Cyclin E



Unlike other Drosophila cyclins, CycE exhibits a dynamic pattern of expression during development. CycE is supplied maternally, but at the completion of the cleavage divisions and prior to mitosis 14, the maternal transcripts are rapidly degraded in all cells except the pole (germ) cells. Two modes of CycE expression are observed in the subsequent divisions. During cycles 14, 15 and 16 in non-neural cells, zygotically expressed CycE mRNA levels show no cell-cycle-associated variation. CycE expression in these cells is therefore independent of the cell cycle phase, and transcripts persist at a low but relatively constant level during this period. CycE expression ceases at the time when cells stop proliferating. In contrast, expression in proliferating peripheral nervous system cells occurs during interphase as a brief pulse that initiates before and overlaps with S phase, demonstrating the presence of a G1 phase in these embryonic neural cell cycles (Richardson, 1993).

During endoreplication cycles during late embryogenesis, the spacial and temporal program of CycE expression mirrors precisely the pattern of endoreplication (Knoblich, 1994).

Entry into S phase of the mitotic cell cycle is normally strictly dependent on progression through the preceding M phase. In contrast, during endoreduplication, a cell cycle phenomenon that accompanies post-mitotic cell growth in many organisms, repeated S phases occur without intervening M phases. Upon transition from mitotic to endoreduplication cycles in Drosophila embryos, expression of the mitotic cyclins A, B and B3 is terminated and Cyclin E expression is changed from a continuous to a periodic mode. Whether or not these changes are required was investigated by continuously expressing Cyclin A, B, B3 or E in the salivary glands of Drosophila throughout late embryonic and larval development. With the exception of Cyclin A, whose expression inhibits endoreduplication effectively but only in a few, apparently randomly distributed cells of the salivary gland, mitotic cyclin expression is found to have no effect. In contrast, Cyclin E expression results in a striking inhibition of endoreduplication and growth, preceded initially by an ectopic S phase occurring just after the onset of ectopic Cyclin E expression. This observation is consistent with previous findings that Cyclin E is required, and pulses of ectopic expression are sufficient, for triggering endoreduplication S phases. These results indicate that Cyclin E activity, which triggers DNA replication, needs to be down-regulated to allow a subsequent S phase in vivo (Weiss, 1998).

The precise cell-cycle alternation of S phase and mitosis is controlled by alternating the competence of nuclei to respond to S-phase-inducing factors. Nuclei acquire competence to replicate at the low point in cyclin-dependent kinase (Cdk) activities that follow mitotic destruction of cyclins. The elevation of Cdk activity late in G1 is thought to drive cells into S phase and to block replicated DNA from re-acquiring replication competence. Whereas mitosis is normally required to eliminate the cyclins prior to another cycle of replication, experimental elimination of Cdk activity in G2 can restore competence to replicate. In this paper, the roles of Cdks in the endocycles of Drosophila have been examined. In these cycles, rounds of discrete S phases without intervening mitoses result in polyteny. Cyclins A and B are lost in cells as they enter endocycles, and pulses of Cyclin E expression drive endocycle S phases. To address whether oscillations of Cyclin E expression are required for endocycles, Cyclin E was expressed continuously in Drosophila salivary glands. Growth of the cells is severely inhibited, and a period of DNA replication is induced but further replication is inhibited. This replication inhibition can be overcome by the kinase inhibitor 6-dimethylaminopurine (6-DMAP), but not by expression of subunits of the transcription factor E2F. These results suggest that the S phase block created by continuous Cyclin E expression acts either downstream of E2F activation or on a parallel pathway that is required for S phase. Since the Cyclin A block to re-replication appears to be bypassed by the expression of E2F subunits, this result suggests that there are two mechanisms for S-phase inhibition, one upstream and one downstream of, or parallel to, E2F activation. These results indicate that endocycle S phases require oscillations in Cdk activity, but in contrast to oscillations in mitotic cells, these occur independently of mitosis (Follette, 1998).

Defects in single minded mutants are characterized by the loss of the gene expression required for the proper formation of the ventral neurons and epidermis, and by a decrease in the spacing of longitudinal and commissural axon tracks. Molecular and cellular mechanisms for these defects were analyzed to elucidate the precise role of the CNS midline cells in proper patterning of the ventral neuroectoderm during embryonic neurogenesis. These analyses have shown that the ventral neuroectoderm in the sim mutant fails to carry out its proper formation and characteristic cell division cycle. This results in the loss of the dividing neuroectodermal cells that are located ventral to the CNS midline. The CNS midline cells are also required for the cell cycle-independent expression of the neural and epidermal markers. This indicates that the CNS midline cells are essential for the establishment and maintenance of the ventral epidermal and neuronal cell lineage by cell-cell interaction. Nevertheless, the CNS midline cells do not cause extensive cell death in the ventral neuroectoderm. This study indicates that the CNS midline cells play important roles in the coordination of the proper cell cycle progression and the correct identity determination of the adjacent ventral neuroectoderm along the dorsoventral axis (Chang, 2000).

The sim mutant shows severe defects in the proper patterning of the ventral neuroectoderm, even though sim is expressed mainly in the midline cells. These defects include the absence of the ventral ectodermal and neural marker expression and the fusion of the longitudinal and commissural connectives of the ventral nerve cord. The lack of the NB and ventral ectodermal marker expression in the sim mutant may originate from (1) defective formation and division; (2) incorrect identity determination, or (3) massive cell death of the ventral neuroectodermal cells during early neurogenesis. Thus, in order to elucidate the molecular and cellular basis of how the CNS midline cells, specified by the sim gene, are required for the proper patterning of the ventral neuroectoderm, the contribution of the above three possibilities to proper patterning of the ventral neuroectoderm was investigated (Chang, 2000).

To investigate whether the absence of NB marker expression is in part due to improper NB formation, panneural NB markers dpn and scrt were used to examine NB formation. scrt and dpn expressing NBs start to form in three columns at stage 9 and give rise to a total of 10-11 S1 NBs at early stage 10. In stage 10 sim embryos, S1 NBs in the three columns of the ventral neuroectoderm are absent, at least in random positions, in 35% of the hemisegments examined. This analysis demonstrates that the CNS midline cells are essential for the proper formation of the ventral NBs in the three columns of ventral neuroectoderm. This result indicates that the absence of the NB marker expression is in part due to the defects in the formation of a correct number of the NBs in the ventral neuroectoderm (Chang, 2000).

To investigate whether the defects of the cell division cycle are responsible for the absence of dpn- and scrt-positive NBs in sim embryos, the mitotic cell division pattern of the ventral neuroectoderm was analyzed by staining with the mitosis markers anti-Cyclin B3 and anti-phosphohistone H3 antibodies. The cyclin E gene was used to examine transition from the G1 to the S phase. In wild-type embryos at stage 8, Cyclin B3 is detected in the eight longitudinal columns of the ventral neuroectodermal cells per hemisegment. In contrast, the mesectodermal cells have no Cyclin B3 since they have already divided. Later at stage 11, a group of Cyclin B3-expressing cells is located medial to the midline. In sim embryos, Cyclin B3 expression is reduced to a width of three to four cells in some segments of the ventral neuroectoderm at stage 8. Later in sim embryos at stage 11, a cluster of four to six Cyclin B3-expressing cells in the medial neuroectoderm show severely reduced Cyclin B3 expression. This analysis suggests that the cycle 14 mitotic cell division is defective, especially during the NB formation in sim embryos. This defect may cause the loss of the dpn and scrt positive NBs, which results in the absence of the ventral neuroectodermal marker gene expression (Chang, 2000).

The expression of cyclin E is detected in the mesoderm, the mesectoderm, and the striped neuroectoderm at stage 8. Later it remains only in the striped ventral neuroectoderm at stage 9. The striped expression of cyclin E in the ventral neuroectoderm, and in the mesoderm, is greatly reduced in the sim mutant. Meanwhile, the expression of cyclin E in the three columns of NBs in each hemisegment of wild-type embryos at stage 10 is fused and disorganized in sim mutant embryos, which may be in part due to the defects in the midline cell development. This result suggests that the ventral neuroectoderm has the defects in promoting continuous NB division, which results in premature NB differentiation and the reduction of the NB number in the sim mutant (Chang, 2000).

The activity of Fizzy-related (Cdh1Fzr) is negatively controlled by Cdk-mediated phosphorylation. Both Cdk1 and Cdk2 kinase activities have been implicated in Cdh1 phosphorylation. During the first 15 divisions, CycE/Cdk2 kinase activity is present throughout the cell cycle. It declines during cell cycle 16, caused by the downregulation of CycE transcription and the upregulation of the Cdk2-specific inhibitor dacapo (dap). Thus, Cdh1Fzr is not inhibited by CycE/Cdk2 activity during later stages of the 16th cell cycle. However, overexpressed CycE is able to suppress the effects of ectopic Cdh1Fzr during cell cycle 16. To analyze whether CycE is also able to compensate for the lack of Regulator of cyclin A1 (Rca1) function, CycE was overexpressed in Rca1 mutant embryos using the prd-Gal drive line. In segments overexpressing CycE, higher cell densities are observed. This demonstrates that CycE is able to suppress the Rca1 mutant phenotype, presumably by its negative influence on Cdh1Fzr activity (Grosskortenhaus, 2002).

These data would suggest that CycE and Rca1 have overlapping functions in Cdh1Fzr inhibition. Thus, the requirement for Rca1 only becomes visible when CycE levels decline during the 16th cell cycle. However, CycE and Rca1 are only partially redundant. This can be concluded from Rca1;CycA double-mutant embryos. In CycA mutants, very low levels of CycA are already present during the 15th cell cycle but these levels are still sufficient to execute mitosis 15, and cells arrest before mitosis 16. Thus, cell numbers are similar to Rca1 mutant embryos and reduced compared with those of wt. Interestingly, Rca1;CycA double mutants have even fewer cells. Apparently, these double-mutant embryos fail to execute mitosis 15, likely caused by a further reduction in CycA levels due to the absence of Rca1. This shows that Rca1 is active at earlier cell cycles and becomes essential when CycA levels are reduced. Under these circumstances, CycE that is present during cycle 15 is apparently not sufficient to prevent excessive CycA degradation (Grosskortenhaus, 2002).

Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms

In newly hatched Drosophila larvae, quiescent cells reenter the cell cycle in response to dietary amino acids. To understand this process, larval nutrition was varied and effects on cell cycle initiation and maintenance were monitored in the mitotic neuroblasts and imaginal disc cells, as well as the endoreplicating cells in other larval tissues. After cell cycle activation, mitotic and endoreplicating cells respond differently to the withdrawal of nutrition: mitotic cells continue to proliferate in a nutrition-independent manner, while most endoreplicating cells reenter a quiescent state. Ectopic expression of Drosophila Cyclin E or the E2F transcription factor can drive quiescent endoreplicating cells, but not quiescent imaginal neuroblasts, into S-phase. Conversely, quiescent imaginal neuroblasts, but not quiescent endoreplicating cells, can be induced to enter the cell cycle when co-cultured with larval fat body in vitro. These results demonstrate a fundamental difference in the control of cell cycle activation and maintenance in these two cell types, and imply the existence of a novel mitogen generated by the larval fat body in response to nutrition (Britton, 1998).

These results suggest that multiple pathways are involved in regulating the onset of cell proliferation in different tissue types in response to the global nutritional cue. Mitotic and endoreplicating cell cycles are regulated differently in response to the nutritional state: the endoreplicating tissues (ERTs) require continuous nutrition to cycle, whereas the mitotic cells cycle in a nutrition-independent manner once activated. In addition, the mechanism of cell cycle arrest in the two types of quiescent cells is different: quiescent ERTs can be driven into S-phase by ectopic expression of either of the G1/S regulators E2F or Cyclin E, while neither of these regulators can induce quiescent neuroblasts to enter S-phase.Conversely, quiescent neuroblasts but not quiescent ERTs are induced to reenter the cell cycle in response to a mitogen produced by the larval fat body (Britton, 1998).

The differential responses of the mitotic and endoreplicative cell cycles to nutrient withdrawal may provide an important mechanism for survival of the organism and reproduction in the face of food shortages in the wild. When nutrients become limiting, available resources can be dedicated to maintaining growth and proliferation in the mitotic tissues which are required to form the reproductive adult. Indeed, larvae are capable of pupating at a much smaller size than they normally do. A 'critical size' has been defined at which larvae are able to pupariate without further feeding. The small pupae which are formed by these larvae produce normal, fertile, but small adult flies (Britton, 1998).

Embryonic neuroblasts have an intrinsic program of cell proliferation. Each type of neuroblast has a specific identity, expresses unique and dynamic combinations of sublineage genes, and will give rise to a precise number and type of progeny before exiting the cell cycle. Interestingly, temporal control of sublineage gene expression in embryonic neuroblasts can be independent of cell cycle progression. Thus arresting a proliferating neuroblast in mid-lineage could lead to the desynchronization of sublineage gene expression and the loss of certain types of progeny, a result which could have disastrous consequences for the developing CNS (Britton, 1998).

In a food withdrawal experiment it was observed that many activated neuroblasts continued to proliferate for up to 7 days after food withdrawal, however a subset of them did not. This observation was most striking in the abdominal region of the VNC. The abdominal neuroblast lineages are much shorter than those of the majority of brain and thoracic neuroblasts, with a single abdominal neuroblast producing as few as four neurons during its postembryonic period of proliferation. Since the abdominal neuroblasts generally complete their entire larval program of proliferation in less than 2 days, it is not surprising that after 7 days of culture on sucrose the majority of these neuroblasts have exited the cell cycle. It is suspected that the reduction in labeled neuroblasts observed in all regions of the CNS over the course of this experiment is due to a subset of neuroblasts completing their intrinsic program of proliferation and exiting the cell cycle (Britton, 1998).

The insect fat body is the source of the majority of hemolymph proteins, including lipid binding proteins, juvenile hormone binding proteins and esterases, peptides which mediate the insect immune response, and vitellogenins involved in oocyte maturation in the adult female. The fat body is also responsible for synthesizing the stores of protein, lipid and glycogen which sustain the animal throughout metamorphosis. Ultrastructurally, the fat body shows a dramatic response to starvation. In Calpodes larvae, starvation leads to a rapid reorganization of the fat body including loss of mitochondria and rough endoplasmic reticulum (RER) by autophagy and depletion of stored metabolites. Refeeding induces mitochondrial divisions andincreases in RER content as well as the eventual replenishment of depleted stores. This study observed dramatic changes in the larval fat body in the course of starvation experiments, including a loss of tissue cohesion and changes in opacity. These changes probably reflect the alteration in composition the fat body cells undergo as stores of metabolites are mobilized to support proliferating mitotic tissues during starvation (Britton, 1998).

Previous studies have demonstrated that the adult female fat body is able to regulate yolk gene transcription in response to the nutritional environment. Interestingly, there is evidence that a component of the adult female abdomen is also capable of supporting the proliferation of larval tissues in a nutrition dependent manner. It has been demonstrated that the proliferation of imaginal disc fragments transplanted into the abdominal cavity of adult female hosts is dependent onnutrition. This study has found that when quiescent central nervous systems from starved larvae are transplanted into the abdomens of fed adult female hosts, larval neuroblasts reenter the cell cycle in what appears to be a normal spatiotemporal pattern. An appealing hypothesis is that production of the neuroblast mitogen in the fat body is regulated at the transcriptional level under the control of nutritional enhancers similar to those identified in the regions upstream of yolk protein genes. The ability of something in the adult female abdomen to activate proliferation in quiescent neuroblasts suggests that similar fat body-derived mitogens are produced in the larval and adult female fat bodies. This adult mitogen could have a role in controlling proliferation in the adult, perhaps functioning to regulate some oogenic process in response to the nutritional state. Indeed, oogenesis is inhibited in adult females fed on sucrose (Britton, 1998).

The dramatic response of the fat body to starvation, the demonstration that there is a mechanism for nutritional controlof transcription in adult female fat body, and the similar abilities of the adult female abdomen and the larval fat body to support nutrition-dependent cell cycle activation lend support to the proposal that the fat body is responsible for mediating the nutritional response in larval neuroblasts. The results of co-culture experiment demonstrate that the fat body supplies a diffusible factor which stimulates larval neuroblasts to enter the cell cycle (Britton, 1998).

A critical role for cyclin E in cell fate determination in the central nervous system of Drosophila

This study examined the process by which cell diversity is generated in neuroblast (NB) lineages in the central nervous system of Drosophila. Thoracic NB6-4 (NB6-4t) generates both neurons and glial cells, whereas NB6-4a generates only glial cells in abdominal segments. This is attributed to an asymmetric first division of NB6-4t, localizing prospero (pros) and glial cell missing (gcm) only to the glial precursor cell, and a symmetric division of NB6-4a, where both daughter cells express pros and gcm. This study shows that the NB6-4t lineage represents the ground state, which does not require the input of any homeotic gene, whereas the NB6-4a lineage is specified by the homeotic genes abd-A and Abd-B. They specify the NB6-4a lineage by down-regulating levels of the G1 cyclin, DmCycE (CycE). CycE, which is asymmetrically expressed after the first division of NB6-4t, functions upstream of pros and gcm to specify the neuronal sublineage. Loss of CycE function causes homeotic transformation of NB6-4t to NB6-4a, whereas ectopic CycE induces reverse transformations. However, other components of the cell cycle seem to have a minor role in this process, suggesting a critical role for CycE in regulating cell fate in segment-specific neural lineages (Berger, 2005).

In Drosophila, individual neuroblasts deriving from corresponding neuroectodermal positions among thoracic and abdominal segments generally acquire similar fates. However, some of these serially homologous neuroblasts produce lineages with segment-specific differences that contribute to structural and functional diversity within the CNS. The NB6-4 lineage was selected as a model to determine how this diversity evolves from a basic developmental ground state. As an experimental system, NB6-4 has an additional advantage, since Eagle (Eg) is expressed in all the cells of both thoracic and abdominal lineages and can thus be used as a lineage marker (Berger, 2005).

First the expression patterns of different homeotic genes were examined in thoracic and abdominal lineages of NB6-4. Antennapedia (Antp) is expressed in NB6-4t lineages of thoracic segments T1-T3. Abdominal A (Abd-A) is expressed in the NB6-4a lineage of abdominal segments A1-A6, whereas Abdominal B (Abd-B) is expressed in the NB6-4a lineage of segments A7-A8. Whereas loss of Antp function does not affect the NB6-4t lineage in any of the thoracic segments, loss-of-function mutations in abd-A and Abd-B cause NB6-4a-to-NB6-4t homeotic transformations in their corresponding segments. Interestingly, Ultrabithorax (Ubx), which is expressed in most of the cells of T3, is specifically absent in the NB6-4t lineage of that segment, and its loss-of-function alleles do not show any thoracic phenotypes. However, overexpression of Ubx as well as abd-A causes NB6-4t-to-NB6-4a transformations. Thus, it seems that the NB6-4t fate is the ground state and the NB6-4a state is imposed by the function of homeotic genes of the bithorax-complex (BX-C). This is consistent with previous reports that the T2 state is the ground state (for epidermis, including adult appendages) and other segmental identities are conferred by the function of homeotic genes (Berger, 2005).

The mechanism was examined by which abd-A or Abd-B specify the NB6-4a lineage compared with the NB6-4t lineage. As the mode and number of mitoses is the most obvious characteristic by which the NB6-4a lineage differs from NB6-4t, it was wondered whether factors regulating the cell cycle might be involved in controlling NB6-4 cell fate. One major factor that regulates the cell cycle is the G1 Cyclin CycE, which is needed for various aspects of the G1-to-S-phase transition (Berger, 2005).

To examine possible effects on cell fate decisions in the NB6-4t lineage, CycEAR95-mutant embryos were stained for gcm transcripts and Pros and Repo proteins. In wild-type embryos, gcm is initially distributed to both daughter cells during the first division of NB6-4t, but subsequently gets rapidly removed in the cell that functions as a neuronal precursor. Pros is transferred asymmetrically into only one cell, where it is needed to maintain and enhance the expression of gcm, thereby promoting glial cell fate. In CycEAR95 embryos, even at late stages (up to stage 14), gcm mRNA is strongly expressed in both daughter cells after the first division of NB6-4t. Even distribution of Pros was observed in both daughter cells, which could be the cause of continued expression of gcm. Furthermore, the glial marker Repo revealed that both cells differentiate as glial cells. The NB6-4a lineage is not affected in CycEAR95-mutant embryos, suggesting that the requirement for zygotic CycE is specific to NB6-4t (Berger, 2005).

Whether ectopic expression of CycE in abdominal lineages causes the opposite effect was tested. The sca-GAL4 line was used to drive UAS-CycE to achieve early expression in the neuroectoderm. An asymmetric distribution of Pros to one of the two progeny cells was observed just after the first division of NB6-4a. At later stages an increase was observed in the number of cells in the NB6-4a lineage (up to 5 cells). Some of these cells migrated medially, as NB6-4 glial cells normally do, maintaining Pros expression at a lower level. They also expressed Repo, which confirmed their glial identity. Other cells stayed in a dorso-lateral position and did not stain for Repo, suggesting neuronal identity (Berger, 2005).

To further investigate if ectopic CycE had indeed induced a neuronal sublineage in NB6-4a, and to test whether CycE can function cell-autonomously, a cell transplantation technique was employed. Single progenitor cells (stage 7) from the abdominal neuroectoderm of horseradish peroxidase (HRP)-labelled donor embryos overexpressing CycE were transplanted into the abdominal neuroectoderm of unlabelled wild-type hosts (at the same stage). The lineages produced by the transplanted cells were identified by morphological criteria. In all six cases, where cell clones were derived from NB6-4a, they were composed of both glial cells and neurons exhibiting their respective characteristic structures and positions. Because the clones are located in a wild-type abdominal environment, this experiment provides evidence that ectopic expression of CycE causes asymmetric division of NB6-4a and confers neuronal identity to one part of the lineage in a cell-autonomous manner. In these single-cell transplantation experiments, similar observations were made for NB1-1 and NB5-4, which also generate segment-specific lineages. Thus, CycE seems to have a general role in establishing segment-specific differences in neuroblast lineages (Berger, 2005).

Next whether the requirement for CycE to specify the neuronal lineage in NB6-4t is due to altered cell-cycle phases was examined. In string mutants, NB6-4t (whose proliferation is blocked before its first division) expresses gcm mRNA, as well as Pros and hunchback protein, although it does not differentiate as a glial cell. The composition of NB6-4t and NB6-4a lineages were further analysed in embryos mutant for other factors that interact with CycE in cell-cycle regulation. dacapo (dap) is the Drosophila homologue of members of the p21/p27Cip/Kip inhibitor family, which specifically block CycE-cdk complexes. Interestingly, in dap-null-mutant embryos an additional glial cell was observed in the NB6-4a lineage, but the appearance of any neuron-like cells was not observed. Consistent with these results, overexpression of Dap resulted in a reduction in the number of neurons in the NB6-4t lineage (from the normal number of 6 to 2-4), but homeotic transformation of the lineage did not occur. The number of glial cells was never affected, neither in the thorax nor in the abdomen. Ectopic expression of p21, the human homologue of the Drosophila dacapo gene, generated similar phenotypes (Berger, 2005).

The influence of the transcription factor dE2F, which mediates the activation of several genes needed for the initiation of S phase, was tested. In dE2F-mutant embryos, unlike in CycE mutants, no homeotic transformation of NB6-4t to NB6-4a was observed, although the number of neurons was reduced from 5-6 to 2-4. Ectopic expression of dE2F resulted in an increase in cell number in some abdominal hemisegments. In only a small percentage of those embryos, cells at lateral positions in abdominal segments did not show expression of gcm or Repo, suggesting their neuronal identity. Thus, although dE2F activation in the CNS depends on CycE, ectopic expression of dE2F cannot fully bypass the requirement for CycE in a NB6-4a-to-NB6-4t transformation. Similarly, ectopic expression of Rbf, a potent inhibitor of E2F target genes, did not cause any changes in the segregation of gcm mRNA, Pros or Repo in the NB6-4t lineage (Berger, 2005).

Whether interfering with another checkpoint of the cell cycle, the transition from G2 to M phase, affects NB6-4 cell fate was tested. Previous studies show that loss of CycA function prevents further mitosis after the first division of NB6-4t. However, the first division of NB6-4t follows the normal pattern; it gives rise to one glial and one neuronal cell. Similar effects were observed in CycA mutants. The cyclin-dependent kinase cdc2 heterodimerizes with CycA and CycB, and high levels of cdc2 expression have been shown to be required for maintaining the asymmetry of neuroblast divisions. In a cdc2 loss-of-function background, NB6-4t generated a normal lineage consisting of two glial cells and five to six dorso-lateral neurons. These observations show that NB6-4 cell fates do not change after manipulation of the transition from G2 to M phase (Berger, 2005).

These results suggest a critical role for CycE per se in regulating the NB6-4t lineage. Therefore whether CycE itself is differentially expressed between thoracic and abdominal NB6-4 lineages was tested. In situ hybridization with CycE RNA on wild-type embryos revealed that CycE is expressed just before the first division in NB6-4t. After the first division, CycE mRNA was detected in the neuronal precursor only and not in the glial precursor. In abdominal segments, no CycE expression was detected in NB6-4a before or after the division. Consistent with the role of CycE in specifying the NB6-4t lineage, notable levels of CycE transcripts were detected in the homeotically transformed NB6-4a lineages in abd-A-mutant embryos. Conversely, overexpression of abd-A caused down-regulation of CycE levels in thoracic segments and homeotic transformation of NB6-4t to NB6-4a. The importance of CycE in generating neuronal cells in the NB6-4t lineage was confirmed in an epistasis experiment involving abd-A and CycE mutants. As described above, loss of abd-A leads to transformation of NB6-4a to NB6-4t. Such homeotic transformation was suppressed by mutations in CycE, suggesting an absolute requirement for CycE in specifying the NB6-4t lineage. Finally, nine potential AbdA-binding sites (five of which are evolutionarily conserved in Drosophila pseudoobscura) were identified in a 5.0-kb enhancer fragment of CycE that is known to harbour cis-acting sequences for driving CycE expression in the CNS (Berger, 2005).

It is concluded that, in addition to its role in cell proliferation, CycE is necessary and sufficient for the specification of cell fate in the NB6-4 lineage. These results suggest that the function of CycE in regulating cell fate in NB6-4 lineages is independent, albeit partially, of its role in cell proliferation. The absence of any cell fate changes in the loss-of-function mutants of string, dap, cdc2 or CycA and in the Dap or Rbf gain-of-function genetic background may be attributed to the presence of CycE, which is strongly expressed in the NB6-4t, but not the NB6-4a, lineage. However, CycE may still function by controlling cell-cycle progression. NB6-4a, which does not express CycE, divides once followed by a cell-cycle arrest, presumably in G1. After the first division in the thorax, one daughter cell expresses high levels of CycE and divides roughly three times to generate neuronal cells. Therefore, this daughter cell presumably progresses through S phase. Chromatin reorganization during S phase might allow cell fate regulators to access their target genes, driving neuronal differentiation. Contrary to this interpretation, the other daughter cell of NB6-4t, which does not express CycE, divides twice but still generates glial cells. Thus, it remains to be investigated whether the role of CycE in neuronal cell fate determination is entirely independent of its role in cell proliferation. The results on the role of CycE in specifying neuronal compared with glial cell fate in the CNS are consistent with data from Xenopus on the role of cyclin-cdk complexes in specifying neuronal cell fate, inhibition of which promotes glial cell fate. In addition, this study shows that homeotic genes contribute to regional diversification of cell types in the CNS through the regulation of CycE levels (Berger, 2005).

APC/CFzr/Cdh1 promotes cell cycle progression during the Drosophila endocycle

The endocycle is a commonly observed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. How the cell cycle machinery is modified to transform a mitotic cycle into endocycle has long been a matter of interest. In both plants and animals, the transition from the mitotic cycle to the endocycle requires Fzr/Cdh1, a positive regulator of the Anaphase-Promoting Complex/Cyclosome (APC/C). However, because many of its targets are transcriptionally downregulated upon entry into the endocycle, it remains unclear whether the APC/C functions beyond the mitotic/endocycle boundary. This study reports that APC/CFzr/Cdh1 activity is required to promote the G/S oscillation of the Drosophila endocycle. Compromising APC/C activity, after cells have entered the endocycle, inhibits DNA replication and results in the accumulation of multiple APC/C targets, including the mitotic cyclins and Geminin. Notably, the data suggest that the activity of APC/CFzr/Cdh1 during the endocycle is not continuous but is cyclic, as demonstrated by the APC/C-dependent oscillation of the pre-replication complex component Orc1. Taken together, these data suggest a model in which the cyclic activity of APC/CFzr/Cdh1 during the Drosophila endocycle is driven by the periodic inhibition of Fzr/Cdh1 by Cyclin E/Cdk2. It is proposed that, as is observed in mitotic cycles, during endocycles, APC/CFzr/Cdh1 functions to reduce the levels of the mitotic cyclins and Geminin in order to facilitate the relicensing of DNA replication origins and cell cycle progression (Narbonne-Reveau, 2008).

The endocycle provides a useful model for determining the minimum cell cycle inputs required to achieve a G/S oscillation and the once-per-cell-cycle replication of the genome. This study demonstrates that APC/C activity is required for endocycle progression. During the endocycle, mitotic activities are repressed. This is accomplished, at least in part, by preventing the accumulation of the mitotic activators Cyclin A, Cyclin B and Cdc25, which function to activate the mitotic kinase Cdk1. During the mitotic cycle, the mitotic cyclins are periodically targeted for regulated proteolysis by the E3-Ubiquintin ligase the APC/C. Yet the transcriptional downregulation of several APC/C targets at the mitotic/endocycle boundary, including the mitotic cyclins and String/Cdc25, suggested that the proteolytic activity of the APC/C might not be necessary during endocycles. However, this study found that compromising APC/C activity, after cells have entered the endocycle, results in the accumulation of Geminin and the mitotic cyclins, and in a block of DNA replication. Thus, the transcriptional downregulation of APC/C targets observed at the mitotic/endocycle transition is either downstream of APC/C activity and/or not sufficient to maintain low levels of these proteins. Taken together, these data suggest a model in which APC/C promotes the G/S oscillation of the endocycle by preventing the unscheduled accumulation of Geminin and the mitotic cyclins (Narbonne-Reveau, 2008).

During endocycles, APC/C activity prevents the inappropriate accumulation of Geminin, an inhibitor of the DNA replication-licensing factor Cdt1/Dup. When directly expressed in endocycling cells, Geminin efficiently inhibits DNA replication. These results strongly suggest that an essential function of the APC/C during the endocycle is to prevent the unregulated accumulation of Geminin. A similar role has been proposed for the APC/C during endoreplicative cycles of mouse trophoblasts (Gonzalez, 2006). However, the current data indicate that Geminin is not the only essential target of the APC/C during endocycles. A candidate for a second important target of the APC/C during endocycles is Cyclin A. Previous studies have shown that the overexpression of Cyclin A in the salivary gland, between the first and second endocycle, results in variable inhibitory effects on endoreplication. Although the majority of salivary gland cells that overexpress Cyclin A appear to be unaffected, a small percentage show a marked decrease in ploidy values. The reason for this variability is not clear. However, if the inhibitory influence of Cyclin A is mediated through binding and activation of Cdk1, this effect may be greatly amplified in the presence of high levels of String/Cdc25, which removes an inhibitory phosphate from Cdk1. Recent studies indicate that String/Cdc25, which contains both a consensus Ken box and D-box, is a target of the APC/C (Barbara Thomas, personal communication to Narbonne-Reveau, 2008). Therefore, an essential function of the APC/C during endocycles may involve restricting the activity of the mitotic kinase Cdk1, by preventing the accumulation of both Cyclin A and String/Cdc25. Finally, it is noted that the APC/C may have additional essential targets during the endocycle, which have yet to be identified (Narbonne-Reveau, 2008).

The periodic accumulation of the Orc1 protein during endocycles strongly suggests that the activity of the APC/CFzy/Cdh1 may not be continuous but cyclical. Previous work indicates that in Drosophila Cyclin E/Cdk2 inhibits the activity of APC/CFzy/Cdh1. These data are consistent with the observation that phosphorylation of Fzr/Cdh1 by Cdks inhibits the ability of Fzr/Cdh1 to bind and activate the APC/C in yeast, Xenopus and mammals. During the endocycle, the levels of Cyclin E oscillate. Taken together, these observations suggest a model in which APC/CFzy/Cdh1 is regulated by the periodicity of Cyclin E/Cdk2 activity, with high levels of Cyclin E resulting in the inhibition of APC/CFzy/Cdh1 activity and low levels of Cyclin E permitting full APC/CFzy/Cdh1 activity. The current data support this hypothesis. First, it was found that the periodicity of Orc1 levels during the endocycle requires a functional O-box, consistent with the cyclic destruction of Orc1 by APC/CFzy/Cdh1. Second, the levels of Orc1 are sensitive to Cyclin E. Specifically, overexpressing Cyclin E after cells have entered the endocycle results in the accumulation of APC/CFzy/Cdh1 targets, including Orc1, Cyclin A, Cyclin B and Geminin. Thus, the regulatory relationship observed between Cyclin E/Cdk2 and Fzr/Cdh1 that has been reported during mitotic cycles is conserved during endocycles. Finally, in endocycling cells the accumulation of Orc1 occurs during periods of high Cyclin E/Cdk2 activity, when APC/CFzy/Cdh1 dependent proteolysis would be predicted to be low. These data support the idea that the oscillations of Cyclin E/Cdk2 activity drive the periodicity of APC/CFzy/Cdh1 activity during the endocycle (Narbonne-Reveau, 2008).

Although a requirement for the oscillation of APC/CFzy/Cdh1 activity during the Drosophila endocycle has not been formally demonstrated, it is interesting to speculate on how the cyclic, rather than the continuous, activity of the APC/C might serve to facilitate endocycle progression. The data indicate that a period of high APC/CFzy/Cdh1 activity is required during the G phase of the endocycle in order to degrade the mitotic cyclins and Geminin, which can function to inhibit the formation of pre-RCs. However, a period of low APC/C activity may also be important. The continuous activation of APC/CCdh1 significantly slows DNA replication in mouse tissue culture cells. This inhibition may reflect the inability of a cell to accumulate adequate levels of proteins required for DNA replication, such as the APC/CCdh1 target and pre-replication complex component CDC6, in the presence of a constitutively active APC/CCdh1. In Drosophila, continuous APC/CFzy/Cdh1 activity might prevent the accumulation of two pre-RC components, CDC6 and Orc1. Intriguingly, APC/C activity also appears to oscillate during mammalian endocycles. In endocycling mouse trophoblasts, the levels of Cyclin A oscillate, consistent with the regulated destruction of the Cyclin A protein by the APC/C. Additionally, the inhibition of APC/C activity in endocycling trophoblasts results in the accumulation of the APC/C targets Cyclin A and Geminin. Taken together, these observations support a model in which the oscillation of APC/CFzy/Cdh1 activity, which is driven by the regulatory influences of Cdks, promotes efficient cell cycle progression during the endocycle (Narbonne-Reveau, 2008).

The data raises important questions. Why do levels of some APC/CFzy/Cdh1 targets, such as Cyclin A, Cyclin B and Geminin, remain below the level of detection while the levels of Orc1 protein oscillate? What might account for these different modes of regulation? Currently, there is no definitive explanation. However, at least three possibilities, which are not mutually exclusive, are envisaged, that may contribute to this differential behavior. First, it was found that relative to the Cyclin A and geminin, the levels of Orc1 transcript are only minimally downregulated upon entry into the endocycle. Transcriptional downregulation, or changes in transcript stability, may help contribute to the low levels of Geminin and Cyclin A proteins observed during the endocycle. Second, the translational efficiency of a subset of transcripts may be reduced upon entry into the endocycle. Finally, it is possible that the Orc1 protein is not as efficiently targeted by the APC/CFzy/Cdh1 as the mitotic cyclins or Geminin. Indeed the cis-acting sequences that target these proteins for destruction show considerable variability. Orc1 is targeted for APC/CFzy/Cdh1 destruction via a novel motif called the O-box (Araki, 2005). By contrast, Cyclin B and Geminin are targeted by a similar but unique sequence called the destruction-box (D-box), while Drosophila Cyclin A is targeted for destruction by a large complex N-terminal degradation sequence. There is precedence for post-translational regulation of APC/CFzy/Cdh1 targets, resulting in differential expression. In mammalian cells the pre-RC component CDC6, which is structurally related to Orc1, is protected from APC/CFzr/Cdh1 degradation by phosphorylation by Cyclin E/Cdk2. One or all of these potential mechanisms may contribute to the differential expression of various APC/CFzr/Cdh1 targets during the endocycle (Narbonne-Reveau, 2008).

Recent evidence from mice indicates that the depletion of the APC/C inhibitor Emi1/Rca1, results in both a strong decrease in E2F target mRNAs, such as geminin and Cyclin A, as well as APC/C activation. This study suggested that the regulation of APC/C activity, by the inhibitor Emi1/Rca1, drives a positive feedback circuit that controls both protein stability and mRNA expression. Thus, the observed decrease in the levels of at least some APC/C targets that occurs upon depletion of Emi1/Rca1, including Geminin and Cyclin A, are controlled at the levels of transcription and protein stability. Developmentally programmed endocycles may provide a natural example where cell cycle progression occurs in the context of increased APC/CFzr/Cdh1 activity. Thus, a similar positive-feedback circuit may be operating during Drosophila endocycles to downregulate the transcription of E2F target genes. Determining the precise regulatory relationships between the upregulation of APC/CFzr/Cdh1 activity and the transcriptional downregulation of genes such as Cyclin A and geminin, during the Drosophila endocycle represents an exciting area for future research (Narbonne-Reveau, 2008).

The requirement for APC/C activity to promote endocycle progression may help answer several longstanding questions concerning the regulation of the Drosophila endocycle. For example, why does the continuous expression of Cyclin E inhibit cell cycle progression during the endocycle but not the mitotic cycle? Several models have been proposed to explain this difference. First, the breakdown of the nuclear envelope that occurs during the mitotic cycle, but not the endocycle, may allow for a transient decrease in local Cyclin E/Cdk2 activity, thus allowing for the relicensing of DNA replication origins. Alternatively, there may be differences in the machinery required to produce a functional pre-RC in mitotic versus endocycling cells. The current results suggest an alternative model for why endocycles are unusually sensitive to continuous Cyclin E expression. This model is based on the demonstration that endocycle progression requires APC/C activity. Both Fzy/Cdc20 and Fzr/Cdh1 function as activators of the APC/C. However, the regulation of these APC/C activators is very distinct. During the mitotic cycle, the binding of Fzy/Cdc20 to the APC/C is dependent on the phosphorylation of several APC/C subunits by the mitotic kinase Cdk1. By contrast, a Cdk-dependent inhibitory phosphorylation on Fzr/Cdh1 relegates APC/CFzr/Cdh1 activity to late M phase and G1. Because of its requirement for Cdk1 activity, APC/CFzy/Cdc20 is unlikely to be active during most endocycles. Indeed, Drosophila endocycles proceed normally in fzy mutants. Thus, the only available activator of the APC/C during the endocycle is Fzr/Cdh1. As previously discussed, Fzr/Cdh1 is inhibited by Cyclin E/Cdk2 activity. Therefore, it is proposed that during the endocycle, continuous Cyclin E/Cdk2 activity results in the permanent inhibition of the only available activator of the APC/C, Fzr/Cdh1. This leads to the accumulation of Geminin, Cyclin A and other potential targets, which act to block cell cycle progression. Thus, the ability of continuous Cyclin E to inhibit DNA replication during the endocycle may reflect differences in the available activators of the APC/C present in mitotic versus endocycling cells (Narbonne-Reveau, 2008).


Ectopic expression of CycE in the eye epithelium induces premature entry of most of these G1 cells into S phase. Thus in these cells, control of DmcycE expression is required for regulated entry into S phase. Significantly, a band of eye imaginal disc cells in G1 phase was not induced to enter S phase by ectopic expression of DmcycE. This provides evidence for additional regulatory mechanisms that operate during G1 phase to limit cell proliferation during development. These results demonstrate that the role of Cyclin E in regulating progression into S phase in mammalian tissue culture cells also applies to some cells (but not all) during Drosophila development (Richardson, 1995).

Three discrete populations of neuroblasts in the larval brain show stereotypic temporal and spatial patterns of cell cycle arrest and activation. All three populations are quiescent upon larval hatching and begin cell division as specific times post hatching (ph): the central brain neuroblasts (CBNBs) begin division at about 8-10 h ph; the optic lobe neuroblasts (OLNBs) at about 10-12 h ph, and the thoracic neuroblasts (TNBs) at about 28 h ph. In contrast, the mushroom body neuroblasts (MBNBs) and the ventral lateral NBs divide continously from larval hatching through pupariation, and provide an internal control to differentiate between developmental regulation of proliferation and the process of cell division. The trol locus of Drosophila regulates the timing of neuroblast proliferation. In trol mutants, quiescent neuroblasts fail to begin division. The trol gene product is required specifically for initiation of OLBN and CBNB proliferation. Activation of OLNB and CBNB cell division by mutation of the proliferation repressor ana bypasses the requirement for trol, consistent with the hypothesis that trol is not required for the maintenance of the proliferative state. This trol mutation induced cell cycle arrest was investigated in order to examine trol function. Induced expression of cyclin E or DP/E2F in trol mutants results in normal levels of dividing neuroblasts, while cyclin B expression has no effect. cyclin E expression is lower in the trol mutant larval CNS as assayed by quantitative RT-PCR, suggesting that trol neuroblasts are arrested in G1 due to lack of Cyclin E. Neither cyclin E nor E2F expression can phenocopy ana mutations, indicating that arrest caused by lack of Trol is different from Ana-mediated arrest. Neither the induction of cyclin E nor DP/E2F is capable of bypassing Ana mediated repression. It is concluded that trol neuroblasts are arrested in mid-G1 stage (Caldwell, 1998).

In the eye disc, cyclin D is expressed in the G1-arrested cells in the morphogenetic furrow; expression precedes that of Cyclin E and entry into S phase. The timing of cyclin D and Cyclin E expression parallels that seen in mammalian cells and is consistent with a model in which entry into the cell cycle involves activation of cyclin dependent kinase-cyclin D complexes, followed by activation of cyclin dependent kinase-Cyclin E complexes (Finley, 1996).

Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. The behavior of MCM proteins was examined in endoreplicating larval salivary glands, to determine whether oscillation of MCM-chromosome localization occurs in conjunction with passage through an endocycle S phase. MCMs in polytene nuclei exist in two states: either associated with or dissociated from chromosomes. DmMCM2, DmMCM4, and DmMCM5 are detected as nuclear proteins in polytene nuclei of salivary glands during the three larval instars. In a majority of polytene nuclei from second- and third- instar larvae (>80%), most of the nuclear MCM stain is excluded from the region occupied by DNA and the nucleolus. It is inferred that most of the nuclear MCMs are not asociated with chromosomes in these nuclei and this pattern is referred to as nucleoplasmic. In contrast, in a small fraction of nuclei (~10%), nuclear MCM staining is coincident with the DNA. This state is interpreted as indicating a chromosomal association of MCMs. Cyclin E is expressed in transient pulses, each of which overlaps the beginning of each endocycle S phase. cyclin E mutants fail to undergo endoreplication. Heat induction of cyclin E produces a synchronous burst of DNA synthesis in polytene cells lasting between 3 and 6 hours. 40% of nuclei display chromosomal DmMCM2 after heat shock, and this association rapidly diminishes. Thus heat induction of Cyclin E drives chromosome association of DmMCM2. Subsequently, DNA synthesis erases this association. To test whether DNA synthesis is required for the dissociation of DmMCM2, DNA synthesis was blocked with an inhibitor, aphidicolin. In the presence of aphidicolin, chromosome-associated DmMCM2 is retained for up to 3 hours after induction of S phase by cyclin E, apparently stabilizing the association of DmMCM2 with chromosomes. It is concluded that DNA replication is required for dissociation of DmMCM2 from chromosomes. Thus, mitosis is not required for oscillations in chromosome binding of MCMs and it is proposed that cycles of MCM-chromosome association normally occur in endocycles. These results demonstrate that the cycle of MCM-chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles (Su, 1998).

The Drosophila Malpighian tubules (MTs), form a simple excretory epithelium comparable in function to kidneys in vertebrates. MTs function as the insect kidney both in the larva and the adult. They consist of two pairs of blind ending tubes that are composed of a single cell-layered epithelium made up of a tightly controlled number of cells. The tubules float in the hemolymph from where they take up nitrogenous waste that is excreted as uric acid. During embryogenesis, MTs evert as four protuberances from the hindgut primordium, the proctodeum. The everting tubules grow by cell proliferation, which takes place in a few cells along the tubules and extensively in a distal proliferation domain located in the tip region of the tubules. Cell ablation experiments and studies on the pattern of cell division have shown that a single large cell at the distal end of each tubule, termed the tip cell, is decisive for controlling the proliferation of its neighboring cells. The tip cell that differentiates into a cell with neuronal characteristics during later stages of development arises by division of a tip mother cell that is selected in the tubule primordium by lateral inhibition involving the Notch signaling pathway and the transcription factor Kruppel (Kr). It has been suggested that the tip cell sends a mitogenic signal to adjacent cells in the distal proliferation zone. It has remained elusive, however, what the signal is or what its target molecules in the signal-receiving cells could be and how cell proliferation during MT morphogenesis is regulated. Seven-up is shown to be a key component that becomes induced in response to mitogenic EGF receptor signaling activity emanating from the tip cell. Seven-up (Svp) in turn is capable of regulating the transcription of cell cycle regulators (Kerber, 1998).

If Svp is expressed ectopically in wild-type MTs, an increased number of tubule cells is obtained. BrdU incorporation studies indicate that this increased cell number results from extra cell divisions, indicating that svp is both necessary and sufficient to induce cell proliferation in the MTs. Analyses were carried out to further elucidate how the EGFR pathway and svp control cell proliferation, and whether these developmental regulators have an impact on components of the cell cycle machinery during MT growth. Two genes are limiting key components of the cell cycle during the period when the MT cells proliferate: string (stg), which encodes a Cdc25 phosphatase involved in the regulation of the G2/M transition, and cyclin E (cycE), which regulates the G1/S transition. In situ hybridization reveals that both genes are expressed asymmetrically in the everting tubules and subsequently in the distal proliferation zone. These expression domains match the svp expression domain. With the onset of the endomitotic cycles, a second phase of cycE expression occurs from proximal to distal in the tubules. In EGFR mutants, the transcriptional activation of stg and cycE, which occurs in the tubule proliferation domains in wild type, cannot be detected. This correlates with a strong reduction of BrdU incorporation and the dramatic reduction of the tubule cell number in Egfr mutants. During the subsequent endomitotic cycles, expression of cycE is not affected, indicating a specific function of EGFR signaling in activating early cycE expression. In svp mutants, the expression of stg and cycE is reduced (most likely reflecting that Svp is only one of the regulators that transmits the mitogenic EGFR signal); however, in MTs in which svp is ectopically expressed, stg becomes transcriptionally misexpressed in the cells that undergo extra cell divisions. Similar (although weaker) misexpression is obtained with cycE. However, extra cell divisions can only be obtained early during MT outgrowth, suggesting that other regulators limit cell proliferation during later stages of MT development (Kerber, 1998).

It is not known whether Svp, whose function has been characterized initially in the context of photoreceptor development in the eye also plays a role for cell proliferation during eye imaginal disc development. In MT there must be other factors in addition to Svp that are dependent on EGF signaling and are involved in MT growth. This is apparent from the finding that the svp mutant phenotype is less severe than that of Egfr mutants. Those predicted factors might include other steroid hormone receptors that interact with Svp as cofactors. Studies on ecdysone signaling pathways show that Svp can heterodimerize with subunits of the ecdysone receptor and regulate gene expression. Whether ecdysone-based signaling pathways also play a role in controlling cell proliferation in the MT is not known. Once cell proliferation is completed, the tubule cells elongate as a result of cell rearrangement and long thin tubes are generated with only two or three cells surrounding the lumen. An additional role of EGFR signaling during later stages of MT development cannot be excluded. This is consistent with recent results obtained with an antibody against the activated form of MAP kinase, which visualizes the activated state of receptor tyrosine kinase (RTK) signaling pathways and shows a rather uniform actived ERK pattern in all of the tubule cells. As there is no apparent tubule elongation defect in svp mutants, other downstream factors must be involved in mediating this potential aspect of EGFR signaling. In summary, these data provide a framework for further analysis of the molecular mechanisms that underlie the control of cell proliferation by developmental regulators during MT morphogenesis (Kerber, 1998).

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).

The Drosophila cyclin E gene gives rise to two transcripts encoding proteins that differ at their N termini, CycEII and CycEI. This study presents the first in vivo dissection of Cyclin E function. Ectopic expression studies using N- and C-terminal deletions of CycEI have revealed that a region of 322 residues surrounding the cyclin box is sufficient to induce entry of G1-arrested larval eye imaginal disc cells into S phase. Ectopic expression of CycEI in the eye disc drives anterior, but not posterior, G1-phase cells within the morphogenetic furrow (MF) into S phase. Significantly, ectopic expression of CycEII and N-terminal deletions of CycEI are able to drive all G1 cells within the morphogenetic furrow into S phase, while a C-terminal deletion of CycEI can not. Using yeast two-hybrid, coimmunolocalization, and in vivo functional studies, the p21 homolog Dacapo was shown not to be the mediator of the CycEI inhibition in the posterior part of the MF. Taken together, these results reveal a novel zone within the posterior region of the MF where CycEI but not CycEII function is inhibited, and suggest that CycEII is a more potent inducer of S phase (Crack, 2002).

To investigate the distribution of CycE protein during embryogenesis, CycE polyclonal antisera raised to the region of CycE present in both of the Cyclin E proteins was used. During the first 16 embryonic cell cycles, maternal CycEII protein (prevalent during 0-3 h AED) and zygotic CycEI protein (prevalent after 3 h AED) are nuclear localized in interphase cells. At the onset of prophase, CycE becomes dispersed throughout the cytoplasm until late in telophase, where again it becomes localized to the nucleus. Although less CycE protein appears to be present in mitotic cells, this is due to dispersion or epitope masking rather than degradation since analysis by immunoblotting has shown that CycE protein levels do not decrease during mitosis (Crack, 2002).

Throughout embryogenesis, CycE protein is present in proliferating cells but is down-regulated as cells stop dividing. After the 16th mitosis, most cells in the embryo enter their first G1 phase and begin to differentiate. At this time, CycE is no longer detected in differentiating epidermal cells but is present in patches of epidermal cells in the thoracic segments, which go through one further cycle. When proliferation of cells in the thoracic patches ceases, CycE is no longer detected in these cells but is still present in the dividing cells of the central and peripheral nervous system. As the nervous system cells cease division, expression of CycE protein also ceases. This is evident in peripheral nervous system cells, where CycE protein is no longer present when these cells cease proliferation. In addition, CycE protein can be detected at low levels in endoreplicating tissues when S phases are occurring. Thus, the pattern of CycE protein distribution during embryogenesis generally correlates with the pattern of CycE mRNA, being expressed in mitotically proliferating and endoreplicating cells and down-regulated when these cells cease replication (Crack, 2002).

To specifically investigate the localization and distribution of the CycEII protein during development, an antibody was raised in rats to the unique N-terminal 119 amino acids of the CycEII protein. Western analysis of Drosophila larval extracts has shown that the antisera detected protein bands at ~80 kDa after heat shock induction of a heat shock-inducible CycEII transgene. To investigate the tissue distribution of CycEII protein during embryonic development, CycE antibody stainings were carried out. In early embryos, CycEII protein is detected at high levels in interphase nuclei, but upon entry into mitosis the protein becomes distributed throughout the cytoplasm. In cellularized cycle 14 embryos, CycEII is also detected at high levels in the nuclei of interphase cells. However, at later developmental times, in embryos undergoing cycles 15 and 16, low levels of the CycEII protein are detected throughout most of the embryo, although higher levels are present in the amnioserosa. At slightly later times (stage 13) when most epidermal cells have arrested in G1, CycEII protein is undetectable in the epidermis or in proliferating cells of the central and peripheral nervous system. The persistence of CycEII protein during cycles 15 and 16 is consistent with the observation that CycE zygotic null mutants complete cycles 15 and 16 and arrest in G1 in cycle 17. In addition, CycEII protein persists in the pole cells until late in embryogenesis, consistent with the presence of the CycEII transcript in these cells (Crack, 2002).

The distribution of CycEII was examined in larval tissues by Western analysis, using the anti-CycEII antibody. Third instar larval eye-antennal discs and wing discs contain CycEII protein. However, the specific localization of CycEII in these tissues could not be determined by antibody staining, due to background problems (Crack, 2002).

The distribution of CycE protein during development correlates with proliferating cells with a few exceptions. CycE mRNA and protein are undetectable in all cases but one in which cells are known to be in G1 phase. CycE protein is down-regulated in embryonic epidermal cells when cells exit into G1 of cycle 17 and in neural cells as they cease proliferation. Likewise, CycE protein abundance in endoreplicating tissues correlates with their known replication pattern. Furthermore, during third instar larval optic lobe and eye imaginal disc development, CycE expression is absent in G1-arrested cells. In all of these cases, down-regulation of CycE may be important in limiting cell proliferation. The one known exception is the zone of nonproliferating cells of the third instar larval wing disc, which transiently arrest proliferation. In this tissue, CycE and the S phase transcription factor E2F are present but inactive, suggesting that they may be regulated by inhibitors such as Ckis or Retinoblastoma (Crack, 2002).

There are three other cases in which CycE is not down-regulated in cells that have ceased proliferating. In two cases, cells are arrested in G2 phase, where they may be refractory to S phase induction by CycE. (1) The amnioserosa cells, which exit from the cell cycle in G2 phase of cycle 14, maintain high levels of CycE for at least 1-2 h after they cease division. CycE in the amnioserosa persists without the presence of detectable CycE transcript, implying that CycE protein is stabilized in these cells. (2) In the nonproliferating G2-arrested pole cells, CycE is also maintained at high levels throughout embryogenesis even though these cells remain dormant for at least 14 h and do not recommence division until the end of embryogenesis. The presence of high levels of CycE protein in the pole cells correlates with high levels of maternally derived transcript localized to these cells during early embryogenesis. This represents maternally derived CycEII protein, since it is present in CycE-deficient embryos that have no zygotic expression of CycE and is detected with the CycEII-specific antibody. For the same reasons, CycE protein persisting in the amnioserosa is likely to be mostly CycEII. (3) CycE is present in nonproliferating cells in the lamina of third instar larval optic lobes but it is not known at which cell cycle stage these cells are arrested (Crack, 2002).

A 322-amino acid region spanning the cyclin box (residues 196-518) is sufficient for ectopic CycE function. The potential bipartite NLS of CycEI is important for nuclear localization, and that the N terminal region of CycEI contains an inhibitory domain. These results, defining the functional region of CycEI to 196-518 amino acids, are consistent with studies of human Cyclin A. Deletion of the N-terminal 172 amino acids of Cyclin A (corresponding to the N-terminal 216 amino acids of CycE) has no effect on Cdk binding or activation compared with full-length Cyclin A. The largest deletion of the C terminus of human Cyclin A that still retains some function, although considerably reduced, is to position 369, corresponding to position 433 of CycE. Furthermore, truncations of the N terminus of human Cyclin E, which presumably initiates at methionine 129 (corresponding to residue 254 of CycE), are functional in yeast (Crack, 2002).

The fact that deletions of the N-terminal regions of CycEI containing the potential bipartite NLS at amino acid positions 5-28 of CycEI are still functional raises questions regarding the nuclear targeting of CycEI. While full-length CycEI is nuclear-localized, it has been shown that the D12N-CycEI is not specifically targeted to the nucleus although some nuclear localization is evident. This suggests that this putative NLS is likely to be mostly responsible for the nuclear targeting of CycEI. The weak nuclear staining observed for D12N-CycEI may be due to the remaining part of the bipartite NLS, but this is unlikely since a 40-amino acid N-terminal deletion completely removing the NLS also shows some nuclear localization (Crack, 2002).

Nuclear localization may occur by the action of a novel NLS or by association with another protein that becomes nuclear localized. Interestingly, the N-terminal-deleted CycEI proteins are apparently as functional as the nuclear localized full-length CycEI and CycEII proteins in inducing G1 cells into S phase. Since nuclear localization of CycE is expected to be required for function, the fact that relatively low levels of nuclear-localized D12N-CycEI is able to effectively induce S phase entry suggests that these low levels of CycE are sufficient for S phase induction or that D12N-CycEI is a more potent S phase inducer than CycEI or CycEII (Crack, 2002).

An important developmental question is whether the two isoforms of CycE, which are present at different developmental stages, have different functions? In this study, it was shown that heat shock-induced expression of CycEII is able to induce G1-arrested cells within the posterior part of the MF into S phase, whereas CycEI can not. Likewise, CycEI constructs lacking the unique 12 amino acids at the N terminus are able to induce all of the G1-arrested cells within the MF into S phase. These results suggest that the unique N-terminal region of CycEI acts as an inhibitory domain. This inhibitory domain may be a target for an inhibitor that is present within the posterior part of the MF. Alternatively, the N-terminal region of CycEI may be an intrinsic inhibitory domain that requires inactivation by the binding of an activator, which is not present in the posterior part of the MF. It is intriguing that the CycEII protein, which differs from CycEI only by replacement of the 12 N-terminal amino acids of CycEI with a novel 119-residue sequence, is able to overcome inhibition in the posterior region of the MF. It should be noted that BrdU labeling of these cells is not as intense and that induction of these posterior MF cells into S phase by CycEII occurs after S phase induction of the anterior MF cells. This suggests that CycEII may be partially sensitive to inhibition within the posterior region of the MF. Interestingly, Drosophila Cyclin A appears to be resistant to inhibition within the posterior region of the MF, since ectopic expression of Cyclin A can induce all cells within the MF into S phase with equal efficacy (Crack, 2002).

What is different about the posterior part of the MF? Differential regulation of G1 arrest occurs in the anterior and posterior regions of the MF. The Drosophila TGFß homolog, decapentaplegic, is expressed in the G1-arrested cells of the MF and is required to establish G1 arrest in the anterior part of the MF. In posterior MF cells, a Dpp-independent mechanism mediates G1 arrest. It is possible that the Dpp-independent mechanism for G1 arrest in the posterior region of the MF is related to the mechanism that have been observed in this study that acts upon the N terminus of CycEI. If this Dpp-independent mechanism occurs by the induction of an inhibitor, it is unlikely that this inhibitor is Dacapo. The results presented here show that Dacapo is not detectable in the posterior part of the MF and does not show higher binding efficiency or inhibition of CycEI/Dm Cdk2 compared with CycEII/Dm Cdk2. Nor is ectopic expression of CycEI able to induce posterior MF cells into S phase in dacapo mutant eye discs. Another Cdk inhibitor that functions within the MF of the eye disc is Roughex (Rux), which has been shown to inhibit Cyclin A/Cdk (but not Cyclin EI/Cdk2), as well as induction of S phase in embryos and eye imaginal discs. Rux functions by down-regulating Cyclin A accumulation, while Cyclin E/Cdk2 binds to, phosphorylates, and down-regulates Rux. Furthermore, CycEI and CycEII bind to Rux with equal affinity in yeast two-hybrid assays. Another mediator of G1 arrest, that can act downstream of Cyclin E function by inhibiting the activity of the S phase transcription factors E2F/DP, is the Retinoblastoma (Rb) protein. The Drosophila Rb (Rbf1) is also unlikely to be involved in this inhibition, since in rbf1 mutant eye discs, although the anterior MF cells do not arrest in G1, posterior MF cells remain G1-arrested. Since Dacapo, Rux, and Rbf1 are unlikely to mediate the inhibition of CycEI in the posterior part of the MF, the mechanism of CycEI inhibition remains to be determined (Crack, 2002).

CycEII has been detected in third instar larval eye and wing imaginal discs. Although the precise cells in which CycEII is expressed in these tissues have not been determined, CycEII may play an important role in specific cells in overcoming an inhibitor to drive entry into S phase. For example, it is possible CycEII may be expressed in the post-MF S phase region of the eye imaginal disc and be specifically required here to overcome a G1-S phase inhibitor that is expressed at high levels within the MF (Crack, 2002).

During embryogenesis, CycEII is present at high levels in the early embryo where cell cycles are very rapid and essentially have no G1 or G2 phases. It is possible that CycEII has a role in allowing the very rapid S phases to occur in these early embryonic cycles. The demonstration that ectopically expressed CycEII is largely resistant to an inhibitor in the eye disc that targets CycEI, suggests one way in which the specific properties of CycEII may be promoting the rapid embryonic cycles. For example, a maternally supplied inhibitor may be unable to effectively inhibit CycEII/Cdk2 function in vivo, allowing for immediate entry into S phase after the completion of mitosis in these early cycles. In contrast, once CycEI protein starts to predominate after postblastoderm cycle 14 expression of the potential inhibitor, a G1 arrest may result, due to a more readily inhibited CycEI/Cdk2 function (Crack, 2002).

It is possible that similar mechanisms exist in mammalian cells to allow different types of cell cycles to occur. Interestingly, three different splice variants of human Cyclin E have been reported. Two of these variants, Es and Et, are unable to activate Cdk2 activity, suggesting that they may act in a dominant-negative manner in the G1 to S phase transition. In contrast, protease cleaved N-terminal truncated forms (deleting ~45 or ~69 N-terminal amino acids) of human Cyclin E have been identified in tumor cells that have higher activity and greater S phase-inducing potential than full-length Cyclin E. These data suggest that human Cyclin E may also be subjected to an inhibitory mechanism that acts at the N-terminal region. Furthermore, a second mammalian cyclin E cDNA, cyclin E2, that encodes a protein differing significantly in its N-terminal region from Cyclin E1, has been identified. It will be interesting to determine whether cyclin E2 and cyclin E1 are differentially expressed during mouse development and whether Cyclin E2 is a more potent inducer of S phase than Cyclin E1 or vice versa (Crack, 2002).

In summary, the results of this study have revealed the existence of a novel inhibitory domain at the N terminus of CycEI that prevents ectopic CycEI from inducing G1 cells within the posterior region of the MF into S phase. Although CycE is normally not expressed at detectable levels within the MF, it is possible that such a mechanism prevents low levels of CycE protein from functioning inappropriately during eye development. The results described here have also demonstrated that CycEII is largely resistant to the inhibitory mechanism that acts on CycEI, highlighting the complexity of G1-S regulation and exposing a novel cell cycle regulatory mechanism that acts during development (Crack, 2002).

During animal development, organ size is determined primarily by the amount of cell proliferation, which must be tightly regulated to ensure the generation of properly proportioned organs. However, little is known about the molecular pathways that direct cells to stop proliferating when an organ has attained its proper size. Mutations have been identified in a novel gene, shar-pei, that is required for proper termination of cell proliferation during Drosophila imaginal disc development. Clones of shar-pei mutant cells in imaginal discs produce enlarged tissues containing more cells of normal size. This phenotype is the result of both increased cell proliferation and reduced apoptosis. Hence, shar-pei restricts cell proliferation and promotes apoptosis. By contrast, shar-pei is not required for cell differentiation and pattern formation of adult tissue. Shar-pei is also not required for cell cycle exit during terminal differentiation, indicating that the mechanisms directing cell proliferation arrest during organ growth are distinct from those directing cell cycle exit during terminal differentiation. shar-pei, also termed salvador, encodes a WW-domain-containing protein that has homologs in worms, mice and humans, suggesting that mechanisms of organ growth control are evolutionarily conserved (Kango-Singh, 2002).

Cyclin E is limiting for S-phase initiation and progression during imaginal disc development and several tumor suppressor genes negatively regulate its activity or levels. Cyclin E levels are upregulated in shrp1 mutant cells in the second mitotic wave and posterior to it. Elevated levels were also observed just anterior to the second mitotic wave, although this effect was not as pronounced. The effect on Cyclin E is cell autonomous and observed in most or all mutant cells, even though only a fraction of them are actively progressing through S phase. Thus, the effect of Shrp on cell proliferation arrest may be mediated by regulating the levels of Cyclin E (Kango-Singh, 2002).

Transformation of eye to antenna by misexpression of a single gene

In Drosophila, the eye and antenna originate from a single epithelium termed the eye-antennal imaginal disc. Illumination of the mechanisms that subdivide this epithelium into eye and antenna would enhance understanding of the mechanisms that restrict stem cell fate. This study shows that Dorsal interacting protein 3 (Dip3), a transcription factor required for eye development, alters fate determination when misexpressed in the early eye-antennal disc, and this observation has been taken advantage of to gain new insight into the mechanisms controlling the eye-antennal switch. Dip3 misexpression yields extra antennae by two distinct mechanisms: the splitting of the antennal field into multiple antennal domains (antennal duplication), and the transformation of the eye disc to an antennal fate. Antennal duplication requires Dip3-induced under proliferation of the eye disc and concurrent over proliferation of the antennal disc. While previous studies have shown that overgrowth of the antennal disc can lead to antennal duplication, these results show that overgrowth is not sufficient for antennal duplication, which may require additional signals perhaps from the eye disc. Eye-to-antennal transformation appears to result from the combination of antennal selector gene activation, eye determination gene repression, and cell cycle perturbation in the eye disc. Both antennal duplication and eye-to-antennal transformation are suppressed by the expression of genes that drive the cell cycle providing support for tight coupling of cell fate determination and cell cycle control. The finding that this transformation occurs only in the eye disc, and not in other imaginal discs, suggests a close developmental and therefore evolutionary relationship between eyes and antennae (Duong, 2008).

Dip3 is able to bind DNA in a sequence specific manner and activate transcription directly. Dip3 possesses an N-terminal MADF domain and a C-terminal BESS domain, an architecture that is conserved in at least 14 Drosophila proteins, including Adf-1 and Stonewall. The MADF domain directs sequence specific DNA binding to a site consisting of multiple trinucleotide repeats, while the BESS domain directs a variety of protein-protein interactions, including interactions with itself, with Dorsal, and with a TBP-associated factor (Bhaskar, 2002).

Antagonism between the N and EGFR signaling pathways influences developmental fate in the eye-antennal disc leading to a loss of eye tissue and the appearance of extra antennae. Although this phenotype was originally suspected to represent eye-to-antennal transformation, subsequent analysis suggests that it most likely represents antennal duplication. Specifically, the absence of the N signal leads to a failure in eye disc proliferation resulting in compensatory over-proliferation of the antennal disc and its subdivision into multiple antennae. Consistent with the idea that the extra antennae result from under-proliferation of the eye field, it was found that the phenotype was largely suppressed by over-expression of CycE to drive the cell cycle (Duong, 2008).

In this study, it was found that inhibition of eye disc growth leads to antennal duplication. But in addition, it was shown that the same treatment that leads to antennal duplication can also direct the transformation of eyes to antennae. These two phenotypes are anatomically distinct. This anatomical distinction is evident in adults: antennae resulting from antennal duplication are found anterior to the antennal foramen, while the antennae resulting from eye-to-antenna transformation are found posterior to the antennal foramen. It is also apparent in larval eye-antennal imaginal discs: antennal duplication discs exhibit multiple circular dac expression domains within a single sac of epithelium (the antennal disc), while eye-to-antennal transformation discs exhibit two or more circular dac expression domains spread over both the eye and antennal discs. The two types of discs display distinct molecular signatures as well: the antennal duplication discs exhibit duplicated Dll expression domains, while the eye discs undergoing transformation to antennae lack Dll expression (Duong, 2008).

Perhaps the most persuasive evidence that Dip3 can direct eye-to-antennal transformation is provided by the observation of eyes that are only partially transformed to antennae since is very difficult to reconcile these partial transformations with the idea of antennal duplication. In some cases, proximal antennal segments tipped with eye tissue are observed. In accord with this phenotype, some third instar larval eye discs display a central domain of Elav-positive differentiating photoreceptors surrounded by a circular dac domain (Duong, 2008).

These arguments support the idea that antennal duplication and eye-to-antennal transformation are mechanistically distinct phenomena, and the remainder of the discussion assumes this to be the case. However, the possibility that these two phenotypes are two manifestations of a single mechanism cannot be excluded. For example, the discs exhibiting duplicated Dll domains may represent complete transformations, while the discs lacking duplicated Dll domains, but containing Elav may represent partial transformations (Duong, 2008).

The data show that discs undergoing antennal duplication as a result of Dip3 expression are comprised of a severely diminished eye region and an enlarged antennal region. As shown by BrdU labeling experiments, these antennal duplication discs most likely result from suppression by Dip3 of cell proliferation in the eye field leading to overproliferation of the antennal disc. This conclusion is supported by the ability of factors that drive cell proliferation (e.g., Cyclin E) to alleviate the Dip3 misexpression defect (Duong, 2008).

Many experimental manipulations that reduce the size of the eye disc (e.g., surgical excision, induction of cell death, or suppression of cell proliferation) lead to enlargement and duplication of the antennal primordium. How might reduction of the eye field lead to antennal field over-growth? One possibility is that the eye field produces a growth inhibitory signal. Alternatively, the eye field and the antennal field may compete with each other for limited nutrients or growth factors. In support of this latter possibility, recent studies of the role of dMyc in wing development have demonstrated growth competition between groups of imaginal disc cells (Duong, 2008).

While the results imply that antennal disc overgrowth is required for antennal duplication, overgrowth is thought not to be sufficient for duplication. This conclusion derives from experiments in which an antennal disc specific driver is used to direct over-expression of CycE or Nact. This resulted in antennal overgrowth without concurrent reduction in the eye disc. In this case, antennal duplication was not observed. Thus, in addition to antennal overgrowth, antennal duplication also appears to require reduction or elimination of the eye disc. Regulatory signals from the eye disc may act to prevent antennal duplication (Duong, 2008).

The eye and antenna discs differ in several respects: (1) Specific expression of the organ-specification genes. The eye disc expresses the retinal determination gene network (RDGN) genes, including eyeless (ey), twin of eyeless (toy), eyes absent (eya), sine oculis (so), and dachshund (dac), while the antennal disc expresses Dll and hth. hth is also expressed in the eye disc but in a distinct pattern from that seen in the antennal disc. In the second instar eye disc, hth is expressed throughout the eye disc, and collaborates with ey and teashirt (tsh) to promote cell proliferation. The hth expression domain later retracts to only the anterior-most region of the eye disc. This pattern is different from the circular expression pattern observed in the antennal disc. (2) In the antennal disc, dpp is expressed in a dorsal anterior wedge and wg is expressed in a ventral anterior wedge. The intersection of Dpp and Wg signaling is required to specify the proximodistal axis in the leg and antenna. In the early eye disc, Wg and Dpp signaling may overlap. But as the disc grows in size, the wg and dpp expression domain are separated, so that there is probably no intersection between high levels of Wg and Dpp signaling. (3) Whereas the partial overlap of Dll and hth expression domains in the antennal disc is important for proximodistal axis specification, there is no Dll expression in the eye disc. Dll expression in the center of the antennal and leg discs is induced by the combination of high levels of Dpp and Wg signaling. Because there is no overlap of Dpp and Wg signaling in the eye disc, Dll is not induced (Duong, 2008).

Therefore, efficient transformation of the eye disc into an antennal disc requires at least three things: (1) repression of the eye fate pathway; (2) activation the antennal fate pathway; and (3) the intersection of Dpp and Wg signaling, mimicking the situation in the antenna and leg disc that induces proximodistal axis formation. Any one of these three conditions by itself is not sufficient. (1) Loss of the RDGN genes leads only to the loss of the eye. However, if apoptosis is blocked, or cell proliferation is induced, in the ey2 mutant (ey>p35 or ey>Nact in ey2), then Dll can be induced in the eye disc and extra antenna are formed. The induction of Dll is not ubiquitous in the eye disc, suggesting that the loss of ey does not autonomously lead to the expression of Dll and the transformation to the antennal fate. (2) Simply expressing the antennal determining genes Dll or hth in the eye disc does not change the eye fate into antennal fate. It was found that uniform expression of Dll in the eye disc (ey>Dll) resulted in mild eye reduction, whereas ey>hth completely abolished eye development. E132>Dll caused the formation of small antenna in the eye in about 46% of flies, whereas ptc>Dll and C68a>Dll induced extra antenna but not within the eye field. Therefore, although Dll and hth are important determinants for antennal identity, it is their specific spatial expression patterns that determine antennal development. (3) Creating the intersection of Wg and Dpp signaling does not change the eye into antenna. Such manipulation in the leg disc turned on vg and transdetermined the leg disc into wing disc. Therefore, the specific genes induced by Dpp and Wg signaling may depend on disc-specific factors. In the eye disc, turning on Wg signaling in the dpp expressing morphogenetic furrow only blocked furrow progression (Duong, 2008).

In this study, it was found that the ectopic expression of a single gene, Dip3, can cause eye-to-antenna transformation. Dip3 apparently satisfied all three requirements. (1) Overexpression of Dip3 repressed (non-cell-autonomously) ey and dac. The repression of ey may be due to the induction of ct. The ability of Dip3 to simultaneously repress multiple retinal determination genes is completely consistent with the many known cross-regulatory interactions between these genes. (2) ey>Dip3 turned on ct and hth. (3) By blocking cell proliferation, ey>dip3 reduced the eye field size and allowed the intersection of Dpp and Wg signaling. Furthermore, ey>Dip3 induced en, which probably created an ectopic A/P border and induced ectopic dpp/wg expression (Duong, 2008).

Interference with cell cycle progression appears to be a common link between the two phenotypes described in this study. In the case of antennal duplication, interference with eye disc growth leads to antennal disc overgrowth, which is a prerequisite for duplication. In the case of eye-to-antenna transformation, eye disc undergrowth allows the required intersection between Dpp and Wg signaling (Duong, 2008).

The observation that Dip3 misexpression can transform the eye field, but not other tissues, to an antennal fate suggests a close evolutionary relationship between the eye and the antenna. Previous studies have emphasized the homology between antennae and legs. The findings presented here that misexpression of a single transcription factor, namely Dip3, can transform eyes to antennae provides support for the notion that the eye and antenna may also, in some sense, be homologous to one another. Previous evidence in support of this idea comes from the observation that similar spatial arrangements of Wg and Dpp signaling along with a temporal cue provided by the ecdysone signal are required for the formation of the eye and the mechanosensory auditory organ. Small mechanosensory sensilla, such as Johnston's organ and the chordotonal organs (stretch receptors) are thought to represent the earliest evolving sense organs. Perhaps the eye resulted from a duplication and specialization of such a sensillum (Duong, 2008).

Effects of Mutation or Deletion

Homozygous mutant embryos at late stage 11 show severely reduced DNA synthesis in the nervous system, and no DNA synthesis in the epidermis. Arrest in Cyclin E mutants occurs in the G1 phase of cycle 17, the first embryonic cell cycle to contain a G1 phase, just after the disappearance of the maternal Cyclin E transcript. Ectopic expression of Cyclin E induces S phase, but fails to do so when cells are in G2 (Knoblich, 1994).

The Drosophila embryo ordinarily undergoes thirteen cycles of rapid syncytial division followed by three rounds of cellular division for most cells. Strict regulation of the number of divisions is believed to be essential for normal patterning and development. To determine how the embryo responds to hyperplastic growth, epidermal development was examined in embryos that experience additional rounds of mitosis as the result of ectopic Cyclin E expression. The cell density in the epidermis nearly doubles within 1 hour of Cyclin E induction. The spacing and width of the Engrailed and wingless stripes is unchanged, but the cell density within the stripes is increased. By 4 hours after Cyclin E induction, the cell density has returned to almost normal values. The embryos develop, albeit more slowly, to produce viable larvae and adults. The excess cells were removed by apoptosis in a reaper-dependent fashion as evidenced by increased reaper expression. Embryos lacking cell death in the abdomen exhibit changes in Engrailed expression. In addition, germband retraction and dorsal closure are slower than normal. Ectopic Cyclin E expression in cell-death-deficient embryos exacerbates the germband retraction and Engrailed-expression defects (Li, 1999).

Simultaneous overexpression of both E2F subunits, E2F and DP, stimulates the expression of multiple E2F-target genes including cyclin E, and also causes the initiation of S phase. Mutation of cyclin E prevents the initiation of S phase after overexpression of E2F/DP without affecting induction of target gene expression. Thus, E2F-directed transcription cannot bypass loss of cyclin E in Drosophila embryos. It is concluded that Cyclin E has an essential in vivo role in S phase induction other than induction of E2F activity (in mammals accomplished by phosphorylation and inactivation of Rb). Reduction of cyclin E expression blocks E2F-induced S phase in epidermis, but not in the midgut. These results suggest that different cell types have different sensitivity thresholds to cyclin E expression, and that as little as a two fold change in Cyclin E levels can have dramatic effects of cell cycle (Duronio, 1996).

To ascertain the extent to which pupal wing cells are in G2 phase, string and cycE were put under heat shock (HS) regulation and mitotic activity in wing cells was examined. Overexpression of HS-stgcauses entry in M phase of G2-arrested cells; overexpression of HS-cycE causes entry in S phase of G1-arrested cells. Overexpression of stg or cycE after puparium formation, that is, at the beginning of pupal development when the wing cells stay mitotically quiescent, causes an almost generalized entry into mitosis of the wing cells, with the exception of wing margin cells. Overexpression of cycE at this pupal stage provokes the entry into S phase of wing margin cells and only a few cells of the distal wing blade. These results indicate the arrest of most wing blade cells at the G2 stage and all the wing margin cells at the G1 stage at the beginning of pupal development. In metamorphosing wing discs, at the latter stages of pupal development, progression through the cell cycle takes place, as in larval discs, in nonclonally derived clusters of cells synchronized in the same cell cycle stage (G1-M transition). At 12-16 hours after puparium formation, String mRNA accumulates, mostly at the wing margin and at the distal part of the wing blade; a little later, from 16-20 hours, there is a large increase in the number of string-expressing cells distributed throughout the wing blade, including hinge and vein trunk regions. During the last 4 hours of the pupal proliferative period (20-24 hours), there is mitotic activity but no DNA synthesis. Contrary to early discs, there are temporal and spatial heterogeneities in cell proliferation associated with wing margin, vein, intervein, and middle intervein territories. Such heterogeneities can be associated with gene expression patterns (i.e., rhomboid [veinlet], blistered, and >extramachrochaetae), known to occur in wing venation and morphogenesis. Within these territories, there are no indications of a wave of cell cycle progression. As in early discs, mitotic orientations are found at random, but there is a preferential allocation of postmitotic cells along the proximodistal axis, thus explaining the elongated shape of the resulting clones along this axis. Shapes of clones in mature discs and in evaginated wings are similar, thus excluding major morphogenetic movements during evagination. After the proliferative period, all the cells are arrested in G1 phase. The final number of cells of the wing is fixed, independent of experimental perturbations that alter the cell division schedule (Milán, 1996).

A Drosophila cyclin E hypomorphic mutation, DmcycEJP, has been generated and characterized that is homozygous viable and fertile, but results in adults with rough eyes. The mutation arose from an internal deletion of an existing P[w+lacZ] element inserted 14 kb upstream of the transcription start site of the DmcycE zygotic mRNA. The presence of this deleted P element, but not the P[w+lacZ] element from which it was derived, leads to a decreased level of DmcycE expression during eye imaginal disc development. Eye imaginal discs from DmcycEJP larvae contain fewer S phase cells, both anterior and posterior to the morphogenetic furrow. This results in adults with small rough eyes, largely due to insufficient numbers of pigment cells. Altering the dosage of the Drosophila cdk2 homolog cdc2c, retinoblastoma, or p21(CIP1) homolog dacapo, all of which encode proteins known to physically interact with Cyclin E, modifies the DmcycEJP rough eye phenotype as expected. Decreasing the dosage of the S phase transcription factor gene, dE2F, enhances the DmcycEJP rough eye phenotype. Surprisingly, mutations in G2/M phase regulators cyclin A and string (cdc25), but not cyclin B1, B3, or cdc2, enhance the DmcycEJP phenotype without affecting the number of cells entering S phase; instead, the mutations decrease the number of cells entering mitosis. This analysis establishes the DmcycEJP allele as an excellent resource for searching for novel cyclin E genetic interactors. In addition, this analysis has identified cyclin A and string as DmcycEJP interactors, suggesting a novel role for cyclin E in the regulation of Cyclin A and String function during eye development. Existing mechanisms do not explain the genetic interaction between DmcycE and string that was observed or between roughex and string observed in another study. In Drosophila embryos at least, phosphorylation of the tyr 15 and thr 14 residues of Cdc2 in Cyclin A/Cdc2 complexes inhibits Cdk activity. String (Cdc25) acts to dephosphorylate these residues and activate Cdk activity. In mammalian cells Cyclin E/Cdk2 phosphorylates and activates the Cdc25A phosphatase in the G1 to S phase transition. One possible explanation, therefore, is that DmcycE acts to phosphorylate and activate String phosphatase activity, leading to the activation of Cyclin A/Cdc2 activity (Secombe, 1998).

In addition to D-type cyclin-cdk complexes, cyclin E-cdk2 has also been implicated in the regulation of cell cycle progression through the G1 phase. The presence of Cyclin E-Cdk2 might explain the relatively mild phenotype observed in flies lacking Cyclin-dependent kinase 4/6 function. To evaluate this notion, the effects of heterozygosity for Cyclin E and Cdk2 mutations was studied in Cdk4 mutants. While heterozygosity for mutations in Cyclin E is readily tolerated in Cdk43 heterozygotes, it results in complete lethality in Cdk43 homozygotes. Similarly, heterozygosity for mutations in Cdk2 results in an almost complete lethality in Cdk43 homozygotes, while it has no effect in Cdk43 heterozygotes. In contrast, mutations in Cyclin A, Cyclin B, Cyclin B3 and Cdk1 have no effect on the survival of Cdk43 homozygotes. These observations demonstrate that Cdk4 mutants are particularly sensitive to reduction in Cyclin E-Cdk2 levels (Meyer, 2000).

Control of endoreduplication domains in the Drosophila gut by the knirps and knirps-related genes

Endoreduplication cycles that lead to an increase of DNA ploidy and cell size occur in distinct spatial and temporal patterns during Drosophila development. Only little is known about the regulation of these modified cell cycles. Fore- and hind-gut development have been investigated and evidence is presented that the knirps and knirps-related genes are key components to spatially restrict endoreduplication domains. Lack and gain-of-function experiments show that knirps and knirps-related, which both encode nuclear orphan receptors, transcriptionally repress S-phase genes of the cell cycle required for DNA replication and that this down-regulation is crucial for gut morphogenesis. Furthermore, both genes are activated in overlapping expression domains in the fore- and hind-gut in response to Wingless and Hedgehog activities emanating from epithelial signaling centers that control the regionalization of the gut tube. These results provide a novel link between morphogen-dependent positional information and the spatio-temporal regulation of cell cycle activity in the gut (Fuss, 2001).

The development of the gut epithelium is accompanied by a stereotyped pattern of cell cycle regulation. The fore- and hind-gut primordia undergo a fixed number of postblastodermal cell divisions until late stage 10/early stage 11. Endoreduplication cycles have been described to occur at stages 13/14 in the hindgut. BrdU incorporation studies have shown that the hindgut epithelium displays a subdivision into replicating tissues (such as the developing large intestine) and quiescent tissues (such as the developing small intestine) and the rectum from stage 11 onwards. The replicative activity is reflected by a specific BrdU incorporation pattern in the hindgut: no incorporation is observed in the small intestine and rectum and but high incorporation is observed in the large intestine primordia in between. Notably, the kni/knrl expression pattern in the hindgut of wild-type embryos is complementary to the BrdU incorporation pattern. This complementarity also applies to the foregut in which kni/knrl are ubiquitously expressed. Endocycles have not been described for the developing foregut and BrdU is not incorporated from stage 11 onwards (Fuss, 2001).

In Df(3L)riXT1 mutant embryos, the analysis of the BrdU incorporation pattern reveals a tissue and time specific defect of cell cycle activity in the hindgut epithelium. An ectopic domain of DNA replication in the rectum and a slight expansion of DNA replication into the small intestine is detectable using the BrdU incorporation assay in stage 13 mutant embryos. The appearance of a G1 phase in the endoreduplicative cycle and the transition from G1 to S phase is accompanied by a molecular network controlling the coordinate transcription of cycE. CycE in turn regulates the activity of the S-phase genes Polalpha, PCNA and RNR2. Since cycE is only weakly expressed in the hindgut whether its expression is changed in Df(3L)riXT1 mutant embryos could not be analyzed (both kni and knrl are unchanged in cycE mutants). On the contrary, the Polalpha, PCNA and RNR2 genes which are activated in response to CycE activity, indeed do have a strong expression pattern in the hindgut that parallels the BrdU incorporation pattern in wild-type embryos. In line with the BrdU experiments, loss of kni/knrl function in Df(3L)riXT1 mutant embryos leads to an ectopic expression of RNR2, PCNA and Polalpha in the rectum and an upregulation of these genes in the small intestine prior to the upcoming defect in these gut regions. To further investigate this, gain-of-function experiments were performed using the UAS-Gal4 system. Ectopic expression of either kni or knrl in the entire hindgut using the 14-3fkh-Gal4 driver and the UAS-Kni or UAS-Knrl effector lines merely leads to a mild reduction of the BrdU incorporation domain in the large intestine. Ectopic expression of both kni and knrl has a strong effect on DNA replication in the hindgut. The BrdU domain is completely abolished, suggesting a combinatorial function of both genes in the suppression of endoreduplication cycles. The expression of various cell cycle components was analyzed. Ectopic kni and knrl activities in the entire hindgut are able to completely repress the transcription of RNR2, PCNA and Polalpha in the large intestine. Notably, cycE mutants in which no endoreduplication occurs in the large intestine, display a mutant phenotype that is similar to the one obtained when kni and knrl are ubiquitously expressed in the hindgut (Fuss, 2001).

The Drosophila Tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation

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).

Tsc1 and Tsc2 may modulate the cell cycle via changes in cyclin levels. In Tsc1 and Tsc2 mutant clones, the levels of both Cyclin E and Cyclin A are elevated. Cell growth driven by dmyc or Target of rapamycin (dTor) elevates Cyclin E levels. It has been postulated that Cyclin E may function as a 'growth sensor' in a manner analogous to CLN3 in yeast and that the translation of Cyclin E is more efficient in cells that have an increased rate of growth. The increased levels of Cyclin E may be responsible for the shortening of G1 in Tsc1 and Tsc2 mutants. It is unclear why Cyclin A and Cyclin B are also elevated in mutant cells. Cyclin A is normally expressed at high levels in G2. In Tsc1 or Tsc2 mutants, Cyclin A levels are elevated in the post-mitotic cells of the eye disc that are clearly not arrested in G2. Thus it seems likely that the increased growth in mutant cells may also lead to increased levels of mitotic cyclins. Alternatively Tsc1 and Tsc2 may function in a pathway that negatively regulates cyclin levels (Tapon, 2001).

While the increased levels of cyclins are likely to be a response to the increased growth rate of mutant cells, the possibility that they are in some way responsible for the increased growth rate cannot be excluded. In Drosophila, the Cyclin D/cdk4 complex serves to promote growth. In such a scenario, the loss of Tsc1 or Tsc2 gene function may lead to elevated levels of cyclins leading to increased growth and proliferation. Surprisingly, increased expression of Cyclin E, which is thought to primarily promote S-phase entry and not growth, is also able to suppress the phenotype induced by overexpression of Tsc1 and Tsc2. This might reflect the existence of feedback loops where Cyclin E might downregulate the levels or activity of the Tsc1/Tsc2 complex. Alternatively, in some circumstances, Cyclin E might assume some of the functions of the growth promoting Cyclin D. Indeed, in mammalian cells, cyclin E has been shown to fully compensate for the loss of cyclin D1 (Tapon, 2001).

Tsc mutant cells fail to maintain a developmentally induced G1 arrest posterior to the second mitotic wave in third instar eye imaginal discs. The establishment of this G1 arrest requires a downregulation of Cyclin E and Cyclin A expression. However, the transient cell cycle arrest in the morphogenetic furrow occurs normally in Tsc1 and Tsc2, suggesting that it is the maintenance of G1 arrest that is perturbed rather than its initial establishment. Postmitotic cells continue to grow abnormally in Tsc1 and Tsc2 mutants and express elevated levels of Cyclin E and Cyclin A. A likely model is that inappropriate and continued growth in postmitotic cells leads to an accumulation of Cyclin E and the mitotic cyclins. This would eventually force cells to overcome a developmentally regulated cell cycle arrest and to reenter the cell cycle. Indeed, many of the lesions in patients with TSC occur in organs that consist predominantly of postmitotic cells such as the heart and brain. A successful therapeutic strategy in tuberous sclerosis is likely to be one that can curtail the inappropriate cell growth (Tapon, 2001).

Drosophila p27Dacapo expression during embryogenesis is controlled by a complex regulatory region independent of cell cycle progression

In the embryonic epidermis, dacapo expression starts during G2 of the final division cycle and is required for the arrest of cell cycle progression in G1 after the final mitosis. The onset of dacapo transcription is the earliest event known to be required for the epidermal cell proliferation arrest. To advance an understanding of the regulatory mechanisms that terminate cell proliferation at the appropriate stage, the control of dacapo transcription has been analyzed. dacapo transcription is not coupled to cell cycle progression. It is not affected in mutants where proliferation is arrested either too early or too late. Moreover, upregulation of dacapo expression is not an obligatory event of the cell cycle exit process. During early development of the central nervous system, Dacapo cannot be detected during the final division cycle of ganglion mother cells, while it is expressed at later stages. The control of dacapo expression therefore varies in different stages and tissues. The dacapo regulatory region includes many independent cis-regulatory elements. The elements that control epidermal expression integrate developmental cues that time the arrest of cell proliferation (Meyer, 2002).

In the embryonic epidermis, dap transcripts start to accumulate during G2 before the final mitosis 16. Within the epidermis, the pattern of dap transcript accumulation anticipates the pattern of mitosis 16. It is first observed in the region of the tracheal pits and in the prospective posterior spiracle region, then in the dorsal epidermis and finally also in the ventral epidermis. To determine whether dap expression is dependent on progression through previous divisions, string (stg) mutant embryos were examined. In these embryos, cell proliferation is prematurely arrested in G2 before mitosis 14. Nevertheless, the accumulation of dap transcripts is not delayed in stg embryos. In Cyclin A Cyclin B double mutant embryos. cell proliferation is prematurely arrested in G2 before mitosis 15. Nevertheless, accumulation of dap transcripts occurs normally in these embryos. In contrast to mutations in stg and Cyclin A and B, which result in a premature cell cycle arrest, overexpression of Cyclin E triggers an additional division cycle, as also observed in dap mutants. To address whether Cyclin E overexpression inhibits dap transcription, embryos carrying prd-GAL4 and UAS-Cyclin E were examined. In these embryos, Cyclin E is overexpressed in alternating segments of the epidermis. However, accumulation of dap transcripts starts normally throughout the entire epidermis. It is concluded therefore that the extra division cycle that occurs in the UAS-Cyclin E-expressing segments does not result from inhibition of dap expression, and it is assumed that p27DAP protein levels are simply insufficient to bind and inhibit all of the Cyclin E/Cdk2 complexes present in the overexpressing regions (Meyer, 2002).

The idea that developmental regulators govern dap expression is consistent with the finding that the onset of dap transcription is not dependent on completion of the embryonic cell proliferation program. The accumulation of dap transcripts occurs at the correct stage also in the epidermis of stg and Cyclin A Cyclin B double mutants where cells arrest prematurely. Moreover, the onset of dap transcription in the epidermis is not affected by overexpression of positive regulators of cell proliferation like Cyclin E, Cyclin D/Cdk4 and E2F1/DP (Meyer, 2002).

During development of multicellular organisms, cell proliferation evidently has to be coordinated with other processes (pattern formation, morphogenesis and growth). In principle, coordination could be achieved by developmental control of a single essential cell cycle regulator, while all others might simply be governed by feedback coupling to cell cycle progression. Analyses in Drosophila embryos have clearly demonstrated that multiple cell cycle regulators are controlled by developmental signals. Apart from this work on dap, previous studies have revealed very similar findings in the case of string and Cyclin E. The embryonic expression of both genes is controlled largely independent of cell cycle progression by many independent enhancers within extensive cis-regulatory regions (Meyer, 2002).

Sister chromatids fail to separate during an induced endoreplication cycle in Drosophila embryos

When mitosis is bypassed, as in some cancer cells or in natural endocycles, sister chromosomes remain paired and produce four-stranded diplochromosomes or polytene chromosomes. Cyclin/Cdk1 inactivation blocks entry into mitosis and can reset G2 cells to G1, allowing another round of replication. Reciprocally, persistent expression of Cyclin A/Cdk1 or Cyclin E/Cdk2 blocks Drosophila endocycles. Inactivation of Cyclin A/Cdk1 by mutation or overexpression of the Cyclin/Cdk1 inhibitor, Roughex (Rux), converts the 16th embryonic mitotic cycle to an endocycle; however, Rux expression fails to convert earlier cell cycles unless Cyclin E is also downregulated. Following induction of a Rux transgene in Cyclin E mutant embryos during G2 of cell cycle 14 (G214), Cyclins A, B, and B3 disappear and cells reenter S phase. This rereplication produced diplochromosomes that segregated abnormally at a subsequent mitosis. Thus, like the yeast CKIs Rum1 and Sic1, Drosophila Rux can reset G2 cells to G1. The observed cyclin destruction suggests that cell cycle resetting by Rux is associated with activation of the anaphase-promoting complex (APC), while the presence of diplochromosomes implies that this activation of APC outside of mitosis is not sufficient to trigger sister disjunction (Vidwans, 2002).

The abrupt disappearance of mitotic cyclins is generally taken as an indication of activation of the mitotic ubiquitin ligase APC and subsequent degradation. Since Rux-mediated resetting from G2 to G1 was accompanied by cyclin disappearance, it is speculated that APC activation occurs during resetting but that it is insufficient to promote sister disjunction in this context. Consistent with this, studies in Saccharomyces cerevisiae have suggested that mitotic phosphorylation acts in conjunction with APC-targeted degradation to promote chromatid disjunction. The widespread occurrence of polytene and diplochromosomes suggests that disjunction can be bypassed in diverse systems. The variety of cell cycle defects and polyploid phenotypes in cancer cell lines has suggested that production of diplochromosomes involves the complete bypass of mitosis. Perhaps the dependence of disjunction on mitotic events is general (Vidwans, 2002).

SNR1 (INI1/SNF5) mediates important cell growth functions of the Drosophila Brahma (SWI/SNF) chromatin remodeling complex

SNR1 is an essential subunit of the Drosophila Brahma (Brm) ATP-dependent chromatin remodeling complex, with counterparts in yeast (SNF5) and mammals (INI1). Increased cell growth and wing patterning defects are associated with a conditional snr1 mutant, while loss of INI1 function is directly linked with aggressive cancers, suggesting important roles in development and growth control. The Brm complex is known to function during G1 phase, where it appears to assist in restricting entry into S phase. In Drosophila, the activity of DmcycE/CDK2 is rate limiting for entry into S phase and the Brm complex can suppress a reduced growth phenotype associated with a hypomorphic DmcycE mutant. The results reveal that SNR1 helps mediate associations between the Brm complex and DmcycE/CDK2 both in vitro and in vivo. Further, disrupting snr1 function suppresses DmcycEJP phenotypes, and increased cell growth defects associated with the conditional snr1E1 mutant are suppressed by reducing DmcycE levels. While the snr1E1-dependent increased cell growth does not appear to be directly associated with altered expression of G1 or G2 cyclins, transcription of the G2-M regulator string/cdc25 is reduced. Thus, in addition to important functions of the Brm complex in G1-S control, the complex also appears to be important for transcription of genes required for cell cycle progression (Zraly, 2004).

The conditional mutant snr1E1 displays wing patterning defects and increased mitotic growth at both the permissive (18° C) and the restrictive (29° C) temperatures. The mutant phenotypes are sensitive to both temperature of incubation and snr1 gene dosage, indicating that they specifically result from reduced or compromised SNR1 function, rather than from complete disruption of Brm complex activities. In contrast to the use of null alleles that may reduce total complex number by half, snr1E1 produces a stable protein that is assembled into Brm complexes at both temperatures, thus allowing complexes to form and bind their targets, but then are defective in some other function of the complex. This point is critical for these studies, since there are significantly different effects resulting from complete loss of functional Brm complexes or activities as contrasted with impaired functions that result from the incorporation of defective subunits. To help understand the functional roles of SNR1 within the conserved Brm ATP-dependent chromatin remodeling complex during metazoan development, advantage was taken of these dosage- and temperature-dependent snr1E1 phenotypes, as well as the brmK804R dominant-negative, both of which result in the incorporation of defective subunits (Zraly, 2004).

This report shows that snr1 can genetically interact with a subset of genes involved in cell cycle control. In addition, co-immunoprecipitation of DmcycE/CDK2 and the Brm complex indicate that stable complexes could form in vivo, while both GST-pulldown and yeast two-hybrid studies suggest that residues within SNR1 might help mediate or stabilize these contacts. SNR1 is strongly conserved with counterparts in yeast (SNF5) and mammals (INI1). The most conserved portions among SNR1-related proteins occur within the ~200-amino-acid C-terminal region comprising two imperfect repeats and a coiled coil. The repeat regions are important for contacts with a variety of cellular factors, including Drosophila Bicoid, the HOX gene regulators TRX and HRX/MLL, c-MYC as well as the viral-encoded proteins HIV integrase and HPV E1. In addition, yeast SNF5 is involved in direct associations with the GAL4 transcriptional activator. Contacts with conserved features of SNR1 are important for recruiting or modulating Drosophila Brm complex functions in vivo. The SNR1/DmCDK2 interaction may also be an important conserved feature, since similar contacts are observed between SNR1 C-terminal residues and mammalian CDK2 using yeast two-hybrid assays (Zraly, 2004).

Components of the mammalian Brm complexes, including the hBrm/BRG-1 and BAF155 (MOR) subunits, are phosphorylated prior to the onset of mitosis and this modification may be important for restricting or modulating complex activity. However, the cell cycle kinase involved and specific target residues within Brm complex components have not been identified. On the basis of work from cultured mammalian cells and the results reported in this study, CycE/CDK2 appears to be among the likely candidates for important regulatory kinase functions during portions of the cell cycle (Zraly, 2004).

CDK2 is capable of forming contacts with SNR1 through the Repeat 2 and coiled-coil regions. What might be the importance of the SNR1-CDK2 interaction? SNR1 and INI1 do not contain any obvious CDK2 phosphorylation sites and SNR1 does not appear to be a phosphoprotein, since assays using a variety of general protein phosphatases produce no detectable change in SNR1 electrophoretic migration on SDS-PAGE gels. This may be misleading, since other putative phosphoproteins, including Drosophila RBF, do not change electrophoretic mobility when treated with phosphatases. However, yeast SFH1p found in the SWI/SNF-related RSC complex and a close relative of SNR1/INI1/SNF5 appears to be phosphorylated during G1 phase. Thus, while SNR1 does not appear to be the likely direct target for DmcycE/CDK2 regulation, the genetic results suggest the possibility that contacts between SNR1 and CDK2 may serve to stabilize or regulate interactions between DmcycE/CDK2 and the Brm complex or help to direct kinase activity, targeted either to other components of the Brm complex or to unknown cellular proteins (Zraly, 2004).

How might interactions between the Brm chromatin remodeling complex and DmcycE/CDK2 contribute to appropriate cell cycle regulation? A growing body of evidence strongly suggests that ATP-dependent chromatin remodeling complexes perform essential functions in controlling normal mitotic cell cycles. For example, the SWI/SNF complex is important for the expression of mitotic genes and DNA replication in yeast. In mammals, the Brm-related complexes functionally interact with histone deacetylases and pRB to block entry into S phase. As a consequence of losing or misregulating chromatin remodeling activities, normal cell cycle control is disrupted. Specifically, loss of INI1 is associated with aggressive cancers, leads to the rapid development of tumors in knockout mice, and results in G1-specific defects. Further, overexpression of Cyclin E can abrogate cell cycle arrest caused by the introduction of BRG1 into SW13 adenocarcinoma cells (Zraly, 2004).

The requirements for ATP-dependent chromatin remodeling activities during the cell cycle are likely to be quite complex, perhaps involving known functions in controlling gene transcription (activation and repression) and/or regulating aspects of chromosome replication. In cultured mammalian cells, INI1 was shown to repress cyclinD1 transcription in G1 phase through collaboration with HDAC1. Unlike mammalian cyclinD, Drosophila DmcycD is not required for entry into S phase, but has been proposed to function during G1 to regulate cell growth. While snr1E1 mutant phenotypes are sensitive to Cyclin D levels, the expression of DmcycD is unaffected in the mutant, consistent with the view that the snr1E1 growth defects are likely due to misregulation of genes downstream of DmcycE, possibly involving targets of E2F regulation (Zraly, 2004).

In addition to demonstrating Brm complex regulation of gene expression during the S and G2 phases, these results also suggest RNA PolII-independent roles in restricting S-phase entry. For example, SNR1 is excluded from mitotic chromatin during the early embryonic nuclear divisions in the absence of zygotic transcription or G1-G2 phases. During these early divisions, type II DmcycE is a potent inducer of S phase and this form exhibits strong in vivo associations with SNR1. One scenario is that the Brm complex is recruited to specific chromosomal sites by sequence-specific repressors where the complex might act to stabilize binding of the repressor and/or remodel nucleosomes in an ATP-dependent manner, thereby establishing a repressive environment to restrict replication initiation. The cellular proteins involved in potentially recruiting the Brm complex to specific loci involved in replication initiation are not presently known, but may include transcription factors, such as RBF/E2F or ORC. Recruitment of CycE/CDK2 to replication origins and interaction with SNR1 might then allow for inactivation of Brm activity and release of the complex from chromatin through phosphorylation of specific subunits. The SNR1E1 mutant protein likely compromises one or more of these interactions, reducing the effective recruitment of the Brm complex to targets that are normally repressed by Brm complex activities. This could possibly lead to compromised S-phase restriction, partly relieving the requirement for DmcycE/CDK2 activity to allow progression into S phase (Zraly, 2004).

A genetic screen for dominant modifiers of a cyclin E hypomorphic mutation identifies novel regulators of S-phase entry in Drosophila

Cyclin E together with its kinase partner Cdk2 is a critical regulator of entry into S phase. To identify novel genes that regulate the G1- to S-phase transition within a whole animal use was made of a hypomorphic cyclin E mutation, DmcycEJP, which results in a rough eye phenotype. The X and third chromosome deficiencies were screened, candidate genes were tested, and a genetic screen of 55,000 EMS or X-ray-mutagenized flies was carried out for second or third chromosome mutations that dominantly modify the DmcycEJP rough eye phenotype. Focused was placed on the DmcycEJP suppressors, S(DmcycEJP), to identify novel negative regulators of S-phase entry. There are 18 suppressor gene groups with more than one allele and several genes that are represented by only a single allele. All S(DmcycEJP) tested suppress the DmcycEJP rough eye phenotype by increasing the number of S phases in the postmorphogenetic furrow S-phase band. By testing candidates several modifier genes were identifed from the mutagenic screen as well as from the deficiency screen. DmcycEJP suppressor genes fall into five general classes: (1) chromatin remodeling or transcription factors; (2) signaling pathways, and (3) cytoskeletal, (4) cell adhesion, and (5) cytoarchitectural tumor suppressors. The cytoarchitectural tumor suppressors include scribble, lethal-2-giant-larvae (lgl), and discs-large (dlg), loss of function of which leads to neoplastic tumors and disruption of apical-basal cell polarity. The genetic interactions of scribble with S(DmcycEJP) genes were further explored and it was shown that hypomorphic scribble mutants exhibit genetic interactions with lgl, scab (alpha PS3-integrin -- cell adhesion), phyllopod (signaling), dEB1 (microtubule-binding protein -- cytoskeletal), and moira (chromatin remodeling). These interactions of the cytoarchitectural suppressor gene, scribble, with cell adhesion, signaling, cytoskeletal, and chromatin remodeling genes, suggest that these genes may act in a common pathway to negatively regulate cyclin E or S-phase entry (Brumby, 2004).

This work has led to the identification of many genes that when mutated have the ability to dominantly modify the DmcycEJP adult rough eye phenotype and S-phase defect in third instar larval eye imaginal discs. In addition to genes already known to be regulators of Drosophila cyclin E or G1-S progression [such as E2F1; retinoblastoma (Rbf); ago (cdc4) encoding a protein involved in Cyclin E degradation; the EGF receptor pathway genes Egfr and Ras85D, which act to promote Cyclin E protein accumulation, and Hh signaling pathway genes, which act to promote cyclin E transcription], this screen led to the identification of many novel cyclin E interactors. This study mainly concentrated on the suppressors of DmcycEJP, although from the deficiency screen and specifically testing candidates, axin (an inhibitor of Wg signaling), rho1, and crumbs as enhancers of DmcycEJP, which therefore may act as novel positive regulators of G1-S progression, were identified. The suppressors of DmcycEJP identified include the following classes: (1) chromatin remodeling genes brm, mor, Trl, or the transcription factor Zn72D; (2) signaling pathway genes phyl, sina, trio, Abl, RpS6, wg and Wg pathway effectors dsh and arm; (3) genes encoding cytoskeletal proteins dEB1 (encoding a microtubule-binding protein) and expanded (encoding a FERM domain cytoskeletal protein and hyperplastic tumor suppressor); (4) genes encoding cell adhesion proteins scab (encoding an alpha-integrin), cadN (N-Cadherin), shg (E-Cadherin), and fat (encoding an atypical-cadherin and hyperplastic tumor suppressor); and (5) cytoarchitectural tumor suppressor genes scribble, lgl, and dlg, required for apical-basal cell polarity and cell proliferation inhibition. While some of these genes (brm, mor, expanded, fat, scribble, and lgl) have been previously shown or implicated to play a role in negatively regulating G1-S, a potential role for Trl, Znf72D, phyl, sina, trio, Abl, RpS6, wg, dsh, arm, dEB1, scab, cadN, and shg in inhibiting G1-S progression in Drosophila is novel. Further studies are required to determine whether Abl, RpS6, wg, dsh, arm, and shg do indeed suppress DmcycEJP by acting at the S-phase level and to understand the mechanism by which these genes act in G1-S regulation. The identification of novel classes of presumptive negative regulators of cyclin E or G1-S progression highlights the power of Drosophila whole-animal genetics as a tool for revealing new cell proliferation pathways (Brumby, 2004)

Chromatin regulation mediated by Domino and PcG-like factors controls E2F activity and cell growth

Regulation of chromatin structure is critical in many fundamental cellular processes. Previous studies have suggested that the Rb tumor suppressor may recruit multiple chromatin regulatory proteins to repress E2F, a key regulator of cell proliferation and differentiation. Taking advantage of the evolutionary conservation of the E2F pathway, a genome-wide RNAi screen was conducted in cultured Drosophila cells for genes required for repression of E2F activity. Among the genes identified are components of the putative Domino chromatin remodeling complex, as well as the Polycomb Group (PcG) protein-like fly tumor suppressor, L3mbt, and the related Scm-related gene containing four mbt domains (CG16975/dSfmbt). These factors are recruited to E2F-responsive promoters through physical association with E2F and are required for repression of endogenous E2F target genes. Surprisingly, their inhibitory activities on E2F appear to be independent of Rb. In Drosophila, domino mutation enhances cell proliferation induced by E2F overexpression and suppresses a loss-of-function cyclin E mutation. These findings suggest that potential chromatin regulation mediated by Domino and PcG-like factors plays an important role in controlling E2F activity and cell growth (Lu, 2007).

This study identified the putative Dom/SWR1 chromatin remodeling complex and the PcG-like MBT domain-containing factors were identified as E2F repressors. These proteins are recruited to E2F target promoters through association with E2F and inhibit E2F in an apparently Rb-independent manner. Depletion of these genes resulted in derepression of some endogenous E2F target genes accompanied by changes in histone modification. More importantly, dom genetically interacts with the E2F pathway. These proteins show an extensive degree of evolutionary conservation, indicating the mechanism of E2F regulation provided by these factors may be well conserved (Lu, 2007).

Regulation of E2F is tightly linked to cell proliferation and differentiation. Existing evidence suggests that perturbation of the Dom and MBT proteins may cause dysregulation of these cellular processes. Apart from the fact that the heterozygous dom mutation modifies cell growth in an E2F-transgenic or a cycE hypomorphic background, fly mutants homozygous for several dom alleles show enlarged lymph glands apparently because of excessive proliferation of prehemocytes. In human, the Dom complex subunit YL1 possesses growth suppressive activity, and the Dom homolog p400 is an essential target for the viral oncoprotein E1A-mediated transformation. Indeed, overexpression of E1A disrupts the association of E2F with the Dom complex in mammalian cells. Furthermore, mutations in the fly tumor suppressor gene l3mbt result in overgrowth of the larval brain lobes and epithelial imaginal discs, and failure of neural differentiation (Wismar, 1995). This is intriguing, because in mammalian cells, many E2F-regulated genes are repressed during quiescence and differentiation, and mammalian MBT proteins are found in an inhibitory E2F complex purified from quiescent cells (Lu, 2007).

Although the mechanism of Rb-mediated repression on E2F is complex, these studies indicate that Dom and MBT possess Rb-independent activities. In support of this view, recent studies suggest that the C. elegans Dom and Rb homologs share redundant functions in vulva development, a process controlled by the E2F pathway (Ceol, 2004). In addition, these proteins may participate in distinct E2F complexes. Mammalian MBT orthologs have been identified from Rb-independent complexes, and they can associate with E2F forms lacking the Rb-binding motif, such as E2F6 and a C-terminal truncated E2F3 mutant. Interestingly, L3mbt is shown to interact with dREAM, a dE2F2-Rb complex, even though it is not a stoichiometric subunit. But unlike L3mbt, RNAi of dE2F2 and several other components of the core dREAM complex had no effect on the E2F reporter. This observation may hence indicate the existence of multiple L3mbt-containing complexes or hint at a potential collaboration among different E2F regulatory activities. So far, there is no evidence linking Dom and CG16975 to Rb. It is likely that both Rb-mediated and -independent chromatin modulations play critical roles in E2F regulation and cell proliferation. Future biochemical and genetic studies may shed light on these potentially independent and collaborative relations (Lu, 2007).

The myeloid leukemia factor interacts with COP9 signalosome subunit 3 in Drosophila melanogaster

The human myeloid leukemia factor 1 (hMLF1) gene was first identified as an NPM-hMLF1 fusion gene produced by chromosomal translocation. In Drosophila, Myelodysplasia/myeloid leukemia factor (dMLF) has been identified as a protein homologous to hMLF1 and hMLF2, which interacts with various factors involved in transcriptional regulation. However, the precise cellular function of dMLF remains unclear. To generate further insights, the behavior of dMLF protein was examined using an antibody specific to dMLF. Immunostaining analyses showed that dMLF localizes in the nucleus in early embryos and cultured cells. Ectopic expression of dMLF in the developing eye imaginal disc using eyeless-GAL4 driver resulted in a small-eye phenotype and co-expression of cyclin E rescued the small-eye phenotype, suggesting the involvement of dMLF in cell-cycle regulation. The molecular mechanism was examined of interactions between dMLF and a dMLF-interacting protein, dCSN3, a subunit of the COP9 signalosome, which regulates multiple signaling and cell-cycle pathways. Biochemical and genetic analyses revealed that dMLF interacts with dCSN3 in vivo and glutathione S-transferase pull-down assays revealed that the PCI domain of the dCSN3 protein is sufficient for this to occur, possibly functioning as a structural scaffold for assembly of the COP9 signalosome complex. From these data the possibility is proposed that dMLF plays a negative role in assembly of the COP9 signalosome complex (Sugano, 2008).

MLF family proteins are novel intracellular factors whose in vivo functions remain largely unknown. This study analyzed the function of Drosophila MLF through its expression pattern, subcellular localization and molecular mechanisms of binding to a dMLF-interacting protein to generate further insights into dMLF and MLF family proteins. It is reported that dMLF proteins are largely cytoplasmic in early blastoderm embryos prior to cellularization, although they progressively accumulate in the nuclei thereafter. In addition, strong cytoplasmic localization of dMLF was observed in cultured Kc cells in a previous study. However, in this study, immunostaining of Drosophila embryos and cultured cells revealed that dMLF primarily localizes in the nucleus in early embryos and mainly in the nuclear envelope with a slight accumulation in cytoplasm and nucleoplasm in cultured cells. Accumulation in the peripheral region of the inner nuclear membrane was also observed in some embryos, consistent with the reported nuclear localization of dMLF with some concentration inside the nuclear envelope and in the perinuclear region in the salivary glands of Drosophila larvae. The discrepancies are probably not due to the antibodies used in the experiments, because these results are found using another antibody. Thus, differences between observations may result from variation in the physiological conditions of cells and/or fixation processes for immunostaining analyses (Sugano, 2008).

In a previous study, MLF1 in cultured mammalian cells was seen to localize in both the cytoplasm and the nucleus. It was also noted that MLF1 was not evenly distributed in the cytoplasm, but was more concentrated in the perinuclear region including centrosomes. In addition, it has been suggested that MLF1 translocates between the nucleus and cytoplasm. In this study, dMLF was detected not only in the nucleoplasm, but also in the nuclear envelope during early embryogenesis. Taking the available information, it is likely that dMLF shuttles between the cytoplasm and the nucleus depending on other protein factors or physiological conditions of cells. Indeed, it has been reported that mammalian MLF1 interacts with 14-3-3zeta, which is involved in subcellular localization and shuttling between the nucleus and cytoplasm. Immunoblot analysis with anti-dMLF IgG suggested that some modified forms of dMLF protein are present in embryonic and larval-pupal stages. It has been reported that a serine kinase recruited by MADM phosphorylates MLF1 at the 14-3-3 binding motif in mammals. As with MLF1, both MADM and 14-3-3 are also conserved in Drosophila, and it is possible that dMLF also interacts with Drosophila MADM to become phosphorylated. It is also conceivable that 14-3-3 interacts with phosphorylated dMLF and affects its subcellular localization, although it remains to be confirmed that dMLF does in fact undergo phosphorylation (Sugano, 2008).

In this study it was observed that the dMLF-induced small-eye phenotype was suppressed by a half-dose reduction in the dCSN3 gene. These observations appear to be consistent with the report describing that knockdown of CSN3 rescued hMLF1-induced growth inhibition of NIH 3T3 cells (Yoneda-Kato, 2005). It has been reported that amino acids 50-125 of hMLF1 are required for CSN3-binding (Yoneda-Kato, 2005). In the case of Drosophila, amino acid region 1-202 is necessary for the interaction between dMLF and dCSN3. Therefore, in both mammals and Drosophila, the C-terminal region of MLF appears to be dispensable for CSN3 binding (Sugano, 2008).

This study has shown that dMLF interacts genetically with cyclin E, and dCSN3 was identified as a dMLF-interacting protein. GST pull-down assays revealed that dCSN3 interacts with dMLF via its PCI domain. It has been reported that the C-terminal half of CSN1 and CSN2 encompassing the PCI domain is required for incorporation of the subunit into the CSN complex in mammals. It has also been suggested that the N-terminal part of CSN3 is not essential for binding to CSN1 and CSN2, in contrast to the PCI domain in the C-terminal part of CSN3. Furthermore, mutational analysis showed that PCI domains are important for assembly of the regulatory particle of the 26S proteasome in budding yeast. Thus the PCI domain may function as a structural scaffold to assemble the CSN complex and its dMLF binding may inhibit the integration of dCSN3. It is reported that a null mutation in csn4 results in a complete loss of the entire CSN complex in Drosophila, indicating that deletion of one subunit of the CSN complex may lead to disassembly of the entire complex. Therefore, it is possible that this might occur with masking of dCSN3 by dMLF. Multiple CSN subunits can be found in subcomplexes of molecular mass lower than the intact 500 kDa CSN complex, and individual CSN subunits are also linked through protein-protein interactions to a broad range of cellular processes. Genetic analyses of flies expressing dMLF revealed a genetic interaction with cyclin E, implying negative regulation of cyclin E functions in vivo. It has been shown previously that the CSN regulates degradation of cyclin E in Drosophila. It is therefore hypothesize that the proper function of CSN requires regulation of integration of the CSN complex by dMLF binding to CSN3 (Sugano, 2008).

To examine the involvement of cell death, flies expressing dMLF were crossed with others expressing an apoptosis inhibitor protein, p35. The small-eye phenotype induced by dMLF overexpression was not suppressed by expression of p35. It is therefore suggested that the phenotype is not due to induction of apoptosis. However, the dMLF-induced small-eye phenotype was enhanced by expression of p35. There are no clear interpretations to these observations at the moment. Further analyses are necessary to address this point (Sugano, 2008).

Cyclin E controls Drosophila female germline stem cell maintenance independently of its role in proliferation by modulating responsiveness to niche signals

Stem cells must proliferate while maintaining 'stemness'; however, much remains to be learned about how factors that control the division of stem cells influence their identity. Multiple stem cell types display cell cycles with short G1 phases, thought to minimize susceptibility to differentiation factors. Drosophila female germline stem cells (GSCs) have short G1 and long G2 phases, and diet-dependent systemic factors often modulate G2. Previous studies have observed that Cyclin E (CycE), a known G1/S regulator, is atypically expressed in GSCs during G2/M; however, it has remained unclear whether CycE has cell cycle-independent roles in GSCs or whether it acts exclusively by modulating the cell cycle. In this study, CycE activity was detected during G2/M, reflecting its altered expression pattern, and it was shown that CycE and its canonical partner, Cyclin-dependent kinase 2 (Cdk2), are required not only for GSC proliferation, but also for GSC maintenance. In genetic mosaics, CycE- and Cdk2-deficient GSCs are rapidly lost from the niche, remain arrested in a G1-like state, and undergo excessive growth and incomplete differentiation. However, it was found that CycE controls GSC maintenance independently of its role in the cell cycle; GSCs harboring specific hypomorphic CycE mutations are not efficiently maintained despite normal proliferation rates. Finally, CycE-deficient GSCs have an impaired response to niche bone morphogenetic protein signals that are required for GSC self-renewal, suggesting that CycE modulates niche-GSC communication. Taken together, these results show unequivocally that the roles of CycE/Cdk2 in GSC division cycle regulation and GSC maintenance are separable, and thus potentially involve distinct sets of phosphorylation targets (Ables, 2013).

Mitotic cell rounding accelerates epithelial invagination

Mitotic cells assume a spherical shape by increasing their surface tension and osmotic pressure by extensively reorganizing their interphase actin cytoskeleton into a cortical meshwork and their microtubules into the mitotic spindle. Mitotic entry is known to interfere with tissue morphogenetic events that require cell-shape changes controlled by the interphase cytoskeleton, such as apical constriction. However, this study shows that mitosis plays an active role in the epithelial invagination of the Drosophila tracheal placode. Invagination begins with a slow phase under the control of epidermal growth factor receptor (EGFR) signalling; in this process, the central apically constricted cells, which are surrounded by intercalating cells, form a shallow pit. This slow phase is followed by a fast phase, in which the pit is rapidly depressed, accompanied by mitotic entry, which leads to the internalization of all the cells in the placode. It was found that mitotic cell rounding, but not cell division, of the central cells in the placode is required to accelerate invagination, in conjunction with EGFR-induced myosin II contractility in the surrounding cells. It is proposed that mitotic cell rounding causes the epithelium to buckle under pressure and acts as a switch for morphogenetic transition at the appropriate time (Kondo, 2013).

The invagination of epithelial placodes converts flat sheets into the three-dimensional structures that form complex organs, and it is a key morphogenetic process in animal development. A major mechanism of invagination is apical constriction, which is driven by actomyosin contraction. However, not all constricted cells invaginate, and some cell internalization occurs without apical constriction, suggesting that additional mechanisms of inward cell movement contribute to invagination (Kondo, 2013).

To obtain three-dimensional information about cell behaviour during invagination, live imaging was performed of the Drosophila tracheal placode. Ten pairs of tracheal placodes, each of which is composed of about 40 cells, are formed in the ectoderm at mid-embryogenesis, and each placode initiates invagination simultaneously. Using an adherens junction marker, DE-cadherin-green fluorescent protein (E-cad-GFP), it was found that the adherens junctions of the central placode cells slowly created a depression by apical constriction, which became the tracheal pit. After 30 to 60 min of slow movement (slow phase), the tracheal pit was suddenly enlarged, and the tracheal cells were rapidly internalized (fast phase) and eventually formed L-shaped tube structures (Kondo, 2013).

After the fast transition, all the tracheal cells and surrounding epidermal cells entered mitosis 16, the final round of embryonic mitosis. It was noticed that the fast invagination was always associated with the mitotic entry of central cells that were frequently the first to enter mitosis 16. Intriguingly, mitotic rounding of the central constricted cells occurred simultaneously with the rapid depression of their apices, followed by chromosome condensation 10 min later. In this study, this atypical mitotic rounding associated with apical depression in an internalized cell is called 'rounding', to distinguish it from canonical surface mitosis (surface cell rounding) (Kondo, 2013).

To determine whether cell rounding is required for invagination, zygotic mutants were examined of the cell-cycle gene Cyclin A (CycA), which fail to enter mitosis 16, and double parkeda3 (dupa3), which show a prolonged S phase 16 and delayed entry into mitosis 16. Tracheal invagination was initiated normally in the CycA and dupa3 mutants, but proceeded more slowly than in controls, indicating that entry into mitosis 16 is required for proper timing of the fast phase (Kondo, 2013).

Although delayed, the accelerated invagination in the CycA or dupa3 mutants eventually occurred, allowing the formation of tube structures and suggesting that additional mechanisms are involved. After invagination, fibroblast growth factor (FGF) signalling is activated in the tracheal cells to induce branching morphogenesis through chemotaxis. To examine the contribution of FGF signalling to invagination, mutants of the FGF ligand branchless (bnl) or the FGF receptor breathless (btl) were analyzed. These mutants invaginated normally, indicating that chemoattraction to FGF is dispensable for invagination (Kondo, 2013).

Next, to assess FGF's role in the mitosis-defective condition, double mutants were analyzed for CycA and bnl or CycA and btl, and it was found that they showed slower invagination than CycA single mutants. Furthermore, the invagination in these double mutants was incomplete, in that the cells failed to form L-shaped tubular structures. Therefore, FGF signalling is critical for invagination when mitosis is blocked, serving a back-up role. Tracheal-specific CycA expression rescued the defects in invagination speed and tube structure in the CycA btl mutants. In addition, mitosis of cells outside the pit was occasionally observed that occurred before the mitosis of the central apically constricted cells and was not correlated with the fast invagination phase. Thus, mitosis of the surrounding epidermal cells is dispensable for tracheal invagination. Taken together, it is concluded that mitotic entry of central cells is a major mechanism for accelerating tracheal invagination (Kondo, 2013).

To distinguish the role of cell rounding from that of cell division in the fast phase, the microtubule inhibitor colchicine was used to arrest the cell cycle after cell rounding. Colchicine treatment after mitosis 15 induced M-phase arrest at mitosis 16, but the fast invagination movement accompanied by cell rounding was not affected. This result indicates that cell rounding, but not cell division, is responsible for the acceleration phase of the tracheal invagination (Kondo, 2013).

Mitosis of cells in the columnar epithelium normally occurs at the apical surface after surface rounding. It was next asked how the apical surface of the central cells becomes depressed during internalized cell rounding. One possible model explains internalized cell rounding as cell-autonomously controlled by the association of the cells with the basement membrane or underlying mesodermal cells. However, genetic removal of basement-membrane adhesion by the maternal and zygotic mutation of βPS-integrin (also known as mys) did not compromise the speed of invagination, and snail-twist double-mutant embryos, which lack mesodermal cells, still showed tracheal invagination with internalized cell rounding. These results suggest that anchoring to the basal side is probably not required (Kondo, 2013).

A second model proposes that the apical depression of the rounding cells is driven by local planar interactions among the tracheal cells. Before and during tracheal invagination, myosin II is enriched at the cell boundaries tangential to the centre of the placode and regulates cell intercalation. It was noted that the myosin II level in the central cells was lower than in the surrounding, intercalating cells. Nevertheless, the apices of the central cells were constricted during the slow phase, strongly suggesting that the surrounding cells exerted centripetal pressure on the central cells through myosin II cables. Myosin II cables fail to form in EGFR signalling mutants (such as rho, the rhomboid endopeptidase required for EGF ligand maturation, and Egfr), and apical constriction is impaired in these mutants. The first few cells undergoing mitosis 16 in the tracheal placode of rho or Egfr mutants showed surface cell rounding with expanded apices, indicating that EGFR signalling is required to couple the mitotic cell rounding with fast apical depression. It is speculated that the columnar shape of the central cells resists centripetal movements, resulting in the accumulation of inward pressure during the slow phase. The existence of such resistance was supported by the results of a physical perturbation experiment using a pulsed ultraviolet lase. The cell rounding associated with mitotic entry would release the stored inward pressure by means of cytoskeletal remodelling that causes rapid depression of apical surface together with the active shortening of cell height, leading to rapid buckling of the apical surface and the fast phase of invagination (Kondo, 2013).

Even with the loss of both EGFR and FGF signalling, the tracheal placodes form moderately invaginated structures, compared to the flat tracheal placode observed in the rho-bnl-CycA triple mutant at the same stage, indicating that cells needed to undergo mitosis 16 to induce invagination, independent of EGFR and FGF signalling. In rho bnl double mutants, although the cells undergoing the earliest mitoses showed surface cell rounding, some of the subsequent mitotic events were coupled to apical depression and internalized cell rounding. Unlike the earlier mitotic events on the surface, the internalized rounding cells in the rho bnl embryos showed constricted apices and were surrounded by apically rounded cells before mitosis. Internalized rounding with a constricted apical surface were shared properties of cells in mitoses leading to invagination, in both control and rho bnl embryos. It is suggested that the first few cells undergoing surface cell rounding compress the adjacent interphase cells and restrict their apical area, so that they are forced to move internally after rounding, causing the epithelial layer to buckle and invaginate (Kondo, 2013).

Although invagination was largely blocked in the rho-bnl-CycA triple mutants, any double mutant combination permitted invagination to some degree, indicating that three qualitatively distinct mechanisms, mitotic cell rounding, myosin II contractility (EGFR) and active cell motility (FGFR), can independently trigger invagination. In the normal context of wild-type development the combination of cell rounding and EGFR signalling may optimize the timing and speed of invagination, and then invaginated tracheal sacs subsequently respond to FGF emanating from several target tissues guiding branching morphogenesis (Kondo, 2013).

These observations demonstrates a new role for mitosis in tissue morphogenesis to generate mechanical force through cell rounding, independent of cell division. This is distinct from previously described invagination mechanisms involving cell-autonomous constriction by the apical activation of actomyosin contractility, which is incompatible with mitosis. Mitosis 16 outside the tracheal placode occurs in clusters on the ectoderm surface, but does not lead to invagination, suggesting that the tracheal placode is sensitized to invaginate upon mitosis, independent of EGFR and FGFR signalling. Future research to uncover the properties of the tracheal placode that enables it to respond to clustered mitosis will explain not only this new mode of morphogenesis, but also the homeostasis mechanisms of epithelial architecture (Kondo, 2013).

The Drosophila endocycle is controlled by Cyclin E and lacks a checkpoint ensuring S-phase completion

The Drosophila endocycle is controlled by Cyclin E. In oogenesis, nurse cells become polyploid, and the major satellite DNAs, associated with heterochromatin, become under-represented, while euchromatic DNA is preferentially replicated. A hypomorphic, female sterile cyclin E mutation increases the amount of satellite DNA propagated in nurse cells. In mutant but not wild-type endomitotic nurse cells, "late S" patterns of DNA synthesis are similar to those in mitotic cells, that is heterochromatic satellite DNAs are replicated. Cyclin E protein still cycles in germ cell cysts of cycE hypomorphic mutants, but at reduced levels, and it is found throughout a polyploid S phase, rather than undergoing the normal process of degredation. Ectopic expression of cyclin E produces late DNA replication in polyploid nuclei, phenocopying the hypomorphic cyclin E mutation. These experiments support the view that oscillating levels of Cyclin E control the polyploid S phase. A checkpoint normally links the presence of unreplicated DNA to the Cyclin E oscillator. This checkpoint is lacking in cyclin E mutants and overproducers, leading to replication of late-replicating sequences such as satellite DNAs. Unexpectedly, two to three of the 16 cells in hypomorphic cycE mutant cysts frequently differentiate as oocytes, implicating cell-cycle programming in oocyte determination (Lilly, 1996).

The p27cip/kip ortholog dacapo maintains the Drosophila oocyte in prophase of meiosis I

Animal oocytes undergo a highly conserved developmental arrest in prophase of meiosis I. Often this marks a period of rapid growth for the oocyte and is necessary to coordinate meiotic progression with the developmental events of oogenesis. In Drosophila, the oocyte develops within a 16-cell germline cyst. Throughout much of oogenesis, the oocyte remains in prophase of meiosis I. By contrast, its 15 mitotic sisters enter the endocycle and become polyploid in preparation for their role as nurse cells. How germline cysts establish and maintain these two independent cell cycles is unknown. This study demonstrates a role for the p21CIP/p27Kip1/p57Kip2-like cyclin-dependent kinase inhibitor (cki) dacapo in the maintenance of the meiotic cycle in Drosophila oocytes. The data indicate that it is through the differential regulation of the cki Dacapo that two modes of cell-cycle regulation are independently maintained within the common cytoplasm of ovarian cysts (Hong, 2003).

In females homozygous for the hypomorphic mutation cycE01672, a fraction of egg chambers contain two cells that have oocyte-like nuclear features, such as low ploidy values, an endobody and a small DNA mass in a very large nucleus. Egg chambers that contain two oocyte nuclei have only 14 polyploid nurse cells, indicating that a cell that was destined to develop as a nurse cell has been partially transformed towards the oocyte fate. The extra oocyte nucleus (which can be distinguished from the true oocyte by its presence in a small cell that lacks signs of cytoplasmic oocyte differentiation), almost invariably is the other four-ring canal cell in the cyst. Interestingly, these transformed nuclei accumulate persistently high levels of Dap protein in a manner similar to the true oocyte. Cells with persistently high levels of Dap have low ploidy values, indicating they have either not entered the endocycle or have prematurely exited the cycle. By contrast, in wild-type egg chambers the other four-ring canal cell develops as a highly polyploid posterior nurse cell in which Dap levels oscillate. Thus, mutations in dap result in germline cysts in which all 16 cells enter the endocycle and develop as nurse cells, while a mutation in cycE has the opposite effect, resulting in two or more cells that have persistently high levels of Dap that cannot enter and/or maintain the endocycle (Hong, 2003).

To examine the relationship between cycE and dap in the regulation of the cell-cycle program of ovarian cysts, whether mutations in dap could dominantly modify the cycE01672 two oocyte phenotype was examined. Reducing the dose of the dap gene by half, results in ~2.5 fold suppression of the cycE01672 two oocyte phenotype. In wild-type egg chambers the four posterior nurse cells, which are connected to the oocyte via ring canals, have the highest ploidy values in the cyst. In cycE01672 females 37±7% of egg chambers contain a cell adjacent to the true oocyte with inappropriately low ploidy values. When a single copy of the null allele dap4 was placed in the cycE01672 background, fewer than 14±7% of egg chambers had a posterior nurse cell with a reduced DNA content. These data indicate that whether a cyst cell enters and/or maintains the endocycle is at least partially determined by the balance of CycE and Dap. In addition, they strongly suggest that, as is observed during embryogenesis, the primary target of Dap in the ovary is the CycE/Cdk2 complex (Hong, 2003).

These observations suggest a model for how the meiotic cycle and the endocycle are independently maintained within Drosophila ovarian cysts. It is proposed that the presence of high levels of the cki Dap in the oocyte (throughout the time the nurse cells are in the endocycle) persistently inhibits cyclinE-Cdk2 kinase activity and prevents inappropriate DNA replication during meiosis. Without the inhibition of cyclinE-Cdk2 kinase activity provided by high levels of Dap, the majority of dap mutant oocytes abandon the meiotic cycle and enter the endocycle with the nurse cells. Importantly, these data indicate that, as has recently been observed in mice, oocytes in prophase of meiosis I are competent to replicate their DNA. In the mouse oocyte, the inhibition of DNA replication during prophase of meiosis I may be accomplished through the downregulation of the G1 cyclins and Cdk2. In the Drosophila oocyte, the inhibition of cyclinE-Cdk2 activity by Dap achieves the same aim. In contrast to the oocyte, the nurse cells require a period when Dap levels are low to allow cyclinE-Cdk2 kinase activity to rise high enough to trigger each endocycle S phase. These low points occur during the oscillations of the Dap protein. The data indicate that it is through the differential regulation of Dap that two apparently incompatible cell cycles are stably maintained within the common cytoplasm of the ovarian cyst (Hong, 2003).

The regulatory relationship between cyclinE-Cdk2 activity and the Dap ortholog p27 suggest a feedback loop that may account for the long-term stabilization of Dap in post germarial oocytes. In mammalian cells, phosphorylation by cyclinE-Cdk2 targets the p27 protein for destruction by the proteasome. Similarly, the Dap protein contains a CDK phosphorylation consensus site (Ser205) and can be phosphorylated by mammalian cyclinE-Cdk2 in vitro. It is proposed that in early stage 1 egg chambers, the balance of cyclinE-Cdk2 activity and Dap protein is slightly different in the 15-nurse cells versus the single oocyte. In the oocyte, the balance is tipped towards the inhibitor Dap, resulting in diminished cyclinE-Cdk2 activity. Lower cyclinE-Cdk2 activity leads to a reduced rate of Dap phosphorylation and proteolysis, thereby increasing the concentration of the Dap protein. The stabilization of the Dap protein ultimately results in the permanent inhibition of cyclinE-Cdk2 activity in the oocyte. In contrast to the oocyte, in stage 1 nurse cells cyclinE-Cdk2 kinase activity reaches high enough levels to trigger the phosphorylation and subsequent destruction of the Dap protein, thus allowing endocycle progression. The above model predicts that additional proteins that are targeted for destruction by cyclinE-Cdk2 phosphorylation should be stabilized in the oocyte but not in the nurse cells. Like p27, the proteolytic destruction of CycE itself is dependent on phosphorylation by the cyclinE-Cdk2 complex. As predicted by the model, CycE is stabilized in the oocyte and accumulates to high levels as oogenesis progresses. This model allows the amplification of a slight difference in the balance of cyclinE-Cdk2 activity and Dap early in oogenesis, resulting in the two cell types of the germline cyst permanently adopting dramatically different cell cycles (Hong, 2003).

Coupling of Hedgehog and Hippo pathways promotes follicle stem cell maintenance by stimulating proliferation

It is essential to define the mechanisms by which external signals regulate adult stem cell numbers, stem cell maintenance, and stem cell proliferation to guide regenerative stem cell therapies and to understand better how cancers originate in stem cells. This paper shows that Hedgehog (Hh) signaling in Drosophila melanogaster ovarian follicle stem cells (FSCs) induces the activity of Yorkie (Yki), the transcriptional coactivator of the Hippo pathway, by inducing yki transcription. Moreover, both Hh signaling and Yki positively regulate the rate of FSC proliferation, both are essential for FSC maintenance, and both promote increased FSC longevity and FSC duplication when in excess. It was also found that responses to activated Yki depend on Cyclin E induction while responses to excess Hh signaling depend on Yki induction, and excess Yki can compensate for defective Hh signaling. These causal connections provide the most rigorous evidence to date that a niche signal can promote stem cell maintenance principally by stimulating stem cell proliferation (Huang, 2014).

Cyclin E: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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