Wrinkled/head involution defective
Patterns of hid expression are highly dynamic and complex throughout embryogenesis. Significantly, hid is expressed in many regions where cell death occurs. For example, in stage 11 embryos, both acridine orange staining, diagnostic for PCD, and HID mRNA hybridization are observed in the head and gnathal segments, as well as being segmentally repeated throughout the extended germ band. In slightly older embryos undergoing early stages of head involution, a correspondence between the patterns of cell death and HID mRNA expression is observed, particularly in the head. There is evidence of hid expression within macrophages, apparently confined to cell corpses that have been engulfed. Although there is significant overlap between the patterns of hid expression and acridine orange staining, these patterns are not entirely coincident. For example, HID mRNA is found throughout the entire optic lobe primordium, but only some of these cells undergo apoptosis. Not all cells may be equally sensitive to the amount of hid expression. Although there is considerable cell death in the ventral nerve cord during late embryogenesis, little or no hid expression can be detected at this time. Perhaps hid is not required for these deaths; alternatively, hid may be expressed in the ventral nerve cord below the level of detection (Grether, 1995).
Sexually dimorphic development of the gonad is essential for germ cell development and sexual reproduction. The Drosophila embryonic gonad is already sexually dimorphic at the time of initial gonad formation. Male-specific somatic gonadal precursors (msSGPs) contribute only to the testis and express a Drosophila homolog of Sox9 (Sox100B: Loh, 2000), a gene essential for testis formation in humans. The msSGPs are specified in both males and females, but are recruited into only the developing testis. In females, these cells are eliminated via programmed cell death dependent on the sex determination regulatory gene doublesex. This work furthers the hypotheses that a conserved pathway controls gonad sexual dimorphism in diverse species and that sex-specific cell recruitment and programmed cell death are common mechanisms for creating sexual dimorphism (DeFalco, 2003).
To investigate when sexual dimorphism is first manifested in the somatic gonad, expression of SGP markers were examined in embryos whose sex could be unambiguously identified, at a developmental stage (stage 15) soon after gonad coalescence has occurred. Analysis of Eya expression reveals anti-Eya immunoreactivity throughout the female somatic gonad, though Eya expression is somewhat stronger in the posterior. In males, anti-Eya immunoreactivity is also found throughout the somatic gonad. However, the expression at the posterior of the gonad is much more intense than in females, as there appears to be a cluster of Eya-expressing cells at the posterior of the male gonad that is not present in females. In blind experiments, the sex of an embryo could be accurately identified by the Eya expression pattern in the gonad. Thus, sexual dimorphism is already apparent in the somatic gonad soon after initial gonad formation. A sex-specific expression pattern is also observed with Wnt-2 at this stage. As is observed with Eya, Wnt-2 is expressed in the SGPs of the female gonad, but its expression is greatly increased at the posterior of the male gonad. The SGP marker bluetail (see Galloni, 1993) exhibits a similar sex-specific pattern as Eya; however, the SGP marker 68-77 is expressed equally in both sexes (see below). Thus, the somatic gonad is sexually dimorphic by stage 15, but only a subset of SGP markers reveals this sexual dimorphism (DeFalco, 2003).
To investigate how programmed cell death might be controlled in the msSGPs, the genes of the H99 region (head involution defective [hid], reaper [rpr], and grim), which are regulators of apoptosis in Drosophila, were examined. A small deletion (DfH99) removes all three of these genes and blocks most programmed cell death in the Drosophila embryo. In DfH99 mutants, an equivalent cluster of Sox100B-positive cells is observed in both males and females. Again, these posterior cells are also Eya positive. Furthermore, XX embryos mutant for hid alone also contain Sox100B-positive cells in the posterior of the gonad, although the posterior cluster of cells is slightly smaller than in the male. It is concluded that the msSGPs are normally eliminated from females through sex-specific programmed cell death, controlled by hid and possibly also other genes of the H99 region. However, if cell death is blocked in females, these cells can continue to exhibit the normal male behavior of the msSGPs, including proper marker expression and recruitment into the gonad. Therefore, the decision whether or not to undergo apoptosis is likely the crucial event leading to the sexually dimorphic development of these cells at this stage (DeFalco, 2003).
Robotic methods and the whole-genome sequence of Drosophila melanogaster were used to facilitate a large-scale expression screen for spatially restricted transcripts in Drosophila embryos. In this screen, scylla (scyl) and charybde (chrb), which code for dorsal transcripts in early Drosophila embryos and are homologous to the human apoptotic gene RTP801, were identified. In Drosophila, both gene products are transcriptionally regulated targets of Dpp/Zen-mediated signal transduction and appear more generally to be downstream targets of homeobox regulation. Gene disruption studies revealed the functional redundancy of scyl and chrb, as well as their requirement for embryonic head involution. From the perspective of functional genomics, these studies demonstrate that global surveys of gene expression can complement traditional genetic screening methods for the identification of genes essential for development: beginning from their spatio-temporal expression profiles and extending to their downstream placement relative to dpp and zen, these studies reveal roles for the scyl and chrb gene products as links between patterning and cell death (Scuderi, 2006).
Based upon the observations that: (1) simultaneous loss of scyl and chrb function leads to a hid-analogous, cell death defective phenotype and (2) scyl and chrb are homologous to the mammalian apoptotic gene RTP801, it was postulated that the scyl and chrb gene products have pro-apoptotic functions in the embryonic Drosophila head. Two lines of experimentation were employed to test this hypothesis. (1) hid expression was examined in scyl chrb double mutant embryos in situ. The scyl and chrb gene products do not function as transcriptional modulators of hid since hid transcription is unaffected in scyl chrb double mutant embryos. (2) A Caspase-3 activity assay was employed to monitor apoptosis in wild-type and scyl chrb double mutant embryos. Activated Caspase-3 has been used previously to specifically label apoptotic cells in Drosophila. Anti-Caspase-3 staining mirrors cell death patterns previously defined by acridine orange and TUNNEL assays in the Drosophila embryo and pupal retina. In this study, dying cells expressing activated Caspase-3 were evident in the head and the nervous system of 95% of embryos derived from matings of Df(3L)vin4/twi:GFP heterozygotes 0-8 h AEL (n = 278). When GFP screening was used to enrich for similarly staged mutant embryos, it was noted that Caspase-3 activity was greatly diminished in mid-stage scyl chrb double mutants. By 8 AEL, 75% of the mutant-enriched population was caspase-negative, in contrast to the unselected population in which only 8% of the embryos were found to be caspase-negative. No gross differences in Caspase-3 activity were found prior to the onset of germ band retraction and head involution. Since cleaved Caspase-3 is a key executioner (and hence marker) of apoptosis, these data support the hypothesis that Scylla and Charybde have pro-apoptotic roles in Drosophila head involution. More generally, Scylla and Charybde likely function as essential death activators in Drosophila since Caspase-3 activation in scyl chrb double mutants is disrupted in the nervous system as well as in the head. The scylla and charybde gene products are not, however, sufficient for cell death since (1) immunostains reveal wild-type patterns of Caspase-3 activation in embryos derived from dl mutant mothers and in which expression of scylla and charybde is greatly expanded and (2) neither scyl nor chrb (alone or in combination) can mimic hid-induced apoptosis in cultured Cos or Hela cells (Scuderi, 2006).
Several lines of evidence indicate that Scylla and Charybde function in the Hid-mediated cell death pathway. (1) A previous phenotypic analysis of scyl chrb mutants revealed their essential roles in regulating cell death in the developing Drosophila eye. Loss-of-function studies have similarly revealed a requirement for Hid in modulating cell death events in early and late stages of Drosophila eye development. (2) In this study, which relied upon deficiencies and RNAi methodologies to generate scyl chrb null double mutants, an earlier developmental requirement for the scyl and chrb gene products was documented. scyl chrb double mutants suffer an embryonic lethality that is associated with defects in the morphogenetic process of head involution. Drosophila homozygous for loss-of-function hid alleles similarly suffer an embryonic lethality and exhibit signature defects in head involution. (3) Molecular characterization of the embryonic lethality in scyl chrb double mutants revealed that Caspase-3 activation is disrupted not only in the morphogenetically aberrant head, but in the CNS as well. In Drosophila, Hid induces apoptosis in midline glia cells failing to activate the EGFR signaling cascade. Together, the significant homologies of scyl and chrb to the mammalian RTP801 gene product that functions as an apoptotic factor in mammalian cell culture systems, as well as the scyl chrb embryonic and eye phenotype studies establish redundant roles for scyl and chrb in Hid-mediated cell death in both embryonic and post-embryonic stages of the Drosophila life cycle (Scuderi, 2006).
Each of the three cell death proteins, hid, rpr and grim, has been implicated in apoptotic events defining segmental boundaries and/or neuronal fates in the CNS, albeit in different paradigms. In the CNS, specificity in neuronal apoptosis is achieved via differential expression of the BX-C Hox gene abd-A, which prevents neuronal apoptosis in posterior segments. Viewed from this perspective, the finding that the Zen and BX-C Drosophila Hox gene products regulate transcription of the scyl and chrb pro-apoptotic genes (and thereby potentially sculpt head and segment boundaries during development) is reminiscent of the Deformed Drosophila Hox protein functioning as a transcriptional activator of the rpr cell death gene. Together, these studies strengthen the idea that Hox-gene-dependent induction of cell death is a general phenomenon in Drosophila (Scuderi, 2006).
Intriguingly, the pro- and anti-apoptotic roles of the Zen and BX-C Homeobox transcription factors in Drosophila embryogenesis correspond to their activation and repression effects on scyl and chrb gene expression. In this regard, scyl, chrb and cell death are activated by Zen in dorsal domains of the developing embryo, whereas ventrally scyl, chrb and cell death are repressed by one or more of BX-C gene products. Hence, in addition to the pro-apoptotic role of Zen, there is evidence for an anti-apoptotic role for the BX-C gene product(s) and in flies as in mouse related transcription factors function in context-specific fashion (Scuderi, 2006).
As a final point, both TGF-β and BMP mammalian members of the TGF-β cytokine superfamily have been documented to induce cell death in numerous developmental contexts. Along these same lines, previous reports in Drosophila have suggested a link between Dpp and cell death but have stopped short of designating this link as direct. Based on molecular and genetic evidence, it is suggested that the Drosophila pro-apoptotic scyl and chrb gene products serve as direct links between Dpp/Zen-mediated patterning and differentiation, in this case, cell death. Thus, in Drosophila as in vertebrates, cytokines of the TGF-β superfamily control both cell death and cell proliferation within the contexts of their cellular environments (Scuderi, 2006).
Given the importance of cell death regulation in development and disease, it is likely that there are several mechanisms by which cell death can be regulated, and, in like fashion, several nodes where independent regulatory pathways may in specific contexts converge. With respect to members of the RTP801 family of apoptotic factors, evidence points to at least two triggers of regulation: cell death can be a pathologic response to stresses such as hypoxia (as is the case for mammalian RTP801) or cell death can be a developmental response to a spatially and temporally restricted cell signaling pathway, such as the Dpp/TGF-β cytokine-mediated signaling pathway (as is the case for Drosophila Scylla and Charybde). Within the context of pathway convergence nodes, it is particularly notable that several reports document cross-talk between the HIF-1 and TGF-β pathways in regulating gene expression and cell death, and thus it is possible that the RTP801/Scylla/Charybde death effectors represent a point of convergence between these two death activating pathways. Consistent with this model is the demonstration that scyl and chrb are hypoxia-inducible in Drosophila (Reiling, 2004). Viewed from this perspective, the genetically defined roles of Scylla and Charybde as pro-apoptotic effectors establish a clear basis for future genetic and biochemical characterization of the mechanism by which activation of cell death programs might occur via Dpp/TGF-β-mediated signaling (Scuderi, 2006).
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).
Ultraviolet (UV) light is absorbed by cellular proteins and DNA, promoting skin damage, aging and cancer. The UV response by cells of the Drosophila retina have been explored. The retina enters a period of heightened UV sensitivity in the young developing pupa, a stage closely associated with its period of normal developmental programmed cell death. Injury to irradiated cells include morphology changes and apoptotic cell death; these defects can be completely accounted for by DNA damage. Cell death, but not morphological changes, is blocked by the caspase inhibitor P35. Utilizing genetic and microarray data, evidence is provided for the central role of Hid expression and for Diap1 protein stability in controlling the UV response. In contrast, Reaper has no effect on UV sensitivity. Surprisingly, Dmp53 is required to protect cells from UV-mediated cell death, an effect attributed to its role in DNA repair. These in vivo results demonstrate that the cellular effects of DNA damage depend on the developmental status of the tissue (Jassim, 2003).
The major inhibitor of caspase activity in Drosophila is Diap1. Stability of Diap1 is the central point of cell death regulation in the developing retina and this is also true during UV irradiation in the retina. Genetic and microarray results further suggest that the retina requires Hid as a primary regulator of Diap1 stability during UV irradiation. Hid may represent the primary regulator of Diap1 during UV (versus ionizing) irradiation response by the fly. Alternatively, the retina utilizes Hid as its major RHG factor during its development, and this preference may simply extend to its response to UV; other tissues may exploit different Diap1 regulators that reflect their use during development (Jassim, 2003).
Together, these results identify two points of regulation during a retinal cell's response to UV irradiation. The early step involves pyrimidine dimers, and requires proper repair from factors that include XPG and p53. The second step involves activation of caspases and requires regulation of Diap1 stability; interommatidial cells utilize Hid at this step, and the remaining cells employ a different (unknown) regulator. One challenge will be to connect these two points of regulation. Multiple signaling pathways are suggested by the microarray data. These include EGFR/Ras1 signaling (a central regulator of Hid), JNK pathway signaling and TGFß pathway signaling. The role of these factors is not known, but understanding them may help to connect early and late events (Jassim, 2003).
In the Drosophila antenna, sensory lineages selected by the basic helix-loop-helix transcription factor Atonal are gliogenic while those specified by the related protein Amos are not. What are the mechanisms that cause the two lineages to act differentially? Ectopic expression of the Baculovirus inhibitor of apoptosis protein (p35) rescues glial cells from the Amos-derived lineages, suggesting that precursors are removed by programmed cell death. In the wildtype, glial precursors express the extracellular-signal regulated kinase (phosphoERK) transiently, and antagonism of Epidermal growth factor pathway signaling compromises their development. It is suggested that all sensory lineages on the antenna are competent to produce glia but only those specified by Atonal respond to EGF signaling and survive. These results underscore the importance of developmental context of cell lineages in their responses to non-autonomous signaling in the choice between survival and death (Sen, 2004).
Several lines of investigation have ascertained that the first cells to divide in the sensory lineages are the secondary progenitors: PIIa, PIIb and PIIc. The numbers of sensory cells undergoing division at different times in the developing antenna were estimated by staining mitotic nuclei with antibodies against phosphorylated histone. A peak of cell division was observed between 16 and 24 h after puparium formation (APF). It has been considered that only in those sensory lineages specified by Ato, PIIb produces a glial cell and a tertiary progenitor, PIIIb, which in turn divides to form the sheath cell and a neuron. In Amos dependent lineages, PIIb is believed to directly give rise to a neuron and a sheath cell. The difference between the two lineages could be entirely dependent on the nature of the proneural genes activated; Amos, for example, could direct a non-gliogenic lineage. Alternatively, the two proneural genes could specify similar division patterns but the glial cell precursor in Amos-lineages could be removed by PCD, resulting in non-gliogenic lineages (Sen, 2004).
To test the latter possibility, cell death profiles were examined in developing pupal antennae using the terminal transferase assay (TUNEL) and attempts were made to correlate the timing of PCD with cell division profiles discussed above. The appearance of TUNEL-positive cells peaked between 22 and 24 h APF consistent with the occurrence of PCD immediately after division of secondary progenitors (Sen, 2004).
TUNEL reactions were performed on 22-24 h APF antennae from lz-Gal4; UAS-lacZnls and ato-Gal4; UAS-lacZnls animals. Double labeling with antibodies against ß-galactosidase marked sensory cells arising from the Lz and Ato lineages. Lz::lacZ overlaps the regions of the antennal disc where amos expression occurs and labels all the basiconic and trichoid sensilla in the mature (36 h APF) antenna. Hence for the purpose of this study, all cells in which lz-Gal4 expresses will be considered to belong to the Amos-dependent lineages. ato-Gal4 drives reporter activity in proneural domains of the disc and subsequently in all cells of the coeloconic sense organs (Sen, 2004).
Most of the apoptotic nuclei observed during olfactory sense organ development co-localized with Lz::LacZ suggesting that death occurred mainly within the 'Amos-dependent' sensory clusters. Only very few TUNEL-positive cells were detected in regions where ato-lacZ expressed and these did not co-localize with the reporter expression. If PCD is the mechanism used to remove glial precursors from Amos lineages, then their rescue would be expected to result in additional peripheral glia in the antenna (Sen, 2004).
The GAL4/UAS system was used to target ectopic expression of baculovirus inhibitor of apoptosis protein (p35) to different cell types within the developing antennal disc. distalless981-Gal4 (henceforth called dll-Gal4), which drives expression in all cells of the antennal disc, resulted in the formation of >300 glial cells as compared to ~100 in the wildtype. Other sensory cells--neurons, sheath, socket and shaft cells--within sense organs were unaffected. Ectopic expression of p35 specifically in Ato lineages (ato::p35) did not alter glial number. This means that the `additional' glial cells rescued in dll::p35 must arise from lineages other than Ato. Mis-expression of p35 in Amos-dependent lineages using lz-Gal4, on the other hand, resulted in a significant increase in glial number. While other explanations are possible, it is believed that the somewhat lower number of glia obtained in lz::p35 as compared to dll::p35 could be accounted for by the strength of the P(Gal4) driver (Sen, 2004) (Sen, 2004).
In order to identify the cell within the Amos lineage that is fated to die, the cellular events during development of sense organs were re-examined. At approximately 12-14 h APF, most sensory cells are associated in clusters of secondary progenitors. Two cells in each cluster -- PIIb and PIIc -- express the homeodomain protein Prospero (Pros). pros-Gal4;UAS-GFP recapitulates Pros expression at this stage and marks PIIb and PIIc and their progeny in all olfactory lineages. In the wildtype, a Repo-positive cell was associated with only a few of the total sensory clusters, these were all located within the coeloconic domain of the antenna. Targeted expression of p35 using pros-Gal4 increased glial number indicating that cells which are the progeny of either PIIb or PIIc could be rescued from apoptosis. In the pros-Gal UAS-2XEGFP/UAS-p35 genotype, a glial cell was associated with most clusters at 18 h APF rather than in Ato lineages alone (Sen, 2004).
In order to directly visualize the cell undergoing apoptosis, 22-24 h APF antenna from the neuA101 strain were stained with antibodies against ß-galactosidase to mark the sensory cells and with TUNEL. Sensory clusters located in basiconic and trichoid domains of the pupal antenna each had a single associated TUNEL positive cell. Since TUNEL reactivity data does not reflect the initiation of the death program, developing antennae were also stained at different time points with an antibody that recognized the activated caspase -- Drice. At 20 h APF, a single Drice-positive cell was found within each sensory cluster within the basiconic and trichoid domains of the pupal antenna. This cell also expressed low levels of Pros suggesting that it could arise from either PIIb or PIIc. This means that the PIIb/c in Amos lineages, like that in Ato, divides to give rise to a PIIIb and its sibling. The sibling in the former lineage was not previously detected because it expresses only low levels of Pros and soon dies. Since this cell is capable of expressing the glial-identity gene repo when rescued from death, it is denoted as a glial precursor (Sen, 2004).
How is apoptosis of a specific cell within the lineage regulated? In Drosophila three genes [reaper (rpr), grim and head involution defective (hid)] which all map under the Df(3L)H99 are necessary for the initiation of the death program. Heterozygotes of Df(3L)H99 show a small but significant increase in glial number over that of normal controls. hid-lacZ was used to follow expression during antennal development; reporter activity occurs at low levels ubiquitously including in glial cells. Levels of reporter expression indicate somewhat higher hid transcription in glia rescued by p35 mis-expression. The presence of Hid in the 'normal' glial precursors suggests a mechanism dependent on possible trophic factors to keep cells alive. In several other systems signaling, mainly through the EGFR pathway, results in an antagonism of Hid action and transcription. The sustained levels of hid transcription in the rescued glia, is not unexpected since inhibitors of apoptosis act by antagonizing a downstream event of caspase activation, rather than on Hid itself (Sen, 2004).
Experiments in both vertebrates and invertebrates have illustrated the competitive nature of growth and have led to the idea that competition is a mechanism for regulating organ and tissue size. Competitive interactions between cells were assessed in a developing organ and their effect on its final size were examined. Local expression of the Drosophila growth regulator dMyc, a homolog of the c-myc proto-oncogene, induces cell competition and leads to the death of nearby wild-type cells in developing wings. Cell competition is executed via induction of the proapoptotic gene hid and both competition and hid function are required for the wing to reach an appropriate size when dMyc is expressed. Moreover, evidence is provided that reproducible wing size during normal development requires apoptosis. Modulating dmyc levels to create cell competition and hid-dependent cell death may be a mechanism used during normal development to control organ size (de la Cova, 2004).
This work leads to three major conclusions. (1) Expression of the c-myc protooncogene homolog dMyc in small populations of wing disc cells induces cell competition, leading to the elimination of nearby cells via induction of the proapoptotic gene hid. (2) The competition induced by dMyc and the elimination of cells that results is required for control of proper wing size. (3) Studies reveal that apoptosis is required for the fidelity of size during normal wing development, suggesting that the modulation of hid expression by competitive interactions between cells may be used as an endogenous mechanism of size control (de la Cova, 2004).
These experiments demonstrate that expression of dMyc in some cells of a developing organ leads to elimination of nonexpressing cells through apoptosis. The growth disadvantage induced by dMyc-expressing cells fulfills the classic definition of cell competition: viable but slower-growing cells in an organ are eliminated by an encroaching faster-growing cell population, proximity to the fast-growing cell population dictates the severity of the disadvantage in the slow-growing cells, cells are protected from cell competition by developmental compartment boundaries, and appropriate organ size is reached at the end of development. Relative differences in dMyc levels lead to competitive situations between cells -- dmyc mutant cells are outcompeted by neighboring nonmutant cells; wild-type cells, with a normal complement of endogenous dmyc, are also subject to competition if surrounded by cells expressing a dMyc transgene. However, wild-type cells appear to be subject to competition only if they lie within about eight cell diameters of dMyc-expressing cells, and they must reside in the same developmental compartment. Thus, proximity, compartmental provenance, and the relative levels of dmyc are particularly important aspects of the competitive effects of dMyc (de la Cova, 2004).
During the process of cell competition induced by dMyc, the proapoptotic gene hid is induced in the growth-disadvantaged cells. Since a reduction of hid function protects cells from competition-induced death, it is believed that hid upregulation is a consequence of the sensing of competitive stress. An intriguing question that remains is how cells are able to sense competition. One possibility is that cells compete for sufficient levels of a survival factor that normally blocks hid expression. Dpp signaling promotes cell survival in the wing disc but appears to be unaffected in discs expressing dMyc. Alternatively, some cells in competition may be deprived of adequate nutrients, although in these experiments, cells at a growth disadvantage retain a normal nucleolar size, arguing that their biosynthetic rates are not abnormally low. However, the results suggest that dMyc provokes competition and hid expression via a short-range signal, since close proximity is required for the perception of competitive effects. Perhaps the most intriguing feature of this signal is that it is not perceived by nearby cells across a compartment boundary, although dMyc induces competition between cells within the posterior compartment as well as within the anterior. One possibility is that cells expressing dMyc acquire adhesive properties that transmit a competitive signal to neighboring cells, which is not compatible with the adhesive barrier that maintains the compartment boundary (de la Cova, 2004).
These studies reveal that cell competition is not invariably induced whenever rapidly growing cells populate regions of a developing organ. Both the PI3K Dp110 and cyclin D/Cdk4 potently promote growth when overexpressed, yet they do not induce competition in any of these assays. These observations also demonstrate that balanced growth -- growth that simultaneously drives cell division and cellular growth -- is not required to induce cell competition. dMyc expression increases clonal mass solely by increasing cell size. Thus, this trait of cell competition may be related to a size-measuring mechanism that recognizes total mass rather than cell number. However, Dp110 also promotes growth primarily by increasing cell size, indicating that qualitative differences exist in the cellular response to expression of dMyc and Dp110. Although both growth regulators increase protein synthesis, Orian (2003) suggests that dMyc probably does so by increasing components of the protein synthetic machinery (initiation factors and ribosomal proteins, etc.) whereas PI3K signaling is thought to function by increasing the utilization of existing machinery. Regardless of the mechanism, these experiments argue against the notion that apposed populations of fast- and slow-growing cells always result in cell competition (de la Cova, 2004).
Three lines of evidence have been provided that indicate that cell competition leading to cell death is required for control of wing size. (1) Growth induced by local expression of either Dp110 or cyclin D + Cdk4 does not induce competition and causes wing overgrowth. (2) When dMyc is expressed in all cells of the wing disc, the wing overgrows, whereas the introduction of clones lacking dMyc leads to cell competition and to wings approaching normal size. (3) Genetic reduction of hid prevents the cell death associated with competition and leads to overgrowth of the compartment in which the dMyc-expressing cells reside (de la Cova, 2004).
An important conclusion of this work is that apoptosis is critical for appropriate wing development. These experiments demonstrate that apoptosis has two roles in regulating wing size. One role is uncovered when the disc is challenged by local changes in dMyc levels, conditions in which cells are exceptionally sensitive to hid gene dosage: the full hid complement is necessary for the disc to respond properly to competition and eliminate cells. However, a second role of apoptosis is revealed when it is abolished: this role regulates uniformity of disc size, and its loss is manifested as a widening of the range of disc sizes within a given population. This second role of apoptosis indicates that organ overgrowth is distinct from loss of organ size control. Wing overgrowth -- observed when cell competition is not executed during local growth perturbations -- occurs such that, although larger than normal, wing size still falls within a uniform range. In contrast, loss of size control is the absence of a discrete and reproducible size population and results from a failure to induce apoptosis during the process of growth. Based on these observations, it is proposed that hid-regulated apoptosis contributes to a disc-intrinsic mechanism that limits variation in size by allowing elimination of cells. This mechanism may serve as negative feedback to the positive aspects of growth during development. Loss of feedback control could allow stochastic variation in size, as has been observed. Although it has been proposed that overall organ mass rather than cell number is sensed by the intrinsic size mechanism, these experiments imply that size control is implemented at least in part by reduction of cell number via apoptosis (de la Cova, 2004).
Is cell competition also part of the intrinsic size control program? If cell competition has a role in normal development, growth rate variations should be observed within developing organs. Indeed, both spatial and temporal differences in cell proliferation rates exist in the wing disc, and cell size also varies across the disc, suggesting differences in cellular growth rates. dmyc is regulated both by Wingless and Dpp, which direct the majority of disc patterning. Minor alterations in their signaling could plausibly cause subtle competitive effects by influencing levels of dmyc expression, which in turn would modulate hid expression and allow for the correction of patterning mistakes that occur during development. In this sense, cell competition, on a small scale, might be a surveillance or 'quality control' mechanism to guarantee that organs reach a body-proportional, reproducible size with the appropriate complement of cell fates (de la Cova, 2004).
Cell competition is likely a common mechanism used in organs under many conditions, including those that are adverse. Competitive mechanisms are known to be important to reestablish homeostasis in lymphoid tissue after an immune response. During tumorigenesis, cancer cells may compete with normal tissue and ultimately overtake the organ, leading to overgrowth of the tumor. In addition, cell competition could prove important therapeutically for many diseases. For example, when liver cells are transplanted into a diseased host liver, cell competition would be critical for the replacement of viable but damaged liver cells with the regenerating donor cells. Although of the three growth regulators tested only dMyc induced cell competition, other growth-promoting genes that induce cell competition probably exist. The identification of these genes holds promise for a further elucidation of the role of cell competition in organ development (de la Cova, 2004).
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 is 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).
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).
Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in Drosophila utilize the REAPER/HID/GRIM family proapoptotic proteins. These proteins promote cell death, at least in part, by disrupting interactions between the caspase inhibitor DIAP1 and the apical caspase DRONC, which is continually activated in many viable cells through interactions with ARK, the Drosophila homolog of the mammalian death-activating adaptor APAF-1. This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC. This study demonstrates that ARK- and HID-dependent activation of DRONC occurs at sites of spermatid individualization and that all three proteins are required for this process. dFADD, the Drosophila homolog of mammalian FADD, an adaptor that mediates recruitment of apical caspases to ligand-bound death receptors, and its target caspase DREDD are also required. A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization. These results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process (Huh, 2004; full text of article).
In multicellular organisms, apoptotic cells induce compensatory proliferation of neighboring cells to maintain tissue homeostasis. In the Drosophila wing imaginal disc, dying cells trigger compensatory proliferation through secretion of the mitogens Decapentaplegic (Dpp) and Wingless (Wg). This process is under control of the initiator caspase Dronc, but not effector caspases. This study shows that a second mechanism of apoptosis-induced compensatory proliferation exists. This mechanism is dependent on effector caspases which trigger the activation of Hedgehog (Hh) signaling for compensatory proliferation. Furthermore, whereas Dpp and Wg signaling is preferentially employed in apoptotic proliferating tissues, Hh signaling is activated in differentiating eye tissues. Interestingly, effector caspases in photoreceptor neurons stimulate Hh signaling which triggers cell-cycle reentry of cells that had previously exited the cell cycle. In summary, dependent on the developmental potential of the affected tissue, different caspases trigger distinct forms of compensatory proliferation in an apparent nonapoptotic function (Fan, 2008).
In developing wing discs in which apoptosis was induced by expression of the pro-apoptotic gene hid, loss of the caspase inhibitor DIAP1, or by X-ray treatment, the accumulation of two major mitogens, Dpp and Wg, has been observed in dying cells. Key for this finding is the simultaneous expression of the caspase inhibitor P35. Under these conditions, the dying cells were kept alive ('undead'), allowing accumulation of Dpp and Wg. This accumulation appears to be dependent on the initiator caspase Dronc, because it cannot be blocked by expression of P35 which inhibits effector caspases but not Dronc. In addition, the Drosophila homolog of the tumor suppressor p53, Dp53, has been implicated downstream of Dronc for compensatory proliferation. Notably, these studies on mechanisms of compensatory proliferation were carried out in developing larval wing imaginal discs in Drosophila. Cells in wing discs proliferate extensively during larval stages, and the majority of these cells does not differentiate before they reach pupal development. Hence, the mechanisms of compensatory proliferation have so far only been investigated in situations where most cells are proliferating. Interestingly, apoptosis-induced compensatory proliferation in differentiating eye tissue of third-instar larvae. However, it is unclear whether this form of compensatory proliferation is controlled by a similar mechanism as reported for larval proliferating wing discs (Fan, 2008).
This study revealed that there are at least two distinct mechanisms that promote compensatory proliferation in response to apoptotic activity. The general difference between these two mechanisms lies in the developmental context of the tissue in which compensatory proliferation occurs. In proliferating wing and eye tissues, compensatory proliferation induced by extensive apoptosis is dependent on Dronc and Dp53, which induce Dpp and Wg expression. In contrast, in differentiating eye tissue, apoptosis induces compensatory proliferation through a novel mechanism requiring the effector caspases DrICE and Dcp-1, which induce Hh signaling in a nonapoptotic function (Fan, 2008).
When cells stop proliferating and become committed to adopt cell fate, dramatic changes in gene expression are occurring. Given these changes in developmental plasticity, it is not surprising that distinct mechanisms of apoptosis-induced compensatory proliferation are employed in proliferating versus differentiating tissues. However, it should be noted that the proliferating capacity of differentiating tissues is rather restricted. In GMR-hid eye discs, although hid is expressed in all cells posterior to the MF, compensatory proliferation occurs only in cells that are still undifferentiated. Yet, even though they are undifferentiated they have withdrawn from the cell cycle and, under normal developmental conditions (i.e., without GMR-hid), they would soon be recruited to adopt cell fate. However, the apoptotic environment causing increased Hh signaling appears to be able to trigger reentry of these cells into the cell cycle (Fradkin, 2008).
Interestingly, the Hh signal is specifically increased in photoreceptor neurons requiring a nonapoptotic activity of effector caspases. Hh signaling can then nonautonomously induce proliferation of undifferentiated cells at the basal side of the eye disc. However, overexpression of Hh posterior to the MF in wild-type eye discs alone is not sufficient to induce a comparable wave of compensatory proliferation as in GMR-hid eye discs. This suggests that cell-cycle reentry requires activation of additional factors/pathways stimulated in apoptotic cells (Fradkin, 2008).
Although hid can stimulate increased Hh expression in photoreceptor neurons throughout the posterior half of the eye disc, compensatory proliferation is restricted to a certain distance (six to ten ommatidial columns) from the MF. This corresponds to approximately 6-15 hr of developmental time, and might be the time required for cell-cycle reentry. Similarly, when mammalian cells that have exited the cell cycle are stimulated to reenter the cell cycle, they need about 8 hr to do this. The reason for this delay is unknown. Studying compensatory proliferation in GMR-hid eye discs might provide a genetic model to address this interesting problem (Fradkin, 2008).
It is not clear whether this novel effector caspase-, Hh-dependent pathway of compensatory proliferation also applies to other, or even all, differentiating tissues. However, what this study shows is that there are at least two distinct mechanisms of apoptosis-induced compensatory proliferation. It is also possible that other mechanisms of compensatory proliferation in different developmental contexts are going to be uncovered in the future. Interestingly, in developing larval wing discs, P35-dependent compensatory proliferation has been implicated in cell competition. This suggests that, even in tissue with the same developmental potential, compensatory proliferation can occur with distinct mechanisms (Fradkin, 2008).
How cells sense different developmental contexts and operate distinct proliferating mechanisms in response to apoptotic stress is unknown. Specifically, where is the specificity and selectivity for distinct caspases coming from in tissues of different developmental potential? What are the mechanisms engaged by these caspases to trigger secretion of either Dpp and Wg or Hh? These are questions which need to be addressed in the future (Fan, 2008).
This study has several implications for tumorigenesis. First, many tumors develop when quiescent cells reenter the cell cycle. The mechanisms for cell-cycle reentry are largely unknown. Second, evasion from apoptosis is a hallmark of cancer. Many tumor cells are induced to undergo apoptosis. However, they do not die, because they downregulate essential components of the apoptotic pathway such as Apaf-1 and caspases. Thus, these undead tumor cells might secrete mitogens which might induce compensatory proliferation similar to the Drosophila case. In this way, undead cells might contribute to the growth of the tumor. A similar argument can be made for chemotherapy, which in many cases attempts to activate the apoptotic program in a tumor cell. If the death of the tumor cell is blocked, or slow, mitogens might be produced and the tumor growth could be even more severe. This is very obvious in the apoptotic wing or anterior eye discs in Drosophila when apoptosis is blocked by P35. Under these conditions, overgrown wing and eye tissues are observed. Thus, evasion of apoptosis might directly contribute to tumor growth. Finally, although increased Hh signaling can lead to various cancers, how Hh induces cellular proliferation and tissue overgrowth is not well understood. Mutations in Patched1, a negative regulator of sonic Hh, frequently give rise to human tumors. The exact cause is unknown. These data imply that Hh signaling might be involved in cell-cycle reentry allowing cells to resume proliferation (Fan, 2008).
Handed asymmetry in organ shape and positioning is a common feature among bilateria (for a review see Huber, 2007), yet little is known about the morphogenetic mechanisms underlying left-right (LR) organogenesis. This study utilized the directional 360° clockwise rotation of genitalia in Drosophila to study LR-dependent organ looping. Using time-lapse imaging, it was shown that rotation of genitalia by 360° results from an additive process involving two ring-shaped domains, each undergoing 180° rotation. The results show that the direction of rotation for each ring is autonomous and strictly depends on the LR determinant myosin ID (MyoID: Myo31DF). Specific inactivation of MyoID in one domain causes rings to rotate in opposite directions and thereby cancels out the overall movement. A specific pattern of apoptosis at the ring boundaries is revealed, and this study also shows that local cell death is required for the movement of each domain, acting as a brake-releaser. These data indicate that organ looping can proceed through an incremental mechanism coupling LR determination and apoptosis. Furthermore, they suggest a model for the stepwise evolution of genitalia posture in Diptera, through the emergence and duplication of a 180° LR module (Suzanne, 2010).
Left-right (LR) asymmetric development is essential to the
morphogenesis of many vital organs, such as the heart. Directional
looping of LR organs is a complex morphogenetic process relying on proper coordination of early LR patterning events with late cell-tissue behaviors. In vertebrates, several developmental models have been proposed for gut coiling
downstream of the Nodal-Pitx2 regulatory pathway, including
intrinsic asymmetric elongation of the gut in Xenopus or
extrinsic force generation by mesenchymal tissue in Zebrafish
and by dorsal mesentery in the chick and mouse embryos. However, the cellular mechanisms underlying LR organ morphogenesis are mostly unknown (Suzanne, 2010).
In Drosophila, directional clockwise (or dextral) rotation of
the genital plate and gut has been shown only recently to be
controlled by the LR determinant myosin ID (MyoID).
In myoID mutant flies, LR morphological markers are inverted,
leading to counterclockwise (or sinistral) looping of the genital
plate, spermiduct, gut, and testis. This indicates that
myoID is a unique situs inversus gene in Drosophila.
Intriguingly, the expression of MyoID is restricted to two
rows of cells within the A8 segment of the genital disc (the
analia and genitalia precursor), with one row of expression in the anterior compartment (A8a) and the other in the posterior compartment (A8p) (Suzanne, 2010).
Removal of myoID function specifically in the A8 segment
is sufficient to provoke the complete inversion of rotation
(360° counterclockwise) of the genitalia and sinistral looping
of the spermiduct to which it is attached. The A8 segment
therefore represents a LR organizer controlling the directional
rotation of the whole genitalia in Drosophila (Suzanne, 2010).
Because circumrotation (the process of 360° rotation) may result from a number of different morphogenetic processes, not deducible from the simple observation of the final adult phenotype, a new and innocuous imaging
method was developed to follow the rotation in living pupae (Suzanne, 2010).
To be able to analyze the movement of distinct domains in
live developing genitalia, time-lapse imaging was coupled with
genital disc 'painting' by expressing different fluorophores in
various regions of the genitalia precursor. Analysis of wildtype
live genitalia through this method revealed their spatial
and temporal organization during rotation. It was first determined
that rotation begins at around 25 hr after puparium formation
(APF) and lasts 12-15 hr. At 25 hr APF, the genital
disc is organized into concentric rings, which, from anterior
to posterior, include an A8a ring, an A8p ring, and a large central
disc composed of A9-A10 tissues. The analysis of
rotation in live pupae coupled to manual tracking allowed the
identification of two distinct moving domains: a large posterior
domain comprising A8p-A9-A10 (hereafter referred to as A8p) and a smaller
anterior domain made of A8a. The A8p domain moves first and is followed by A8a, which starts moving later on. During the entire process, cells from the abdomen, to which the genital disc is connected, remain immobile. The finding of two rotating
domains, A8a and A8p, was unexpected. It reveals a complex
rotational activity of the genitalia and rules out a simple model
in which the genital plate would rotate by 360° as a whole. To
further understand how rotation occurs, timelapse
imaging of the full, 15-hr-long rotation was performed. This analysis
revealed that each ring had a different rotational activity.
When viewed from the posterior pole, the A8p ring undergoes
360° clockwise rotation, while the A8a ring makes a 180° clockwise
rotation. Whereas the rotation of the central part (A8p-A10) of the disc was inferred from the looping of the spermiduct around the gut, the 180° rotation
of A8a was not predicted and could only be revealed by
time-lapse analysis because this compartment solely gives
rise to a tiny and colorless part of the cuticle. Altogether,
these in vivo analyses show that rotation of genitalia in
Drosophila is a composite process involving two compartments
of the A8 segment, A8a and A8p, each expressing a row of MyoID at its anterior boundary and having its own rotational behavior (Suzanne, 2010).
These findings raise the questions of the contribution of
each of the two rings to the entire rotation and of how they
interact during rotation. In order to address this question, the intrinsic or real rotational activity of A8a and A8p was determined. So far, each ring movement was analyzed relative to the same immobile referential: the abdomen. Although this
referential allows the real movement of A8a to be determined, it cannot be used to determine that of A8p, because A8p moves relatively to a mobile referential, i.e.,
A8a, to which it is attached. To determine the real
movement of A8p, it is thus essential to analyze its angular
movement relative to A8a, in other words A8a contribution to
motion must be subtracted from the apparent A8p movement.
To do so, movies were analyzed by setting A8a as a referential
and by determining the angular movement of A8p. Reassessing
A8p movement through this approach revealed that
A8p rotates clockwise only by 180° relative to A8a.
The new angular velocity curve of A8p fits almost perfectly with
that of A8a, indicating that both movements have similar
features. Importantly, these data also indicate
that the observed 360° clockwise rotation is the result of a
composite process involving two additive 180° clockwise
components: a 180° rotation of the A8a relative to the
abdomen and an 180° rotation of A8p relative to A8a (Suzanne, 2010).
To further determine the autonomy of each ring relatively to
the other, the role of the LR determinant MyoID in
this process was dissected by specifically inactivating myoID in either A8a or
A8p or in both. By convention, the presence or absence of
myoID is represented by a + or - sign, respectively.
Accordingly, the wild-type context is noted 'A8a+A8p+' and
the myoID mutant 'A8a-A8p-.' Upon specific inactivation of myoID in the A8a domain (A8a-A8p+ context), the adults showed an apparent 'nonrotation
phenotype' (0°, no spermiduct looping and genitalia correctly
oriented). However, time-lapse imaging revealed that both rings
were spinning, although in opposite directions: the A8a domain
rotated counterclockwise by 180° (-180°), whereas the A8p
domain rotated clockwise by 180° (+180°, real movement). Reciprocally, the inactivation of myoID in the A8p domain (A8a+A8p- context) also led to an apparent nonrotation phenotype. In this context, the behavior of each
domain was inverted compared to the previous condition,
with the A8a domain rotating clockwise by 180° (+180°) whereas
the A8p domain rotated anticlockwise by 180° (-180°, relative
or real movement). In both cases, the movement of each ring is consistent with its myoID genotype and the 'dextralizing' activity of this gene. The strict dependence on MyoID for the direction of the rotation is further
confirmed in flies where both A8a and A8p were mutants for
myoID (myoIDk1). The rotation is often
incomplete in this genotype because of the hypomorphic nature
of the myoIDk1 allele analyzed; however, both domains show
an anticlockwise movement. Therefore, in all genetic contexts
analyzed, all parameters of the rotation remain unaffected
except the direction of rotation, as illustrated by the perfect
mirror image of the angular velocity curves (Suzanne, 2010).
These experiments reveal that each ring adopts an independent
180° movement relative to more anterior structures
(A8a relative to the abdomen and A8p relative to A8a):
clockwise in the presence of MyoID, anticlockwise in its
absence. When both movements are unidirectional, the net
rotation is circumrotation (± 360°), whereas upon opposite
movements of A8a and A8p, the net rotation is zero (0°),
leading to an apparent nonrotation phenotype. Therefore, the
net rotation (or apparent rotation = R) can be modeled through
a simple equation in which R equals the addition of A8a and A8p movements, with MyoID acting as a sign function (Suzanne, 2010).
It was next of interest to characterize potential cellular mechanisms
acting downstream of LR determination during genitalia
rotation. In particular, the cellular events responsible for uncoupling rings at the onset of their rotation was determined. Initial insights came from blocking apoptosis, which leads to genitalia rotation defects,
but the role of apoptosis in the process is not completely
understood. To determine the morphogenetic function of the
apoptotic pathway during genitalia rotation,
the spatial and temporal requirements for apoptosis were first characterized by
analyzing the expression pattern of hid and reaper (rpr) in
the genital disc, using two reporter lines. Both reporters
were strongly expressed in the A9 and A10 segments. However,
in the A8 segment, only hid expression is observed. This coincides with the phenotype of misrotated genitalia observed specifically when hid function
is altered but not in rpr mutants. Then the pattern and timing of cell death was determined in the genital disc. To do so, nuclear fragmentation was followed, and an in vivo reporter of caspase activation (the apoliner
construct) was used. At the onset of rotation, a large number of
apoptotic cells was detected on the most ventral part of the
genital disc, first within the A8p ring bordering A8a, coinciding
with the beginning of A8p movement. These data indicate an
overlap between the apoptotic field and the domain of MyoID
expression. These results have been further confirmed by the detection of
apoptotic cells by TUNEL staining of fixed pupal genital discs. Later on, a new wave of apoptosis was detected in the most anterior part of the A8a ring, at the junction between A8a and the abdomen. In contrast, only marginal if
any apoptosis was detected before and at the end of rotation. Therefore, two waves of cell death are taking place in the A8 segment, coinciding spatially and temporally with the rotation of A8a and A8p rings (Suzanne, 2010).
Given that rings are initially part of the same epithelium and
move independently later, it was reasoned that local cell death
may be a mechanism to provide the degree of liberty
necessary for proper movement. To test this hypothesis, cell death was inhibited in each compartment separately by expressing the caspase inhibitor p35. Interestingly, inactivation of apoptosis in either A8p or A8a leads to a similar
phenotype, with flies showing a high proportion of half-rotated
genitalia (180° rotation), suggesting that rotation was blocked in the ring deficient for apoptosis. This has been further demonstrated by following
the rotation process in vivo, when apoptosis is specifically
blocked in the A8a. In this genetic context, the A8a ring stayed
mostly still during the whole process, whereas A8p rotated
normally. The resulting 180° rotation is thus exclusively due
to the movement of one ring, i.e., A8p, in which apoptosis is
unaffected. Inhibiting apoptosis in both domains
strongly aggravates the phenotype, with 40% of the flies
showing nonrotated genitalia (0°), suggestive of an additive
phenotype. The rest of the population had 90° rotated genitalia,
which may be due to incomplete inhibition of apoptosis. Alternatively, it is possible that some rotation occurs without apoptosis thanks to tissue elasticity. In any case, the results indicate that cell death is required in each
ring for separating them from the neighboring tissues and
allowing their free rotation. Consistently, nuclei fragmentation
and cell death occur normally in a myoID mutant background. Because local cell death is not likely to provide a direct force for rotation, it is proposed that it contributes to the release of rings from neighboring tissues (Suzanne, 2010).
This study has revealed that organ looping can proceed
through discrete steps, breaking down circumrotation into the
simple building blocks of 180° each. The incremental nature of
genitalia rotation is indeed based upon two 180° LR modules,
sharing identical angular velocity and range as well as requirement
for MyoID and apoptosis. Modularity in
morphogenesis provides interesting control mechanisms
(through addition or substraction) and therefore plasticity to
the process, both at the organism level and during evolution.
Entomologists have described different patterns of genitalia
rotation in Diptera, ranging from 0° to 360°, that evolved
together with changes in mating position. Interestingly,
in the Brachycera suborder, to which Drosophilidae belong, we
notice that most ancestral species have a nonrotated genitalia
(Stratiomyomorpha and Tabanomorpha), whereas 180° and
360° rotation have appeared progressively later in evolution
(in Muscomorpha and Cyclorrhapha, respectively).
Together with this sequential organization of rotation amplitude
in the phylogenetic tree, these data strongly support a model
by which the 360° rotation observed in Brachycera ('modern
Diptera') would result from the emergence (transition from
0 to 180°) and duplication (transition from 180° to 360°) of a
180° L/R module (Figure S3), thus providing a simple additive
model for both the origin of circumrotation and the evolution of
genitalia rotation and mating position. However, it should be
noted that alternative mechanisms maylead to a similar pattern
of genitalia rotation among Diptera (Suzanne, 2010).
The incremental model presented here also offers a solution
to the apparent paradox of circumrotation and the question of
its elusive utility, illustrated by the fact that both 360° rotation
and the absence of rotation lead to the same final posture of
genitalia. A facultative role of 360° rotation is further supported
by the finding that D. melanogaster males with nonrotating
genitalia (A8a-A8p+ or A8a+A8p-) are normally fertile (data
not shown). An incremental origin of 360° rotation in which
a second half-turn would be added to the existing 180° rotation
would well explain this paradox. Thus, circumrotation can be
viewed as recapitulating the evolutionary history of genitalia
rotation in Brachycera, and its logic would reveal a case of
'retrograde evolution,' in which duplication of a functional
module is used to revoke a previous evolutionary step (Suzanne, 2010).
Finally, this analysis of genitalia rotation highlights a new mechanism of morphogenesis relying on a combination of LR patterning and apoptosis. In this process, a new role for apoptosis is revealed as a releasing mechanism
allowing the sliding of two parts of an organ. It will be interesting
to test in the future whether this releasing role of
apoptosis is used more generally, in other morphogenetic
movements requiring important cellular rearrangement (Suzanne, 2010).
The head involution defective locus is located within the
chromosomal region 75B8-C1,2. During the morphogenetic reorganization of the embryonic head
region, hid+ function is necessary for the movement of the dorsal fold across the procephalon and
clypeolabrum, a process that forms the frontal sac. The absence of the frontal sac in the hid mutant
embryos affects the formation of the dorsal bridge and disrupts the development of the larval
cephalopharyngeal skeleton. In addition to its embryonic role, this same hid function is also required
during pupal development for the 360 degrees rotation about the
anterior-posterior body axis of the male terminalia, and for a late step of wing blade morphogenesis. Although the abnormal
wing phenotype caused by the Wrinkled (W) mutation is quite different from the one resulting from the
loss-of-function hid mutations, the characterization of EMS-induced W revertants reveals that W is
actually an antimorphic allele of hid (Abbott, 1991).
Deletions of chromosomal region 75C1,2 block virtually all programmed cell death (PCD) in the Drosophila embryo. A second gene, in addition to reaper, has now been identified in this region. head involution defective (hid) plays a similar role in PCD. hid mutant embryos have decreased levels of cell death and contain extra cells in the head. hid mutant embryos have extra cells in the head region, in particular, extra larval photoreceptor cells. There are also extra cells in the abdominal segments. Expression of the hid gene is sufficient to induce PCD in cell death defective mutants. The hid gene appears to encode a novel 410-amino-acid protein, and its mRNA is expressed in regions of the embryo where cell death occurs. Ectopic expression of hid in the Drosophila retina results in eye ablation. This phenotype can be suppressed completely by expression of the anti-apoptotic p35 protein from baculovirus, indicating that p35 may act genetically downstream from hid (Grether, 1995).
Expression of the cell death regulatory protein Reaper (Rpr) in the developing Drosophila eye
results in a smaller than normal eye owing to excess cell death. Mutations in thread (th) are
dominant enhancers of Rpr-induced cell death. thread encodes a protein homologous to
baculovirus inhibitors of apoptosis (IAPs), called Drosophila IAP1 (DIAP1). Overexpression of DIAP1 (or a related protein, DIAP2) in the eye suppresses normally occurring cell death as well as death due to overexpression of rpr or head involution defective. IAP death-preventing activity localizes to the N-terminal baculovirus IAP repeats, a motif found in both viral and cellular proteins associated with death prevention (Hay, 1995).
A new activator
of apoptosis, grim, maps between two previously identified cell death genes in this region: reaper
and head involution defective. Expression of Grim RNA coincides with the onset of programmed cell
death at all stages of embryonic development, whereas ectopic induction of grim triggers extensive apoptosis
in both transgenic animals and in cell culture. Cell killing by Grim was blocked by coexpression of p35, a viral
product that inactivates ICE-like proteases, and does not require the function of either rpr or hid. The predicted Grim
protein shares an amino-terminal motif in common with RPR. However, Grim is sufficient to elicit apoptosis in
at least one context, where Rpr is not. The grim gene product might thus function in a parallel circuit of cell
death signaling that ultimately activates a common set of downstream apoptotic effectors (Chen, 1996).
The neuropeptide eclosion hormone (EH) is a key regulator of insect ecdysis. The role of the two
EH-producing neurons in Drosophila was determined by using an EH cell-specific enhancer to activate cell death genes reaper and
head involution defective in order to ablate the EH cells. In the EH cell knockout flies, larval and adult ecdyses are disrupted,
yet a third of the knockouts emerge as adults, demonstrating that EH has a significant but nonessential role in
ecdysis. The EH cell knockouts have discrete behavioral deficits, including slow, uncoordinated eclosion and an
insensitivity to ecdysis-triggering hormone. The knockouts lack the lights-on eclosion response despite having a
normal circadian eclosion rhythm. This study represents a novel approach to the dissection of neuropeptide regulation
of a complex behavioral program (McNabb, 1997).
In Drosophila, the chromosomal region 75C1-2 contains at least three genes (reaper,
head involution defective, 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).
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).
Three genes---reaper, grim, and hid---are crucial to the regulation of programmed cell death in Drosophila. Mutations involving all three genes
virtually abolish apoptosis during development, and homozygous hid mutants die as embryos with extensive defects in apoptosis. Although Hid is central to
apoptosis in Drosophila, it has no mammalian homolog identified to date. Evidence is presented that expression of Drosophila Hid in mammalian cells
induces apoptosis. This activity is subject to regulation by inhibitors of mammalian cell death. The N terminus of Hid, which is a region of
homology with Reaper and Grim, is essential for Hid's function in mammalian cells. Hid is localized to the mitochondria via a
hydrophobic region at its C terminus and functionally interacts with BclXL. This study shows that the function of Hid as a death inducer in Drosophila is
conserved in mammalian cells and argues for the existence of a mammalian homologue of this critical regulator of apoptosis (Haining, 1999).
Some Bcl2 family members have potent antiapoptotic effects. The antiapoptotic members include BclXL and the
adenoviral protein E1B19k. Although homologs of this family exist in C. elegans and mammals, no Drosophila counterpart has yet been identified. It was
therefore of interest to ascertain whether the apoptosis pathway triggered by Hid in mammalian cells is susceptible to Bcl2-family inhibition. BclXL shows a
pronounced effect on reducing Hid-induced apoptosis (35% to 11%), whereas E1B19k shows a more modest effect (to 23%). These results demonstrate
that Bcl2-type antiapoptotic genes can inhibit Hid-induced apoptosis in mammalian cells (Haining, 1999).
Given the functional interaction between Hid and BclXL, a protein that can target the mitochondria, it was of
interest to determine the cellular localization of Hid. A monoclonal antibody was raised to full-length Hid protein and used to label transfected cells immunohistochemically. Hid immunostaining is predominantly punctate and perinuclear. To better identify the subcellular distribution of Hid, transfected cells were colabeled with a
fluorescent dye that accumulates inside mitochondria. The pattern of mitochondrial staining is very similar to that of Hid. Merged images of Hid- and
mitochondrially stained cells show a striking concordance in the distribution of these two stains. This result demonstrates that Drosophila Hid localizes to
mitochondria when expressed in mammalian cells. Further magnified views of dually stained cells shows that, although the pattern of staining is very similar, it is not
overlapping; rather, the Hid-staining appears on the outside of the mitochondrion whereas the mitochondrial dye labels the inner portion.
Despite the lack of Hid-induced apoptosis in 293 cells, it is noteworthy that Hid's distribution in these cells is also mitochondrial (Haining, 1999).
Because the mitochondrial localization of Hid had not been previously demonstrated in insect cells, Hid was expressed by transient transfection in the insect cell line
SF9. This cell line was found to be susceptible to apoptosis from Hid overexpression. Cells colabeled with mitochondrial dye and Hid antibody again showed a
predominantly mitochondrial pattern of Hid staining (Haining, 1999).
To assess the effect of apoptosis inhibition on the pattern of Hid staining, immunohistochemistry was performed on HeLa cells cotransfected with Hid and BclXL.
The mitochondrial localization of Hid is disrupted in these cells, and Hid fluorescence is found in a diffuse pattern, suggestive of cytoplasmic
distribution. This effect is not seen in cells cotransfected with p35, DIAP1, or XIAP or in those treated with the inhibitor of apoptosis BOC-D-fmk (Haining, 1999).
To investigate which portions of the Hid molecule are
required for its proapoptotic activity and subcellular localization, two Hid mutant proteins encoded by alleles A206 and A329 were studied. These mutations in the hid
gene locus were induced in flies by chemical mutagenesis, and they cause a mild reduction in Hid function in Drosophila. Each mutation is the result of a single
nucleotide change that causes a premature stop codon at amino acid position 261 and position 304 in alleles A206 and A329, respectively. Both of
these prematurely truncated proteins induce apoptosis in HeLa cells at levels comparable to those caused by wild-type Hid. This may be because of the
high levels of Hid expression achieved in HeLa cells. A reduction of Hid function that may be sufficient to reduce its proapoptotic activity in Drosophila cells may not
be noticeable in HeLa cells because of the large amounts of Hid protein expressed.
Immunohistochemistry of cells transfected with each of these mutants, however, shows a marked alteration of cellular localization. Whereas levels of expression
are comparable, the mitochondrial targeting of wild-type Hid is completely lost, and the mutant Hid-transfected cells shows a diffuse cytoplasmic pattern of
staining. Although Hid appears to have neither a signal sequence nor a mitochondrial localization signal, close scrutiny of the C terminus reveals a stretch of
hydrophobic residues (amino acid position 393-409). Deletion of these residues is sufficient to abolish mitochondrial localization. However, this mutation
does not impair apoptosis induction. These results suggest that when expressed at high levels in HeLa cells, Hid does not require mitochondrial localization to
effect cell death. However, the fact that mutations that delete the C terminus of Hid were identified as loss-of-function in Drosophila suggests that this domain, and
possibly mitochondrial localization, is important for Hid's proapoptotic function (Haining, 1999).
Sequence analysis of Hid, Reaper, and Grim reveals similarities among the three proteins restricted to their
N-terminal 14 amino acids. Deleting residues 2-14 of Hid abolishes its ability to initiate apoptosis in mammalian cells. Immunostaining of
mutant-transfected cells shows levels of expression comparable to cells transfected with wild-type Hid. The deletion does not impair the mutant's ability to
localize to the mitochondria. Because the deleted region is that required for DIAP1 binding, one interpretation of this result is that binding to IAPs (presumably endogenous mammalian IAPs) in these experiments is essential for Hid's ability to induce cell death in HeLa cells (Haining, 1999).
During development, signaling pathways coordinate cell fates and regulate the choice between cell survival or programmed
cell death. The well-conserved Wingless/Wnt pathway is required for many developmental decisions in all animals. One
transducer of the Wingless/Wnt signal is Armadillo/ß-catenin. Drosophila Armadillo not only transduces Wingless signal,
but also acts in cell-cell adhesion via its role in the epithelial adherens junction. While many components of both the
Wingless/Wnt signaling pathway and adherens junctions are known, both processes are complex, suggesting that unknown
components influence signaling and junctions. A genetic modifier screen was carried out to identify some of these
components by screening for mutations that can suppress the armadillo mutant phenotype. Twelve regions of the genome were identified that have this property.
From these regions and from additional candidate genes tested, four genes were identified that suppress arm: dTCF, puckered, head involution defective
(hid), and presenilin. The interaction with hid, a known regulator of programmed cell death, was further investigated. The data suggest that Wg signaling
modulates Hid activity and that Hid regulates programmed cell death in a dose-sensitive fashion (Cox, 2000).
It has been known for more than a decade that PCD plays an important role in the segment polarity phenotype resulting from inactivation of either the Hedgehog or Wg pathways. Detailed analysis of this process has been carried out, quantitating cell death in wg, arm, gooseberry, and naked. The elevation in cell death affects particular cells. Since the first reports of cell death in segment polarity mutants, the machinery that drives PCD in embryos has begun to be identified. Homozygosity for the small chromosomal Deficiency, Df(3L)H99, blocks essentially all PCD. Within this interval, three genes play roles in PCD: grim, reaper, and hid. Ectopic expression of any of these can trigger PCD, but loss-of-function mutations are only available for hid (Cox, 2000 and references therein).
Given the role of PCD in the segment polarity phenotype, it is perhaps not surprising that elimination of PCD would alter it. Several aspects of the effect of PCD reduction were unexpected, however. First, and most striking, the phenotypes of arm and wg mutants were very sensitive to relatively small changes in the dose of hid and the other cell-death promoters. For example, while heterozygosity for hid has no known effects on normal development, it strongly suppresses arm. Further reductions in the levels of hid or the other cell-death regulators have no additional effect on arm, suggesting that reducing the Hid dose by half eliminates the relevant ectopic PCD that occurs in an arm mutant. The wg phenotype is also suppressed in a highly dose-sensitive fashion, but in a different dosage range. A 50% reduction of hid causes slight but detectable effects; a 50% reduction in all three death promoters causes greater suppression, while homozygosity for the deletion removing all three genes results in the strongest wg suppression (Cox, 2000).
Recent observations regarding the role of Hid in PCD in the eye may explain this. Signaling through the ras/mitogen-activated protein kinase (MAPK) pathway promotes cell survival by antagonizing Hid. It has been suggested that Hid serves as a rheostat, with its levels determining the probability of PCD. It has been further suggested that Hid activity has to exceed a threshold to trigger PCD; the accumulation of hid mRNA in cells that are not programmed to die is consistent with this. Current observations further support this model. Wg signaling may normally antagonize Hid, potentially by regulating its expression. In embryos where Wg signaling is attenuated, elevated Hid activity may trigger PCD when it rises above a critical threshold. A threshold model could explain why the segment polarity phenotype is so sensitive to the dose of Hid and its partners (Cox, 2000 and references therein).
Another surprise was the qualitative difference in the effect of cell death reduction on wg and arm mutants. While the resulting cell number is likely increased in both double-mutant genotypes in the arm; hid double mutant, the reduction in PCD restored an almost wild-type-length cuticle, while in the wg;hid double mutant, the increase in cell number is not reflected in an increase in cuticle length. The reason for this remains a mystery. One possible explanation for this discrepancy is the difference in the degree to which Wg signal is compromised in the two situations and the embryonic stage at which this disruption occurs. In the wg null, Wg signaling is totally eliminated from the beginning of development. In contrast, perdurance of maternal Arm substantially rescues early defects in Wg signaling in arm zygotic nulls. arm mutants remain more normal in morphology than wg mutants through the onset of germband retraction and retain remnant denticle diversity. Thus when one eliminates PCD in an arm mutant a more normal pattern is restored. The difference in amount and timing of Wg signaling in the two backgrounds may also explain why arm mutants are affected by smaller alterations in Hid level. The remaining Wg signaling in an arm zygotic mutant may promote cell survival to some extent, meaning that a smaller reduction in Hid activity prevents ectopic PCD (Cox, 2000).
It is also surprising that reduction in cell death alleviates arm's dorsal closure defect. It has been suspected that this defect is due solely to Arm's role as a catenin. However, recent data suggest that dorsal closure is promoted by Wg signaling. It is now suspected that defects in Wg signaling and catenin function combine to block dorsal closure in arm mutants. Restoring either rescues the arm dorsal closure defect. However, blocking PCD alone should not restore Wg signaling or catenin function. Perhaps the excess cell death in the head region or in the amnioserosa of an arm mutant contributes to its dorsal closure defect (Cox, 2000).
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).
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).
Genetic and microarray analyses have been used to determine how
ionizing radiation (IR) induces p53-dependent transcription and
apoptosis in Drosophila melanogaster. IR induces
MNK/Chk2-dependent phosphorylation of p53 without changing p53
protein levels, indicating that p53 activity can be regulated without
an Mdm2-like activity. In a genome-wide analysis of IR-induced
transcription in wild-type and mutant embryos, all IR-induced
increases in transcript levels required both p53 and the Drosophila
Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper,
sickle, and the tumor necrosis factor family member EIGER.
Overexpression of Eiger is sufficient to induce apoptosis, but
mutations in Eiger do not block IR-induced apoptosis. Animals
heterozygous for deletions that span the reaper, sickle, and
hid genes exhibited reduced IR-dependent apoptosis, indicating
that this gene complex is haploinsufficient for induction of
apoptosis. Among the genes in this region, hid plays a
central, dosage-sensitive role in IR-induced apoptosis. p53 and
MNK/Chk2 also regulate DNA repair genes, including two components of
the nonhomologous end-joining repair pathway, Ku70 and Ku80. These
results indicate that MNK/Chk2-dependent modification of Drosophila
p53 activates a global transcriptional response to DNA damage that
induces error-prone DNA repair as well as intrinsic and extrinsic
apoptosis pathways (Brodsky, 2004).
Programmed cell death is a critical process for the patterning and sculpting of organs during development. The Drosophila arista, a feather-like structure at the tip of the antenna, is composed of a central core and several lateral branches. A homozygous viable mutation in the thread gene, which encodes an inhibitor of apoptosis protein, produces a branchless arista. Mutations in the proapoptotic gene hid led to numerous extra branches, suggesting that the level of cell death determines the number of branches in the arista. Consistent with this idea, it was found that thread mutants show excessive cell death restricted to the antennal imaginal disc during the middle third instar larval stage. These findings point to a narrow window of development in which regulation of programmed cell death is essential to the proper formation of the arista (Cullen, 2004).
Analysis of the th1 mutant has revealed a decrease in cell number by pupal stages, suggesting that excessive apoptosis could have occurred earlier in development. Indeed, TUNEL analysis revealed that th1 mutants show a dramatic increase in apoptosis compared to wild-type at a specific developmental timepoint, the middle third larval instar. Interestingly, caspase activity was found to be more extensive than TUNEL labeling, suggesting that caspases are activated in many antennal cells, but only a fraction succumb to apoptosis. This may indicate that there are other protectors acting downstream of caspase activation when inhibition by Thread fails. Alternatively, because this antibody detects processed effector caspases, the th1 mutant may not be able to inhibit caspase processing but may be able to inhibit enough caspase activity to prevent apoptosis. The increase in caspase activity that was observed is limited both spatially and temporally, such that by the late third larval instar, th1 discs show wild-type levels of immunolabeling. The ectopic caspase activity is also limited to the antennal portion of the eye-antennal disc, suggesting that thread activity or caspase inhibition is regulated differently in the eye and the antenna (Cullen, 2004).
One of the best-characterized activators of apoptosis in Drosophila is head involution defective or hid. Hid is thought to promote apoptosis by binding to Th, displacing it from caspases and triggering its auto-ubiquitination. hid mutants have been shown to have excessive cell numbers in the embryonic CNS and the adult eye. Here, hid mutants have numerous ectopic lateral branches in the posterior antennal arista. Mitotic clones of Df(3L)H99 dp not appear to have more branches than hid mutants alone, suggesting that hid is the primary regulator of cell death in the arista, as it is in the eye. Consistent with this idea, reaper mutants show only a mild aristal phenotype. Attempts were made to alter the amount of cell death in the arista by expression of reaper, hid, grim, or dcp-1, but high levels of expression tended to result in lethality and lower levels of expression did not produce phenotypes (Cullen, 2004).
hid mutants or H99 mosaics did not show any ectopic laterals on the anterior side of the arista, indicating that the anterior laterals could be regulated by a distinct apoptotic activator, or may be formed through an apoptosis-independent mechanism. However, because th1 mutants lack anterior laterals, and dark; th1 double mutants show ectopic anterior laterals, it is likely that an apoptotic mechanism is indeed involved. Rescue experiments indicate that a higher level of thread expression is required for anterior lateral formation, suggesting that there may be a potent apoptotic activator that can overcome low levels of Th present in the cells that give rise to the anterior laterals. Alternatively, different cohorts of caspases may be activated in the anterior and posterior cells, and the caspases in the anterior cells could require a higher level of Th for inhibition (Cullen, 2004).
The results with hid and th mutants suggest that an inhibition of cell death is required for lateral formation. This could be a direct effect, with a particular dying cell influencing the fate of a neighboring cell. Alternatively, the role for cell death could be more indirect, simply affecting the total number of cells, which in turn could determine whether a lateral will form or not. Indeed, th1 mutants have reduced cell numbers in the pupal aristae compared to wild-type, consistent with the observation of considerable apoptosis in the mid-third instar larval stage. Further support for the cell number hypothesis comes from observations of non-autonomy in H99 mitotic clones in the arista. While it was possible to detect ectopic branches in the H99 mosaics, these branches were not always marked with yellow, suggesting that they arose from heterozygous (or homozygous) yellow+ cells. Thus, the H99 clones may increase the total cell number in the developing aristae, but the specific cells that give rise to branches could be either homozygous or heterozygous for H99. How cell number influences lateral formation is unclear. It could involve lateral inhibition or lateral specification, where signaling cells induce adjacent cells to produce branches, and branch-producing cells block that fate in their neighbors. There may be a minimal number of cells required for basic support and extension of the arista; th1 mutants may have only this minimal number of cells, with no extra cells available for branch production. Alternatively, the th1 cells could be unable to produce lateral extensions due to cellular damage from insufficient caspase inhibition (Cullen, 2004).
The ectopic laterals observed in hid mutant aristae are intermediate in length and thickness compared to the long and short wild-type laterals. In addition, the normal longer branches in hid mutants are often shorter and thinner than wild-type. Since the laterals are thought to be formed as actin-rich projections of single cells, it is unclear how perturbing apoptosis could influence the length of the lateral. One possibility is that an increased cell number could lead to crowding or an overall decrease in cell size. The cell size could then influence the amount of cellular material available for the lateral projection. Alternatively, the 'undead' cells that survive abnormally may have ill-defined cell fates or lack sufficient cytoskeletal proteins to generate long lateral branches. Several caspase targets are regulators of the actin cytoskeleton, so increases in caspase activity might perturb the cytoskeleton, even though the caspase activity is not high enough to cause apoptosis. Similarly, the split laterals seen in the dark; th1 mutants could arise from cellular abnormalities (Cullen, 2004).
th1 antennal imaginal discs show increased apoptosis at a specific developmental timepoint, suggesting that regulation of th is critical in these cells. This developmental stage is characterized by rapid cell divisions and the establishment of cell fates. Key regulators of distal antennal fates are the transcription factors Distalless (Dll) and Homothorax (Hth). Coexpression of Dll and hth is sufficient to induce aristal transformations in leg, wing, head, and genital disc derivatives, accompanied by misexpression of spalt, a gene normally expressed in antennal but not leg discs. spalt and several other genes have been identified as targets of Dll and/or hth, however, most of these genes appear to be expressed in the proximal antenna, largely excluded from the presumptive arista. One exception is spineless, which is expressed in the aristal primordia during larval stages. spineless mutants show antennal to leg transformations, suggesting that its normal function is to repress leg and promote antennal fates. It remains unclear how such patterning genes could produce cell fates that are specifically susceptible to loss of Th. Perhaps these genes could directly regulate th levels transcriptionally or post-transcriptionally, and the th1 mutant may have a mutation in a corresponding region (Cullen, 2004).
The molecular nature of the th1 mutation is currently unknown. The th1 mutation behaves like a loss-of-function allele, displaying the aristal phenotype in trans to a deficiency and being rescued by a duplication for the chromosomal region. The coding sequence of the th1 allele is reported to lack any obvious mutations, although the appropriate background strain is unknown. Further investigation will be required to determine if any observed amino acid changes affect the protein function. There are three reported transcripts of th initiating from distinct promoters, but the tissue-specificity of these transcripts has not been reported in detail. Perhaps, the th1 mutation could disrupt one of the transcript variants that is primarily expressed in the presumptive arista, lowering the Th protein levels below a certain level necessary for maintaining caspase inhibition. The spontaneous nature of the th1 mutation suggests that it could be caused by the insertion of a transposable element, which could potentially disrupt specific transcripts. Future molecular analysis of the th1 mutation will contribute to the understanding of the role of cell death in patterning the antennal arista (Cullen, 2004).
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).
Regulated cell death and survival play important roles in neural development. Extracellular signals are presumed to regulate seven apparent caspases to determine the final structure of the nervous system. In the eye, the EGF receptor, Notch, and intact primary pigment and cone cells have been implicated in both survival and death signals. An antibody (CM1) raised against a peptide from human caspase 3 was used to investigate how extracellular signals control spatial patterning of cell death. The antibody crossreacts specifically with dying Drosophila cells and labels the
activated effector caspase Drice. The initiator caspase Dronc and the proapoptotic gene head involution defective are important
for activation in vivo. Dronc may play roles in dying cells in addition to activating downstream effector caspases. Epistasis experiments ordered the EGF
receptor, Notch, and primary pigment and cone cells into a single pathway that affects caspase activity in pupal retina through hid and Inhibitor of
Apoptosis Proteins (IAPs). None of these extracellular signals appear to act by initiating caspase activation independently of hid. Taken together, these
findings indicate that in eye development spatial regulation of cell death and survival is integrated through a single intracellular pathway (Yu, 2002).
A particularly useful feature of the CM1 antiserum is the detection of cells that would otherwise be marked for death but which are protected by baculovirus p35 expression. The morphological protection provided by p35 may permit better investigation of the location and autonomy of death and survival signals. On Western blots the CM1 antibody detects activated Drice but not the Drice zymogen; Drice is the Drosophila sequence most similar to the immunizing peptide. Definitive evidence that CM1-stained cells are apoptotic comes from the dependence of embryonic CM1-staining on the 75C1,2 chromosome region, and from the p35-sensitivity of larval and pupal CM1-stained cells. The morphology of all CM1-stained cells is altered by p35 expression. In the presence of p35, CM1-stained cells become indistinguishable from normal cells by morphological criteria, and are not distinguishable except by CM1 staining. Since baculovirus p35 blocks cell death by inhibiting caspase activity, p35-dependent morphology of CM1-stained cells shows that such morphology depends on caspase activity in the cells, which are therefore apoptotic. These results show that only apoptotic cells are labelled by the CM1 antiserum. Apoptotic cells that are unlabelled by CM1 might also exist, although none have been noticed (Yu, 2002).
The results indicate that effector caspases such as Drice can be processed in the presence of baculovirus p35. The initiator caspase Dronc is responsible for effector caspase processing in p35 expressing cells. p35-insensitive caspases are also thought to initiate cell death in other insect cells. In Drosophila eye development, Dronc always functions redundantly with other, p35-sensitive initiator caspases. Such redundancy explains why Dronc-DN had no effect in earlier studies of eye development. Dronc has been implicated in embryonic cell death by RNA interference studies (Yu, 2002).
If feedback of effector caspases on their own activation is essential for cell death, it would be expected that Dronc alone is insufficient for CM1 labelling in p35-expressing cells. By contrast, CM1 labelling persists in most cells, indicating that feedback of effector caspases is dispensable for the activation of at least one effector caspase. However, the results do not exclude the possibility that feedback makes a quantitative contribution to the pace of death or is required for a subset of effector caspases. Results are different for neuronal photoreceptor cells. R8 photoreceptor survival is rescued in the absence of EGFR by p35 expression, but such rescued R8 cells are not labelled strongly by CM1. It is possible that amplification of the apoptotic cascade might be more important for apoptosis of R8 precursors than for unspecified cells. It is also possible that R8 cell apoptosis involves different effector caspases or is initiated independently of Dronc (Yu, 2002).
These findings suggest that Dronc may cleave other substrates in dying cells in addition to activating p35-sensitive effector caspases. Mild eye roughening, seen when p35 is expressed in the eye, is found to depend on Dronc activity. It seems unlikely that the rough eye could be due to some downstream effector caspases escaping the p35 inhibition, because DIAP1 overexpression blocks cell death less effectively than p35 but does not cause eye roughening. It is speculated that Dronc might have cellular targets other than downstream caspases and that cleavage of such targets affects eye morphology. However, these data provide no evidence for Dronc activity, except in cells that would normally die. The simplest model is that Dronc might have another role in cell death in addition to activating effector caspases. The data do not support any effect of p35 other than its inhibition of caspases, since the eye roughening caused by p35 is suppressed by co-expression of DIAP1 or Dronc-DN (Yu, 2002).
Mutations of hid reduce cell death in pupal eye development. hid is absolutely required for caspase activation in both eye disc and pupal retina. Cell death is reduced even in hid/+ heterozygotes, consistent with dominant effects of hid in modifier screens. hid is required for caspase processing, which is redundantly mediated by Dronc and other initiator caspases. Therefore, in principle, Hid is a candidate for regulating initiator caspases, however, in embryos, hid functions to sequester DIAPs. In pupal retinas, DIAP overexpression mimics hid mutation, consistent with sequestration of DIAPs by Hid. In the eye imaginal disc, however, proapoptotic Hid function is not overcome by DIAP1 overexpression, since targeted DIAP1 expression neither reduces cell death in normal eye discs nor protects against cell death when EGFR function is removed in egfr mutant clones. It seems unlikely that endogenous Hid levels are too high for DIAP1 to be effective, because hid is haploinsufficient for eye disc cell death. Instead, these findings raise the possibility of a proapoptotic activity of Hid that is not blocked by DIAP1. This could involve inhibiting a pathway parallel to DIAP1 that also inhibits caspase activation, or promoting activation of caspase zymogens in other ways, for which there is precedent in vertebrates (Yu, 2002).
Two other proapoptotic genes, rpr and grim, induce eye cell death on ectopic expression. Whether rpr or grim are required for cell death in normal eye development is uncertain because point mutants are not available. The absolute requirement for hid may indicate that rpr and grim are not active during normal eye development. Since hid has been shown to be required for eye death in response to ectopic rpr, however, it is also possible that rpr and grim have activities that depend on hid function (Yu, 2002).
Experiments using the egfrts1a allele have confirmed that Egfr is required for survival of pupal retinal cells, as suggested by misexpression experiments. Egfr is also required for survival of eye imaginal disc cells. Consistent with the model that Egfr prevents cell death by inactivating hid, hid is absolutely required for caspase activation in egfr mutant clones. Similar results have been obtained using TUNEL experiments to assess Egfr-DN-induced cell death (Yu, 2002).
Survival in pupal retina is regulated by two further extracellular signals that are not involved in eye imaginal discs. In principle, such signals might act to modulate Egfr signaling, to regulate Hid or DIAP activity in parallel to Egfr, or to activate initiator caspases. Notch (N) is required for caspase activation in the pupal retina. Epistasis experiments show that N is not required for pupal cell death in the absence of Egfr function, and therefore that the normal function of N is to inhibit the Egfr survival signaling pathway in pupae. Such results place N upstream of Egfr and indicate that N acts ultimately through hid and the anti-apoptotic DIAP proteins that prevent caspase activation, rather than through N-mediated caspase activation. Survival in pupal retinas also depends on signals from primary pigment cells and/or cone cells. Such signals must antagonize proapoptotic N activity, since N is epistatic to the primary pigment cell/cone cell signal. The data now imply a pathway in which primary pigment cells and/or cone cells promote survival by inhibiting activation of N, thus preventing N antagonism of Egfr activity in the interommatidial cells (Yu, 2002).
The essential role of Egfr now seems to be downstream of N, whereas the cone cell/primary pigment cell signal must act upstream. Downstream Egfr function raises anew the question of identity of the primary pigment cell/cone cell signal. Primary pigment cells or cone cells do not seem essential for Egfr activation, because N is still required for apoptosis after ablation of these cells. Pupal photoreceptor cells express the Egfr ligand SPI and its processing/presenting factor Rhomboid, and are one possible source of Egfr activation. One model suggests that primary pigment cells and/or cone cells are the source of an unidentified signal or mechanism that prevents N activation (in particular interommatidial cells) so that Egfr survival signaling can continue (Yu, 2002).
According to one view, survival signals are the critical extracellular regulators of developmental cell death. By contrast, results from C. elegans and mammals indicate that cell death depends on activation of initiator caspases to trigger the apoptotic cascade. Homologs of the activatory components exist in Drosophila. Studies of eye development place three extracellular signals in a pathway acting through Egfr and hid to regulate survival, in part through IAPs. The only evidence consistent with positive regulation of apoptosis is that in eye imaginal discs, hid appears to promote cell death through an unidentified mechanism independent of DIAPs, and, in this case, the role of EGF receptor signaling is still to promote survival by inhibiting Hid (Yu, 2002).
These findings do not rule out other pathways that activate initiator caspases during eye development, or that such activation might be required for cell death. Since hid is essential for cell death, however, pathways that activate initiator caspases independently of hid cannot be sufficient for any of the cell death that normally occurs during eye development. Because loss of Egfr survival signaling is sufficient for cell death, and Egfr survival signaling is only important to inhibit Hid, these data imply that release of hid is sufficient as well as necessary for normally occurring cell death. The data do not rule out any parallel Egfr-dependent signal to suppress caspase activation independently of hid, but such a pathway cannot be sufficient for cell death in the absence of hid. These findings suggest that positive activators of caspase processing may not be the direct targets of extracellular regulation. However, it will be important to investigate survival and death signals in other organs, including cell deaths that occur independently of reaper, grim and Hid in ovarian nurse cells and during autophagy, the mechanisms of which have yet to be determined (Yu, 2002).
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).
Apoptosis in the pupal retina requires hid function, since hid mutants display additional interommatidial cells. Hid is thought to induce caspase activation by binding to the DIAP1 protein and preventing it from inhibiting caspase function. Overexpression of hid using the eye-specific GMR promoter generates a small eye. The induction of cell death by hid is severely impaired in sav mutant clones. As a consequence, eyes derived from GMR-hid-expressing discs that contain sav mutant clones are larger than those derived from wild-type discs that express GMR-hid. Since sav function is required for hid-induced cell death, sav is likely to function either downstream of hid or in a parallel pathway (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. 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).
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).
Programmed cell death (PCD) is utilized in a wide variety of tissues to refine
structure in developing tissues and organs. However, little is understood about
the mechanisms that, within a developing epithelium, combine signals to
selectively remove some cells while sparing essential neighbors. One popular
system for studying this question is the developing Drosophila pupal retina,
where excess interommatidial support cells are removed to refine the patterned
ommatidial array. Data is presented indicating that PCD occurs earlier within
the pupal retina than previously demonstrated. As with later PCD, this death is
dependent on Notch activity. Surprisingly, altering Drosophila Epidermal
Growth Factor Receptor or Ras pathway activity has no effect on this death.
Instead, a role for Wingless signaling is indicated in provoking this cell
death. Together, these signals regulate an intermediate step in the selective
removal of unneeded interommatidial cells that is necessary for a precise
retinal pattern (Cordero, 2004).
In the course of examining
hid mutant retinae, it was noticed that blocking cell death in the earliest
pupal stages -- prior to known stages of cell death -- led to a clear
increase in the number of interommatidial cells. With this in mind,
pupal retinas was examined at earlier developmental stages from 14 to 24 h
APF by using an antibody to the junction protein Armadillo; apoptotic cell death
was also directly assessed with TUNEL staining.
Prior to approximately 20 h APF, the retina is composed
of a loosely patterned array of ommatidia consisting of photoreceptor neurons
and cone cells; primary pigment cells (1°s)
first emerge and enwrap the cone cells at 20 h APF, and secondary and
tertiary pigment cells (2°/3°s) begin organizing at about this stage
as well. Approximately one third of the
interommatidial cells observed at 24 h APF (25°C)
are selected to die by PCD during the following
10-12 h (Cordero, 2004).
Prior to 18 h APF, no significant amount of death
was observed. At 18 h APF, a sharp band of
death was observed in the anterior portion of the retina; some of this death is within the retina, and some is just outside the
retinal field. Between 20 and 24 h
APF, additional death is observed towards the middle of the retina in addition
to the anterior death band. Levels of apoptosis are highest in anterior regions
of the eye, but the center of the eye, for example, also contains significant levels of
death. At 24 h APF, this early wave of death
rapidly declines; the remaining interommatidial
cells have reorganized end-to-end by this stage. At 26 h
APF, the known, previously described burst of death commences.
The increasing amount of TUNEL staining correlates
with a decrease in the number of interommatidial cells. These
results indicated that the pupal retina undergoes
two separate surges of cell death that occur between 18-24 and
26-36 h APF; for convenience, these events are referred to as
'early-stage' and 'late-stage' cell death events in
the pupal eye, respectively. During the early-stage death 1.8 cells are removed
per ommatidia. The early-stage
has not been described previously, and it was examined whether the pathways
known to regulate late-stage death also regulate its predecessor (Cordero, 2004).
The baculovirus protein P35 interferes with apoptosis by binding to
and inhibiting caspase activity; it is effective in inhibiting cell death
including late-stage
death in the Drosophila eye. Targeted
over-expression of P35 with the eye-specific promoter GMR led to a near complete
block of early-stage death: only a line of anterior cell
death remained in GMR-p35 retinas. This result indicates that the
early-stage cell death occurred by caspase-dependent apoptosis. In addition, it
confirmed the assessment, based on TUNEL staining, that some of the
anterior-most apoptotic cell death occurs in a region of future head cuticle
just anterior to the retina (and is therefore outside of the expression domain
of the GMR promoter (Cordero, 2004).
The head
involution defective hid gene is a central regulator of cell death in
Drosophila including late-stage cell death pathway in the retina.
Hid induces PCD
through activation of caspases. Retinas lacking
functional hid activity looses all evidence of early-stage PCD.
The number of cells within the GMR-p35 and
hid-/- retinas at 20 and 21 h APF,
respectively was in fact higher than the number of cells in a 18 h APF
control retina. In these
mutant genotypes the ommatidia are disorganized when compared with the control
retinas due to the excess of cells. It was often found that
hid-/- retinas are attached to what seems to be
the antennal disc, suggesting that this
early-stage death may include events required for separation of the eye-antennal
discs. Together these results suggest that, similar to late-stage death,
early-stage death is regulated by a caspase-mediated apoptosis pathway (Cordero, 2004).
The Egfr/Ras-1 pathway has been implicated in
multiple stages of fly eye development including cell proliferation, survival
and differentiation. Loss of function
mutations in the Egfr leads to excessive cell death
of the interommatidial cells. Activation of Egfr
leads to activation of dRas1, which promotes cell survival by repressing the
activity and expression of hid (Cordero, 2004).
Activated Egfr
and dRas1V12 was expressed under the control of an inducible,
heat shock promoter. As expected, late-stage cell death
(26 h APF) is almost completely blocked by each transgene.
Surprisingly, no alteration was seen in either the
pattern of death or the cell number in 21 h APF retinas,
suggesting that early-stage death is insensitive to the Egfr/dRas1 pathway.
Consistent with these results, no effect on cell death was seen
upon over-expression of the Egfr antagonist Argos. These findings
are especially surprising because of the results indicating that hid is
required for early-stage death: unlike larval or late-stage death, hid
activity appears to be regulated by a Egfr-independent mechanism during
early-stage cell death (Cordero, 2004).
Incorrectly specified or mis-specified cells often undergo cell death or
are transformed to adopt a different cell fate during development. The
underlying cause for this distinction is largely unknown. In many
developmental mutants in Drosophila, large numbers of mis-specified
cells die synchronously, providing a convenient model for analysis of this
phenomenon. The maternal mutant bicoid is a particularly useful model
with which to address this issue because its mutant phenotype is a combination
of both transformation of tissue (acron to telson) and cell death in the
presumptive head and thorax regions. A subset of these
mis-specified cells die through an active gene-directed process involving
transcriptional upregulation of the cell death inducer hid.
Upregulation of hid also occurs in oskar mutants and other
segmentation mutants. In hid bicoid double mutants, mis-specified
cells in the presumptive head and thorax survive and continue to develop, but
they are transformed to adopt a different cell fate. Evidence is provided that
the terminal torso signaling pathway protects the mis-specified
telson tissue in bicoid mutants from hid-induced cell death,
whereas mis-specified cells in the head and thorax die, presumably because
equivalent survival signals are lacking. These data support a model whereby
mis-specification can be tolerated if a survival pathway is provided,
resulting in cellular transformation (Werz, 2005).
Although this study largely focus on the maternal effect mutants
bicoid and oskar, it is likely that the principles
uncovered are of broader significance. Segmentation mutants acting downstream
of bicoid and oskar, including mutants of gap genes
(Krüppel, knirps), pair-rule genes (odd, fushi-tarazu)
and segment polarity genes (wg, hedgehog, engrailed) induce
expression of hid. These mutants are characterized by loss of larval tissue. As
in the case of bicoid and oskar, hid expression is
upregulated during stage 9 of embryogenesis in the regions of the mutant
embryos that are later deleted in the larvae. In addition, hid
mutants rescue the cuticle phenotype of armadillo mutants. Finally,
hid expression accompanied by TUNEL-positive cell death was found
in dorsal and Toll10b mutants, which cause
dorsalizing and ventralizing phenotypes, respectively, along the dorsoventral
axis of Drosophila embryos. Thus, these data support
the notion that upregulation of hid appears to be a common trigger
for a caspase-dependent cell death program in mis-specified cells of
patterning mutants (Werz, 2005).
Furthermore, mutations affecting imaginal disc development result in loss
of the adult appendage due to inappropriate cell death.
It is currently being determined whether these mutants also require hid
expression to develop the final phenotypes. Moreover, many gene disruptions in
mice result in inappropriate cell death in the tissue that requires the
function of the disrupted gene, suggesting that similar mechanisms might exist in mammalian
development. Finally, cell death may be an important contributing factor to
human congenital birth defects. Thus, an understanding of the underlying
mechanisms is of general interest (Werz, 2005).
Interestingly, not all segment polarity mutants analyzed induce
hid expression and cell death. Embryos mutant for patched,
which encodes the hedgehog receptor, were not found to express
hid and do not exhibit increased cell death,
although hedgehog mutants both upregulate hid and contain
increased amounts of cell death. The reasons for these differences are not
known, but partial redundancy might account for lack of hid
expression in patched mutants. The Drosophila genome encodes
another patched homolog, patched-related, which
might provide the survival requirement for mis-specified cells in patched mutants (Werz, 2005).
Mis-specified cells in bicoid and oskar mutants induce
expression of hid. No increased reaper or
grim expression was observed in these mutants. However, expression of
reaper has been reported in crumbs mutants, which affect
epithelial integrity. X-ray-treated embryos also preferentially respond by
upregulation of reaper, rather than hid. Although crumbs mutants
were not analyzed for hid
expression, it appears that cells contain several developmental checkpoints,
which activate different cell death-inducing regulators depending on the type
of abnormal cellular development (Werz, 2005 and references therein).
Mis-specified cells can survive if an alternative survival pathway is
provided. The example presented here is the acron into telson transformation
in bicoid mutants, which is mediated by the torso signaling
pathway. Although the cells giving rise to telson structures at the anterior
tip are mis-specified based on Abd-B-labeling experiments, they survive
because they receive a survival signal from the torso signaling
system. In this case, transformation rather than cell death is favored. It has
been shown that activation of the Ras/Mapk pathway protects cells
from hid-induced apoptosis, both by transcriptional repression of
hid and by phosphorylation of Hid protein by Mapk.
Because Torso, which encodes a receptor tyrosine kinase (RTK), is
known to activate Ras and Mapk, tests were performed to see
whether manipulation of active Mapk levels using
a gain-of-function allele, MapkSem, can suppress
hid expression and cell death in bicoid mutants. However,
this was found not to be the case. Thus, torso
appears to protect mis-specified cells independently of Mapk activation (Werz, 2005).
The hid bicoid double mutant analysis reveals that the
transformation of anterior into posterior identity expands beyond the telson,
and that this expansion undergoes hid-induced cell death in
bicoid single mutants. The rescued cells secrete larval cuticle
elements, suggesting that mis-specified cells have the developmental capacity
to terminally differentiate. However, in hid+ background,
they instead die, presumably because equivalent survival signals are lacking.
It is proposed that mis-specified cells undergo cell death if no alternative
survival pathway is provided to protect them (Werz, 2005).
An alternative survival mechanism might also operate in other developmental
mutants where transformation rather than cell death occurs. Mutations in the
sev RTK and its ligand boss result in transformation of the
R7 photoreceptor cell into a non-neuronal cone cell.
Survival of this cell could be mediated by the Drosophila Egf
receptor (Egfr), another RTK, which is required to maintain cell survival in
the developing eye disc. Accordingly, activation of the Ras/Mapk pathway by Egfr
would inhibit hid expression and support survival of the presumptive
R7 photoreceptor cell. This interpretation is also consistent with
observations that egfr- clones are small and undergo cell
death, and that this death can be suppressed in hid
mutants. Thus, transformation of the R7 photoreceptor to a cone cell rather than R7
cell death in sev and boss mutants could occur because of
survival signaling by the Egfr (Werz, 2005).
The hid bicoid double mutant analysis suggests that mis-specified
cells can continue to develop and differentiate. Yet, they die. Presumably,
this cell death protects the organism from potentially dangerous cells. For
example, it is conceivable that in mammals, surviving mis-specified cells
might lie dormant in the host organism for years. During this time, they might
acquire additional genetic alterations that could drive the progressive
transformation of these cells into malignant cancer. In wild-type embryos,
mis-specification probably occurs in cells in isolation, and elimination of
these cells does not interfere with development and survival of the organism.
Only in extreme situations, such as the patterning mutants analyzed in this study, is
the mis-specification caused by aberrant development so severe that the
affected organism dies (Werz, 2005).
The cause of mis-specification in each segmentation mutant is different.
Usually, the expression of other segmentation genes is shifted and expanded,
resulting in flattened gradients. Yet, irrespective of the cause of mis-specification, most
of these mutants have in common that they induce hid expression. It
is currently unknown how the mis-specified fate of cells is recognized, and
how hid expression is induced. One possibility might be that the
protein gradients established by bicoid+ and
oskar+, as well as other segmentation genes
are used as readout for proper cellular
specification. The steepness of protein gradients as a means to determine life
or death decisions has recently been proposed. Such
a model would imply that cells are able to determine their position in a
graded field and compare this readout with their neighbors. Because in
bicoid and oskar mutants these gradients do not form, the
concentration difference between neighboring cells would be zero. If the
concentration difference between two neighboring cells is below a crucial
threshold, they induce the expression of hid and undergo cell death.
This model could also explain embryonic pattern repair, which was described in
embryos that express six copies of the bicoid gene. In these
embryos, the head and thorax primordia are expanded because of the presence of
six copies of bicoid. However, this expansion is corrected for by
induction of cell death, and relatively normal larvae develop. In this
case, the Bicoid protein gradient does form, but would be flatter compared
with wild type. Thus, the concentration difference between neighboring cells
would be below a critical threshold, sufficient to induce
hid-dependent cell death. However, it is largely unknown how cells
compare their position in a graded field with those of their neighbors. It has
been proposed that short-range cell interactions mediated via the cell-surface
proteins Capricious and Tartan provide cues that support cell survival during
wing development. Cells unable to participate in these interactions are
eliminated by cell death. It is unclear, however, whether short-range
interactions are sufficient to explain the cell death phenotype in
bicoid and oskar mutants (Werz, 2005).
Irrespective of the underlying mechanism for sensing mis-specification, the current results highlight the role of an active gene-directed process that removes
mis-specified cells during development. However, if a survival mechanism is
provided, mis-specified cells can survive and adopt a different fate. In
wild-type embryos, mis-specification probably occurs in cells in isolation,
and hence is difficult to study. However, in bicoid and
oskar mutants, large regions of neighboring cells are mis-specified
and undergo cell death simultaneously, providing a unique opportunity to
clarify the signals that initiate cell death in situations where cells are
developmentally mis-specified (Werz, 2005).
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).
Appropriate cell-cell signaling is crucial for proper tissue homeostasis. Protein sorting of cell surface receptors at the early endosome is important for both the delivery of the signal and the inactivation of the receptor, and its alteration can cause malignancies including cancer. In a genetic screen for suppressors of the pro-apoptotic gene hid in Drosophila, two alleles of vps25, a component of the ESCRT machinery required for protein sorting at the early endosome, were identified. Paradoxically, although vps25 mosaics were identified as suppressors of hid-induced apoptosis, vps25 mutant cells die. However, evidence is provided that a non-autonomous increase of Diap1 protein levels, an inhibitor of apoptosis, accounts for the suppression of hid. Furthermore, before they die, vps25 mutant clones trigger non-autonomous proliferation through a failure to downregulate Notch signaling, which activates the mitogenic JAK/STAT pathway. Hid and JNK contribute to apoptosis of vps25 mutant cells. Inhibition of cell death in vps25 clones causes dramatic overgrowth phenotypes. In addition, Hippo signaling is increased in vps25 clones, and hippo mutants block apoptosis in vps25 clones. In summary, the phenotypic analysis of vps25 mutants highlights the importance of receptor downregulation by endosomal protein sorting for appropriate tissue homeostasis, and may serve as a model for human cancer (Herz, 2006).
The inactivation of signaling pathways is as important for appropriate
tissue homeostasis as its activation. Interference with the inactivation
process often gives rise to malignant phenotypes, including cancer. Several
strategies to restrict signaling exist, including receptor sequestration,
receptor inactivation, production of inhibitory signaling proteins and
inactivation of intracellular signaling proteins. The phenotypic analysis of
vps25 mutants highlights the importance of receptor downregulation by
endosomal protein sorting. Lack of vps25 function causes at least
three phenotypes: non-autonomous proliferation, non-autonomous resistance to
cell death and autonomous apoptosis. The cause of these phenotypes and the
potential role of class E Vps proteins for tumorigenesis will be
discussed (Herz, 2006).
Vps25 is a component of the ESCRT-II complex required for internalization of cell surface receptors into MVBs at the early endosome. The signal for protein sorting into MVBs is provided by mono-ubiquitylation. In yeast, vps25 mutants cause aberrant endosomal structures and the accumulation of ubiquitylated proteins. A similar phenotype in vps25 clones in Drosophila, suggesting the conserved function of vps25 (Herz, 2006).
The lack of appropriate protein sorting at early endosomes in
vps25 clones causes the accumulation of cell surface receptors
including N and Dl. Genetic analysis using a dominant-negative N transgene
(NDN) suggests that the strong overgrowth phenotype of vps25 mosaics is largely due to inappropriate N signaling, which is known to induce
proliferation non-autonomously through activation of the JAK/STAT pathway (Herz, 2006).
It is unclear whether N exerts this function in a ligand-dependent manner.
Dl protein also accumulates in vps25 clones, and endocytosis of Dl is
required for N activation. Thus, blocking MVB formation in vps25 clones
may lead to the accumulation of active Dl, resulting in increased N activity.
However, it was also shown that N is required for Dl accumulation in vps25
clones. Therefore, Dl accumulation is either directly or indirectly the result of increased N activity in vps25 clones. This conclusion infers that N activation occurs before Dl accumulation and would argue in favor of a ligand-independent mechanism for N activation in vps25 clones, although Dl may be
required for maintaining N activity. N activity is also controlled by several
proteolytic cleavages, which lead to translocation of the intracellular domain of
N to the nucleus where it regulates the expression of target genes. Thus, a
potential ligand-independent mode of N activation may include inappropriate
cleavage of N at the vps25 endosome. Further studies are needed to
clarify this point (Herz, 2006).
Mutations in erupted, the vps23 homolog that encodes a
component of ESCRT-I, give rise to similar phenotypes to those observed for
vps25. However, in hrs mosaics in Drosophila, non-autonomous cell proliferation has not been observed, although signaling receptors including N and Dl accumulate in hrs clones. This is a puzzling observation as hrs encodes a class E Vps protein acting immediately upstream of the ESCRT complexes. It is possible that N and Dl are not in an environment in the hrs endosome that permits signaling. Alternatively, it has been shown
showed that hrs controls the steady-state levels of non-activated
receptors at the plasma membrane. Although this function may apply to
vps25, it may also indicate that there are inherent differences
between the different class E proteins regarding protein sorting at the early
endosome (Herz, 2006).
Paradoxically, although vps25 clones die by apoptosis, the vps25 alleles were identified as being recessive suppressors of
GMR-hid-induced cell death. This analysis demonstrates that the
wild-type tissue accounts for this suppression even though these cells are
exposed to GMR-hid. The initial explanation for this observation was
that non-autonomous proliferation mediated by JAK/STAT signaling in
vps25 mosaics overrides the apoptotic activity of GMR-hid.
However, overexpression of Upd, the ligand of the JAK/STAT pathway, does not
significantly suppress GMR-hid, although GMR-upd flies have
a similar overgrowth phenotype to vps25 mosaics. This
finding excludes non-autonomous proliferation for the suppression of
GMR-hid by vps25. However, Diap1 protein levels are
increased in tissue abutting vps25 clones. GMR-hid
is sensitive to altered levels of Diap1, suggesting
that the increase of Diap1 outside of vps25 clones may account for
the suppression of GMR-hid. Thus, in addition to non-autonomous
proliferation, vps25 clones also increase the apoptotic resistance of
adjacent wild-type tissue in a non-autonomous manner. The signaling pathway
that can induce non-autonomous survival by increasing Diap1 protein levels is
currently unknown (Herz, 2006).
The data suggest that apoptosis in vps25 mutant tissue is not
only executed via the Hid/Diap1/Dronc/Ark pathway. vps25 ark clones
still died, suggesting that in addition to Ark at least one other cell death
pathway is activated in vps25 clones. It has been shown that a
Dronc/Ark-independent cell death pathway exists in Drosophila, but this pathway has not been identified. The data in this study implicates JNK as potential mediator of the alternative cell death pathway. vps25 ark/Puc mosaic eye discs are extremely overgrown and the clones occupy a large area of the disc. Anti-cleaved Caspase-3 *-dependent apoptosis is blocked in these clones. Only at the clonal boundaries is Caspase-3* activity still detectable, suggesting that at the interface between vps25 clones and wild-type tissue a third potential apoptotic pathway is activated (Herz, 2006).
The data show that cell competition is not sufficient to induce cell death
in vps25 clones. By contrast, given the extremely large size of cell
death-inhibited vps25 clones, it appears that vps25 clones have no intrinsic growth disadvantage, and have the capability to overgrow and outcompete the surrounding wild-type tissue if cell death is blocked. Thus, cell competition
does not contribute significantly to the apoptotic phenotype of vps25
clones (Herz, 2006).
Hippo signaling is increased in vps25 clones. Hippo
signaling can induce cell death, and, consistently, hippo mutants
block cell death in vps25 clones. It is unknown how Hippo signaling
is activated in vps25 clones. However, in analogy to N, a putative
receptor that controls Hippo signaling may be deregulated in vps25
clones and triggers Hippo signaling. This receptor is currently unknown, but
has been postulated previously.
However, it should be pointed out that ESCRT components have additional
functions outside of MVB protein sorting. Certain ESCRT-II members have been
shown to bind to the transcriptional elongation factor ELL in order to
derepress transcription by RNA polymerase II. Thus,
in the absence of Vps25, transcriptional control of components of the Hippo
pathway may be deregulated and contribute to cell death (Herz, 2006).
In summary, the data suggest that impaired ESCRT function leads to the
accumulation of N and Dl, and possibly of a receptor controlling the Hippo
pathway. These receptors control non-autonomous proliferation and autonomous
apoptosis, respectively. In addition, a signaling pathway is postulated that
induces non-autonomous cell survival by controlling Diap1 protein levels.
Further characterization of the vps25 mutant phenotype may help to
identify the postulated receptor of the Hippo pathway and the cell survival
signaling pathway (Herz, 2006).
Human ESCRT components, most notably TSG101 (Vps23p), have been
implicated in tumor suppression. NIH3T3 cells, depleted of Tsg101 by
an antisense approach, formed colonies on soft agar and produced metastatic
tumors in nude mice. However, the conditional Tsg101 knockout in mouse
mammary glands did not cause the formation of tumors over a period of two
years, making a role of TSG101 as tumor suppressor controversial.
However, Tsg101 mutant cells are very sensitive to apoptotic death,
implying that they die before they become harmful to the organism (Herz, 2006).
The phenotypical characterization of vps25 mutants in
Drosophila provides an explanation for the failure to confirm
TSG101 as tumor suppressor. vps25 clones need to survive
over extended periods of time in order to sustain growth. Even though they
induce non-autonomous proliferation, after they have died, N signaling is
turned off and proliferation stops. Furthermore, the size of the adult eye of
vps25 mosaics is only slightly increased when compared with wild
type, and does not match the strong overgrowth phenotype of larval imaginal
discs, which can be twice as large as wild-type discs. Thus, as long as
vps25 clones are not resistant to their own apoptotic death, tissue
repair during pupal stages may partially regress the size of the imaginal disc
back to almost normal. Instead, it appears that inhibition of cell death is
the triggering event for a tumorous phenotype of vps25 clones.
vps25/Diap1 and vps25 ark/Puc clones can make up a large
fraction of the tissue of imaginal discs, and the entire discs can be five
times as large as wild-type discs (Herz, 2006).
Tumorigenesis requires multiple genetic alterations that transform normal
cells progressively into malignant cancer cells.
Thus, additional genetic 'hits' may be necessary to inhibit apoptosis of
Tsg101 mutant cells, which may then be able to induce a similar
growth phenotype to that observed for vps25. Thus, although a tumor
suppressor function for Tsg101 was not confirmed in a mouse model, it
still is possible that Tsg101 and other mammalian ESCRT members have
tumor suppressor properties (Herz, 2006).
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 specifically 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).
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).
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).
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Wrinkled/head involution defective:
Biological Overview
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 15 December 2011
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