between 12 and 18 hr after irradiation in p53 mutants. The basis for these differences is not understood. More detailed time courses as well as deletion analysis of the H99 locus to determine the contribution of each proapoptotic gene to Chk2-/p53-independent apoptosis needs to be performed to address these issues (Wichmann, 2006).

The data presented in this study establish Drosophila as a model for studying p53-independent apoptosis. p53 is the most commonly mutated gene in human cancers. Loss of p53 poses an immense clinical problem because p53-deficient cancer cells no longer stimulate p53-dependent apoptosis in response to radiation or genotoxic chemotherapy drugs. In this scenario, p53-independent apoptotic pathways become key for inducing cancerous cells to die because they provide potential therapeutic targets. In mammals, a p53-independent apoptosis pathway that is mediated by p73, another member of the p53 family, has been identified. In Drosophila, Dmp53 is the only known p53 family member. Therefore, the p53-independent apoptosis that was identified and characterized in this article is likely to represent a previously unknown process. An important goal in the future will be to dissect the Chk2-/p53-independent pathway that links DNA damage to the proapoptotic genes of the H99 locus (Wichmann, 2006).

Several candidates were tested and eliminated as regulators of Chk2-/p53-independent cell death. Mei-41 (ATR) is not required for Chk2-/p53-independent cell death because mei-41, p53 double mutants actually exhibit more cell death than p53 alone. Recent work showed that ectopic induction of eiger, a TNF ligand homolog, can induce apoptosis in Drosophila. Chk2-/p53-independent cell death still occurs in p53, eiger double mutants, suggesting that the TNF pathway is not involved in the induction of cell death characterized in this study. Work in mammalian cells showed that overexpression of c-Myc can induce p53-independent apoptosis. Chk2-/p53-independent apoptosis still occurs in Dmyc, p53 double mutants, indicating that Dmyc is not required for this response (Wichmann, 2006).

A classical genetic screen may identify components of the Chk2-/p53-independent apoptosis pathway, as well as testing more candidates, such as the transcription factor de2f1, grapes (DmChk1), DmATM, and genes required for autophagy. Autophagic cell death, in which a cell lyses itself, occurs during Drosophila metamorphosis to lyse polyploid tissues such as the salivary glands and the fat body and provide nutrients for diploid cells of the imaginal discs; autophagy has been described in larvae only in the polyploid cells and only in response to starvation. Nonetheless, autophagy shares characteristics with apoptosis, including being detectable by AO staining and being dependent on caspases and the H99 locus, and for this reason remains a formal possibility (Wichmann, 2006).

In conclusion, studies have shown that IR-induced apoptosis in two key models for apoptosis, C. elegans and Drosophila, depends on p53. This study has provided evidence that, contrary to the accepted view, Chk2 and p53 are not required for radiation-induced cell death in Drosophila. Furthermore, normal timing of apoptosis that depends on Chk2 and p53 is also not required for ensuring survival after irradiation. Radiation-induced cell death that is independent of Chk2 and p53 depends on radiation dose, has characteristics of apoptosis and is likely to rely on a novel mechanism(s) because no other members of the p53-family are known in Drosophila. This work is the first to establish Drosophila as a model for p53-independent apoptosis. Identification of genes required for Chk2-/p53-independent cell death in Drosophila is of potential therapeutic value because protein products of their human homologs may represent novel targets that can be activated clinically to eliminate p53-deficient cancer cells (Wichmann, 2006).

Programmed cell death in the embryonic central nervous system of Drosophila

Although programmed cell death (PCD) plays a crucial role throughout Drosophila CNS development, its pattern and incidence remain largely uninvestigated. This study provides a detailed analysis of the occurrence of PCD in the embryonic ventral nerve cord (VNC). The spatio-temporal pattern of PCD was traced and the appearance of, and total cell numbers in, thoracic and abdominal neuromeres of wild-type and PCD-deficient H99 mutant embryos were compared. Furthermore, the clonal origin and fate of superfluous cells in H99 mutants was examined by DiI labeling almost all neuroblasts, with special attention to segment-specific differences within the individually identified neuroblast lineages. These data reveal that although PCD-deficient mutants appear morphologically well-structured, there is significant hyperplasia in the VNC. The majority of neuroblast lineages comprise superfluous cells, and a specific set of these lineages shows segment-specific characteristics. The superfluous cells can be specified as neurons with extended wild-type-like or abnormal axonal projections, but not as glia. The lineage data also provide indications towards the identities of neuroblasts that normally die in the late embryo and of those that become postembryonic and resume proliferation in the larva. Using cell-specific markers it was possible to precisely identify some of the progeny cells, including the GW neuron, the U motoneurons and one of the RP motoneurons, all of which undergo segment-specific cell death. The data obtained in this analysis form the basis for further investigations into the mechanisms involved in the regulation of PCD and its role in segmental patterning in the embryonic CNS (Rogulja-Ortmann. 2007).

In this analysis of PCD distribution it was found that, macroscopically, the CNS of wt and PCD-deficient (H99) embryos do not show large differences. These observations indicate that the supernumerary cells do not disturb developmental events in the CNS of H99 embryos, such as cell migration and axonal pathfinding. The glial cells mostly find their appropriate positions accurately. The DiI-labeled NB lineages were, in the majority of cases, easily identifiable based on their shape, position and axonal pattern, despite the supernumerary cells. The FasII pattern showed that the axonal projections form and extend along their usual paths. In fact, the supernumerary cells themselves are capable of differentiating i.e. expressing marker genes and extending axons, as shown by clones of several NBs and by cell marker expression analysis in H99 (e.g. NB7-3) (Rogulja-Ortmann. 2007).

It has been shown that a large number of CNS cells undergo PCD during embryonic development. The distribution of activated Caspase-3-positive cells in wt embryos suggests that the death of some cells is under tight spatial and temporal control, as revealed by their regular, segmentally repeated occurrence. Other dying cells were rather randomly distributed, suggesting a certain amount of developmental plasticity. The overall counts of Caspase-3-positive cells give an estimate of the numbers of dying cells at a given time. They indicate that PCD becomes evident in the CNS at stage 11 and is most abundant in the late embryo (from stage 14). It is however difficult to estimate the total number of apoptotic cells throughout CNS development by anti-Caspase-3 labeling, because the cell corpses are removed fairly quickly. Therefore the total number of cells were counted per thoracic and abdominal hemineuromere in the late embryo. Comparison between stage 16 and stage 17 wt embryos indicates that 25-30 % of all cells are removed in both tagmata after stage 16, which in turn suggests that the total percentage of removed cells must be high, since PCD occurs at high levels already from stage 14 on. In comparison to the developing nervous system of C. elegans, where PCD removes about 10% of cells, and of mammals, where this number can be as high as 50-90%, PCD in the fly CNS appears to show an intermediate prevalence. This lends support to the hypothesis of an increasing contribution of PCD in shaping more advanced nervous systems during evolution (Rogulja-Ortmann. 2007).

Comparisons between wt and H99 reveal, as expected, a greater number of cells in both tagmata of H99 embryos (151% increase in the thorax and 162% in the abdomen at stage 17). These additional cells in H99 may reflect the total number of cells normally undergoing cell death until stage 17. However, there is a large variability in the total number of cells, especially within the H99 strain. In wt embryos, it seems to be more pronounced in the thorax and at stage 17, which might be a consequence of variable amounts of PCD occurring until this stage. The even higher variability within the H99 strain (both in thorax and abdomen) is likely to reflect variable numbers of additional cell divisions. The great majority of abdominal NBs are normally removed by PCD after they have generated their embryonic progeny, whereas in the thoracic neuromeres most of the NBs enter quiescence at the end of embryogenesis and continue dividing as postembryonic NBs in larval stages. Thus, there are few mitoses occurring in the wt CNS from stage 16 onwards. BrdU labeling experiments revealed a high number of BrdU-positive cells in some H99 embryos injected at early stage 17. It is assumed that these are progeny of mitotic NBs and/or GMCs that survive and continue dividing, generating cells that do not exist in wt. Clones obtained by DiI labeling in H99 confirm this conclusion. The finding that surviving cells divide already in the embryo complement results that showed that, in reaper mutants, NBs in the abdominal neuromeres survive and generate progeny in larval stages (Rogulja-Ortmann. 2007).

Among the DiI-labeled clones in H99 embryos, very few NB lineages were obtained which did not differ from their wt counterparts. The majority contained, as expected, supernumerary cells. In some cases axons projected by these cells could be identified, showing that they are specified as neurons. In fact, in three cases (NB4-2, NB5-3 and NB7-3), these additional cells were found to be specified as motoneurons. As additional axons within a fascicle were generally difficult to identify, it is possible that these are not the only lineages which make additional motoneurons in H99. Whether these cells are normally born and apoptose, or originate from additional divisions of surviving NBs or GMCs, cannot be determined from these experiments, but similar observations have been made for both cases. It is interesting that none of these cells, regardless of their origin, are specified as glia. No additional glia were observed in the NB clones in H99 embryos, and equal numbers of Repo-expressing glial cells were found in wt and H99. It is concluded that PCD occurs almost exclusively in neurons and/or undifferentiated cells, and that lateral glia are not produced in excess numbers in the embryo. Furthermore, because it is likely that NBs, which normally die, stay in a late temporal window in H99, one could speculate that NBs in this window normally do not give rise to glia. These results are not in agreement with the notion that LG are overproduced, and their numbers adjusted through axon contact. Occasional apoptotic LG have been observed and it is possible that the current method of counting does not allow a resolution fine enough to account for an occasional additional Repo-positive cell in H99 embryos. However, if LG were consistently overproduced, a higher number of glia in would be expected H99 embryos. It is assumed that LG cell death may reflect a small variability in the number of cells needed, and not a general mechanism for adjusting glial cell numbers (Rogulja-Ortmann. 2007).

Generally, no difference was found between Repo-expressing glia numbers in wt and H99. However, a small difference does become apparent when one separates the total cell counts into those in the CNS and those in the periphery: 25.67±0.45 cells/hs and 28.42±0.64 cells/hs for wt and H99, respectively, were counted in the CNS, whereas 8.50±0.28 cells/hs and 6.35±0.82 cells/hs for wt and H99, respectively, were found in the periphery. The reasons for this difference might be the greater width of the CNS in H99 embryos, and that the cues required for proper migration of the peripheral glia are disturbed by additional cells. Alternatively, the difference might be due to differentiation defects in these cells (Rogulja-Ortmann. 2007).

In addition to NB clones with too many cells and wild-type-like axon projections in H99, some lineages were obtained whose clones exhibited atypical projection patterns. These projections were found to belong both to motoneurons (e.g. in NB4-2) and interneurons (e.g. NB5-3, NB7-2 and NB-7-4). NB4-2 normally produces two motoneurons (RP2 and 4-2Mar) and 8-14 interneurons. In two out of three NB4-2 clones in H99 two additional motoneurons that project anteriorly were found, similar to RP2. One of the two clones was found in the thorax and had a normal cell number (16), whereas the other was abdominal and had too many cells (25). Thus, the two additional motoneurons are likely to be the progeny of divisions occurring in the wt, and not of an additional NB or GMC mitosis. The fact that the third NB4-2 clone (found in the abdomen and comprising 17 cells) did not show the same motoneuronal projections could be due to these cells not being differentiated at the time of fixation (clones of different ages were occasionally observed in the same embryo), or they may not have differentiated at all. It would be interesting to determine the target(s) of these additional motoneurons and thereby perhaps gain insight into physiological reasons for their death. However, such an experiment has to await tools that allow specific labeling of the NB4-2 lineage, or these motoneurons, in the H99 mutant background (Rogulja-Ortmann. 2007).

The other three lineages (NB5-3, NB7-2 and NB7-4) all have atypical interneuronal projections. The cells which these atypical axons belong to may represent evolutionary remnants that are not needed in the Drosophila CNS. Alternatively, they might have a function earlier in development and be removed when this function is fulfilled. Such a role has been shown for the dMP2 and MP1 neurons, which are born in all segments and pioneer the longitudinal axon tracts. At the end of embryogenesis these neurons undergo PCD in all segments except A6 to A8, where their axons innervate the hindgut. It is known that some cells of the NB5-3 lineage express the transcription factor Lbe, and that H99 mutants show about three additional Lbe-positive neurons per hemisegment, which mostly likely belong to NB5-3. The DiI-labeling results complement this finding in that four or more additional neurons were also found in H99 clones. The supernumerary Lbe-positive neurons in H99 could possibly be the ones producing the atypical axonal projections (Rogulja-Ortmann. 2007).

In the wt embryo, only eight NB lineages show obvious tagma-specific differences in cell number and composition. Tagma-specific differences among serially homologous CNS lineages have been shown to be controlled by homeotic genes. Therefore, these lineages provide useful models for studying homeotic gene function on segment-specific PCD. In H99 embryos, further lineages were observed that were differently affected in the thorax and abdomen. How these tagma-specific differences arise in a PCD-deficient background is an interesting question. For example, NB4-3 shows a wild-type cell number in the thorax (8 and 12-13), but has too many cells in the abdomen (15, 15 and 22). There are a couple of plausible scenarios to explain this observation. (1) The development of the NB4-3 lineage, including the involvement of PCD, could actually differ in the thorax and abdomen of wt embryos, with the final cell number being similar by chance. The DiI-labeled clones allow determination of the final cell number, but do not reveal how this number is achieved. The difference would become obvious in an H99 mutant background, at least regarding the involvement of PCD. (2) This possibility does not exclude the first one, the thoracic NB4-3 could become a postembryonic NB (pNB) and the abdominal NB4-3 might undergo PCD after generating the embryonic lineage. In H99, the abdominal NB would be capable of undergoing a variable number of additional divisions to generate a variable number of progeny. This would easily explain larger discrepancies in cell number between individual clones in H99 (e.g. the abdominal NB4-3 clone with 22 cells), and is in agreement with occasional observations of H99 embryos with a very high CNS cell number per segment, and with the two observed classes of H99 embryos with high and low numbers of BrdU-positive cells (Rogulja-Ortmann. 2007).

NB6-2 is another lineage whose clones differ in the two tagmata of H99 embryos. In this case, the abdominal clones showed no difference to their wt counterparts, whereas the thoracic clones did (18 and 19 cells). Although no difference in cell number between thoracic and abdominal clones was reported for this lineage, a rather large count range (8-16 cells) was given, which would allow for a thorax-specific PCD of two to three postmitotic progeny. Alternatively, the thoracic NB6-2 might undergo cell death upon generating its progeny, which would make it the first identified apoptotic NB in the thorax. When PCD is prevented, this NB may undergo a few additional rounds of division. The data obtained in these experiments do not counter this notion, but the number of clones obtained in the thorax was not sufficient to draw a definite conclusion. As the abdominal NB6-2 lineage in H99 did not differ from the one in wt, its NB may be one of the few abdominal postembryonic NBs (Rogulja-Ortmann. 2007).

A specific set of NBs undergoes PCD in the late embryo, whereas surviving NBs resume proliferation in the larva as pNBs, after a period of mitotic quiescence. The identities of the individual NBs undergoing PCD versus those surviving as pNBs are still unknown. The sizes of NB lineages obtained in H99 embryos may provide hints for identifying candidate pNBs in the abdomen [12 NBs/hs in A1, four in A2 and three in A3 to A7, and NBs that undergo PCD in the thorax at the end of embryogenesis [seven NBs/hs in T1 to T3. In the abdomen, NB1-1a and NB6-2 are obvious candidates for pNBs, as they remained consistently unchanged in H99 embryos. Two other NBs, NB1-2 and NB3-2, are also potential abdominal pNBs as they mostly did not differ from their wt counterparts, and only occasionally contained one additional cell. On the other hand, clones which showed more than twice the cell number in H99 (NB2-1, NB5-4a and NB7-3) than in wt, strongly suggest that these NBs normally undergo PCD in the abdomen (but perform additional divisions in H99), because, even if one daughter cell of each GMC undergoes PCD, they still cannot account for all cells found in H99 clones (Rogulja-Ortmann. 2007).

Regarding thoracic NBs, it can only be speculated on account of low sample numbers. NBs which seem to become pNBs in the thorax, as they showed no difference between wt and H99 clones, are NB3-2, NB4-3 and NB4-4. Potential candidates for NBs which do not become pNBs, but undergo PCD in the thorax, are expected to consistently have a significant increase in cell number in H99. These are NB5-1 and NB5-5. In addition, lineages for which one clone was obtained in H99 but which also showed many more cells in the thorax than normal are NB2-2t, NB5-4t and NB7-3 (Rogulja-Ortmann. 2007).

In order to investigate the developmental signals and mechanisms involved in the regulation of PCD in the embryonic CNS, some of the apoptotic cells were identified which will be used as single-cell PCD models. These are the dHb9-positive RP neuron from NB3-1, Lbe-positive neurons from NB5-3, the Eg-positive GW neuron from NB7-3 and the Eve-positive U neurons from NB7-1. As not much is known about the dying RP motoneuron or the Lbe-positive neurons, the first goal will be to characterize each of these cells more closely, based on the combination of expressed molecular markers (Rogulja-Ortmann. 2007).

Some of the dying NB7-3 cells are already known to be undifferentiated daughter cells of the second and third GMC, which undergo PCD shortly after birth. Notch has been identified as the signal initiating PCD. The surviving daughters receive the asymmetrically distributed protein Numb, which counteracts the PCD-inducing Notch signal. The same had been shown in a sensory organ lineage of the embryonic peripheral nervous system, where cells produced in two subsequent divisions undergo Notch-dependent PCD. Both the PCD in the NB7-3 lineage and in the sensory organ lineage require the Hid, rpr and grim genes. It will be interesting to see whether the Notch-Numb interaction also plays a role in the segment-specific PCD of the differentiated GW motoneuron, or if another signal is used for the removal of this, and possibly other, differentiated cells (Rogulja-Ortmann. 2007).

The U motoneurons also show a segment-specific cell death pattern (they apoptose in A6 to A8), thus somewhat resembling the MP1 and dMP2 neurons. However, in contrast to MP1 and dMP2, the U neurons survive in the anterior segments and undergo PCD in the posterior ones. Whether homeotic genes play any role in the survival or death of these cells remains to be investigated (Rogulja-Ortmann. 2007).

In summary, this study has presented descriptions of PCD in the developing CNS of the wt Drosophila embryo, and of the CNS of PCD-deficient embryos. The pattern of Caspase-dependent PCD is partly very orderly, suggesting tight spatio-temporal control of cell death, and partly random, which suggests a certain amount of plasticity already in the embryo. The CNS of PCD-deficient embryos is nevertheless well organized, despite the presence of too many cells. These superfluous cells come from both a block in PCD and from additional divisions that surviving NBs go through. It was possible to link the occurence of cell death to identified NB lineages by clonal analysis in PCD-deficient embryos, to uncover segment-specific differences, and to establish single-cell PCD models that will be used in further studies to investigate mechanisms responsible for controlling PCD in the embryonic CNS (Rogulja-Ortmann. 2007).

Drosophila muscleblind is involved in troponin T alternative splicing and apoptosis; Identification of dominant suppressors and enhancers of a muscleblind overexpression phenotype

Muscleblind-like proteins (MBNL) have been involved in a developmental switch in the use of defined cassette exons. Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D) are coded by the unique Drosophila muscleblind gene. This study used evolutionary, genetic and cell culture approaches to study muscleblind (mbl) function in flies. The evolutionary study showed that the MblC protein isoform was readily conserved from nematodes to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors leads to an externally rough eye morphology. This phenotype has been used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog arrest, exon junction complex components tsunagi and always early, and pro-apoptotic genes Traf1 and reaper. This study further investigated Muscleblind implication in apoptosis and splicing regulation. Missplicing of troponin T was found in muscleblind mutant pupae, and Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing was confirmed in human HEK cells. MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner. Bioinformatics analysis identified a conserved FKRP motif, weakly resembling a sumoylation target site, in the MblC-specific sequence. Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression. Taken together these genetic approaches identify cellular processes influenced by Muscleblind function, whereas in vivo and cell culture experiments define Drosophila troponin T as a new Muscleblind target, reveal a potential involvement of MblC in programmed cell death and recognize the FKRP motif as a putative regulator of MblC function and/or subcellular location in the cell (Vicente-Crespo, 2008).

Using Drosophila as a model organism, this study reports the first screen specifically addressed to identify gene functions related to the biomedically important protein Muscleblind. In support of the relevance of the results, the strong functional conservation between fly and vertebrate Muscleblind proteins is shown. Furthermore, data is presented supporting that Muscleblind can induce apoptosis in vivo in imaginal disc tissue, and a conserved motif in the MblC protein isoform was identified that conferred pro-apoptotic activity in Drosophila cell culture when mutated. Noteworthy, this is the first conserved motif (besides CCCH zinc fingers) that is associated with a particular function in Muscleblind proteins (Vicente-Crespo, 2008).

Whereas most vertebrates include three muscleblind paralogues in their genomes, a single muscleblind gene carries out all muscleblind-related functions in Drosophila. These functions are probably accomplished through alternative splicing, which generates four Muscleblind protein isoforms with different carboxy-terminal regions. An evolutionary analysis was performed with isoform-specific protein sequences in order to assess conservation of alternative splicing within protostomes. MblC-like isoforms have been detected even in the nematodes C. elegans and Ascaris suum but not MblA, B or D, that were only consistently found within Drosophilidae. Interestingly, also vertebrate Mbnl1 genes included MblC-like sequences. This finding, together with previous studies that shown that mblC is the isoform with the strongest activity in a muscleblind mutant rescue experiment and α-actinin minigene splicing assay point to mblC as the isoform performing most of muscleblind functions in the fly. Despite this, Muscleblind isoforms are partially redundant. Both mblA and B partially rescue the embryonic lethality of muscleblind mutant embryos and were able to similarly promote foetal exon exclusion in murine TnnT3 minigene splicing assays. MblD showed no activity in splicing assays or in vivo overexpression experiments. However, we show a marginal increase in cell viability in cell death assays. Using isoform-specific RNAi constructs we plan to re-evaluate the function of Muscleblind isoforms both in vivo and in cell culture (Vicente-Crespo, 2008).

Although the regulation of alternative splicing by Muscleblind proteins is an established fact, the cellular processes in which the protein participates are largely unknown. Genetic screens provide a way to approach those processes as they interrogate a biological system as a whole. Overexpression of MblC in the Drosophila eye originated an externally rough eye phenotype that is temperature sensitive, thus indicating sensitization to the muscleblind dose. A deficiency screen was performed, and several candidate mutations were tested for dominant modification of the phenotype. Nineteen were identifed genes of which more that half can be broadly classified as involved in apoptosis regulation (rpr, th and Traf1), RNA metabolism (Aly, tsu, aret and nonA) or transcription regulation (jumu, amos, Dp, CG15435 and CG15433), whereas the rest do not easily fall into defined classes. muscleblind has been shown to regulate α-actinin and troponinT alternative splicing both in vivo and in cell culture. The genetic interaction with the Drosophila homolog of human splicing factor CUG-BP1 (aret) and nonA supports a functional relationship in flies. The antagonism between MBNL1 and CUG-BP1 has actually been shown in humans, whereas RNA-binding protein NonA might be relevant to Muscleblind sequestration by CUG repeat RNA in flies (Vicente-Crespo, 2008 and references therein).

Reduction of dose of exon junction complex (EJC) components tsunagi and Aly also modify MblC overexpression phenotype. EJC provides a binding platform for factors involved in mRNA splicing, export and non-sense mediated decay (NMD). This suggests a previously unforeseen relationship between Muscleblind and EJC, perhaps helping to couple splicing to mRNA export. Consistently, Aly mutations enhanced a CUG repeat RNA phenotype in the Drosophila eye. A similar coupling between transcription and splicing might explain the identification of a number of transcription factors in the screen. Of these, the effect of jumu alleles in the eye and wing MblC overexpression phenotypes were studied in some detail. Loss of function jumu mutations suppress both wing defects and rough eye, whereas they have no effect on unrelated overexpression phenotypes thus suggesting that the interaction is specific (Vicente-Crespo, 2008).

Mutations in the Drosophila homolog of vertebrate Inhibitor of Apoptosis (Diap1 or thread) dominantly enhanced the rough eye phenotype. Consistently with the specificity of the interaction, a second Drosophila paralog, Diap2, did not interact. Also, a deficiency that removes the Drosophila proapoptotic genes hid, reaper and grim (which inhibit thread) was a dominant suppressor while reaper overexpression in eye disc enhanced the phenotype. Interestingly the human homolog of Drosophila Hsp70Ab, Hsp70, has been related to apoptosis as it directly interacts with Apaf-1 and Apoptosis Inducing Factor (AIF) resulting in the inhibition of caspase-dependent and caspase-independent apoptosis. All these genetic data are consistent with MblC overexpressing eye discs being sensitized to enter apoptosis, although no increase in caspase-3 activation was detected in third instar eye imaginal disc overexpressing MblC (Vicente-Crespo, 2008).

Human MBNL1 and CUB-BP1 cooperate to regulate the splicing of cardiac TroponinT (cTNT). The current study detected splicing defects in Drosophila troponinT mRNA in muscleblind mutant pupae. Interestingly, an abnormal exclusion of exon 3 was detected in muscleblind mutant pupae, encoding a glutamic acid-rich domain homologous to the foetal exon of cTNT regulated by human MBNL1. Drosophila exon 3 is only absent in the troponinT isoform expressed in TDT and IFM muscles and probably confers specific functional properties much like the foetal exon does in humans. This identifies troponinT as a new target of Muscleblind activity in flies (Vicente-Crespo, 2008).

CUG-BP1 protein antagonizes MBNL1 exon choice activity in IR and cTNT pre-mRNAs. Moreover, a genetic interaction has been detected between MblC overexpression and aret loss of function mutations. In order to further characterize the functional interaction between Muscleblind and Bruno proteins, their ability to regulate murine TnnT3 was examined in human cell culture. MblA, B and C showed strong activity on TnnT3 mRNA but no significant activity was detected for any Bruno protein. This shows a strong functional conservation between fly and vertebrate Muscleblind proteins as Drosophila isoforms can act over a murine target in a human environment. In contrast, Bruno proteins might not conserve the regulatory activity over troponinT mRNA described for their vertebrate homologues or at least they were not functional in the cellular environment used in this assay. Because GFP-tagged Bruno proteins were only weakly expressed in HEK cells under the experimental conditions used, the level of expression might be insufficient to overcome endogenous Muscleblind activity in cell culture. Furthermore, Bruno proteins might antagonize Muscleblind on a different subset of RNA targets. Although bruno1 has been shown to regulate splicing of some transcripts in S2 cell culture and Bruno3 binds the same EDEN sequence than human CUG-BP, no in vivo experiments have addressed the functional conservation between fly and vertebrate Brunos. Bruno1 is expressed in the germ line where it acts as translational repressor of oskar and gurken mRNAs (Vicente-Crespo, 2008).

Wing imaginal discs stained with anti-caspase-3 and with TUNEL showed that activation of apoptosis was not general in cells expressing MblC but restricted to defined regions within the disc, in particular the wing blade. The spatial constraints that were observed within the imaginal disc might explain the small effect detected when expressing Muscleblind proteins in S2 cells. MblC might require the presence of other factors to be able to unleash programmed cell death. Alternatively, the level of overexpression may be critical and transfected Muscleblind proteins may not reach a critical threshold in Drosophila S2 cells. MblC activation of apoptosis could reveal a direct regulation of apoptotic genes at RNA level or be an indirect effect. Several apoptotic genes produce pro-apoptotic or anti-apoptotic isoforms depending on the regulation of their alternative splicing. MblC could be similarly regulating protein isoforms originating from one or a number of key apoptotic genes at the level of pre-mRNA splicing. Alternatively, MblC could be regulating isoform ratio of a molecule indirectly related to programmed cell death, for example a cell adhesion molecule causing apoptosis by inefficient cell attachment to the substrate. Furthermore, human MBNL proteins are implicated not only in splicing but also in RNA localization, a process that if conserved in flies can potentially impinge in apoptosis regulation (Vicente-Crespo, 2008).

The analysis of MblC-specific sequence revealed a region conserved in Muscleblind proteins from nematodes to humans. Post-translational prediction programs found a motif (FKRP) weakly resembling a sumoylation target site. However, results in S2 cells suggest that sumoylation, if actually taking place, modifies only a small fraction of MblC proteins. FKRP may alternatively participate in an interaction with a Muscleblind partner potentially regulating activity or location in cell compartments, assist in protein dimerization, or others functions. The FKRP site was mutated and a number of functional assays were performed using the mutant MblC. Whereas MblCK202I excluded foetal exon in TnnT3 minigene splicing assays and bound CUG repeat RNA like its wild type counterpart, the mutant protein showed a different preferential distribution in human cells and significantly increased cell death activation upon overexpression. The mechanism by which the FKRP site influences subcellular distribution and cell death-inducing activities is currently unknown, but nevertheless constitutes the first motif, other than zinc fingers, that is associated with a function within Muscleblind proteins (Vicente-Crespo, 2008).

The role of apoptosis in shaping the tracheal system in the Drosophila embryo

The tubular network of the tracheal system in the Drosophila embryo is created from a set of epithelial placodes by cell migration, rearrangements, fusions and shape changes. A designated number of cells is initially allocated to each branch of the system. The final cell number in the dorsal branches is not only determined by early patterning events and subsequent cell rearrangements but also by elimination of cells from the developing branch. Extruded cells die and are engulfed by macrophages. These results suggest that the pattern of cell extrusion and death is not hard-wired, but is determined by environmental cues (Baer, 2010).

In live studies of the tracheal system using embryos expressing GFP under the control of the breathless (btl) promoter GFP-expressing motile cells were observed that were not attached to the tracheal system. The btl gene, which encodes an FGF receptor homolog, is mainly expressed in the tracheal system, but also in glial cells and a few other cell type. However, it had not previously been described as being expressed in individual cells that were dispersed in the embryo. Since it was not clear what the individual cells outside the tracheal system were, other tracheal markers were used to determine whether they indeed derived from the tracheal system, or rather represented an as yet undiscovered cell type in which the btl promoter is active. When the tracheal system was marked with lacZ expressed under the control of the promoter of another tracheal gene, trachealess (trh-lacZ), it was observed that lacZ was also expressed not only in the tubular tracheal epithelia but also in single cells detached from the tracheal system (Baer, 2010).

In the live observations using GFP it was noticed that these cells appeared to be moving around in the embryo, preferentially along the dorsal trunk of the trachea. Previously hemocytes had been described to move along the tracheal system. Thus it was asked whether these cells might be a subpopulation of hemocytes that shared expression of some genes with the tracheal system. To assess their cell type, the cells were analyzed in embryos in which markers for the tracheal system and for hemocytes were simultaneously visualized. Hemocytes were visualized by using a croquemort-GAL4 transgene to control the expression of GFP. Croquemort is expressed in a subpopulation of hemocytes, the macrophages. For the tracheal system trh-lacZ was used (Baer, 2010).

It was found that the individual lacZ-expressing cells that are detached from the tracheal system also expressed the crq-controlled GFP. In addition, there were also many cells that showed only crq-GFP staining and no lacZ. There are two possible explanations for these findings. Either a subset of macrophages can activate transcription of the btl and trh promoters, or the doubly-stained objects are not single cells. Consistent with the latter explanation, it was noticed that the two fluorescent markers were often localized in non-overlapping parts of the cells, suggesting that lacZ-expressing cells or cell fragments might have been engulfed by macrophages. If this were the case, then the doubly-stained objects should contain two nuclei. Indeed, in triple stainings with the nuclear marker TOTO-3 two TOTO-3 signals were observed in the majority of the doubly-stained objects, indicating the presence of two nuclei. It is concluded therefore that the objects are hemocytes that have engulfed tracheal cells that have left the tracheal epithelium (Baer, 2010).

To test directly whether these single cells originate from the developing tracheal tree, more detailed live observations were performed. In previous experiments it was noticed that the disconnected cells can be preferentially found near the branches that undergo cellular rearrangements, for example, at the bases of dorsal branches. The areas around these sites were chosen for further studies. Live imaging of dorsal branches of the embryos expressing α-cat-GFP or sqh-GFP revealed that during the outgrowth of the dorsal branches individual tracheal cells detach from the tracheal system at, or near the site where the dorsal branches emerge from the dorsal trunk. The numbers of cells leaving the system from the base of branches and from the interbranch-regions of the dorsal trunk were compared in 12 videos of wildtype embryos. The 'base of the branch' is described as those cells that bordered at least on one side on a cell that was part of the dorsal branch. 42 branches were evaluated and cells were found leaving from the base in 20 cases, whereas in 40 interbranch-regions seven cells leaving the system were found. Thus, cells are almost threefold less likely to be extruded in the large interbranch area than from the small area at the base of the branch (Baer, 2010).

Since the detached cells appeared to be engulfed by macrophages and one function of macrophages is to clear up apoptotic cells, whether the trh-lacZ positive cells seen inside the hemocytes displayed markers for apoptosis was tested. It was found that some but not all of the trh-lacZ cells that were detached from the tracheal system gave a positive signal in a TUNEL reaction. Since the TUNEL assay only detects cells in a brief phase of apoptosis it was not surprising that only a subset of the detached cells were labelled. Thus this result is consistent with the notion that the engulfed cells are dying or dead cells. A further hallmark of apoptosis is the activation of caspase 3, which can be detected with an antibody that specifically recognizes the activated form. Trh-lacZ-expressing cells that were detached from the tracheal system also gave a positive signal when stained with antibodies against activated caspase3 (Baer, 2010).

Next, it was asked whether the detached cells undergo apoptosis because they have lost contact with the tracheal epithelium, or, conversely, whether they die within the epithelium and are subsequently extruded. This question was addressed by two approaches: tracing caspase activity in vivo and blocking apoptosis. To examine when the apoptotic program is induced in tracheal cells leaving the system an in vivo fluorescent sensor of caspase activity, the 'Apoliner' transgene (Bardet, 2008), was used. The Apoliner consists of two fluorophores, a membrane-anchored RFP and a GFP with nuclear localisation signal (NLS), which are linked by a caspase sensitive fragment of the DIAP1 protein. Upon caspase activation within the cell, the sensor is cleaved, resulting in translocation of GFP into the nucleus, whereas RFP stays at the membrane. The Apoliner was expressed using btl-GAL4 and the development of dorsal branches was followed in vivo. Nuclear GFP can be observed in individual cells in the tracheal system. Shortly after they begin to accumulate nuclear GFP these cells detach from the system. Thus apoptosis precedes cell removal from the tissue (Baer, 2010).

If death is a prerequisite for expulsion, then suppressing apoptosis should reduce the number of detached cells. To test this, the tracheal system was examined in embryos homozygous for a deficiency Df(3L)H99, which removes the three pro-apoptotic genes, grim, reaper and hid, and in which apoptosis therefore cannot occur. Since homozygous Df(3L)H99 embryos show strong overall developmental defects, the analysis was restricted to the dorsal branches in segments that do not show gross morphological abnormalities, i.e. segments 3-7. Whereas wild type stage 14/15 embryos show approximately 2-4 detached cells in segments 3-7 on each side, no detached cells were observed in embryos that were homozygous for Df(3L)H99. To exclude the possibility that this was due to a non-autonomous effect of the mutant phenotype of surrounding tissues in Df(3L)H99 mutant embryos, an alternative way of blocking apoptosis only in the tracheal system was used. The baculoviral inhibitor of apoptosis p35 was expressed in tracheal cells using the btl-GAL4 driver line. To confirm that apoptosis was blocked efficiently, the Apoliner was co-expressed together with p35. In these embryos no Apoliner signal was detected, indicating the absence of caspase activity, and no cells were seen to leave the tracheal system. This shows that the completion of the apoptotic program is necessary for detachment of the tracheal cells (Baer, 2010).

The fact that the detached cells were frequently found near the base of the dorsal branches of the tracheal system raises the question whether their leaving the epithelium might be associated with a specific developmental programme, such as the morphogenesis of the dorsal branches. Since each dorsal branch arises from six cells, but the final branch typically consists of only five cells, it has been suggested that the sixth cell migrates back to the dorsal trunk. The results point to an alternative possibility suggesting that apoptosis might be a mechanism that contributes to the determination of cell number in the dorsal branches. If the assumption that cell death is involved in determining the number of cells is correct, then dorsal branches should contain more cells if apoptosis cannot occur. To test this, cell numbers were counted in the dorsal branches of Df(3L)H99 mutant embryos and in embryos expressing p35 in the tracheal system. The embryos were stained with an antibody against Trachealess to mark specifically the nuclei of tracheal cells. The nuclei in dorsal branches were then counted under the microscope by focusing through the entire depth of the dorsal branch. Branches with an unclear course were not included in the analysis (Baer, 2010).

All tested genotypes showed variability in cell number in the dorsal branches, but there were clear differences between the mutant and the wild type embryos. 76.3% of the 93 dorsal branches evaluated in 10 wild type embryos consisted of 5 cells, 8.6% consisted of six cells and 15.1% consisted of 3 or 4 cells. This shows that branches with six cells are possible, but 5 cells are preferred. By contrast, embryos with blocked apoptosis had 51.6% (btl-Gal4, UAS-p35 embryos) or 56.6% (Df(3L)H99 mutant embryos) dorsal branches with six cells. These results indicate that removal of the cells by apoptosis indeed contributes to the determination of the final cell number in the dorsal branches (Baer, 2010).

It is unclear how the emigrating cell is determined, and why it is not determined in every one of the branches. The fact that this is so shows that, in contrast to many other situations in which apoptosis eliminates unwanted cells during development, cell death in this case is not the result of a hard-wired developmental programme. Instead it is more likely to be a response to unfavourable conditions in the cell's environment. Changes in the microenvironment have been shown to induce cell death in mammalian endothelial cells, with signals from the extracellular matrix influencing the balance between cell survival and apoptosis. Adhesion via integrins can protect cells from FAS mediated apoptosis, and integrins may act as mechano-transducers in this context. Thus it is possible that weakening of cell-cell junctions resulting from tissue remodelling in the tracheal system could trigger the apoptotic pathway. Junction rearrangement also affects cell death in the wing disc, where an imbalance in the junctional forces within the epithelium was found to be rebalanced by the elimination of cells. In this system, the initial irregularity arose through cell proliferation, but it is imaginable that in the tracheal system cell intercalation might affect local junctional forces, and cell extrusion can be triggered to obtain a geometrically optimal structure (Baer, 2010).

The fact that only half of the dorsal branches in the mutant embryos had six cells shows that apoptosis cannot be the only mechanism for the removal of supernumerary cells. There must be other ways of losing cells, perhaps by re-integration into the dorsal trunk, as previously suggested. Such cases were indeed observed. More stringently, even embryos in which a larger number of branches have six cells, namely btl-Gal4, UAS-p35 embryos, can develop normally and hatch. It is of course possible that subtle defects, for example a reduced efficiency in oxygen delivery, would not manifest themselves in easily measurable phenotypes in a laboratory setting (Baer, 2010).

In summary, the results are consistent with a scenario in which cells that find themselves in a sub-optimal epithelial context either move away, or die and leave the epithelium. If the apoptotic pathway is blocked, they may be forced to use the option of moving elsewhere, or induce neighbouring cells to rearrange further, or they remain in place, and the system is able to tolerate a sub-optimal structure (Baer, 2010).

Inactivation of both Foxo and reaper promotes long-term adult neurogenesis in Drosophila

Adult neurogenesis occurs in specific locations in the brains of many animals, including some insects, and relies on mitotic neural stem cells. In mammals, the regenerative capacity of most of the adult nervous system is extremely limited, possibly because of the absence of neural stem cells. This study shows that the absence of adult neurogenesis in Drosophila results from the elimination of neural stem cells (neuroblasts) during development. Prior to their elimination, their growth and proliferation slows because of decreased insulin/PI3 kinase signaling, resulting in nuclear localization of Foxo. These small neuroblasts are typically eliminated by caspase-dependent cell death, and not exclusively by terminal differentiation as has been proposed. Eliminating Foxo, together with inhibition of reaper family proapoptotic genes, promotes long-term survival of neuroblasts and sustains neurogenesis in the adult mushroom body (mb), the center for learning and memory in Drosophila. Foxo likely activates autophagic cell death, because simultaneous inhibition of ATG1 (autophagy-specific gene 1) and apoptosis also promotes long-term mb neuroblast survival. mb neurons generated in adults incorporate into the existing mb neuropil, suggesting that their identity and neuronal pathfinding cues are both intact. Thus, inhibition of the pathways that normally function to eliminate neural stem cells during development enables adult neurogenesis (Siegrist, 2010).

These findings demonstrate that two pathways act in concert to eliminate mb neuroblasts and terminate neurogenesis. Downregulation of insulin/PI3 kinase signaling occurs first and may activate both autophagy and a program of caspase-dependent cell death. In the absence of one of these cell death pathways, mb neuroblasts persist, but only transiently. Thus a fail-safe mechanism likely exits to ensure mb neuroblast elimination, similar to salivary gland cells (Siegrist, 2010).

The reduction in growth that precedes neuroblast apoptosis appears to be developmentally regulated since it occurs at an earlier time in central brain neuroblasts than in mushroom body neuroblasts. This may be due to either local differences in microenvironments or differences in the ability of neuroblasts to respond to circulating insulin-like peptides. Moreover, the extended survival of mb neuroblasts under these conditions, but not other central brain neuroblasts, suggests that additional mechanisms such as terminal differentiation still function to ensure elimination of most neuroblasts. Indeed, during mammalian development, neural progenitors are eliminated via cell death and by terminal differentiation. The relative importance of death and differentiation for neuroblast elimination may be lineage dependent. Finally because cricket adult mb neuroblasts proliferate in response to insulin in explant cultures, a common mechanism may regulate adult neurogenesis among insects and possibly in more distantly related metazoans. These findings may represent an important first step towards devising ways to manipulate the regenerative capacity of adult brains in diverse species and provide insight into how aberrantly persisting neural stem cells behave in vivo (Siegrist, 2010).

reaper : Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions and parallel pathways | Developmental Biology | References

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