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).

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).

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date revised: 10 April 2008



 

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