diminutive

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of dm at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

DMYC transcripts, presumably maternal, can be detected ubiquitously from the earliest stages. Later the zygotic transcripts accumulate in a changing pattern in various tissues. In preblastoderm embryos, DMYC mRNA is present throughout the embryo, with the highest levels at the anterior and posterior termini, but is absent from the pole cells. In early gastrulation, additional DMYC mRNA can be detected in the presumptive mesoderm along the ventral midline. This mesodermal staining intensifies during germband extention and remains until late embryogenesis. The terminally located expression follows the posterior and anterior midgut primordia during the germband extended stages. Additional staining is found in salivary placodes. DMAX transcript is less abundant than DMYC, particularly during the earliest stages. In addition dmax is expressed in tissues with undetectable levels of DMYC, such as the developing central nervous system. Hence, whereas tissues containing the highest levels of DMYC and DMAX transcripts are undergoing DNA replication, not all actively proliferating tissues have detectable levels of DMYC.

Myc and wing development

The Ras GTPase links extracellular mitogens to intracellular mechanisms that control cell proliferation. To understand how Ras regulates proliferation in vivo, Ras was either activated or inactivated in cell clones in the developing Drosophila wing. Cells lacking Ras are smaller, have reduced growth rates, accumulate in G1, and undergo apoptosis due to cell competition. Conversely, activation of Ras increases cell size and growth rates and promotes G1/S transitions. Ras upregulates the growth driver dMyc, and both Ras and dMyc increase levels of cyclin E posttranscriptionally. It is proposed that Ras primarily promotes growth and that growth is coupled to G1/S progression via cyclin E. Interestingly, upregulation of growth by Ras does not deregulate G2/M progression or a developmentally regulated cell cycle exit (Prober, 2000).

It is proposed that there is parallel and independent control of G1/S and G2/M transitions in Drosophila wing disc cells. Cellular growth due to Ras or dMyc drives G1/S transitions by promoting translation of cyclin E. Ras may also drive growth via proteins other than dMyc; this could feed back to upregulate translation of dmyc mRNA. Alternatively, Ras may regulate cellular growth and the G1/S cell cycle machinery in parallel. Stg/Cdc25, which is regulated primarily at the transcriptional level, drives G2/M transitions. Signaling molecules capable of regulating coordinated growth and patterning such as Vein may regulate G1/S transitions via Ras, dMyc, or other growth-promoting proteins and regulate G2/M transitions via transcription factors that modulate transcription of Stg/Cdc25 (Prober, 2000).

Other proteins that promote growth, such as the Drosophila homologs of dMyc and Phosphoinositide 3-Kinase (dPI3K), have effects on cell cycle progression similar to Ras. Upregulating these proteins in the developing wing truncates G1, elongates G2, and increases growth rates, while downregulating them cause the opposite effects. Furthermore, the resulting growth rates are inversely proportional to the length of G1. Given these similarities, it is proposed that cellular growth is rate limiting for G1/S progression in wing imaginal cells (Prober, 2000).

The data suggest that the effects of Ras on cellular growth and the cell cycle are at least partially mediated by dMyc. Mammalian Myc transcription factors activate expression of many genes involved in cellular growth and metabolism, and Drosophila dMyc is a potent growth driver in vivo. Upregulation of dMyc by Ras appears to be posttranscriptional. Ras might act by inhibiting degradation of dMyc protein, as has been demonstrated in mammalian cell culture. Alternatively, Ras might stimulate growth via other proteins, such as components of the dPI3K/dAkt/dS6 Kinase pathway, which promote cellular growth in Drosophila. Increased growth due to these proteins could then feed back to promote translation of extant dmyc mRNA. However, dMyc and dPI3K cannot be mediating all of Ras's effects, since unlike Ras they do not affect cell fate or cell adhesion. These additional functions of Ras, along with the ability to increase Myc protein levels, likely contribute to the strong synergistic action of Ras and Myc in oncogenesis (Prober, 2000).

Ras^V12 accelerates G1/S transitions but fails to accelerate rates of cell division. This is similar to findings with overexpressed dMyc. However, coexpressing either Ras^V12 or dMyc with String (Stg), the G2/M rate limitor, does accelerate cell division. This suggests that regulation of Stg is independent of both Ras and dMyc. It is therefore proposed that there is parallel and independent control of G1/S and G2/M transitions during wing development. Signaling molecules capable of regulating coordinated growth and patterning, such as Vein, Decapentaplegic, and Wingless might control G1/S transitions by regulating growth via Ras, dMyc, or other growth-promoting proteins. These signaling molecules might also, unlike Ras and dMyc, control G2/M transitions by modulating transcription of stg. Analysis of more than 40 kb of the stg promoter has revealed an extensive array of regulatory modules that respond to different patterning signals and thus integrate complex patterning information. A model in which cyclin E acts as a growth sensor and Stg acts as a 'pattern sensor' is attractive, as it allows coordination of independent growth and patterning signals by the cell cycle machinery (Prober, 2000).

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).

Myc and eye development

Ectopic expression of transcription factors in eye-antennal discs of Drosophila strongly interferes with their developmental program. Early ectopic expression in embryonic discs interferes with the developmental pathway primed by Eyeless and generates headless flies, which suggests that Eyeless is necessary for initiating cell proliferation and development of both the eye and antennal disc. Interference occurs through a block in the cell cycle that for some ectopic transcription factors is overcome by D-CycE or D-Myc. Late ectopic expression in cone cell precursors interferes with their differentiation. It is proposed that this developmental pathway interference is a general surveillance mechanism that eliminates most aberrations in the genetic program during development and evolution, and thus seriously restricts the pathways that evolution may take (Jiao, 2001).

The eye-antennal discs of ey-GAL4/+; UAS-Gsb/+ third instar larvae are absent or strongly reduced in size. Evidently, developmental pathway interference induced by the ectopic expression of transcription factors eventually results in the inhibition of cell proliferation and/or apoptosis in these discs. To investigate which of these two processes is responsible for the generation of headless flies, attempts were made to inhibit apoptosis or to stimulate cell proliferation in eye-antennal discs. While inhibition of apoptosis by the expression of the baculovirus P35 protein is unable to suppress the headless phenotype, stimulation of cell proliferation by the expression of D-Myc suppresses it in spontaneously eclosing adults (5%-20%), producing adults of variable eye size, from eyeless adults to adults whose eye size is only slightly reduced. The headless phenotype is rescued even more dramatically by D-CycE, which restores a wild-type phenotype in up to 50% of the adults and only rarely generates small-eyed flies. Rescue of the headless phenotype by CycE is not restricted to ey-GAL4/+; UAS-Gsb/+ flies, but is achieved for all Pax proteins and transcription factors whose potency to interfere with ey function in the eye-antennal disc was tested. However, in contrast to headless flies produced by Gsb, Prd, Poxm, D-Pax2 or Dac, many of which were rescued by CycE to adults that eclosed spontaneously, those generated by Mef2, Sim or Poxn were incompletely rescued. D-Myc is not as efficient in its rescue ability, except in the case of D-Pax2, in which nearly all flies were rescued to wild-type adults. It is concluded that developmental pathway interference through ectopic expression of transcription factors results in the inhibition of cell proliferation that is at least partially overcome by co-expression of D-Myc or D-CycE (Jiao, 2001).

Oogenesis

dmyc is expressed in a dynamic pattern in ovaries. At early stages, high levels of DMYC transcripts accumulate in the germarium with lower levels in either stage 1 or stage 2 egg chambers. By stage 3, DMYC mRNA can be detected in all cell types of the chamber: the nurse cells, oocyte, and follicle cells. This expression pattern is maintained throughout oogenesis. dmax expression can still be detected in diminutive (see below) mutant ovaries (Gallant, 1996).

The Notch signaling pathway controls the follicle cell mitotic-to-endocycle transition in Drosophila oogenesis by stopping the mitotic cycle and promoting the endocycle. To understand how the Notch pathway coordinates this process, a functional analysis was performed of genes whose transcription is responsive to the Notch pathway at this transition. These genes include String, the G2/M regulator Cdc25 phosphatase; Hec/Cdh^Fzr, a regulator of the APC ubiquitination complex and Dacapo, an inhibitor of the CyclinE/CDK complex. Notch activity leads to downregulation of String and Dacapo, and activation of Fzr. All three genes are independently responsive to Notch. In addition, Cdh^Fzr, an essential gene for endocycles, is sufficient to stop mitotic cycle and promote precocious endocycles when expressed prematurely during mitotic stages. In contrast, overexpression of the growth controller Myc does not induce premature endocycles but accelerates the kinetics of normal endocycles. F-box/WD40-domain protein Ago/hCdc4 (Archipelago), a SCF-regulator is dispensable for mitosis, but crucial for endocycle progression in follicle epithelium. CycE oscillation remains critical for endocycling; continuous high level of CycE expression blocks the cell cycle in G2. The regulation of CycE levels is achieved by the function of Ago that presumably binds to auto-phosphorylated CycE and directs it to SCF-complex degradation: high levels of CycE and no endocycling is observed in ago-clones. The results support a model in which Notch activity executes the mitotic-to-endocycle switch by regulating all three major cell cycle transitions. Repression of String blocks the M-phase, activation of Fzr allows G1 progression, and repression of Dacapo assures entry into the S-phase. This study provides a comprehensive picture of the logic that external signaling pathways may use to control cell cycle transitions by the coordinated regulation of the cell cycle (Shcherbata, 2004).

The data suggest that a component regulating growth and thereby the kinetics of G1/S transition in follicle cell endocycles is the Myc oncogene instead and independent of CycD. In mammals c-Myc controls the decision to divide or not to divide and thereby functions as a crucial mediator of signals that determine organ and body size. Interestingly, overexpression of Myc in follicle cells does not affect the mitotic cycles but induces, instead, extra endocycles. Because the timing for entering and exit from the endocycles has not changed, however, increased ploidy is observed; therefore, it is suggested that the rate of endocycles is increased because of the overexpression of Myc. This finding is in accordance with recent loss-of-function analysis on myc in follicle cells, suggesting that myc mutant follicle cells can make the transition from mitosis to the endocycle, but that they can only very inefficiently support the endocycle. Therefore, both loss-of-function and overexpression experiments suggest that Myc is an essential component for the proper rate of endocycles in follicle cells (Shcherbata, 2004).

Integration of Insulin receptor/Foxo signaling and dMyc activity during muscle growth regulates body size in Drosophila

Drosophila larval skeletal muscles are single, multinucleated cells of different sizes that undergo tremendous growth within a few days. The mechanisms underlying this growth in concert with overall body growth are unknown. The size of individual muscles correlates with the number of nuclei per muscle cell and with increasing nuclear ploidy during development. Inhibition of Insulin receptor (InR; Insulin-like receptor) signaling in muscles autonomously reduces muscle size and systemically affects the size of other tissues, organs and indeed the entire body, most likely by regulating feeding behavior. In muscles, InR/Tor signaling, Foxo and dMyc (Diminutive) are key regulators of endoreplication, which is necessary but not sufficient to induce growth. Mechanistically, InR/Foxo signaling controls cell cycle progression by modulating dmyc expression and dMyc transcriptional activity. Thus, maximal dMyc transcriptional activity depends on InR to control muscle mass, which in turn induces a systemic behavioral response to allocate body size and proportions (Demontis, 2009).

Therefore, interplay between InR/Tor signaling, Foxo and dMyc activity regulates muscle growth that occurs during Drosophila larval development, in part via the induction of endoreplication. Interestingly, the extent of muscle growth is sensed systemically, regulates feeding behavior and, in turn, influences the size of other tissues and indeed the whole body. Thus, the growth of a single tissue is sensed systemically via modulating a whole-organism behavior (Demontis, 2009).

dMyc, as well as activation of InR signaling, can promote endoreplication in muscles, whereas Foxo and inhibitors of dMyc and of InR/Tor have the opposite effect. dMyc is likely to regulate the expression of genes required for multiple G-S and S-G transitions during endoreplication, similar to vertebrate Myc, which regulates key cell-cycle regulators including cyclin D2, cyclin E, and the cyclin kinase inhibitors p21 and p27 (Cdkn1a and Cdkn1b, respectively). Indeed, aberrant levels of Cyclin E block muscle growth, indicating that proper muscle growth requires tight control of the expression and activity of endoreplication genes. Further, endoreplication is also modulated by Foxo, which is activated in conditions of nutrient starvation, impaired InR/Tor signaling and by other cell stressors. Foxo presumably regulates cell cycle progression at least in part by modulating the expression of evolutionarily conserved Foxo/Myc-target genes, such as dacapo (the Drosophila p21/p27 homolog) and Cyclin E, that regulate the G1-S transition. Interestingly, Foxo and Myc might control different steps in the activation of common target genes (Demontis, 2009).

In addition, it was found that active Foxo can also inhibit dMyc protein activity and regulates dmyc gene expression. Mechanistically, Foxo could influence dMyc activity in several ways. First, it might physically interact with dMyc, although no evidence was found to support this notion. Second, Foxo could regulate the expression of genes that target dMyc for proteasomal degradation, including several ubiquitin E3 ligases that are induced by Foxo during muscle atrophy in mice and humans. However, by analyzing dMyc protein levels by western blot, no significant dMyc protein instability was found upon Foxo overexpression. Third, Foxo might promote the expression of transcriptional regulators that oppose dMyc function, including Mad/Mnt, although no substantial increase in dmnt mRNA levels was detected upon Foxo activation in muscles. Possibly, the expression of other dMyc regulators might be affected by Foxo. Future experiments will be needed to dissect the Foxo-dMyc interaction (Demontis, 2009).

Finally, by manipulating muscle growth and/or endoreplication, it was found that in muscles the ratio of cell size to nuclear size is not constant, and increased nuclear size and DNA content, indicative of ploidy, is necessary but not sufficient to drive growth. Usually, an increase in cell size is matched by an increase in nuclear size, which commonly parallels increases in nuclear ploidy. However, the current findings indicate that in muscles, dMyc-driven variation in nuclear size and ploidy is permissive but not sufficient for substantial growth, even in the presence of increased biogenesis of nucleoli and expression of genes involved in protein translation. This is different from fat body cells, in which dmyc overexpression induces endoreplication and proportional cell growth. Thus, additional instructive signals, possibly modulating protein synthesis, mitochondriogenesis, ribosome biogenesis, sarcomere assembly, and other anabolic responses must be concomitantly received to promote maximal muscle growth. Therefore, increases in cell size and nuclear ploidy are surprisingly uncoupled during muscle growth (Demontis, 2009).

Little is known about the mechanisms that control and coordinate cell, organ and body size, and in particular how muscle growth is matched with the growth of other tissues and of the entire organism. Inhibition of InR/Tor signaling and dMyc activity in muscles impairs, in addition to muscle mass, the size of the entire body and of other internal organs. Similarly, overexpression of Cyclin E in muscles also results in autonomous and systemic growth defects, indicating that, at least in some cases, modulation of muscle growth by means independent from InR signaling can be sensed systemically. In the larva, endoreplicating tissues and organs (gut, salivary glands, epidermis, fat body) are severely affected, whereas non-endoreplicating tissues (brain and imaginal discs) are less affected, indicating distinct tissue responsiveness to this regulation. Similarly, inhibition of Tor signaling in the fat body also primarily affects the size of endoreplicating tissues (Demontis, 2009).

Non-autonomous regulation of tissue size may rely on humoral factors (e.g. hormone-binding proteins, hormones, metabolites) produced by muscles in response to achieving a certain mass. However, alternative models are possible. In particular, decreased and increased larval feeding, respectively, were observed upon inhibition and activation of InR signaling in muscles. This whole-organism behavioral adaptation is possibly due to decreased and increased efficiency of smaller and bigger muscles, respectively, and to regulated expression of neuropeptides that hormonally control feeding behavior. As a consequence of the regulation of feeding behavior, nutrient uptake is decreased and larval growth is blocked in the cells of endoreplicating tissues, which are extremely sensitive to poor nutritional conditions, and to a lesser extent in non-endoreplicating tissues, which are more resistant to limited nutritional supply. In turn, increased or decreased size of non-muscle tissues arise as a consequence of abnormal feeding. Thus, muscle size coordinates with the size of other organs and of the entire body, at least in part via a systemic, behavioral response. Distinct tissues are differently sensitive to this regulation, resulting in altered body proportions (Demontis, 2009).

Understanding the mechanisms regulating muscle mass is of special interest because they underline the etiology of several human diseases. Directly relevant to these studies, both MYC and InR (INSR) signaling have been found to regulate muscle growth and maintenance in humans. Further, muscle atrophy is triggered by FOXO activation in several pathological conditions. In addition, MYC function has been implicated in heart hypertrophy, a process that is conversely regulated by FOXO (Demontis, 2009).

The findings that Foxo functionally antagonizes dMyc during the growth of Drosophila muscles suggest that these factors might also interact similarly in humans. Consistent with this hypothesis, FOXO and MYC regulate, in opposite fashions, the atrophic and hypertrophic programs in human skeletal muscles and cardiomyocytes, and display complementary gene expression and activity in these contexts (Demontis, 2009).

Finally, the finding that during larval development, inhibition of InR signaling in muscles has profound systemic effects might also reflect physiological conditions found in humans. Indeed, defective responsiveness of muscles to Insulin during type II diabetes has autonomous effects on muscle maintenance that are associated with systemic effects on the metabolism of the entire organism, contributing to the improper control of glycemia and the development of metabolic syndrome. This study has identified feeding behavior as part of the systemic response that in Drosophila senses perturbations in muscle mass. These findings might help further elucidate the signals involved in metabolic and growth homeostasis, which may be conserved across evolution (Demontis, 2009).

Effects of Mutation or Deletion

The diminutive (dm) mutation is a strong candidate for dmyc. The dm hypomorphic mutation results from insertion of a gypsy transposable element. Phenotypically, dm mutant flies are viable but of smaller body size and females are sterile as a result of defective oogenesis. Defects are observed in both germ cell nuclei and follicle cells, which fail to migrate and undergo the transition to columnar epithelium. This phenotype suggests dysfunction in either follicle or nurse cells, or in communication between these two cell types. Interestingly the degeneration of the egg chamber in dm mutant females occurs at stage 8, when cell division does not occur. It is thought that a stage-specific downregulation of dmyc expression in diminished mutants due to the gypsy insertion results in a loss of the capacity of the follicle cells to grow and migrate (Gallant, 1996).

Biochemical and biological activities of Myc oncoproteins are highly dependent on their association with another basic region helix-loop-helix/leucine zipper (bHLH/LZ) protein, Max. The observation that the DNA-binding/dimerization region of Max is absolutely conserved throughout vertebrate evolution provided the basis for a yeast two-hybrid interaction screen that has led to the isolation of the Drosophila Myc (dMyc1) protein. Structural conservation in regions of known functional significance is consistent with the ability of dMyc1 to interact with vertebrate Max, to transactivate gene expression in yeast cells, and to cooperate with activated H-RAS to effect the malignant transformation of primary mammalian cells. The ability of P-element-mediated ectopic expression of dmyc1 to reverse a subset of the phenotypic alterations associated with the diminutive mutation suggests that diminutive may correspond to dmyc1. This finding, along with the localization of dmyc1 expression to zones of high proliferative activity in the embryo, implicates dMyc1 as an integral regulator of Drosophila growth and development (Schreiber-Agus, 1997).

A low level of maternally derived dmyc1 RNA is observed throughout early embryos before cellular blastoderm formation and appears to be particularly concentrated in the pole plasm. Zygotic expression is detected first during the cellular blastoderm stage in the endodermal anlagen of the anterior and posterior midgut at the two poles of the embryo. Soon after, at the onset of gastrulation, expression is apparent in the mesoderm as it forms the invaginating ventral furrow. During germ-band extension, dmyc1 expression continues in both the anterior and posterior midgut, as well as in the mesoderm. At the end of germ-band retraction, expression remains detectable in the fusing midgut and in tissues that appear to be developing somatic musculature. Expression levels decline during subsequent stages of embryogenesis. Together, this pattern of expression is consistent with a role for dMyc1 in the proliferative phase of the development of the midgut and the somatic mesoderm (Schreiber-Agus, 1997).

Mutations have been characterized in the Drosophila Tsc1 and Tsc2/gigas genes. Inactivating mutations in either gene cause an identical phenotype characterized by enhanced growth and increased cell size with no change in ploidy. Overall, mutant cells spend less time in G1. Coexpression of both Tsc1 and Tsc2 restricts tissue growth and reduces cell size and cell proliferation. This phenotype is modulated by manipulations in cyclin levels. In postmitotic mutant cells, levels of Cyclin E and Cyclin A are elevated. This correlates with a tendency for these cells to reenter the cell cycle inappropriately as is observed in the human lesions (Tapon, 2001).

The enhanced growth observed in the Tsc1 or Tsc2 mutants most resembles the results of inactivating PTEN or increasing Ras1 or dmyc activity. In each of these situations, there is a reduction in the length of the G1 phase. In contrast, increased growth driven by Cyclin D/cdk4 does not alter the distribution of cells in different phases of the cell cycle. The effects of the combined overexpression of Tsc1 and Tsc2 displays genetic interactions with multiple pathways. The phenotype is influenced by alterations in the levels of dS6K, PTEN, Ras1, dmyc, cyclin D, and cdk4. Thus, Tsc1 and Tsc2 may function downstream of the point of convergence of these pathways. Alternatively, Tsc1 and Tsc2 may primarily antagonize one of these pathways, but this effect could be overcome by increasing the activity of one of the others (Tapon, 2001).

Members of the Myc family of proto-oncogenes have long been implicated in regulating proliferation, apoptosis and oncogenesis. Recently, transcriptional and biological studies have suggested a direct role for Myc in regulating growth. dm⁴, a new null allele of the Drosophila diminutive (dm) gene, which encodes dMyc on the X chromosome, has been used to investigate a role for dMyc in larval endoreplicating tissues, where cellular growth and DNA replication occur in the absence of cell division. Hemizygous dm⁴/Y mutants arrest as second instar larvae, and fat body nuclei of dm⁴/Y mutants fail to attain normal size and normal levels of DNA, resulting from a reduced frequency of S-phase. Thus, dMyc is required for endoreplication and larval growth. In support of this, dMyc, as well as its antagonist dMnt (see Drosophila Mnt for information about Mad/Mnt family members), are expressed in larval tissues in a pattern consistent with their involvement in regulating endoreplication. Overexpression of dMyc in endoreplicating cells results in dramatic increases in nuclear DNA content and cell and nucleolar size, whereas dMnt overexpression has the opposite effect. BrdU incorporation and Cyclin E protein levels continue to oscillate in dMyc-overexpressing cells, indicating that the normal cell cycle control mechanisms are not disrupted. dMyc driven growth and endoreplication are strongly attenuated when the endocycle is blocked with Cyclin E or the cdk inhibitor p21. By contrast, the ability of dMyc to promote growth and endoreplication is only partly reduced when PI3K activity is blocked, suggesting that they influence distinct growth pathways. These results indicate that larval growth and endoreplication are coupled processes that, although linked to cell cycle control mechanisms, are regulated by dMyc and dMnt (Pierce, 2004).

To determine whether there is a requirement for dMyc in driving endoreplication, dm⁴, a null allele of dm, was isolated. The failure of dm⁴ mutants to grow beyond the second instar indicates that dMyc is required for growth at the organismal level. It is presumed that maternally deposited dm gene products, or other maternal products, are sufficient for development of the embryo. The fact that maternal dm transcripts and protein are undetectable by the time of hatching suggests that dMyc is not required for the initiation of larval growth and may not be required for completion of embryogenesis, although it is possible that a small amount of residual maternal dMyc supports the growth of dm⁴ mutant larvae prior to their arrest. The massive growth that takes place during larval development is tightly coupled to the endoreplication that takes place in all larval tissues except the imaginal discs and nervous system. The finding that dMyc and dMnt are expressed in distinct groups of cells in these tissues is suggestive of roles in promoting (dMyc) and limiting (dMnt) endoreplication and suggests that the failure of dm⁴ mutants to grow is the result of loss of dMyc in endocycling tissues (Pierce, 2004).

Larval growth involves both cytoplasmic growth and DNA endoreplication. The reduced rate of BrdU incorporation in early larval tissues and the failure of dm⁴ mutant nuclei to grow suggests that these mutants undergo reduced DNA replication. The fact that mutant cells and larvae are smaller than age-matched controls indicates that that there is also a growth defect. Thus, directly or indirectly, dMyc is required for both growth and DNA replication during endoreplication (Pierce, 2004).

Consistent with findings that dMyc loss of function negatively affects endoreplication and growth, overexpression of dMyc drives both cellular growth and DNA replication. By contrast, overexpression of the Drosophila ortholog of Mad, dMnt, blocks cellular growth and DNA replication. These results are consistent with a model in which dMyc and dMnt act antagonistically, with dMnt binding and repressing the genes required for endoreplication that dMyc activates. dMnt is normally highly expressed in the third instar salivary gland and other tissues that have exited the endocycle, indicating that dMnt-mediated gene repression may be necessary for this transition. However, dMnt, the only Mad family ortholog in Drosophila, is non-essential. dMnt null mutants develop normally into adults with modestly increased body weights and shorter lifespans. Although the increased body weight of dMnt mutants is consistent with negative regulation of growth by dMnt, no altered endoreplication was observed in mutants, suggesting that negative regulators of endoreplication other than dMnt must exist (Pierce, 2004).

In endoreplicating cells ectopic expression of dMyc results in increased cytoplasmic and nuclear volume, as well as in enlarged nucleoli, as detected by increased anti-fibrillarin staining. Fibrillarin has been implicated as a Myc target gene in both vertebrate and Drosophila cells and its augmented expression is consistent with the notion that dMyc/Myc promotes ribosome biogenesis. The pitchoune gene, which encodes a putative RNA-helicase localized to the nucleolus, is also induced by ectopic dMyc, and pit null mutants have a severe larval growth defect similar to dm⁴ mutants. The mammalian ortholog of pit, MrDB (DDX18), has been identified as a direct target of c-Myc. In addition, many other known and suspected targets of the Myc family are involved in this process (Pierce, 2004).

S-phase of the endocycle is initiated by the activity of Cyclin E/cdk2 but endoreplication can be blocked by continuous ectopic expression of Cyclin E or the human cdk inhibitor p21. It is thought that Cyclin E levels must drop after each S-phase and then increase again prior to the next S-phase to allow reinitiation of DNA replication. In mitotic cells, this prevents more than one round of DNA replication from occurring during each cell cycle. In endoreplicating cells it results in discrete S-phases separated by a gap phase. Ectopic p21 is likely to inhibit the activity of cdk2 even in the presence of Cyclin E. The extra endocycles driven by ectopic dMyc appear to be normal, in that there are discrete periods of DNA replication and Cyclin E appears to oscillate. As ectopic dMyc induces cells to accumulate high levels of DNA earlier in development, it is presumed that S-phases and Cyclin E oscillations occur more frequently than normal. It is also possible that the S-phases are shorter and that Cyclin E peaks at higher levels when ectopic dMyc is present. When co-expressed with ectopic unregulated Cyclin E or p21, dMyc drives very little endoreplication, suggesting that the cell cycle control exerted by oscillating Cyclin E/cdk2 activity is downstream of dMyc function. Consistent with this, ectopic dMyc can post-transcriptionally increase Cyclin E levels in wing discs and studies in mammalian cells suggest that Myc can indirectly induce Cyclin E expression. Microarray analysis did not identify Cyclin E as a transcriptional target of dMyc, suggesting that the transcriptional oscillation of Cyclin E is not directly regulated by dMyc. Thus, dMyc is unable to drive endoreplication in the absence of normal cdk activity. Although the level of fibrillarin staining was not quantified, dMyc appears to drive somewhat more nucleolar growth than DNA accumulation when co-expressed with Cyclin E or p21, indicating that dMyc may be able to drive a limited amount of nucleolar growth in the absence of DNA replication (Pierce, 2004).

The Drosophila insulin signaling pathway is also essential for growth. Mutations in the receptor InR and downstream components of the pathway, including Dp110, a PI3 kinase homolog, cause larval growth defects. When PI3 kinase signaling is blocked by ectopic expression of p60, dMyc is still able to induce a significant amount of cellular growth and DNA replication. This suggests either that dMyc is downstream of PI3 kinase signaling or that dMyc and dDp110 represent independent pathways that are both essential for growth. Recent studies have found that Dp110 or InR overexpression do not result in increased dMyc transcription and that activated Ras increases dMyc levels and PI3 kinase activity via independent effector pathways, suggesting that dMyc transcription is not downstream of the insulin signaling pathway. In addition, although ectopic expression of either dMyc or Dp110 leads to increased cell growth, the increase in nuclear size is more pronounced in response to dMyc whereas the increase in cytoplasmic volume is more pronounced in response to Dp110, further supporting the idea that dMyc and Dp110 regulate growth and endoreplication independently (Pierce, 2004).

dMyc overexpression augments cell growth in mitotic wing disc cells by shortening the mass doubling time. Such cells display a decrease in the length of G1 and a compensatory increase in the length of G2/M, resulting in a division time equal to that of control cells. They retain their normal ploidy and show little effect on the length of S phase. In endoreplicating cells, dMyc drives both cellular growth and DNA replication. What is the relationship of dMyc function to these processes? dMyc transcriptionally activates a wide range of genes involved in ribosome biogenesis, translation and metabolism, suggesting that the relationship of dMyc to growth is likely to be very direct. The absence of an effect of dMyc on S phase length and cell division rate in mitotic cells argues that perhaps the only role of dMyc is to regulate cell growth. Interestingly, the division rate of dMyc-overexpressing mitotic cells is increased by introduction of String, which accelerates G2/M, resulting in the generation of a larger number of cells. Perhaps in endoreplicating cells, which lack G2/M entirely, dMyc simply increases the growth rate thereby shortening the G1-S transition and leading to a higher rate of S phase entry. Because such cells are incapable of division, the net effect observed is larger cells with increased ploidy. In this model, dMyc is thought to augment endoreplication indirectly, through its promotion of growth. dMyc is also required during oogenesis for somatic and germ cell growth and endoreplication, but not for proliferation prior to the onset of endoreplication. The finding that dMyc mutant follicle cells exhibit reduced growth prior to endoreplication suggests that the defect in endoreplication may be secondary to the defect in cellular growth (Pierce, 2004).

Alternatively, dMyc might affect endoreplication more directly. Although both mammalian and Drosophila Myc target genes are predominantly growth related, a smaller number of gene targets are involved in cell cycle control and DNA replication. Importantly, dMyc does not increase transcript levels of Cyclin E or the Drosophila E2F1 transcription factor, the only known limiting factors for endocycles in endoreplicating tissues. The finding that the effect of dMyc on growth is attenuated when the cell cycle is blocked by continuous expression of Cyclin E or p21 indicates that dMyc-induced growth is tightly coupled to DNA replication, at least in endoreplicating cells. The large number and diversity of the genes identified as likely targets of Myc genes indicates that Myc activity impinges on a broad range of cellular functions that must be highly coordinated for proper cell behavior. Interestingly Myc overexpression has been reported to lead to endoreplication and polyploidy in human kertinocytes. Furthermore, in murine fibroblasts treated with colcemid, Myc overexpression leads to abrogation of the G2/M checkpoint and marked polyploidy. These results suggest that Myc function is involved in controlling S-phase entry and G2/M in diverse vertebrate cell types. The Drosophila endoreplicating cell system should provide a good model for better defining the precise role of Myc in coordinating growth and cell cycle (Pierce, 2004).

Drosophila Myc is required for ovary cell growth and endoreplication

Although the Myc oncogene has long been known to play a role in many human cancers, the mechanisms that mediate its effects in both normal cells and cancer cells are not fully understood. A genetic analysis has been undertaken of the Drosophila homolog of the Myc oncoprotein (dMyc), which is encoded by the dm locus. Mosaic analysis was carried out to elucidate the functions of dMyc in the germline and somatic cells of the ovary during oogenesis, a process that involves cell proliferation, differentiation and growth. Germline and somatic follicle cells mutant for dm exhibit a profound decrease in their ability to grow and to carry out endoreplication, a modified cell cycle in which DNA replication occurs in the absence of cell division. In contrast to its dramatic effects on growth and endoreplication, dMyc is dispensable for the mitotic division cycles of both germline and somatic components of the ovary. Surprisingly, despite their impaired ability to endoreplicate, dm mutant follicle cells appear to carry out chorion gene amplification normally. Furthermore, in germline cysts in which the dm mutant cells comprise only a subset of the 16-cell cluster, strictly cell-autonomous growth defects are observed. However, in cases in which the entire germline cyst or the whole follicular epithelium is mutant for dm, the growth of the entire follicle, including the wild-type cells, is delayed. This observation indicates the existence of a signaling mechanism that acts to coordinate the growth rates of the germline and somatic components of the follicle. In summary, dMyc plays an essential role in promoting the rapid growth that must occur in both the germline and the surrounding follicle cells for oogenesis to proceed (Maines, 2004).

A newly-isolated lethal allele was used to study the role of dMyc in Drosophila oogenesis, a process that involves cell proliferation, differentiation and growth. The results indicate that the primary consequence of the loss of dMyc activity from either nurse cells or follicle cells is a reduced ability to grow and support DNA endoreplication. The results are consistent with a previous report that loss of dMyc function in the developing wing disc retards cellular growth and reduces final cell size. dMyc is also required for endoreplication and growth of larval cells (Maines, 2004).

The phenotype of dm² mutant cells in the ovary is consistent with a requirement for dMyc function in both cell growth and the endocycle, two processes that are interdependent. Conditions that block cell growth invariably perturb endocycle progression. Conversely, the inhibition of DNA synthesis during the endocyle, through the expression of inhibitors of DNA replication or by mutating genes essential for DNA replication, leads not only to a slowed increase in DNA content but also to a decrease in overall cell growth. The idea is favored that dMyc activity is required initially to enhance growth, with the effects of dMyc loss-of-function on endoreplication being a secondary consequence of impaired growth. At oogenic stages prior to the onset of endoreplication in the follicular epithelium, dm² mutant follicle cells are detectably smaller than their wild-type neighbors. This indicates that reduced dMyc function affects cell growth prior to, and independently of, its effect on endoreplication. Because of the reciprocal relationship between growth and endoreplication, the initial growth defect in dm² cells may lead to endoreplication defects that then feed back and contribute to further reductions in cell growth. Ultimately this growth defect could have the effect of greatly reducing the number of endoreplication cycles that dm² mutant cells can complete. However, a direct effect of dMyc on endoreplication, in addition to its influence on growth cannot be ruled out (Maines, 2004).

Although endoreplication is severely impaired when dMyc activity is reduced, dm mutant cells maintain a limited ability to endocycle. During a one-hour pulse of BrdU, dm² mutant germline cells were 3- to 5-fold less likely than wild-type cells to be in the S phase of the endocycle, as measured by BrdU incorporation. This suggests that relative to wild-type cells, dm² mutant cells spend a longer fraction of the endocycle in G1 phase and a shorter proportion in S phase. A simple explanation for this observation would be that endoreplicating cells remain in G1 until they reach a critical metabolic or growth threshold required for the onset of DNA synthesis, and the rate at which they reach this threshold depends on the level of dMyc activity. This implies that an important function of dMyc in the ovary is to promote growth during G1 so that the G1/S progression can occur. This interpretation would be consistent with the observation that overexpressing dMyc in wing disc cells decreases the proportion of the cell cycle spent in G1 (Maines, 2004).

In contrast to its dramatic effects on cell growth and endoreplication, dMyc appears to be largely dispensable for the mitotic proliferation of both germline and somatic cells. Not only were germline and follicle cell clones recovered that resulted from mitotic recombination subsequent to egg chamber formation, but clones were also identified that had been produced during the division of the stem cell precursors of these two cell types. This indicates that ovarian cells are capable of dividing many times in the absence of wild-type dMyc activity, which strongly argues against a role for dMyc in mitotic cell cycles in the ovary. Although the notion that mitotic proliferation of these cells can occur in the total absence of dMyc activity is favored, the possibility cannot be currently ruled out that the mitotic proliferation observed is supported by residual activity associated with the truncated protein produced by the dm² mutant allele (Maines, 2004).

In contrast to these observations, investigations carried out in mammalian tissue culture have suggested a crucial role for Myc in cell cycle progression. Exposure of quiescent cells to growth factors rapidly induces the expression of Myc, and forced expression of Myc in various cell types can induce them to enter S phase, accelerate their rate of cell division, and alter their requirements for growth factor stimulation. Correspondingly, reduction of Myc expression is correlated with exit from the mitotic cycle and cell differentiation. In the developing Drosophila wing disc, overexpression of dMyc dramatically shortens the length of G1, but a concomitant increase in the length of S phase and G2 resulted in no change in the length of the cell cycle overall. Thus, in Drosophila, the primary function of dMyc may be to promote cell growth rather than cell proliferation (Maines, 2004).

Overexpression of dMyc has been proposed to accelerate the G1/S transition in wing disc cells by activating Cyclin E through a post-transcriptional mechanism. Because Cyclin E has been identified as an important regulator of the endocycle in nurse cells, the protein expression of both Cyclin E and Dacapo, and the Drosophila p27 Cip/Kip homolog that specifically inhibits Cyclin E/Cdk2 activity were examined. Both Cyclin E and Dacapo protein expression continued to cycle in dm² mutant nurse cells. These observations suggest that the effect of the dm² mutation on the endocycle does not result from an influence on the pattern of Cyclin E expression. Consistent with this conclusion, Cyclin E protein levels also continue to oscillate in larval fat body and salivary gland cells that overexpress dMyc. Perturbations in Cyclin E expression can also lead to the differentiation of multiple oocytes in a single cyst. The finding that only one oocyte differentiated in dm² mutant cysts provides further evidence that Cyclin E regulation was relatively normal (Maines, 2004).

In mammalian cells, expression of the Dacapo homologs p21CIP1 and p27KIP1 is repressed by Myc. In the Drosophila ovary, Dacapo present in the oocyte nucleus prevents it from undergoing DNA endoreplication, helping to maintain it in prophase I of meiosis. These findings suggested a possible mechanism whereby dMyc-mediated inhibition of dacapo gene expression in nurse cells might facilitate their endoreplication. However, the fact that Dacapo protein did not show an obvious increase in expression in dm² mutant germ cells suggests that in contrast to what is seen in mammals, dacapo expression is not directly influenced by dMyc in these cells, nor is dMyc-mediated repression of dacapo expression an important mechanism regulating nurse cell endoreplication (Maines, 2004).

In Drosophila, cell growth is known to be regulated by two distinct but interacting signalling pathways: one mediated through the insulin receptor (InR) and phosphatidylinositol-3-OH kinase (PI3K), and the other through the nutrition-sensing protein kinase TOR (target of rapamycin). The dm mutant phenotype closely resembles that of mutants affecting the dTOR effector protein ribosomal S6 kinase. In mammals, S6 kinases have been shown to promote the translation of ribosomal proteins and translation factors. In a recent study that examined the binding to DNA of the Drosophila Myc network proteins Myc, Max and Mad/Mnt, a number of genes involved in ribosome biogenesis and proteins synthesis were identified as dMyc targets. Taken together, these findings suggest that dMyc, like dS6K, may exert its effect on growth through the enhancement of protein translation (Maines, 2004).

One protein through which dMyc might exert its effects on translation is the product of the pitchoune (pit) gene, a putative DEAD-box RNA helicase whose human homolog, MrDB, has been shown to be a transcriptional target of Myc-Max heterodimers. pit mutants exhibit a constellation of phenotypes similar to that observed for dm² mutants, and constitutive expression of dMyc induces expression of pit in embryos and third instar larvae. The identity of Pitchoune protein as a DEAD-box RNA helicase and its subcellular localization in the nucleolus suggests that it may be involved in rRNA processing or ribosome biogenesis (Maines, 2004).

Perturbation of Delta/Notch signaling between the germline and the follicle cells has been observed to disrupt follicle cell endoreplication. Follicle cells homozygous for loss-of-function alleles of Notch exhibit a delay in exiting the mitotic division cycle, which leads to overproliferation of the follicular epithelium and the formation of abnormally small mutant cells with small nuclei. This phenotype has been interpreted as an inability of the Notch mutant cells to undergo their normal program of differentiation. By contrast, the observations suggest that dm mutant follicle cells exhibit reduced levels of postmitotic DNA synthesis not because they cannot make the transition from mitosis to the endocycle, but because they are unable to grow sufficiently well to support the endocycle. In addition, the execution of chorion gene amplification by dm² follicle cells suggests that they adopt at least some of the characteristics of mature follicle cells. This observation may reflect the possibility that dMyc regulates distinct effectors of endocycle DNA replication that do not participate in gene amplification. Alternatively, the metabolic and synthetic needs of cells undergoing gene amplification may be much lower than those of endocycling cells, and may not require the concerted action of the dMyc-activated gene network (Maines, 2004).

In addition to the effects of loss of dMyc activity on the growth of homozygous mutant germline or follicle cells, a profound effect on the growth and development of the entire egg chamber was observed when either the complete germline or the entire follicular epithelium was mutant for dm². In both cases, the follicles were delayed in their development and rarely progressed to vitellogenic stages, in which yolk uptake can be detected in the oocyte. These results suggest that the growth of the somatic and germline components of the ovary are tightly coordinated. In addition to their failure to grow, the genotypically wild-type follicle cells surrounding dm² mutant germline clones exhibit some signs of immaturity, such as perdurance of uniform FasIII expression. Surprisingly, expression of the BR-C by these cells appears to be determined by their age, based upon their position within the ovariole, rather than on the maturity of the egg chamber, as judged by its size. This raises the interesting possibility that follicle cells can assess their age by a mechanism that is independent of the growth state of the egg chamber in which they are contained. Alternatively, the ability of the follicular epithelium to respond to ecdysone signaling may depend in part on influences external to an individual follicle. It has been well documented that signaling between the soma and the germline is required for the establishment of the dorsoventral and anteroposterior axes of both the follicle and the future embryo. However, the signals that communicate the growth status of one tissue to the other are less well understood. The finding that the loss of dMyc function in either the germline or in the soma is sufficient to prevent the growth of the complementary soma or germline, respectively, may provide a useful tool for investigating how this information is transferred between the two tissues (Maines, 2004).

In contrast to clones comprising entire germline cysts, the loss of dMyc activity from a subset of nurse cells led to an apparently autonomous deficiency in growth that did not affect the wild-type nurse cells present in the same cyst. The recovery of such mixed-phenotype mosaic cysts also led to the conclude that the reduced growth and impaired endoreplication detected in dm² mutant nurse cells did not result from the failure of a general cyst-wide developmental transition that requires the function of dMyc. Indeed, the ability to generate such mixed phenotype cysts was quite surprising, because the nurse cells are interconnected by cytoplasmic bridges and are typically considered to share a common cytoplasm. The restriction of hGFP to the nurse cells in which it was synthesized, and the phenotypic differences between the dm²/dm² and +/+ nurse cells in the same cyst, convincingly demonstrate that there are restrictions that limit the intercellular movement of at least some gene products between nurse cells. The use of the hGFP marker will make it possible to investigate whether the products of other genes expressed by the nurse cells are similarly confined to their cells of origin (Maines, 2004).

In summary, this analysis of the function of dMyc in the Drosophila ovary is consistent with the conclusions of other recent work that indicates that Myc family proteins profoundly influence the ability of cells to grow. By combining the information obtained from genomics-based molecular studies with the genetic analysis of putative target genes, it should be possible to elucidate the role of dMyc in different tissues, and to identify those genes that act as its downstream effectors. The dm ovarian phenotype will provide a useful framework in which to investigate the function of the dMyc network in growth and endoreplication (Maines, 2004).

Drosophila growth and development in the absence of dMyc and dMnt

Myc oncoproteins are essential regulators of the growth and proliferation of mammalian cells. In Drosophila the single ortholog of Myc (dMyc), encoded by the dm gene, influences organismal size and the growth of both mitotic and endoreplicating cells. A null mutation in dm results in attenuated endoreplication and growth arrest early in larval development. Drosophila also contains a single ortholog of the mammalian Mad/Mnt transcriptional repressor proteins (dMnt), which is thought to antagonize dMyc function. Animals lacking both dMyc and dMnt display increased viability and grow significantly larger and develop further than dMyc single mutants. Increased endoreplication and growth of larval tissues is observed in these double mutants, along with disproportionate growth of the imaginal discs. Gene expression analysis indicates that loss of dMyc leads to decreased expression of genes required for ribosome biogenesis and protein synthesis. The additional loss of dMnt partially rescues expression of a small number of dMyc and dMnt genes that are primarily involved in rRNA synthesis and processing. These results indicate that dMnt repression is normally overridden by dMyc activation during larval development. Therefore the severity of the dm null phenotype is likely due to unopposed repression by dMnt on a subset of genes critical for cell and organismal growth. Surprisingly, considerable growth and development can occur in the absence of both dMyc and dMnt (Pierce, 2008).

These experiments show that Drosophila larvae lacking both dMyc and dMnt display increased viability, grow significantly larger, and advance considerably further in larval development than dMyc single mutants. dm⁴dmnt¹ mutant larvae reached a maximum size that was much larger than the maximum size of dm⁴ mutants, which are terminally growth arrested at an early larval stage. In addition, a substantial fraction of dm⁴dmnt¹ mutant larvae can continue to develop and form pupae, although they fail to metamorphose (Pierce, 2008).

The most dramatic defect in larvae lacking dMyc is the failure of endoreplicating cells to attain both wild-type size and DNA levels. Similar effects have been observed in germline and somatic follicle cells mutant for dm. Because endoreplicating cells comprise the bulk of the early larva and are responsible for most of the 200-fold increase in mass that occurs between embryogenesis and pupariation, it is likely that the defect in endoreplicating tissues accounts to a large extent for the very limited larval growth and the inviability in dm⁴ animals. The additional removal of dMnt results in increased cell and nuclear growth, which is accompanied by a modest increase in the fraction of cells undergoing endoreplication in the early larva as well as increased survival. It is surmised that this increase in endoreplication and growth, combined with the extended larval period, is likely to account for the overall growth rescue. These results indicate that the strong larval growth arrest phenotype in the dMyc single mutant (dm⁴) is due, in part, to the presence of dMnt, whose activity as a repressor would be unopposed by dMyc in dm⁴. As a result, when dMnt is removed in the dm⁴ background, a significant amount of growth can occur in the absence of dMyc. A recent study in mammalian cells and Xenopus extracts has demonstrated that c-Myc plays a regulatory role in initiation of DNA replication through direct association, together with Max, with the pre-replication complex (Dominguez-Sola, 2007). It is possible that dMyc could drive endoreplication directly rather than as a secondary consequence of increased growth. Further experiments will be required to determine if dMyc is involved in DNA replication in Drosophila and, if so, whether dMnt plays an antagonistic role (Pierce, 2008).

In Drosophila, dMyc has been demonstrated to drive the growth of mitotic as well as endoreplicating cells. dMyc overexpression results in dramatically larger wing disc cells and analysis of dMyc overexpressing clones has demonstrated that dMyc drives cell growth with little effect on cell division time. Clones of mitotic cells with reduced dMyc activity are smaller than clones of wild-type cells, indicating that the mutant cells grow more slowly. As expected, overexpression of dMnt results in small cells and cell clones while dMnt loss of function produces larger cells in the adult wing as well as heavier animals. In mammals, primary mouse embryo fibroblasts (MEFs) and other cell types lacking c-Myc are unable to grow and proliferate in response to mitogenic signals. However, MEFs lacking both c-Myc and Mnt display accelerated proliferation following selection for cells that have escaped apoptosis. While no reports to date have addressed loss of both Myc and Mnt function in tissues or whole organisms, the MEF studies are generally consistent with the finding that the imaginal discs, which consist of mitotically dividing cells, are rescued in larvae lacking dMyc and dMnt (Pierce, 2008).

It is likely that the increased growth of the endoreplicating tissues plays a significant role in the growth rescue of the whole animal and the discs. In particular, the fat body is a critical tissue for mediating the response to nutritional signals and is required to promote growth and proliferation in mitotic cells and larvae in a cell non-autonomous fashion. However, if larvae are starved after growth begins, mitotic cells continue to proliferate after endoreplication stops. Thus is it likely that the increased growth of the endoreplicating tissues, particularly the fat body, promotes the growth of the entire animal to a point at which the imaginal discs can continue to proliferate despite the limited growth of the endoreplicating tissues. In animals that lack only dMyc, there may be sufficient growth of the endoreplicating tissues to initiate growth of the animal as a whole, but not enough to sustain that growth to a point at which the discs can proliferate more independently (Pierce, 2008).

Given the strong rescue of overall imaginal disc growth it was surprising to find that clones of imaginal disc cells lacking dMyc alone or lacking both dMyc and dMnt are the same size and significantly smaller than their sibling wild-type clones. This indicates that these cells are at a proliferative disadvantage. Because such mutant clones are surrounded by cells with higher levels of dMyc, their dramatically smaller clone size can be ascribed to cell competition, a process resulting in elimination of more slowly growing cell populations from the disc epithelium. It has been well established that cell clones with lower dMyc levels relative to the surrounding population are at a competitive disadvantage and die through activation of apoptotic pathways. In this regard it is interesting that dMnt loss of function has no discernable effect on the size of dm⁴ clones in a heterozygous background. One interpretation is that the growth rescue resulting from dMnt loss is simply not sufficient to avoid cell competition induced apoptosis. Alternatively it is possible that dMnt does not influence the pathway through which reduced dMyc levels stimulate apoptosis in cells targeted for elimination (Pierce, 2008).

The signals required for the growth and maturation of multicellular organisms include extrinsic environmental cues, such as nutrition, and intrinsic developmental signals, which may have cell-autonomous and cell non-autonomous effects. The major pathways that regulate growth in response to nutrition in Drosophila are the insulin receptor and TOR signaling pathways. The TOR pathway in particular is responsive to amino acids and, with input from the phosphoinositide 3-kinase (PI3K) branch of the insulin receptor pathway, regulates ribosome biogenesis and translation. In Drosophila dMyc and dMnt have been shown to influence cell and organismal growth primarily through effects on transcription. Yet many Myc- and Mnt-regulated transcriptional targets in flies and vertebrates are themselves involved in the regulation of the translational machinery. For example Myc directly binds to and stimulates RNA PolI transcription of rDNA in mammalian cells and indirectly stimulates RNA PolI transcription in Drosophila. Interestingly, strong mutations in, or inhibition of, components of the insulin and TOR pathways result in larval growth arrest phenotypes that are similar to the effect of amino acid starvation and the phenotype of the dMyc null mutant, dm⁴. A weaker TOR mutant resembles the dm⁴dmnt¹ mutant in that it grows larger and the wing discs overgrow at the expense of the endoreplicating tissues. Indeed it is also noted that growth of the dm⁴dmnt¹ mutant was sensitive to environmental conditions such as nutrient source and presence of competition. However, clear molecular connections between the insulin and Tor pathways and dMyc/dMnt function have yet to be established (Pierce, 2008).

Although the dm⁴dmnt¹ larvae display delayed pupariation, the pupae are all very uniform in size, suggesting that they have reached a threshold for growth or size. The transition from feeding and growth to pupariation and metamorphosis is normally triggered by induction of the steroid hormone ecdysone. This occurs after larvae reach a minimum viable weight at which they possess sufficient nutritional stores to survive metamorphosis. However, experiments indicate that treatment with ecdysone is unable to rescue the delay in pupariation suggesting that ecdysone is not limiting for dm⁴dmnt¹ pupariation. Since the dm⁴dmnt¹ pupae are abnormal and never result in the formation of adult animals, their extended larval period and other modulatory factors in larvae may interact with ecdysone to trigger the initiation of metamorphosis without the larvae reaching the minimum weight required to sustain metamorphosis. Thus the failure of dm⁴dmnt¹ larvae to survive metamorphosis could be explained by their failure to accumulate sufficient nutritional stores. In addition, there may be a specific requirement for dMyc during pupal development that cannot be rescued by removing dMnt function. This latter possibility would be consistent with earlier work showing that a subset of genomic binding sites for dMyc do not bind dMax or dMnt as well as a study indicating dMax independent functions of dMyc. Because dMax coordinates binding of both dMyc and dMnt to common E-box sites it is unlikely that genes activated by dMyc independent of dMax would be repressed by dMnt (Pierce, 2008).

This is the first analysis of gene expression changes in animals that are null for dMyc, and it was found that the affected genes are primarily involved in protein synthesis. Although it was previously known that dMyc was required for pre-rRNA transcription and that PolI function is required for dMyc-induced cell growth, this study has demonstrated that dMyc is also necessary for the expression of a wide range of genes involved in ribosome biogenesis and translation. This is consistent with what is known about Myc target genes in Drosophila and mammals, in which Myc proteins have been shown to regulate the transcription of genes from all three polymerases thus controlling both ribosome biogenesis and subsequent translation. Because larvae are primarily made up of endoreplicating non-dividing cells, it is not surprising that particularly strong overlap was found between genes for which expression decreased in the absence of dMyc and those for which expression increased when dMyc was overexpressed in larvae and less overlap with dMyc direct binding sites. Note that some of the genes detected are not direct targets of dMyc but are likely to be induced as a secondary response to Myc (Pierce, 2008).

Given the growth rescue seen when dMnt function is removed in the context of the dMyc mutant, it was particularly interesting to find that the expression of only a subset of dMyc-responsive genes was altered in the double mutants relative to the dMyc single mutant. There was a modest, but significant, increase in pre-rRNA, which can in part be explained by an increase in PolI components such as Rpl1. There was a more significant increase in the expression of genes that are known or predicted to be involved in rRNA processing. Interestingly, c-Myc has been shown to promote rRNA processing in a human B-cell line. It is surmised that an increase in both rRNA synthesis and processing leads to more efficient ribosome biogenesis and, ultimately, protein synthesis. Although transcription of only one of four ribosomal protein genes that was tested by qPCR was significantly rescued, the availability of ribosomal proteins may be increased through post-transcriptional mechanisms or existing ribosomal proteins may be utilized more efficiently due to increased levels of processed rRNA (Pierce, 2008).

Although it has been shown that dMyc and dMnt are capable of acting antagonistically, in a manner similar to their mammalian counterparts, the lack of a dramatic phenotype in flies lacking dMnt might suggest that dMnt is not normally playing a major regulatory role at dMyc target genes. Nonetheless, a modest increase in expression of growth related genes is seen in dmnt¹ larvae indicating that in wild-type flies dMnt is likely functionally required to limit cell growth during normal development. The data showing that dMnt loss produces partial alleviation of the repression of a subset of growth related genes in dm⁴ larvae is consistent with the notion that dMnt is involved in the downregulation of growth gene expression upon loss of dMyc. In the absence of both dMyc and dMnt, active repression by dMnt is relieved and gene transcription is likely to be dependent on the presence of other transcription factors that may be gene-specific and normally act to regulate expression of specific genes in collaboration with Myc and Mnt. It is also possible that other Drosophila bHLH class activators that do not normally interact with dMyc-dMnt regulated promoters may play a role in the rescue of growth related gene expression when both dMnt and dMyc are absent. It is proposed that it is this rescue of growth related gene expression that leads to increased viability and development (Pierce, 2008).

A similar model has been proposed to explain the effects of deletion of another antagonistic pair of transcription factors dE2F1 and dE2F2. dE2F1 is a transcriptional activator that is essential for normal proliferation and larval growth. dE2F2 is a transcriptional repressor that can inhibit dE2F1-dependent transcription by binding to the same DNA binding sites. The growth defects in larvae lacking dE2F1 can largely be suppressed by also removing dE2F2, indicating that the dE2F1 mutant phenotype is largely due to unchecked repression by dE2F2. Interestingly the E2F proteins are also subunits of the Myb-MuvB/dREAM complex that additionally comprises Drosophila Myb, required for the selective amplification of the chorion genes, and the Mip130, Mip120, and Mip40 repressors of DNA replication (Georlette, 2007). Loss of function mutations of Dm-Myb are lethal, while single Mip mutants and the double Dm-Myb;Mip mutants are viable, indicating that the Mip proteins are primarily responsible for the lethality of the single Dm-Myb mutants (Pierce, 2008).

The results suggest that, in order to activate genes required for growth and development, dMyc must overcome the repressive effect of dMnt. It is therefore likely that dMnt serves to refine or limit the activity of dMyc. Although the weak phenotypes associated with the dMnt single mutant suggest that this role is minor, it is important to note that dMnt mutants as well as mutants in the C. elegans ortholog of dMnt (mdl1) have a reduced lifespan, indicating that the modulation of dMyc activity by dMnt is necessary for wild-type fitness (Pierce, 2008).

Persistent competition among stem cells and their daughters in the Drosophila ovary germline niche

Cell competition is a short-range cell-cell interaction leading to the proliferation of winner cells at the expense of losers, although either cell type shows normal growth in homotypic environments. Drosophila Myc (dMyc) is a potent inducer of cell competition in wing epithelia, but its role in the ovary germline stem cell niche is unknown. This study shows that germline stem cells (GSCs) with relative lower levels of dMyc are replaced by GSCs with higher levels of dMyc. By contrast, dMyc-overexpressing GSCs outcompete wild-type stem cells without affecting total stem cell numbers. Evidence is provided for a naturally occurring cell competition border formed by high dMyc-expressing stem cells and low dMyc-expressing progeny, which may facilitate the concentration of the niche-provided self-renewal factor BMP/Dpp in metabolically active high dMyc stem cells. Genetic manipulations that impose uniform dMyc levels across the germline produce an extended Dpp signaling domain and cause uncoordinated differentiation events. It is proposed that dMyc-induced competition plays a dual role in regulating optimal stem cell pools and sharp differentiation boundaries, but is potentially harmful in the case of emerging dmyc duplications that facilitate niche occupancy by pre-cancerous stem cells. Moreover, competitive interactions among stem cells may be relevant for the successful application of stem cell therapies in humans (Rhiner, 2009).

In an analysis of mosaic niches, it was found that stem cells mutant for the hypomorphic allele dmyc^P0 (dmyc^P0/dmyc^P0) were gradually outcompeted by neighboring GSCs with higher levels of dMyc (dmyc^P0/+). The same effect was observed with stem cells overexpressing dMyc relative to their counterparts, resulting in the niche conquest by such dMyc-overexpressing stem cells. Comparison of mosaic niches with niches harboring a homogenous population of stem cells regarding dMyc levels showed that dMyc was able to induce competition, measurable in shorter decay times of the outcompeted cells (Rhiner, 2009).

The results contrast, however, with a recent study by Jin, 2008, who also examined dmyc in GSC competition. Jin tested the lethal dmyc alleles dm² and dm⁴, which behave genetically as null alleles and found that such dMyc-deficient GSCs were not outcompeted by control GSCs. Strikingly, Jin also obtained different results with stem cells carrying two copies of the nos-gal4VP16 driver to overexpress UASdmyc in the germline. Such GSCs did not compete with control GSCs carrying only one copy of nos-gal4VP16, rather they were lost slightly faster from the niche (Rhiner, 2009).

Part of the explanation for the discrepancies might lay in differences in experimental design and chosen dmyc alleles. In the current study, GSCs carrying strong or null mutations in dmyc (dmyc^PG45 or dm⁴, respectively) showed poor viability and, in the few mosaic niches found, dmyc-deficient stem cells were not lost from the niche. Therefore it wasdecided to study hypomorphic dmyc^P0 stem cells that express dMyc, but at lower levels - an ideal case of viable, but suboptimal stem cells. GSCs devoid of any dmyc (dm4 deletion) may not enter competitive interactions because some basal dMyc levels might be required to permit an intercellular comparison of relative competitiveness. Wing pouch cells of the imaginal disc have been proposed to be able to compare their Dpp signaling levels with those of cells outside of the pouch. Intriguingly, this differential behavior coincides with the dMyc expression pattern. It is also believed that dMyc-induced cell competition acts only within a certain range of dMyc fluctuation, and will be overrun by apoptosis as a consequence of the well-characterized effect of dMyc to induce cell-autonomous apoptosis when expressed at high levels (Rhiner, 2009).

Both genetic systems employed here to raise dMyc levels (dmyc duplication and tub>cd2>dmyc flip-out cassette) are compatible with GSC viability. The overexpression of dMyc using Gal4 amplification systems with two copies of nos-gal4VP16 driver may not be so well tolerated by stem cells, compromising their competitiveness (Rhiner, 2009).

In contrast to studies that have focused on cell adhesion and mechanical models of GSC extrusion from the niche, this study provides evidence that Dpp signaling and cell-cell communication play a role for dMyc-induced competition. It is not completely understood how cells compare the Myc levels of one another, evidence that the ability to compete for 'stemness' factors, like Dpp, is important: when GSCs with more dMyc contact GSCs with less dMyc, the cells with higher levels become more sensitive to Dpp and accumulate pMad, which ensures a long-lasting stem cell fate, whereas the cells with lower relative levels of dMyc lose responsiveness and eventually differentiate. A possible connection between dMyc and the acquisition of an extracellular ligand(s) has been proposed before. This relationship seems to be indirect and is likely to involve the coordinated control of several target genes, resulting in a gain of several aspects of biometabolic functions, e.g. modifying rates of endocytosis (Rhiner, 2009).

The identification of paracrine and/or juxtacrine signals that are specific to loser and/or winner cells is likely to become a fascinating line of future research, as in the case of the apoptotic elimination of 'loser' cells. In fact, the possibility that cells can compare their relative Dpp signaling levels has been postulated to explain both apoptotic cell competition and the regulation of cell proliferation, but the molecules that mediate this comparison are unknown. Further studies will also clarify how similar apoptotic and non-apoptotic cell competition really are (Rhiner, 2009).

The results with dmyc^P0 mutant stem cells have demonstrated that suboptimal GSCs are efficiently recognized and eliminated from the stem cell pool, suggesting that dMyc-induced cell competition plays a physiological role in controlling tissue quality. Because dMyc is always expressed at high levels in GSCs, the quality control system seems to be especially suited to detecting subtle drops in dMyc levels in suboptimal GSCs. In a similar way, fluctuation in the repression of the differentiation factor bam by Dpp signaling has been suggested to regulate stem cell quality, which would be consistent with the model that competitive stem cells show high pMad levels and, hence, tightly repressed bam expression (Rhiner, 2009).

Although cell competition seems an excellent tool with which to select the 'fittest' stem cell when compromised GSCs are present, it bears the risk that stem cells with modest dMyc overexpression are selected as being preferable over the wild type. Niches occupied by stem cells harboring dMyc duplications will give rise to differentiated tissue containing identical genetic alterations. Such 'pre-cancerous' fields are then more likely to accumulate secondary or tertiary hits that will lead to tumor formation. Given that stem cells are long-lived, it is believed that the characteristics and consequences of dMyc-induced competition are relevant for cancerous transformation, especially for tissues with a high turnover. The most notable features of niche occupancy by dMyc-overexpressing stem cells are (1) the replacement of normal GSCs, which was not observed by other proliferation-promoting mutations (Pten, sty), and (2) the absence of tissue alteration. The property to outcompete wild-type stem cells renders dMyc mutations potentially dangerous because they are capable of establishing a mutant stem cell population that can remain long enough to accumulate further cooperating mutations. This could be a reason why myc is the target of early mutations in cancers and Pten inactivation occurs only at later stages (Rhiner, 2009).

So far, cell competition has been analyzed extensively using artificial means to create mosaic tissues, but few attempts have been undertaken to reveal competitive interactions in a more physiological situation. An exception is the study on the competitive interactions of cells during liver repopulation after hepatectomy (Rhiner, 2009).

This study tested the novel idea that developmentally regulated expression of dMyc in the germline triggers competition for the stem cell factor Dpp between high dMyc-expressing stem cells and low dMyc-expressing progeny. This competition might be classified as 'low level', compared with stem cell-stem cell interactions in the niche, as the daughter cells compete with the handicap of being located more distally from the source of Dpp than are the stem cells. Nonetheless, the experiments, in which the physiological dMyc pattern is perturbed and GSCs and CBs are equalized in terms of dMyc levels, strongly suggested a contribution of competition in the initial step of differentiation. The ability of dMyc to activate a variety of genes encoding components of protein synthesis pathways indicates that it may have the capacity to stimulate protein translation in a coordinate manner. A model is proposed in which high dMyc levels in stem cells stimulate high metabolic rates, including increased protein synthesis and endocytosis, through the activation of multiple target genes. This enables the stem cells to compete efficiently and turn over a high amount of niche secreted Dpp, resulting in elevated pMad levels, which in turn ensure a tight repression of the differentiation gene bam. During the pre-CB to CB transition, dMyc levels are downregulated, probably because of the oncoming expression of Mei-P26, which lowers the efficiency of pre-CBs/CBs to take up Dpp. The consequence is a steep decline of the Dpp gradient across the niche, where remaining Dpp input in CBs is too low to activate pMad and Bam-mediated differentiation is fully initiated. In the absence of competition, achieved by imposing equivalent dMyc levels in all three cell types, available Dpp molecules distribute more uniformly over several cell diameters because cells compete on a more similar basis. As a consequence, Dpp signaling thresholds are still attained in cells distal from the niche, which still present stem cell-like morphology owing to repressed bam transcription by pMad. This study does not intend to play down the role of Bam, which is the main trigger for differentiation, but it is suggested that dMyc-induced competition for Dpp reinforces the tight repression of Bam in GSCs and the efficient derepression of Bam in the differentiating daughter cells. If competition is impaired, the differentiation process is delayed and less defined, occasionally leading to the mixing of cystoblasts at different stages of differentiation (Rhiner, 2009).

The competitive interaction between stem cells and their daughters containing different relative levels of dMyc described in this study is of particular interest because the interface along which the competition takes place is created through gene regulation. Therefore, the term ;programmed cell competition' is proposed to distinguish it from competitive interactions that arise as a result of genetic alterations. Programmed cell competition will occur along boundaries of gene expression that are epigenetically defined (i.e., by gene regulation) and does not require a mutational event, as in previously described forms of competition (Rhiner, 2009).

Because Myc proteins play important roles in the adult stem cells of several mammalian niches, it is possible that the interactions described in this study are conserved, at least in certain tissues. In tissues where stem cells are not grouped together within the same niche, the process may be aided by migration of the stem cells from one niche to the other, as has been recently described for the somatic stem cell niches of the Drosophila ovary. More generally, the concept of competition among cells could be of use to describe several aspects of development and homeostasis, an idea nicely supported, for example, by the competition that occurs between the soma and the germline for lipid phosphate uptake. Stem cell interactions such as those described in this study significantly contribute to the balance between differentiation and self-renewal, and may be relevant for diverse processes such as aging, the accumulation of pre-cancerous mutations and the successful application of stem cell therapies (Rhiner, 2009).

Control of wing size and proportions by Drosophila myc

Generation of an organ of appropriate size and shape requires mechanisms that coordinate growth and patterning, but how this is achieved is not understood. This study examined the role of the growth regulator dMyc in this process during Drosophila wing imaginal disc development. It was found that dMyc is expressed in a dynamic pattern that correlates with fate specification of different regions of the wing disc, leading to the hypothesis that dMyc expression in each region directs its growth. Consistent with this view, clonal analysis of growth in each region demonstrated distinct temporal requirements for dMyc that match its expression. Surprisingly, however, experiments in which dMyc expression is manipulated reveal that the endogenous pattern has only a minor influence on wing shape. Indeed, when dMyc function is completely lacking in the wing disc over most of its development, the discs grow slowly and are small in size but appear morphologically normal. These experiments indicate, therefore, that rather than directly influence differential growth in the wing disc, the pattern of dMyc expression augments growth directed by other regulators. Overall, however, an appropriate level of dMyc expression in the wing disc is necessary for each region to achieve a proportionately correct size (Wu, 2010).

By examining the expression of dMyc over the course of wing development, this study demonstrates that dmyc mRNA and protein are expressed in a temporally and spatially dynamic manner that corresponds to the subdivision of the wing-blade primordium from the hinge primordium. This relationship raised the possibility that dMyc is specifically deployed, presumably by factors that specify regional fates, to control the growth of each region as it develops, thereby contributing to sculpting the adult wing shape. This work makes three major findings. First, the experiments indicate that the intricate pattern of dMyc expression in the wing disc helps cells proliferate at an appropriate rate at any given time during wing development. Second, an adult wing can form in the absence of this pattern, although it is mis-proportioned and rudimentary in size. Finally, the absolute level of dMyc expression determines the rate at which the developing wing grows and also the rate of pattern maturation (Wu, 2010).

The expression pattern of dMyc in the wing is strikingly dynamic. Prior to the subdivision of the distal wing into hinge and blade, dMyc is expressed fairly uniformly, but as these regions are specified its expression undergoes transient up- and downregulation before stabilizing in a WP predominant pattern that prevails until the end of L3. Clonal experiments indicate that the level of dMyc expression in a wing disc cell at any given time determines its rate of proliferation, and the changes in the dMyc expression pattern correlate well with changes in relative functional need. Clear region-specific differences are detected in the functional requirement for dMyc that corresponded to the specification of proximal and distal wing fates. Moreover, it was found that once the wing blade and hinge primordia are specified, they grow with distinct kinetics, such that midway through L2 these regions of the disc switch from isometric to allometric growth, resulting in a considerably larger WP than hinge by late L3 (Wu, 2010).

Despite these correlations, however, modification of the endogenous expression pattern in whole animals demonstrated that the spatial and temporal components are less important than the absolute level of dMyc expressed. The conservative interpretation of these data is that dMyc's role in wing growth is permissive rather than instructive and that it augments a growth rate set by other mechanisms. However, it is puzzling why dMyc is expressed in an extravagant pattern that is not necessary. This pattern could be merely a remnant of evolution. Alternatively, compensatory post-transcriptional control of dMyc could occur. dMyc protein is highly regulated (Galletti, 2009) and is notably increased in the absence of Archipelago, a homolog of the vertebrate Fbw7 F-box protein. However, within the limits of detection, no difference was observed between the expression patterns of dMyc mRNA and protein at any time during wing development in these experiments. Given the high degree of flexibility during wing growth, it is possible that redundancy among growth regulatory factors that function in the wing allows formation of a small but correctly shaped wing when dMyc is expressed ubiquitously or not at all. Indeed, as a whole, the results illustrate the inherent robustness of wing development (Wu, 2010).

The permissive role of dMyc ensures that cells proliferate at stage-appropriate rates, determines overall size, and allows the development of a wing of correct proximal and distal proportions. The results complement those of Pierce (2008), who reported that wing discs carrying null mutations of both dmyc and dmnt, the dMyc antagonist, reach a size comparable to wild type after an extended L3 (3-7 days longer than wild type). In that case, loss of dMnt derepressed a subset of genes that rescued the dm4 mutant phenotype. The fact that wings grow reasonably well under those conditions supports the hypothesis that the growth program of the disc is augmented rather than determined by dMyc. The larval delay in those and in and the current experiments is due to reduced endoreplication of larval cells, which is dMyc dependent. In the VgM experiments, dMyc expression was maintained in most tissues while selectively removing it from the wing. Under these conditions, larval development progressed at the same rate as Tub-dmyc-rescued dm4 mutants, but wing disc growth was significantly slowed. The uncoupling of larval and disc growth rates resulted in an altered size relationship between the wing and hinge, implying that coordination between larval growth and imaginal growth is important for wing size and shape. Growth regulators such as dMyc thus contribute to body and organ proportionality by promoting a rate of wing disc growth that is compatible with the rate of endoreplication and growth of larval cells. Moreover, control of wing size by dMyc is dose dependent. Together, the data suggest that the dm gene could be an evolutionary target that contributes to the wide variability of wing size among Drosophila species. Consistent with this possibility, evidence of strong selection at the dm locus has been documented (Wu, 2010).

Although the pattern of dmyc expression does not appear to instruct overall wing shape, wing cell requirements for dMyc change throughout development, possibly reflecting region-specific responsiveness to dMyc function or expression. What predisposes hinge or WP cells to respond to dMyc differently during wing disc development? Understanding how growth is governed in the different regions of the wing disc should help answer this question. Region-specific cues may be provided by the hinge selector Hth and the wing selector Vg and by Fat/Hippo signaling. The cadherins Fat and Dachsous regulate proximal (hinge) wing growth via Hippo signaling, whereas a feed-forward auto-regulatory loop Vg brings about expansion of distal wing (blade) fates. Regulation in both cases appears to be in response to signaling from Wg and Dpp. Wing growth appears therefore to be controlled quite indirectly. One possibility is that the amplitude of dMyc expression or activity is changed in response to modulation of Hippo and/or Vg activity by signals such as Wg and Dpp. This idea is supported by results showing that dmyc transcripts are significantly upregulated in fat mutant eye discs, in which Hippo activity is deregulated. Experiments to address how dMyc expression is directly regulated in the wing disc are an important goal for the future (Wu, 2010).

A striking finding of these experiments is that the rate of wing disc patterning is directly influenced by the rate of its growth: complete loss of dmyc in the wing disc dramatically slows its growth and also slows the rate at which pattern formation matures. It is generally assumed that growth occurs downstream of patterning. This assumption is based on a variety of experimental models in which reorganization of pattern is always accompanied by growth. Consistent with this idea, Myc expression is regulated by several conserved factors that control pattern formation, whereas Myc itself controls the growth and proliferation of cells by regulating numerous genes required for ribosome biogenesis and protein synthesis. However, the current experiments suggest that the hierarchy between pattern and growth is not absolute. Impaired cellular biosynthesis when dMyc is limiting may affect a cell's ability to produce proteins required for pattern specification as well as those required for cell division, cell survival, and mass accumulation. These studies reveal an unappreciated relationship between patterning and growth that influences their coordination and is worthy of further study (Wu, 2010).

Tuberous sclerosis complex and Myc coordinate the growth and division of Drosophila intestinal stem cells

Intestinal stem cells (ISCs) in the adult Drosophila melanogaster midgut can respond to damage and support repair. This study shows that the tuberous sclerosis complex (TSC) plays a critical role in balancing ISC growth and division. Previous studies have shown that imaginal disc cells that are mutant for TSC have increased rates of growth and division. However, this study shows that loss of TSC in the adult Drosophila midgut results in the formation of much larger ISCs that have halted cell division. These mutant ISCs express proper stem cell markers, do not differentiate, and have defects in multiple steps of the cell cycle. Slowing the growth by feeding rapamycin or reducing Myc is sufficient to rescue the division defect. The TSC mutant guts have a thinner epithelial structure than wild-type tissues, and the mutant flies are more susceptible to tissue damage. Therefore, this study has uncovered a context-dependent phenotype of TSC mutants in adult ISCs, such that the excessive growth leads to inhibition of division (Amcheslavsky, 2011).

This study provides evidence demonstrating that TSC is an essential regulator of ISC growth and division. In the absence of TSC function, ISCs have unrestricted cell growth, which halts cell division and leads to the formation of extremely large cells. Although stem cell markers are still expressed, these ISC-like cells are nonfunctional and can no longer divide or differentiate. As a consequence, the TSC mutant gut has a thinner epithelium and the mutant fly is more susceptible to tissue-damaging agents. This study has uncovered a tissue context-dependent phenotype of TSC mutants, such that unrestricted cell growth can lead to a stop of cell division, and thus, TSC does not function all the time as a classical tumor suppressor (Amcheslavsky, 2011).

The TSC-TOR and other growth regulatory pathways, such as InR and Myc, have intricate interactions. It has been suggested that the InR pathway directly represses TSC, whereas others and the current study suggest that the two pathways act in parallel. The TSC-TOR pathway also has a negative feedback into upstream components of the InR pathway. Recent identification of TORC2 in addition to the original TORC1 further complicates these pathways. However, the current results clearly show that TORC2 mutants and TSC mutants have different phenotypes in the adult Drosophila midgut, suggesting that TSC does not function through TORC2 to regulate ISC division. Previous studies have demonstrated that Myc can modulate TSC-TOR in controlling the growth of mammalian and fly cells, which is consistent with what was observed (Amcheslavsky, 2011).

In normal development and adult tissue homeostasis, cells need to grow in size by approximately twofold before they divide to maintain the original cell size. Reduction in cell growth below a certain threshold can lead to a halt of division. Therefore, the balance between cell growth versus division is a complex process requiring delicate coordination. This study has shown that, in TSC mutants, the increase in midgut ISC size is >10-fold, whereas the increase in larval disc cell size is less than twofold. A possible reason for this difference is that the mutant larval disc cells continue to divide, thereby maintaining a moderate cell size. One key question that remains is why the larval disc cells that contain a TSC mutation have somewhat coordinated growth and division, whereas the adult mutant ISCs have completely stopped their division. It is possible that because imaginal discs are developing organs, they are designed to have faster growth and division. Adult midgut ISCs have a slower intrinsic cell cycle of >24 h, and adult cells have differences in checkpoint controls. These may allow the excessive growth to take place until it passes a critical point that blocks division (Amcheslavsky, 2011).

Phenotypes manifested in TSC patients are mostly benign tumors that rarely progress into higher-grade cancers. TSC1 and 2 have expression in the intestine, and adult patients have occasional intestinal polyps. Mouse embryonic fibroblasts from mutant TSC animals can also enter senescence, which is equivalent to a cessation of cell division. The adult midgut ISC phenotypes shown in this study are consistent with these phenotypes. It is speculated that excessive cell growth leading to a block in cell division is a common phenotype in slowly dividing adult tissues when TSC is mutated. The phenotype of increased cell growth and increased cell division may be applicable to rapidly dividing cells, including developing Drosophila disc cells, mammalian hematopoietic stem cells, and tumor cells. A recent study demonstrates that in TSC mutants, there is loss of adult female germline stem cells because of differentiation. The ISCs and germline stem cells have different niche compositions that may contribute to the observed differences in the mutant phenotype. Moreover, it underscores the idea of a tissue context-dependent phenotype exhibited in TSC mutants (Amcheslavsky, 2011).

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date revised: 28 December 2011

 

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