Regulation of Dacapo expression

Expression of the cyclin-dependent kinase inhibitor Dacapo is regulated by Cyclin E

The Cip/Kip family of cyclin-dependent kinase inhibitors (CKIs) has been implicated in mediating cell cycle arrest prior to terminal differentiation. In many instances, increased expression of CKIs immediately precedes mitotic arrest. However, the mechanism that activates CKI expression in cells that are about to stop dividing has remained elusive. This issue was addressed by investigating the expression pattern of dacapo, a Cip/Kip CKI in Drosophila. The accumulation of Dacapo RNA and protein requires Cyclin E and ectopic expression of Cyclin E can induce dacapo expression. The oscillation of the Cyclin E and Dacapo proteins is tightly coupled during ovarian endocycles. These results argue for a mechanism where Cyclin E/Cdk activity induces Dacapo expression but only within certain windows that are permissive for dacapo expression (de Nooij, 2000).

In a number of different tissues, expression of Dap occurs precisely during the last mitotic division that cells undergo before they terminally differentiate. For instance, in the embryonic epidermis, a rapid accumulation of DAP mRNA and Dap protein is detected only following S-phase 16, just before these cells arrest in the G1-phase of cycle 17. Exit from the cell cycle also requires alterations in the levels of Cyclins and the activity of Cyclin-Cdk complexes. Is the induction of dap directly regulated by the developmental cues that dictate cell cycle exit? Or alternatively, is the induction of DAP RNA and protein a response to an alteration in the activity of one of the other cell cycle regulators (de Nooij, 2000)?

To help distinguish between these possibilities, whether alterations in the levels of one of the known cell cycle regulators would affect the normal pattern of Dap expression was examined in different embryonic tissues. Embryos that were mutant for the G1-S regulators, cyclin E, E2F1 and DP and for the regulators that mediate the G2-M transition, string (stg), cyclin A, rca1, cyclin B and cdc2 were examined. With the notable exception of cyclin E mutants, no obvious abnormalities in the Dap expression pattern could be observed in any of these mutants. Analysis of cyclin E (cycE) mutant embryos, however, shows significant differences in the expression pattern of Dap. Dap protein levels are severely reduced in the cells of the PNS and are almost absent in the cells of the CNS of embryos homozygous or hemizygous for the cycE null allele cycEAr95. The level of DAP RNA is also reduced in the cells of the PNS and the CNS. This suggests that Cyclin E may regulate dap expression at least in part at the transcriptional level. Immunostaining of cycE mutant embryos with an anti-Elav antibody does not show significant abnormalities in the cellularity of the PNS and CNS, suggesting that the loss of dap expression is not a secondary consequence of a failure to form these tissues (de Nooij, 2000).

Interestingly, no reduction in Dap expression levels is observed in the epidermis of cycE mutant embryos. However, cycE mutants have been shown to complete all 16 epidermal divisions normally, possibly due to the activity of residual maternal supplies of Cyclin E. It is therefore likely that the residual supply of Cyclin E may also be sufficient to trigger Dap expression in the epidermis. Alternatively, the apparently normal expression of Dap in the cycE mutant epidermal cells may reflect a difference between the mechanisms that regulate Dap expression in the epidermis and in the nervous system (de Nooij, 2000).

These results also show that normal Dap expression is not contingent upon progression through the cell cycle. Embryos mutant for string, which encodes a Cdc25-like phosphatase, arrest in the G2 phase of cycle 14. Despite the absence of S-phase progression and cell division in string mutants, the onset of Dap expression occurs at the appropriate developmental stage in both the epidermis and the cells of the nervous system. These observations also show that Dap expression is not normally contingent upon cell cycle progression or on reaching cycle 16 in the epidermis (de Nooij, 2000).

The reduced level of dap RNA in cycE mutant embryos seems to indicate a role for Cyclin E in the transcriptional regulation of dap. A potential mechanism for the transcriptional regulation of dap by Cyclin E could involve the E2F transcription factor. It has been demonstrated previously that E2F can mediate the activation of a 'G1-S transcriptional program' that, in the CNS, is dependent upon cyclin E function. E2F can also activate stg expression during G2. However, while E2F1 and DP mutant embryos clearly show a reduction in the RNA levels of the E2F responsive genes PCNA and RNR2, Dap expression appears to be normal in both E2F and DP mutant embryos (de Nooij, 2000).

Cyclin E is also expressed normally in the CNS in E2F and DP mutant embryos. Thus, a redundant function for E2F in regulating dap expression cannot be ruled out, these experiments do not provide any evidence for E2F as a regulator of dap expression. Taken together, these results show that when Cyclin E activity is abolished in the nervous system, Dap expression is no longer observed thus indicating a requirement for Cyclin E activity in regulating Dap expression. This activity of Cyclin E does not appear to involve the activity of the E2F transcription factor. Moreover, Dap expression in either the epidermis or the nervous system is not contingent upon cell cycle progression (de Nooij, 2000).

In both eye and wing imaginal discs, ectopic Cyclin E can induce ectopic expression of Dap. To determine whether the induction of Dap is due to an increase in DAP RNA levels, RNA levels of DAP were examined in whole-mount preparations by in situ hybridizations in eye and wing discs in which Cyclin E was ectopically expressed. In contrast to the dramatic increase in Dap protein, relatively modest increases in the levels of DAP RNA are observed in both the eye and wing discs. Nonetheless, these results are consistent with the notion that Cyclin E regulates DAP RNA levels, and furthermore, suggest that dap transcription may be responsive to Cyclin E levels (de Nooij, 2000).

To test whether dap regulatory elements are responsive to Cyclin E levels, several lines of transgenic flies were generated that contain a 2.7 kb fragment of the dap promoter region linked to a beta-galactosidase reporter. Immunohistochemical analysis of wing discs from flies that contain the pCasdap2.7kb-lacZ transgene either alone, or in the presence of the enGAL4 driver, shows no significant lacZ staining in the posterior compartment of the wing disc. In the presence of both the enGAL4 driver and a UAScycE transgene, increased beta-galactosidase activity is clearly detected in the region where Cyclin E is expressed. Thus Cyclin E can activate the expression of a reporter gene under the control of dap regulatory elements (de Nooij, 2000).

Since Cyclin E appears to be a requirement for the expression of the Dap protein, Dap expression was examined in the context of oscillating levels of Cyclin E in the nurse cell nuclei in the ovary. Oogenesis normally starts with four mitotic germ cell divisions which generate a 16-cell cyst. Surrounded by somatically-derived follicle cells, each cyst forms an individual egg chamber that ultimately gives rise to a mature egg. One of the 16-germ cell nuclei arrests in the prophase of meiosis I, becomes transcriptionally silent and is specified as the oocyte nucleus. The remaining 15 cells, the nurse cells, proceed through a series of endocycles. In wildtype ovaries, Dap expression is first detected in the germ cell nuclei in the germarium at a time when the four mitotic cyst cell divisions are about to be completed (region 2A of oogenesis). In the maturing egg chambers Dap is detected in the endocycling nurse cell nuclei at levels which, within an individual egg chamber, vary from very high to undetectable. This is likely to reflect an oscillation in the level of Dap protein during the endocycles, as has been postulated for Cyclin E. In contrast to the nurse cell nuclei, high levels of Dap are observed in the oocyte nucleus throughout oogenesis (de Nooij, 2000).

Consistent with the immunostaining, DAP RNA can be observed in the germarium early in oogenesis, and subsequently, high levels of DAP RNA are found in the future oocyte throughout oogenesis. However, the pattern of DAP RNA expression in the nurse cells differs significantly from the pattern of Dap protein. In contrast to the dramatic oscillation of Dap protein levels in the nurse cell nuclei, no oscillation of DAP RNA is evident. Only a uniform low level of RNA can be detected in the endocycling nurse cells. Although relative differences may exist but may be beyond the level of detection, this difference in the expression pattern between DAP RNA and protein suggests that post-transcriptional mechanisms may regulate the levels of Dap protein in the individual nurse cell nuclei (de Nooij, 2000).

The expression pattern of Dap protein is reminiscent of the expression pattern of Cyclin E in the ovary, i.e. a strong oscillation in the nurse cell nuclei, and a persistently high level of protein in the oocyte nucleus. Confocal images of ovaries double labeled with antibodies against both Cyclin E and Dap show that the expression of Cyclin E and Dap are largely overlapping. However, some of the nurse cell nuclei in the individual egg chambers do differ in the relative levels of Cyclin E and Dap protein, suggesting that the oscillations of Cyclin E and Dap are slightly out of phase. To assess the regulatory relationship between Cyclin E and Dap in the nurse cells more directly, Dap expression in ovaries was obtained from females homozygous for the cycEfs(2)01672 allele. This allele of cycE specifically perturbs the oscillation of Cyclin E in the nurse cell nuclei and hence ovaries from homozygous cycEfs(2)01672 females show a severe reduction in the amplitude of Cyclin E oscillation. Interestingly, these mutant ovaries also show a strong reduction in Dap oscillation, and most nurse cell nuclei show intermediate levels of Dap expression; nuclei with high or undetectable levels of Dap were almost never observed in mutant egg chambers. Thus in the nuclei of the endocycling nurse cells, expression of Cyclin E and Dap appears to be tightly linked. Dap protein levels oscillate strongly and these oscillations are dampened by a reduction in the extent of Cyclin E oscillation. Since DAP RNA levels in the nurse cells seem to remain relatively constant during oogenesis, it appears likely, that in these cells, Cyclin E controls the level of Dap protein predominantly by post-transcriptional mechanisms (de Nooij, 2000).

The regulation of dap at the transcriptional level seems unlikely to account fully for the dynamic expression pattern of Dap protein. In the situations where Cyclin E was overexpressed, the increase in Dap protein is much more dramatic than the increase in DAP RNA. Likewise, in the ovary, dramatic oscillations in Dap levels are observed without appreciable fluctuations in the levels of DAP RNA. Thus, in these situations, Cyclin E seems to regulate dap at a post-transcriptional level. This is perhaps reminiscent of the type of regulation observed for mammalian p27. Accumulation of p27 has been shown to depend largely on an increased translation of p27 mRNA. Although mammalian Cyclin E has, so far, not been implicated in the translational regulation of p27 or any of the other CKIs, such a mechanism could potentially operate in mammalian cells. Another mode of post-transcriptional regulation that may involve Cyclin E activity is a regulation at the level of protein stability (de Nooij, 2000).

The pan-neural bHLH Proteins Deadpan and Asense regulate mitotic activity and cdk inhibitor dacapo expression in the Drosophila larval optic lobes

Developmental regulators and cell cycle regulators have to interface in order to ensure appropriate cell proliferation during organogenesis. An analysis of the roles of the pan-neural genes deadpan and asense defines critical roles for these genes in regulation of mitotic activities in the larval optic lobes. Loss of deadpan results in reduced cell proliferation, while ectopic deadpan expression causes over-proliferation. In contrast, loss of asense results in increased proliferation, while ectopic asense expression causes reduced proliferation. Consistent with these observations endogenous Deadpan is expressed in mitotic areas of the optic lobes, and endogenous Asense is expressed in cells that will become quiescent. Altered Deadpan or Asense expression results in altered expression of the cyclin dependent kinase inhibitor gene dacapo. Thus, regulation of mitotic activity during optic lobe development may, at least in part, involve deadpan and asense mediated regulation of the cyclin dependent kinase inhibitor gene dacapo (Wallace, 2000).

Cdk inhibitors have been shown to represent key regulators of mitotic activity. In Drosophila a cdk inhibitor gene, dap, has been identified that is transiently expressed during embryogenesis in cells prior to entering their last mitosis and at the onset of terminal differentiation. Ectopic expression of dap results in G1 arrest, while loss of dap function has been shown to cause one extra cell division in embryonic epidermal cells. Dpn appears to promote the continuation of mitotic activity, while Ase has a role in ending cell proliferation in the developing optic lobes. Therefore, it was asked whether altered expression of Dpn and Ase can modulate the expression of the dap. In wild-type third instar larva, optic lobe expression of dap occurs in specific domains. dap is expressed in cells of the lamina furrow and scattered cells of the lamina. There is also strong expression of dap in a subset of cells in the IPC throughout third instar. In contrast, dap expression is virtually absent from the cells of the OPC (Wallace, 2000).

The effects were determined of the loss of dpn function on the expression of dap. In homozygous dpn1 mutant third instar larva, expression expands into the area of the OPC. Also, cells of the lamina begin to express dap more strongly. In contrast, in larvae with ectopic Dpn expression, dap expression is strongly reduced or absent in the optic lobes of third instar larva. Thus, dpn activity has a negative regulatory effect on the dap RNA level (Wallace, 2000).

In homozygous ase mutant third instar larvae, there is a strong reduction of dap RNA throughout the entire developing optic lobe while dap expression in the developing eye disk appears normal. The ase loss of function phenotype demonstrates that ase activity is necessary for the expression of dap throughout the developing optic lobe. When Ase is ectopically expressed in third instar optic lobes, ectopic activation of dap expression becomes evident. Therefore, ase activity has a positive regulatory effect on the dap RNA level (Wallace, 2000).

It was asked whether the phenotypical effects on cell proliferation produced by alterations of Dpn and Ase expression may be caused, at least in part, by changes in the levels of dap transcript. During embryogenesis, alteration in the levels of dap expression through either ectopic expression or by loss of function, result in dramatic changes in mitotic activity. Therefore, the mitotic activity in optic lobes of homozygous dap6 mutant third instar larvae were analyzed. While predominately recessive lethal, a few dap6 homozygous escapees can be viable to adulthood. Therefore, the larval optic lobes of homozygous dap6 mutant third instar larva can be analyzed. In such homozygous dap6 mutant larvae over-proliferation of the cells of the optic lobes is evident. There is a significant increase in the number of mitotically active cells and break down of mitotic domains, as compared to the wild type (Wallace, 2000).

The over-proliferation phenotype of dap6 null mutants can be compared to the over-proliferation phenotype in larvae with ectopic dpn expression, and the associated suppression of dap expression. Although the over-proliferation in both cases is similar, there are clearly more cells produced in the dpn over-expressing brain lobes. This strongly indicates that other cell cycle regulators are also likely to be affected by the ectopic expression of dpn in the optic lobes (Wallace, 2000).

A model is proposed for mitotic control in the developing third instar optic lobe in which cell proliferation is modulated by a positive regulator of mitotic activity such as Dpn and a negative regulator of mitotic activity such as Ase. In this model, one role of Dpn and Ase would be to interface with cell cycle regulation through the direct or indirect modulation of dap expression. Mitotic control during optic lobe development may involve the following events. Cells that give rise to the optic lobe delaminate from the neuroectoderm during embryogenesis and remain quiescent until first instar with the help of proteins such as Anachronism. The mitotic activity is then initiated through a process that requires the trol gene product and the developmental regulator and transcription factor Eve to begin the proliferation of neuroblasts to form the OPC. The mitotically active state of OPC cells would be maintained in part by Dpn. In the absence of Dpn, the cells in the OPC have a greater chance of exiting mitosis by allowing Dap to be expressed. As cells arrive at the edge of the OPC, Ase is expressed at high levels, allowing the neuroblasts to become quiescent only after they pass out of the region where Dpn is expressed. Suppression of dap by Dpn in the OPC would allow the neuroblasts to be mitotically active while the increased expression of Ase at the posterior edge of the OPC allows the neuroblasts to exit mitosis and begin differentiation. In addition, the resulting quiescent state needs to be maintained in the lamina; otherwise the cells may reenter mitosis (Wallace, 2000 and references therein).

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 pattern of stg expression anticipates and determines the embryonic cell division pattern. stg expression in the embryonic epidermis before mitosis 16, therefore, is highly similar to the pattern of dap expression, which also precedes this terminal mitosis 16. To analyze whether stg and dap expression before mitosis 16 are mechanistically coupled, the distribution of stg transcripts in ventral veins lacking (vvl) and trachealess (trh) embryos was examined. vvl and trh are expressed within the prospective tracheal pit regions and are known to co-operate for the specification of tracheal cell fate. The characteristic early dap expression in tracheal pits is not detected in vvl embryos and it is severely decreased in trh embryos. Interestingly, while the characteristic early expression of stg is not observed in trh embryos, it is normal in vvl mutants. As expected, progression through mitosis 16 also occurred in the normal pattern in these vvl mutants. The absence of the characteristic early dap expression in tracheal pits of vvl embryos, therefore, is not preceded by a change in the proliferation program. These findings indicate that the control of stg and dap expression is mechanistically distinct. Moreover, they suggest that regulators of developmental fates control dap expression directly and not indirectly by controlling the cell proliferation program via other cell cycle regulators (Meyer, 2002).

In the epidermis, cell cycle exit is preceded by dap expression. Is cell cycle exit in all tissues preceded by dap expression? Does the same rule apply during nervous system development? Development of the central nervous system starts with delamination of single neuroblasts from the neuroectoderm. These neuroblasts divide asymmetrically. One daughter cell continues with asymmetric neuroblast divisions. The other, the ganglion mother cell (GMC), divides just once to produce two post-mitotic neurons. As in the epidermis, where dap expression starts before the terminal division, dap might be induced in GMCs to prevent further proliferation after its division. To evaluate this idea, embryos were double-labeled with antibodies against p27DAP and Prospero, a pan-neural transcription factor. Prospero is localized to the cell membrane of neuroblasts and segregated asymmetrically into GMCs during neuroblast divisions. In GMCs, Prospero translocates to the nucleus. Because of this asymmetric segregation, Prospero is more than a useful marker for GMCs. It is also an attractive candidate transcription factor that might activate dap expression in all GMCs. Embryos were double-labelled during the stages where the first GMC divisions are initiated. During these stages, p27DAP was not detected in Prospero-positive GMCs. This result indicates that cell cycle exit in the early CNS neurons is not accompanied by p27DAP accumulation before the final division, as in the epidermis (Meyer, 2002).

However, p27DAP expression is detected in the Prospero-positive MP2 neuroblast. MP2 is an exceptional neuroblast that accumulates Prospero in the nucleus. Moreover, MP2 divides just once to produce two postmitotic neurons. The final division of this unusual neuroblast therefore is preceded by dap expression, as in the epidermis. However, anti-p27DAP labeling of prospero mutant embryos, indicates that dap expression in MP2 is not the result of Prospero translocation into the nucleus. At later stages, the pattern of anti-p27DAP labeling observed in the CNS is complex and highly dynamic. Anti-p27DAP signals of non-uniform intensity were detected in both Prospero-positive as well as Prospero-negative cells (Meyer, 2002).

To identify conserved sequence elements in the dap regulatory region, a dap homolog was isolated from Drosophila virilis. The gene product shares 65% amino acid sequence identity with p27DAP from Drosophila melanogaster. This comparison revealed several blocks of high sequence similarity not only within the coding region but also within the regulatory region. The most distal of these blocks (A8) is located within the region containing regulatory elements controlling expression in the epidermis. The significance of these blocks was examined, comparing the expression of transgenes with and without these blocks (dap-15l and dap-15l-A8). The results indicate that the A8 block is required for the early high level expression in a region of the epidermis within the abdominal segment A8. Wild-type dap expression occurs early and at high levels within this prospective posterior spiracle region. Interestingly, the A8 block contains binding sites for the homeodomain transcription factor encoded by Abdominal-B (Abd-B) which is expressed within the posterior abdominal region and required for the specification of posterior spiracles. prd-GAL4 driven expression of UAS-Abd-B induces expression of the endogenous dap gene (Meyer, 2002).

These results indicate that Abd-B is involved in the control of dap expression within the abdominal segment A8. Moreover, they indicate that dap expression is regulated by multiple control elements even within a tissue like the embryonic epidermis. While the regulatory sequences present in the dap-12l construct drive relatively uniform expression throughout the epidermis, the conserved block A8 adds an earlier onset within abdominal segment A8. Another element in dap-1l adds the early onset within the region of the tracheal pits. In summary, these analyses demonstrates that the dap regulatory region is composed of a complex array of stage- and tissue-specific enhancers (Meyer, 2002).

Proximal parts of the dap regulatory region control expression in the peripheral and central nervous system. Even though this regulation has not been dissected in detail, it appears that the expression in the nervous system is controlled by several independent cis-regulatory elements. The 1.3 kb deletion present in dap9 eliminates many but not all aspects of dap expression in the nervous system. Similarly, the 0.7 kb fragment in the dap-3l reporter construct is sufficient to direct expression in some but not all of the peripheral nervous system lineages (Meyer, 2002).

Limited analyses of dap expression during CNS development in wild-type embryos demonstrates that cell cycle exit is not always preceded by dap expression. When the first GMCs are generated during embryonic CNS development and progress through their terminal division cycle, p27DAP cannot be detected in these cells, while expression in the unusual MP2 neuroblast is readily observed. This dap expression in the MP2 neuroblast occurs also in prospero mutant embryos. All the findings therefore argue against the suggestion favored by previous studies, which argues that the timely arrest of cell proliferation in GMC progeny might depend on the induction of dap expression by the transcription factor Prospero. The idea that nuclear Prospero might trigger dap expression in GMCs to bring about the G1 arrest observed in the two MP2 neurons generated by GMC division appeared very attractive. Moreover, in principle this hypothesis is also suggested by the correlation that MP2, an exceptional neuroblast that behaves like a GMC in that it divides just once to produce two postmitotic neurons, accumulates Prospero in the nucleus and expresses dap (Meyer, 2002).

The findings argue against a mechanism that operates generally in all GMCs to prevent further cell cycle progression after the terminal division by Prospero-mediated induction of dap expression; more complex mechanisms might have to be considered that might even vary in different neuroblast lineages. The regulation of dap expression in the nervous system certainly entails such complexity. This work does not exclude a dap-independent, general cell cycle arrest mechanism that operates in all GMCs and is perhaps even triggered by nuclear Prospero (Meyer, 2002).

At least in some CNS lineages, dap expression is required to limit cell proliferation. dap mutants have excess Even-skipped and Eagle-positive neurons in the CNS at stage 14. The proliferation-limiting function of dap therefore is not restricted to the embryonic epidermis, where it has been most convincingly demonstrated. However, apart from the absence of DAP in early GMCs of the CNS, there are additional observations indicating that G1 arrest is not always accompanied by induction of dap expression. dap expression is detectable neither in the zone of non-proliferating cells formed in third instar wing imaginal discs nor in the region just anterior to the morphogenetic furrow in eye imaginal discs where cells become arrested in G1 (Meyer, 2002).

The findings concerning dap expression in the embryonic epidermis further support and extend the notion that dap expression is controlled by a complex array of cis-acting elements. The initial epidermal expression in regions where the tracheal pits invaginate requires regulatory sequences different from those found in subsequent expression in the remainder of the epidermis. Similarly, the early expression in the prospective posterior spiracle region in the abdominal segment A8 requires a specific region that includes binding sites for the HOX-protein Abd-B, which acts at the top of a regulatory cascade directing posterior spiracle development. The Abd-B-binding sites are required for the early expression of dap reporter constructs in the prospective posterior spiracle region and they are conserved in the dap homolog of Drosophila virilis. Transcription factors that specify developmental fates during Drosophila embryogenesis are therefore likely to regulate dap expression directly in the same way as Abd-B (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).

These results therefore demonstrate that the activation of dap expression in the embryonic epidermis is not coupled to cell cycle progression. It is also not a downstream event of a cellular program, which is realized invariably whenever cell proliferation is arrested in various tissues and developmental stages. Instead, the control of dap expression involves a complex regulatory region composed of many cis-acting elements that direct distinct aspects of the intricate expression pattern. Specific combinations of transcription factors which specify various developmental fates appear to act directly at the dap regulatory region (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).

Analysis of Ras-induced overproliferation in Drosophila hemocytes

The Drosophila larval hematopoietic system has been used as an in vivo model for the genetic and functional genomic analysis of oncogenic cell overproliferation. Ras regulates cell proliferation and differentiation in multicellular eukaryotes. To further elucidate the role of activated Ras in cell overproliferation, a collagen promoter-Gal4 strain was generated to overexpress RasV12 (Ras-act) in Drosophila hemocytes. Activated Ras causes a dramatic increase in the number of circulating larval hemocytes (blood cells); this increase is caused by cellular overproliferation. This phenotype is mediated by the Raf/MAPK pathway. The mutant hemocytes retain the ability to phagocytose bacteria as well as to differentiate into lamellocytes. Microarray analysis of hemocytes overexpressing RasV12 vs. Ras+ identified 279 transcripts that are differentially expressed threefold or more in hemocytes expressing activated Ras. This work demonstrates that it will be feasible to combine genetic and functional genomic approaches in the Drosophila hematopoietic system to systematically identify oncogene-specific downstream targets (Asha, 2003).

One overall finding is that many of the genes that are upregulated in Ras-act cells include genes that function in cell cycle regulation and DNA replication. These genes include both positive and negative regulators of cell proliferation. The cyclin-dependent kinase inhibitor dacapo (which antagonizes the function of cyclin E/cdk2 complexes), as well as the wee1 kinase (which inactivates cdc2), are both induced. There is currently no known function for either gene in promoting cell cycle progression. Thus the induction of these genes may represent a negative feedback mechanism that attempts to reduce cell proliferation under conditions of excessive cell proliferation. Another possibility is that these two genes have currently unknown roles in promoting cell cycle progression. The microarray data also show that regulators that promote all stages of cell cycle progression are induced, not only those that promote the G1/S transition. These data therefore suggest that both the G1/S and G2/M cell cycle transitions may be influenced by an increase in Ras activity (Asha, 2003).

The mitotic-to-endocycle switch in Drosophila follicle cells is executed by Notch-dependent regulation of G1/S, G2/M and M/G1 cell-cycle transitions

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

Why is the function of Ago/hCdc4/Fbw7 critical to endocycles but not to mitotic cycles in follicle epithelial cells? A potential answer might reside in Dacapo, a CIP/KIP-type inhibitor of Cyclin E/Cdk2 complexes that is regulated in the mitotic to endocycle transition by activation of Notch pathway. dacapo is downregulated at mitotic-to-endocycle transition because of Notch activation and ectopic expression of dacapo represses endocycle progression. It is plausible that during mitotic phases Ago and Dacapo share a redundant role for regulating the Cyclin E activity level, however, dacapo is downregulated by Notch pathway at the time of mitotic-to-endocycle transition and at that point Ago gains the critical role of sole regulator of Cyclin E protein activity level. However, downregulation of Dacapo does not readily explain the reduction of CycE levels observed in mitotic-to-endocycle transition. Elevation of CycE protein level is detected in response to Dacapo overexpression, pointing out that this CKI may stabilize CycE in an inactive form. One possibility therefore is that less CycE protein is observed after the Dacapo downregulation because Dacapo is no longer stabilizing it (Shcherbata, 2004).

Why is Dacapo downregulated at the time of endocycle transition? Expression of Dacapo is important for proper cell cycle regulation. For example, during vertebrate development, members of the CIP/KIP family of CKIs are often upregulated as cells exit the mitotic cycle and begin to terminally differentiate. Also, reduced expression of p27Kip1 is frequently shown to correlate with a poor prognosis in various cancers, and in the absence of p21, DNA-damaged cells arrest in a G2-like state, but then undergo additional S-phases without intervening normal mitoses. They thereby acquire grossly deformed, polyploid nuclei and subsequently die through apoptosis. Also, p21 elimination causes centriole overduplication and polyploidy in human hematopoietic cells. In the Drosophila germ line Dap is differentially regulated in the nurse cells versus the oocyte. High Dap levels in the oocyte are critical to the maintenance of the prophase I meiotic arrest and ultimately to later events of oocyte differentiation, and in the nurse cells the oscillations of Dap drive the endocycle. In contrast to all these examples, in endocycling follicle cells reduction of p21/Dacapo is a requirement for normal endocycle progression. Similarly, in a megakaryocytic cell line, differentiation is correlated with a downregulation of p27. It is proposed that the downregulation of Dacapo is a reasonable strategy to bypass the G1/S transition and to enter endocycling when mitosis is not completed, however, how these endocycling cells escape possible centrosome amplification and apoptosis that could be consequences of the lack of Dacapo/p21-activity is not clear. This diversity in the processes, that allow cells to exit from mitotic cell cycle, is generating or representing regulatory multiplicity that might be reflected in the ways eukaryotic cells acquire tumor formation capacity (Shcherbata, 2004).

Dicer-1-dependent Dacapo suppression acts downstream of Insulin receptor in regulating cell division of Drosophila germline stem cells

It is important to understand the regulation of stem cell division because defects in this process can cause altered tissue homeostasis or cancer. The cyclin-dependent kinase inhibitor Dacapo (Dap), a p21/p27 homolog, acts downstream of the microRNA (miRNA) pathway to regulate the cell cycle in Drosophila germline stem cells (GSCs). Tissue-extrinsic signals, including insulin, also regulate cell division of GSCs. Intrinsic and extrinsic regulators intersect in GSC division control; the Insulin receptor (InR) pathway regulates Dap levels through miRNAs, thereby controlling GSC division. Using GFP-dap 3'UTR sensors in vivo, this study shows that in GSCs the dap 3'UTR is responsive to Dicer-1, an RNA endonuclease III required for miRNA processing. Furthermore, the dap 3'UTR can be directly targeted by miR-7, miR-278 and miR-309 in luciferase assays. Consistent with this, miR-278 and miR-7 mutant GSCs are partially defective in GSC division and show abnormal cell cycle marker expression, respectively. These data suggest that the GSC cell cycle is regulated via the dap 3'UTR by multiple miRNAs. Furthermore, the GFP-dap 3'UTR sensors respond to InR but not to TGF-beta signaling, suggesting that InR signaling utilizes Dap for GSC cell cycle regulation. The miRNA-based Dap regulation may act downstream of InR signaling; Dcr-1 and Dap are required for nutrition-dependent cell cycle regulation in GSCs and reduction of dap partially rescues the cell cycle defect of InR-deficient GSCs. These data suggest that miRNA- and Dap-based cell cycle regulation in GSCs can be controlled by InR signaling (Yu, 2009).

Previous studies have shown that miRNAs may regulate Dap, thereby controlling the cell division of GSCs. This study shows that the dap 3'UTR directly responds to miRNA activities in GSCs. Using luciferase assays, miR-7, miR-278 and miR-309 were identified as miRNAs that can directly repress Dap through the dap 3'UTR in vitro. Although miR-278 and miR-7 play a role in regulating GSC division and cell cycle marker expression, respectively, neither of these mutants showed as dramatic a defect in the GSC cell cycle as Dcr-1-deficient GSCs. Thus, the dap 3'UTR may serve to integrate the effect of multiple miRNAs during cell cycle regulation. It remains possible that some miRNAs involved in this process remain to be identified. It was further shown that InR signaling controls the dap 3'UTR in GSCs. This led to an exploration of the interaction between InR signaling and miRNA/Dap cell cycle regulation. GSCs deficient for InR or Dcr-1 show similar cell cycle defects. Using starvation to control InR signaling, it was shown that both Dcr-1 and dap are required for proper InR signaling-dependent regulation of GSC division. Further, reduction of dap partially rescues the cell division defect of the InR mutant GSCs, suggesting that InR signaling regulates the cell cycle via Dap. These results suggest that miRNAs and Dap act downstream of InR signaling to regulate GSC division (Yu, 2009).

The data suggest that multiple miRNAs can regulate the 3'UTR of dap: miR-7, miR-278 and miR-309 can regulate the dap 3'UTR directly, whereas bantam and miR-8 may regulate it indirectly, or through cryptic MREs in the dap 3'UTR. Using GFP sensor assays, it was also shown that the dap 3'UTR may be directly regulated by miRNAs in the GSCs in vivo. However, which specific miRNAs control endogenous Dap levels in Drosophila GSCs remains unknown. Mammalian p21cip1 has also been shown to be a direct target for specific miRNAs of the miR-106 family, including miR-290s and miR-372. Further, the mouse miR-290 family has recently been identified as regulating the G1-S transition. In addition, miR-221 and miR-222 have been shown to regulate p27kip1, thereby promoting cell division in different mammalian cancer cell lines. Neither the miR-290 nor miR-220 family is conserved in Drosophila. Together, these results indicate that the CKIs (Dap) might be a common target for miRNAs in regulating the cell cycle in stem cells. However, the specific miRNAs that regulate the CKIs might vary between organisms (Yu, 2009).

This study reveals novel regulatory roles for miR-7 and miR-278 in the GSC cell cycle. miR-7 and miR-278 can directly target Dap. GSCs deficient for miR-278 show a mild but significant reduction in cell proliferation. Ectopic expression of miR-7 in follicle cells reduces the proportion of cells that stain positive for Dap. Furthermore, ablation of miR-7 in GSCs results in a perturbation of the frequency of CycE-positive GSCs. However, the cell division kinetics of miR-7 mutant GSCs is not reduced, by contrast with the dramatic reduction of cell division in Dcr-1-deficient GSCs. It is plausible that miR-7 and miR-278 act in concert with other miRNAs to regulate the level of Dap in GSCs and thereby contribute to cell cycle control in GSCs (Yu, 2009).

The interaction of multiple miRNAs with the dap 3'UTR might integrate information from multiple pathways. Further studies will reveal what regulates miR-7 and miR-278 expression in GSCs and which other miRNAs might act together in Dap regulation. It is known that miR-7 and the transcriptional repressor Yan mutually repress one another in the eye imaginal disk. In this model, Yan prevents transcription of miR-7 until Erk in the Egfr pathway downregulates Yan activity by phosphorylation, thereby permitting expression of miR-7. Conversely, miR-7 can repress the translation of Yan. Thus, a single pulse of Egfr signaling results in stable expression of miR-7 and repression of Yan. Whether similar regulation will be observed between miR-7 and the signaling pathways that regulate GSC division remains to be seen. It has been suggested that miR-7 might regulate downstream targets of Notch, such as Enhancer of split and Bearded. Thus, miR-7 may have a mild repressive effect on multiple targets in GSCs. Further experiments might illuminate this possibility (Yu, 2009).

miR-278, on the other hand, has been implicated in tissue growth and InR signaling. Overexpression of miR-278 promotes tissue growth in eye and wing imaginal discs. Deficiency of miR-278 leads to a reduced fat body, which is similar to the effect of impaired InR signaling in adipose tissue. Interestingly, miR-278 mutants have elevated insulin/Dilp production and a reduction of insulin sensitivity. Furthermore, miR-278 regulates expanded, which may modulate growth factor signaling including InR. Since InR signaling plays important roles in tissue growth and cell cycle control, it will be interesting to further test how miR-278 may regulate InR signaling, and whether InR signaling might regulate miR-278 in a feedback loop in GSCs (Yu, 2009).

Other miRNAs or miRNA-dependent mechanisms might also play roles in Drosophila GSCs. For example, the miRNA bantam is required for GSC maintenance. A recent study has shown that the Trim-NHL-containing protein Mei-P26, which belongs to the same family as Brain tumor (Brat), affects bantam levels and restricts cell growth and proliferation in the GSC lineage (Neumuller, 2008). Interestingly, most miRNAs are upregulated in mei-P26 mutant flies. By contrast, overexpression mei-P26 in bag of marbles (bam) mutants broadly reduces miRNA levels. This suggests that Mei-P26 regulates proliferation and maintenance of GSC lineages via miRNA levels. Since InR signaling cell-autonomously regulates GSC division but not maintenance, the possible interaction between Mei-P26 and InR signaling might be complex (Yu, 2009).

The systemic compensatory effect of insulin secretion in mammals with defective InR signaling is well documented. Insulin levels in mice with liver-specific InR (Insr - Mouse Genome Informatics) knockout are ~20-fold higher than those of control animals owing to the compensatory response of the pancreatic β-cells and impairment of insulin clearance by the liver. Knockout of the neuronal InR also leads to a mild hyperinsulinemia, indicating whole-body insulin resistance. Furthermore, the knockout of components in the InR signaling pathway, such as Akt2 and the regulatory and catalytic subunits of PI3 kinase, also leads to hyperinsulinemia and glucose intolerance. Therefore, a systemic decrease in InR signaling may lead to compensatory responses (Yu, 2009).

To understand the roles of InR signaling in the GSCs while avoiding any systemic compensatory effect the phenotypes of GSC clones were analyzed. Using a panel of cell cycle markers, it was found that InR mutant GSCs show cell cycle defects similar to those of Dcr-1 mutant GSCs: a reduction of cell division rate, an increased frequency of cells staining positive for Dap and CycE, and a decreased frequency of cells staining positive for CycB. Using GFP-dap 3'UTR sensors, it was shown that the dap 3'UTR responds to InR signaling in GSCs, suggesting that InR signaling can regulate Dap expression through the dap 3'UTR. This, together with genetic data indicating that InR/starvation-dependent cell cycle regulation requires Dcr-1 and dap, has led to the proposalthat InR signaling regulates the cell cycle through miRNAs that further regulate Dap levels. Since a reduction in dap only partially rescues the cell cycle defects of InR mutant GSCs, it is possible that InR signaling might also regulate GSC division by additional mechanisms (Yu, 2009).

InR signaling regulates the cell cycle through multiple mechanisms, mainly through the G1-S, but also partly through the G2-M, transition. Recent work has shown a delay in the G2-M transition in GSCs during C. elegans dauer formation. Starvation and InR deficiency may also affect the G2-M checkpoint in Drosophila GSCs (Hsu, 2008). This study has dissected one possible molecular pathway that InR signaling utilizes to regulate the Drosophila GSC G1-S transition and show that InR signaling can control the cell cycle through miRNA-based regulation of Dap (Yu, 2009).

Many studies have connected InR and CKIs to Tor (Target of rapamycin) or Foxo pathways downstream of InR signaling. In S. cerevisiae, the yeast homolog of p21/p27 is upregulated when Tor signaling is inhibited. Foxo, a transcription factor that can be repressed by InR signaling, is known to play important roles in nutrition-dependent cell cycle regulation by upregulating p21 and p27. In C. elegans, starvation causes L1 cell cycle arrest mediated by InR (daf-2) and Foxo (daf-16): InR represses the function of Foxo, thereby downregulating the CKI (cki-1) and upregulating the miRNA lin-4. This study has shown that a miRNA-based regulation of Dap can be coordinated by InR in Drosophila GSCs (Yu, 2009).

Insulin and insulin-like growth factors (Igf1 and Igf2) are known to play important roles in regulating metabolic and developmental processes in many stem cells. In mammals, Igf signaling is required by different stem cell types, including human and mouse ES cells for survival and self-renewal, neural stem cells for expediting the G1-S transition and cell cycle re-entry, and skeletal muscle satellite cells for promoting the G1-S transition via p27kip1 downregulation. This study has dissected the molecular mechanism of the InR pathway in another adult stem cell type, tDrosophila GSCs, showing that InR signaling can regulate stem cell division through miRNA-based downregulation of the G1-S inhibitor Dap. Further studies will reveal whether miRNAs also mediate InR signaling in other stem cell types (Yu, 2009).

A transient expression of Prospero promotes cell cycle exit of Drosophila postembryonic neurons through the regulation of Dacapo

Cell proliferation, specification and terminal differentiation must be precisely coordinated during brain development to ensure the correct production of different neuronal populations. Most Drosophila neuroblasts (NBs) divide asymmetrically to generate a new NB and an intermediate progenitor called ganglion mother cell (GMC) which divides only once to generate two postmitotic cells called ganglion cells (GCs) that subsequently differentiate into neurons. During the asymmetric division of NBs, the homeodomain transcription factor Prospero is segregated into the GMC where it plays a key role as cell fate determinant. Previous work on embryonic neurogenesis has shown that Prospero is not expressed in postmitotic neuronal progeny. Thus, Prospero is thought to function in the GMC by repressing genes required for cell-cycle progression and activating genes involved in terminal differentiation. This study focused on postembryonic neurogenesis and shows that the expression of Prospero is transiently upregulated in the newly born neuronal progeny generated by most of the larval NBs of the OL and CB. Moreover, evidence is provided that this expression of Prospero in GCs inhibits their cell cycle progression by activating the expression of the cyclin-dependent kinase inhibitor (CKI) Dacapo. These findings imply that Prospero, in addition to its known role as cell fate determinant in GMCs, provides a transient signal to ensure a precise timing for cell cycle exit of prospective neurons, and hence may link the mechanisms that regulate neurogenesis and those that control cell cycle progression in postembryonic brain development (Colonques, 2011). During development, cell cycle progression must be coordinated with the regulation of cell specification and differentiation. The underlying mechanisms of coordination are likely to be particularly complex during neural development due to the enormous cell diversity in the brain. In Drosophila, these mechanisms have been well studied during embryonic CNS development. In embryonic neurogenesis, the homeodomain transcription factor Pros is expressed in the NB but it does not enter the nucleus due to its binding to the carrier protein Mira, which localizes to the cell cortex. This interaction facilitates the segregation of Pros from the parent NB to the GMC during asymmetric NB division. In the GMC, Pros is released from its carrier and translocates to the nucleus where it plays a binary role as a cell fate determinant, and as a promoter of terminal differentiation (Colonques, 2011).

It has been reported that Pros is similarly expressed and asymmetrically segregated during the proliferative activity of (type I) NBs in the larval CB although it does not seem to be expressed in CB dorso-medial lineages (type II) NBs. However, as this study shows, during postembryonic neurogenesis, in the majority of larval CB and OPC (outer proliferation center) neuronal lineages, pros expression is transiently upregulated in new born prospective neurons (GCs), in addition to its earlier expression and asymmetric segregation in some larval NBs. This is clearly different from the situation in embryonic lineages where pros is only transcribed in NBs, and Pros protein is downregulated in GCs after the division of their parent GMC (see Summary of cellular expression pattern of PROS in the larval CNS and lineage alterations in pros mutant clones) (Colonques, 2011).

This transient expression in most newborn postembryonic neurons shortly after the division of the GMC implies a novel role of Pros in postmitotic cells. It is postulated that this role is to inhibit cell cycle progression and promote cell cycle exit. The Pros GoF and LoF experiments support this notion. Pros GoF induces proliferation arrest and Pros LoF results in supernumerary cells with sustained expression of cell cycle markers, indicating an inability to withdraw from the cell cycle (Colonques, 2011).

In addition to the marked difference in Pros expression in postmitotic GCs during embryogenesis versus postembryonic neurogenesis (Pros is undetectable in embryonic GCs and high in postembryonic GCs), there are other functional differences in Pros action during embryonic versus postembryonic CNS development. For example, in pros mutant embryos, overproliferation is followed by abundant apoptotic cell death among the supernumerary cells. By contrast, no increas cell death was found in the larval OL of pros mutants. Moreover, while Pros and Dap seem to act in parallel to end the cell cycle in the embryonic CNS, Dap appears to act downstream of Pros in larval CNS neurons. These initial findings suggest that further differences between the functions of Pros during embryonic and postembryonic CNS neurogenesis may exist and should be considered (Colonques, 2011).

The fact that Pros protein is present in embryonic GMCs (intermediate progenitors) but not in embryonic GCs (prospective neurons), suggests that in the embryonic CNS, Pros initiates the end of mitotic activity in the GMC rather than in the GC. Accordingly, it has been proposed that the GMC is a transition state between the proliferating NB and the differentiating neuron that provides a window in which Pros represses stem cell-specific genes and activates differentiation genes. Nevertheless, it is not well understood how the GMC can go through its terminal cell cycle in spite of the repressive action of Pros on cell cycle regulators (Colonques, 2011).

The results strongly suggest that in postembryonic neurogenesis Pros acts not only in the GMC progenitor but also in the postmitotic GCs produced by the GMC. Thus, this analysis indicates that there are two main pros expression pattern subclasses among CB type I and OPC NB lineages. For the shake of simplicity they have been called them A and B. In type A, Pros is expressed in GCs after the division of GMCs while in type B, Pros is first expressed at low level in the NB and asymmetrically segregated to the GMC, and afterwards, upregulated in new born GCs. These two subsets of expression patterns correlate well with the two main phenotypes found in pros mutant clones. Thus, the LOF of pros in NBs with type A Pros expression appears to preclude cell cycle exit of GCs which, consequently, continue dividing and do not differentiate, yielding a type A clone composed of a single NB, a GMC and several small mitotic cells. By contrast, in lineages with type B Pros expression, the LOF of pros seems to cause primarily a change in the fate of the putative GMC that behaves like a NB maintaining the expression of asymmetric division genes (such as Mira) and overproliferating, to yield a type B clone composed of multiple large NB like cells (Colonques, 2011).

Hence, it is postulated that during postembryonic neurogenesis Pros functions in two sequential phases in type I NB lineages, first as cell fate determinant in some GMCs and later as cell cycle repressor in most GCs. Furthermore, the idea is favored that the different roles of Pros in postembryonic GMCs versus postembryonic GCs might be related to the higher level of expression observed in GCs compared to GMCs. Thus, high levels of Pros might be required to definitively withdraw the GCs from the cell cycle, while low levels might be sufficient to specify GMCs and modulate their cell cycle. The higher level of Dap protein in postembryonic GCs in relation to their parent GMCs and NBs is consistent with this hypothesis. The strong burst of Pros at the end of NB proliferation in ventral ganglia of early pupae is also in agreement with the idea that high levels of Pros are required to stop proliferation. Furthermore, it has been recently shown that the missexpression of Pros at high level suppresses proliferation in type II larval brain NBs lineages without apparent change in their identity (Colonques, 2011).

Taken together, all of these findings imply that different developmental strategies have been selected to couple cell fate decisions and cell cycle regulation during embryonic and postembryonic neurogenesis through the same effector, Pros. It is possible that this change in strategy is a consequence of the evolutionary adaptation to regulate the production of a large number of equivalent neurons in postembryonic lineages in contrast to embryonic neurogenesis where a much more limited set of specific neurons are generated in each lineage through GMC divisions (Colonques, 2011).

This study has shown that Pros is coexpressed with Dap in new born prospective neurons and, moreover, it was found that pros is sufficient and it is required for the expression of dap in these larval brain neuronal precursors. The dap gene encodes a member of the Cip/Kip family of CKIs with homology to mammalian p27kip1. This family of CKIs has been implicated in mediating cell cycle exit prior to terminal differentiation. They function by binding and inhibiting G1/S cyclin dependent kinase complexes. There is compelling data supporting a role of Dap in cell cycle exit during Drosophila embryogenesis. In Drosophila embryonic NB lineages, dap expression becomes apparent just before the terminal neurogenic division of the GMC. In contrast, this study has shown that dap is upregulated in new born postembryonic neurons. Consistent with a role in the termination of cell proliferation, dap expression in the larval OL has been tightly correlated with cells ending proliferation. Interestingly, Pros is required to terminate cell proliferation during embryonic neurogenesis and it has been shown to be involved in the regulation of dap expression in the embryonic nervous system. Thus, the results provide support to the idea that Pros promotes the cell cycle exit of post-embryonic GCs by upregulating the expression of dap. The data also suggest that this upregulation of dap is mediated by inhibiting the expression of Dpn. Dpn is an essential panneural bHLH transcription factor, which has been previously shown to be a suppressor of dap expression in the larval OL. Indeed, the dpn gene contains consensus Pros binding sites and Pros has been shown to be required to terminate the expression of dpn in the embryo (Colonques, 2011).

Sumoylation is tumor-suppressive and confers proliferative quiescence to hematopoietic progenitors in Drosophila melanogaster larvae

How cell-intrinsic regulation of the cell cycle and the extrinsic influence of the niche converge to provide proliferative quiescence, safeguard tissue integrity, and provide avenues to stop stem cells from giving rise to tumors is a major challenge in gene therapy and tissue engineering. This question was explored in sumoylation-deficient mutants of Drosophila. In wild type third instar larval lymph glands, a group of hematopoietic stem/progenitor cells acquires quiescence; a multicellular niche supports their undifferentiated state. However, how proliferative quiescence is instilled in this population is not understood. This study showed that Ubc9 protein is nuclear in this population. Loss of the SUMO-activating E1 enzyme, Aos1/Uba2, the conjugating E2 enzyme, Ubc9, or the E3 SUMO ligase, PIAS, results in a failure of progenitors to quiesce; progenitors become hyperplastic, misdifferentiate, and develop into microtumors that eventually detach from the dorsal vessel. Significantly, dysplasia and lethality of Ubc9 mutants are rescued when Ubc9(wt) is provided specifically in the progenitor populations, but not when it is provided in the niche or in the differentiated cortex. While normal progenitors express high levels of the Drosophila cyclin-dependent kinase inhibitor p21 homolog, Dacapo, the corresponding overgrown mutant population exhibits a marked reduction in Dacapo. Forced expression of either Dacapo or human p21 in progenitors shrinks this population. The selective expression of either protein in mutant progenitor cells, but not in other hematopoietic populations, limits overgrowth, blocks tumorogenesis, and restores organ integrity. An essential and complex role for sumoylation in preserving the hematopoietic progenitor states for stress response and in the context of normal development of the fly is discussed (Kalamarz, 2012).

In a quest to identify the source of microtumors in Ubc9 mutants, this study discovered that even though Ubc9 protein is ubiquitously expressed, it plays a specific and essential, niche-independent function in maintaining proliferative quiescence within progenitors of the medullary and transition zones. Reduction of sumoylation via knockdown of any of the other core enzymes of the pathway also leads to progenitor dysplasia and tumorogenesis. Once detached from the dorsal vessel, the microtumors float in the hemolymph (Kalamarz, 2012).

The progenitor population that serves as the source of microtumors is heterogeneous with respect to Dome>GFP and ZCL2897 expression. One of the earliest detectable effects of the mutation is on the differential expression of Dome>GFP and ZCL2897 or 76B>GFP in the expanding population. The onset of the effects of Ubc9 mutation coincides with the period when the progenitors in the medulla of the anterior lobes undergoes proliferative restraint. At the same time, cells of the posterior lobes lag behind; they continue to divide and follow a defined heterochronic developmental pattern. It is somewhat surprising that even though the Ubc9 mutation has differential effects on cells of the anterior versus posterior lobes, the overproliferation defects in both are largely rescued by ectopic expression of p21/Dap. This observation suggests a fundamental role for the enzyme in inhibiting cell cycle progression and conferring quiescence to progenitors. Since the decline in Dome>GFP expression precedes overproliferation in mutant lobes and each defect can be rescued by the expression of wild type Ubc9, it is possible that Dome>GFP expression marks the quiescent cell state. The inability of p21 or Dap to restore normal Dome>GFP expression attests to the notion that the sequential series of events, even at the earliest stages of tumorogenesis, can be genetically teased out in vivo (Kalamarz, 2012).

While the changes in cell identities in mutant lobes are complex, the discovery of heterogeneity in the medullary zone populations of anterior and first posterior lobes is consistent with recent reports that this population has distinct fate-restricted cell populations. The current results suggest that lymph gland progenitors are similar to mammalian transit amplifying cells or those in the Drosophila testis, that have limited proliferative capacity and possess a restricted differentiation potential relative to their multipotent stem cells. With an appropriate immune or developmental cue, Drosophila hematopoietic progenitors may re-enter the cell cycle to produce differentiated progeny (Kalamarz, 2012).

What is the physiological significance of retaining some cells in quiescence at this stage in larval life? One possibility is that mitotic exit shelters progenitors from precocious development and provides a mechanism that determines the number of times they must divide before they differentiate. Additionally, a reserve of progenitors, ready to divide and differentiate rapidly guards larvae against natural enemies such as parasitic wasps that attack them at this stage of the life cycle. This tactic parallels mitotic exit of hematopoietic stem cells (HSCs) in mice about three weeks after birth, or in humans, at about four years of age, when they become adult HSCs. The dormant adult HSCs are activated as the organism recovers from injury (Kalamarz, 2012).

This similarity in strategies between flies and humans in normal hematopoiesis is further reinforced even when the process becomes aberrant. Like in dUbc9 mutants, uncontrolled proliferation of progenitors in human leukemias can occur independently of the signals from the niche. It is intriguing that Antp, a niche marker, is also expressed in the dorsal vessel. Furthermore, Dome>GFP expression, undetectable in normal cells, is strongly activated in mutant cells of the dorsal vessel. Thus, it is possible that cues from the cells of the dorsal vessel influence the state of the hematopoietic progenitors and integrity of the lobes. Conversely, the status of the progenitors themselves may determine the association of the lobes to the dorsal vessel. Further analysis of Ubc9 mutants will clarify the role of the microenvironment in supporting progenitor quiescence and maintaining tissue integrity (Kalamarz, 2012).

A key mechanism by which sumoylation maintains proliferative quiescence in larval hematopoiesis is cell cycle regulation through Dacapo/p21. In the embryo, Dap/p21 binds to cyclin E/Cdk2 complexes to block the G1/S transition in cell cycle. Furthermore, the human p21 protein can block mitosis in the Drosophila eye. This function of Dap/p21 in larval hematopoiesis is similar to the roles of p27KIP1 or p21CIP1/WAF1 in enforcing HSC quiescence (Kalamarz, 2012).

Dap is expressed in Dome>GFP progenitors in wild type and mutant glands, and is reduced shortly after Dome>GFP is downregulated in mutant glands. Overexpression of Dap/p21 in these cells leads to decrease in progenitor number. It is noteworthy that dap mutants do not exhibit apparent tumorous overgrowth, a trait that is similar to young p21 null mice. However, with age, or in the presence of other mutations (e.g., oncogenic Ras), p21 null mice are prone to developing tumors. It is therefore very likely that tumorogenesis in Ubc9 mutants is supported not only by loss of Dap/p21 but also by the activation of other oncogenic and pro-inflammatory proteins (Kalamarz, 2012).

The mechanism by which Ubc9 controls Dap protein levels is not known. dap transcription has been studied in embryonic development where it regulates mitotic exit. High dap transcript levels in stage 16 embryonic central and peripheral nervous system, or in differentiating postmitotic cells of a developing eye disc, correlate with exit from mitosis. These observations suggest that regulation of dap transcription is coupled with mitotic exit, and it is therefore possible that its transcription in the lymph gland progenitors is similarly synchronized. Microarray experiments of whole Ubc9 larvae compared to their heterozygous siblings indicate dap transcript downregulation. An intriguing possibility is that Dacapo itself, or another protein in complex with Dap, is a sumoylation target. In high throughput yeast two-hybrid assay, Dap was found to physically interact with Ubc9. Future experiments including biochemical analyses of Dap and interacting proteins are required to test this idea (Kalamarz, 2012).

The causal relationship between cancer and inflammation is now widely accepted, even though the mechanisms that establish and sustain this relationship remain unresolved . Drosophila Toll-Dorsal pathway not only manages immunity, but also governs hematopoietic development. Ubc9 microtumor development requires Rel/NF-kappa B family transcription factors Dorsal and Dif. Aberrant activation of NF-kappa B signaling in Ubc9 mutants resembles hematopoieitic malignancies in vertebrates that arise due to ectopic germline or somatic disruption of the pathway (Kalamarz, 2012).

It has recently been discovered that sumoylation provides a homeostatic mechanism to restrain systemic inflammation in the fly larva, where it keeps the Toll/Dorsal-dependent immune response in check. Ubc9 controls the 'set point' by maintaining normal levels of IkappaB/Cactus protein in immune tissues (Paddibhatla, 2010). The Ubc9 cancer-inflammation model offers novel opportunities to examine the dynamics of tumor growth, its relationship to metastasis, and the links between cancer and inflammation. Ubc9 tumors are sensitive to aspirin. This model is well-suited for identifying and testing drugs that target highly-conserved biochemical mechanisms, such as sumoylation, which oversee self-renewal pathways in progenitor populations (Kalamarz, 2012).

PRC2 Controls Drosophila Oocyte Cell Fate by Repressing Cell Cycle Genes

The oocyte is a unique cell type that undergoes extensive chromosome changes on its way to fertilization, but the chromatin determinants of its fate are unknown. This study shows that Polycomb group (PcG) proteins of the Polycomb repressive complex 2 (PRC2) determine the fate of the oocyte in Drosophila. Mutation of the enzymatic PRC2 subunit Enhancer of zeste [E(z)] in the germline abolishes spatial and temporal control of the cell cycle and induces sterility via transdetermination of the oocyte into a nurse-like cell. This fate switch depends on loss of silencing of two PRC2 target genes, Cyclin E and the cyclin-dependent kinase inhibitor dacapo. By contrast, the PRC1 component Polycomb (Pc) plays no role in this process. These results demonstrate that PRC2 plays an exquisite role in the determination of the oocyte fate by preventing its switching into an endoreplicative program (Iovino, 2013).

Together, the data show that PRC2 controls oogenesis by direct corepression of CycE and dap in a time window that stretches from region 2b to region 3. This prevents improper endocycling, before the oocyte becomes fully silenced and determined by stage 3. Interestingly, E(z) levels rapidly drop in the germline at stage 4, suggesting that the continuous presence of PRC2 is no longer required after oocyte determination. This observation is consistent with the fact that depletion of E(z) after stage 3 using a late Gal4 driver does not affect oocyte fate (Iovino, 2013).

Although differences between PRC1 and PRC2 have been previously reported in mammals (Lessard, 1999; Sauvageau, 2010) and Drosophila (Richter, 2011), it is crucial to understand whether the molecular differences reflect specific biological roles for each of the two complexes. This study has shown that PRC2 controls oocyte cell fate determination, whereas PRC1 components, such as Pc, had no obvious function in the oocyte, consistent with absence of Pc from the germline. Intriguingly, a different situation is seen in the fly male testis, where PRC1 components are required in the germline (Chen, 2011) while PRC2 is dispensable. Therefore, the production of gametes is a critical biological function that separates the function of these two complexes (Iovino, 2013).

Although a few genes that are responsible for oocyte determination have been identified previously, none is known to act on chromatin. The identification of PRC2 as a chromatin effector complex that is required to fix the oocyte fate offers the possibility of starting to dissect the molecular mechanisms that transduce the early asymmetry between the preoocyte and the surrounding cells into a terminally determined fate (Iovino, 2013).

MicroRNA miR-7 contributes to the control of Drosophila wing growth

The control of organ growth is critical for correct animal development. From flies to mammals, the mechanisms regulating growth are conserved and the role of microRNAs in this process is emerging. The conserved miR-7 has been described to control several aspects of development. This study has analyzed the function of miR-7 during Drosophila wing development. Loss of miR-7 function results in a reduction of wing size and produces wing cells that are smaller than wild type cells. It was also found that loss of miR-7 function interferes with the cell cycle by affecting the G1 to S phase transition. Further, evidence is presented that miR-7 is expressed in the wing imaginal discs and that the inactivation of miR-7 increases the expression of Cut and Senseless proteins in wing discs. Finally, these results show that the simultaneous inactivation of miR-7 and either cut, Notch, or dacapo rescues miR-7 loss of function wing size reduction phenotype. The results from this work reveal that miR-7 functions to regulate Drosophila wing growth by controlling cell cycle phasing and cell mass through its regulation of the expression of dacapo and the Notch signaling pathway (Aparicio, 2015).

The function of miR-7 in eye morphogenesis and ovariole development has been analyzed using the miR-7Δ1 and Df(2R)exu1 deletions and the genetic allelic combination Df(2R)exu1/miR-7Δ1. In contrast, the function of miR-7 in wing development has only been studied analyzing the phenotypes of miR-7 over-expression in the wing disc. These wing studies indicated that miR-7 regulates Notch signaling pathway targets like Enhancer of split to control sensory organ precursor determination. The analysis of the loss of miR-7 function using the Df(2R)exu1/miR-7Δ1 allelic combination has not been performed. Furthermore, the use of miR-7 sponges has been uninformative. As the Df(2R)exu1/miR-7Δ1 allelic combination, used by this study, might also partially inactivate the function of the bancal gene where miR-7 is located, attempts were made to analyze the contribution of miR-7 to the Df(2R)exu1/miR-7Δ1 wing phenotype. This study shows that the over-expression of miR-7 and the constitutive and ubiquitous expression of miR-7 rescue, at least partially, the wing phenotype of Df(2R)exu1/miR-7Δ1, thereby demonstrating that lack of miR-7 function contributes to the wing phenotype observed in Df(2R)exu1/miR-7Δ1 flies and indicating that miR-7 is involved in the control of wing growth. This is consistent with the findings that miR-7 is expressed in the wing imaginal discs and that the G1 to S cell cycle transition is arrested in Df(2R)exu1/miR-7Δ1 wing imaginal discs. Expression of mature miR-7 in the wing imaginal discs has been previously reported. The current results show that in situ miR-7 expression in the wing disc is weak but still significant compared to Df (2R) exu1/miR-7Δ1 wing discs. Moreover, miR-7 expression in the 'non-boundary cells' along with the absence of its expression in the 'boundary cells' suggests that miR-7 is involved in the regulation of the factors that control the cell interactions regulating growth at the D/V boundary (Herranz, 2008). Although these results strongly suggest that miR-7 has a function in wing growth control, definitive proof will require either a precise miR-7 deletion or an effective miR-7 sponge (Aparicio, 2015).

Bioinformatic analyses have predicted at least 150 miR-7 putative target genes in Drosophila genome. Among these, only a few have been validated using in vivo experiments. To date, miR-7 has been shown to promote photoreceptor differentiation by regulating the expression of yan as well as to control chordotonal organ differentiation through the control of the Enhancer of split-complex genes (Stark, 2003; Li, 2009). Moreover, miR-7 has been shown to regulate dacapo expression to maintain the GSCs proliferation as well as to regulate bag of marbles expression to control the GSCs testis differentiation. The current results show that the Df (2R) exu1/miR-7Δ1 wing phenotype is rescued by reducing the levels of expression of the genes Notch, cut, and dacapo. These results indicate that miR-7 functions together with the Notch pathway and with dacapo to control wing growth. Curiously, the shape of the Df (2R) exu1/miR-7Δ1 wings appears slightly different from wild type wings perhaps due the Insulin-like receptor signaling control of dacapo expression. Neither the Notch nor cut genes have been predicted to be targets of miR-7. However, effectors of the Notch pathway such as components of the Enhancer of split complex genes have been shown to be regulated by high levels of miR-7 to control chordotonal organ differentiation (Aparicio, 2015).

How could miR-7 be involved in the control of wing growth and the regulation of the Notch pathway? This study has shown that the G1-S cell cycle transition is delayed in miR-7 loss of function mutants perhaps as a consequence of the reduced cell size to compensate for the loss of cell mass. Also, this study showed that miR-7 regulates Notch activity. Considering that Notch has been shown to negatively regulate the G1-S transition, a mechanism consistent with all these results is that miR-7 regulates Notch activity, which in turn regulates cell cycle progression and ultimately wing growth (Aparicio, 2015).

dacapo is an inhibitor of the cell cycle progression that is expressed ubiquitously in the wing imaginal cells and is required for normal wing growth. The dacapo 3'UTR contains functional miR-7 binding sites that are required for the maintenance of GSC division. Interestingly, reducing the activity in the wing imaginal discs of dicer, the central element in miRNA biogenesis, produces small wings whose size is rescued when dacapo levels of expression are decreased. Thus, wing growth control mediated by dacapo depends on microRNAs activity. The current results show that reducing the levels of dacapo expression rescues the small wing phenotype produced by miR-7 loss of function. Therefore, the results strongly suggest that miR-7 regulates dacapo expression to control wing growth and, furthermore, that this control is direct as it has been shown using luciferase assays that miR-7 directly regulates the expression of dacapo (Aparicio, 2015).

In the current model, miR-7 controls the expression of Notch pathway components as well as dacapo to allow cell cycle progression and regulate wing growth. A role of miR-7 in the direct regulation of dacapo as well as the direct or indirect regulation of Notch activity is supported by these results. Additionally, Notch has also been proposed to regulate dacapo expression in the follicle cells and it is not unreasonable to think that this regulation might also take place in the wing to control organ growth. These investigations add a new component, the microRNA miR-7, to the group of factors modulating proliferation/growth. Thus, the study of their interactions may help to resolve the intricate relationship between cell cycle and cell growth. Further analysis will be necessary to study the mechanisms involved in this regulation and the factors that together with miR-7, dacapo, and Notch control both the initiation and the termination of cell growth of the wing imaginal disc (Aparicio, 2015).

Protein Interactions

Purified DAP protein inhibits the histone H1 kinase activity of Drosophila cyclin E/cdk2 (also known as cyclin E/cdc2c) complexes; a mutant version of DAP, lacking the first cluster of conserved amino acids, is a less efficient inhibitor. Cdk1, also known as cdc2, cyclin A and cyclin B complexes are inhibited by neither mutant nor wild type DAP. With coimmunoprecipitation experiments, Cdk2 and cyclin E (but not cdk1, cyclin A and cyclin B) are coimmunoprecipitated with anti-DAP antibodies. Dacapo can also inhibit the kinase activity of human cyclin E-cdk2 complexes. Dacapo itself is phosphorylated by mammalian cyclin E-cdk2 complexes (Lane, 1996 and de Nooij, 1996).

Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. Three Drosophila MCM proteins have been characterized: DmMCM2, DmMCM4, and DmMCM5. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Cyclin:cdks can prevent an assembly of proteins called the "prereplicative complex" on origins of DNA replication. The prereplicative complexes are thought to contain MCMs. Their absence from chromatin is thought to contribute to the inability of the post S phase nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM-chromatin interaction, a test was carried out as to whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B shows that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. Thus MCM-chromosome association is delayed when mitosis is followed by a prolonged G-1 phase. dacapo mutant embryos lack an inhibitor for cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. It is proposed that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. Thus, it is suggested that cyclins have both positive and negative roles in controlling MCM-chromatin association (Su, 1997).

Involvement of CUL4 ubiquitin E3 ligases in regulating CDK inhibitors Dacapo/p27Kip1 and cyclin E degradation

The CUL4 (cullin 4) proteins are the core components of a new class of ubiquitin E3 ligases that regulate replication and transcription. To examine the roles of CUL4 in cell cycle regulation, the effect of inactivation of CUL4 was examined in both Drosophila and human cells. Loss of CUL4 in Drosophila cells causes G1 cell cycle arrest and an increased protein level of the CDK inhibitor Dacapo. Coelimination of Dacapo with CUL4 abolishes the G1 cell cycle arrest. In human cells, inactivation of CUL4A induces CDK inhibitor p27Kip1 stabilization and G1 cell cycle arrest which is dependent on the presence of p27, suggesting that this regulatory pathway is evolutionarily conserved. In addition, it was found that the Drosophila CUL4 also regulates the protein level of cyclin E independent of Dacapo. Evidence is provided that human CUL4B, a paralogue of human CUL4A, is involved in cyclin E regulation. Loss of CUL4B causes the accumulation of cyclin E without a concomitant increase of p27. The human CUL4B and cyclin E proteins also interact with each other and the CUL4B complexes can polyubiquitinate the CUL4B-associated cyclin E. These studies suggest that the CUL4-containing ubiquitin E3 ligases play a critical role in regulating G1 cell cycle progression in both Drosophila and human cells (Higa, 2006).

The CUL1 (cullin 1; see Drosophila Cul1) containing SCF (SKP1, CUL1/CDC53, F-box proteins) ubiquitin E3 ligases are key regulators of cell cycle progression from yeast to human. The SCF E3 ligases use different F-box proteins to bind and target various cell cycle regulators for ubiquitin-dependent proteolysis. In mammalian cells, it has been shown that SKP2, an F-box protein, primarily binds and targets phosphorylated CDK inhibitors p27Kip1 and p21Cip1 for ubiquitin-dependent proteolysis, while another F-box protein, human CDC4/AGO/FBXW7 regulates the proteolysis of phosphorylated cyclin E protein. In mammalian cells, the G1 cell cycle is regulated by the relative abundance of G1 cyclin/CDKs and CDK inhibitors such as p27 and p21. Similarly, the Drosophila G1 cell cycle is regulated by the balance between the CDK inhibitor Dacapo, which shares substantial homology to p27, and cyclin E. While cyclin E is regulated by the conserved Drosophila SCFAgo E3 ligase, it is not clear how the level of Dacapo is regulated in the cell cycle (Higa, 2006).

Like other cullin family members, CUL1 is regulated by the covalent linkage of an ubiquitin like protein, NEDD8, through the neddylation activating enzyme E1 (APPBP1 and UBA3) and the E2 enzyme, UBC12. Neddylation of CUL1 dissociates CAND1, an inhibitor of SCF, from CUL1, and consequently promotes the binding of SKP1 and F-box proteins such as SKP2 to CUL1 and the assembly of the SCF E3 ligase complex. The neddylation of CUL1 is removed (deneddylated) by the peptidase activity of the COP9-signalosome complex (CSN; see Drosophila COP9 complex homolog subunit 5). Many lines of evidence suggest that the activity of cullins is regulated by the elegant balance between the neddylation and deneddylation activities (Higa, 2006).

Cullin 4 (CUL4) is a conserved core component of a new class of ubiquitin E3 ligase that also contains the UV-damaged DNA-binding protein 1 (DDB1) and Ring finger protein ROC1 (also called RBX1 or HRT1). Unlike Drosophila or other metazoans, mammals encode two paralogues of CUL4, CUL4A and CUL4B. CUL4A and CUL4B are coexpressed in many cells but the functional differences between them remain unclear. Like other cullin E3 ligases, the CUL4 proteins also bind to CAND1 and CSN, and are regulated by neddylation and deneddylation processes. Previous studies suggest that CUL4-containing E3 ligase complexes and CSN regulate the proteolysis of replication licensing protein CDT1 (see Drosophila Cdt/Double parked) in response to UV or gamma-irradiation. Additional studies suggest that DDB1, a potential SKP1-like adaptor for CUL4 E3 ligase is also involved in UV-induced CDT1 proteolysis. The CUL4ADDB1 complex also regulates the proteolysis of c-Jun and DDB2. However, the roles of CUL4-containing ubiquitin E3 ligases in cell cycle regulation remain uncharacterized. This study has investigated the regulation of cell cycle regulators by neddylation and CAND1 and reports the unexpected finding that CUL4 E3 ligase plays a critical role in regulating G1 cell cycle progression (Higa, 2006).

Loss of CUL4 E3 ligases causes a G1 cell cycle arrest that is dependent on CDK inhibitors Dacapo in Drosophila and p27 in human cells. The regulation of Dacapo and p27 by CUL4 E3 ligases occurs at the post-transcriptional levels of protein stability. Although it has not been demonstrated that p27 can be directly polyubiquitinated by the CUL4 E3 ligase complexes in vitro due to technical difficulties, this study raises the possibility that CUL4 E3 ligases may regulate Dacapo or p27 by directly targeting them for ubiquitin-dependent proteolysis. Several lines of evidence support this hypothesis. Dacapo protein is regulated by CUL4 but not by CUL1 in Drosophila cells. Although in human cells, SCFSKP2 regulates p27, there is no structural and functional evidence that SKP2 is conserved in Drosophila cells. In addition, although Dacapo shares substantial homology to p27 or p21 in the core region that mediates cyclin or CDK binding, it diverges greatly at the carboxy terminal end with p27 in which the critical threonine 187 is located for the SCFSKP2- dependent proteolysis of p27 (this threonine is absent in Dacapo). Furthermore, it was found that there are no significant differences in the SCF-dependent p27 degradation between extracts derived from the control and DDB1 or CUL4A siRNA treated cells, suggesting that reduced levels of DDB1 and CUL4A proteins does not significantly affect SCFSKP2 activity. However, these experiments do not completely rule out the possibility that CUL4A/DDB1 are catalytically involved in SCFSKP2-mediated p27 degradation since small amounts of DDB1 and CUL4A proteins remain in the siRNA treated cells. Moreover, although SKP2 represents a major proteolysis pathway for regulating p27 degradation in S phase of human cells, substantial evidence suggests there are additional pathways that regulate the stability of CDK inhibitors. For example, it was found that the Xenopus p27 homologue p27Xic1 is polyubiquitinated on chromatin only when DNA replication starts in the Xenopus egg extracts. Replication licensing protein CDT1 is proteolyzed by CUL4/ROC1 E3 ligase in response to UV or gamma-irradiation. CDT1 is also degraded in S phase in mammalian cells and such an event can be reproduced in Xenopus egg extracts in which CDT1 was found to undergo ubiquitin-dependent proteolysis once DNA replication starts. In C. elegans, loss of CUL4 stabilizes CDT1 in S phase and causes the accumulation of polyploid nuclei containing 100C DNA content. It is possible that CUL4 may also regulate the proteolysis of Dacapo or p27 in similar processes in Drosophila or human cells (Higa, 2006).

Cyclin E protein accumulates in CUL4 silenced Drosophila and human cells often in the absence of CDK inhibitors Dacapo or p27. Although this effect is more pronounced in Drosophila cells, the CUL4 E3 ligase may represent one of several pathways that regulate cyclin E in response to certain signals in mammalian cells. It has been shown that CUL1- and CUL3-containing E3 ligases regulate cyclin E stability in mammalian cells. Cyclin E expression and its protein stability are also regulated by an E2F/DP-1 dependent process. This study found that cyclin E directly interacts with Drosophila CUL4 and human CUL4B and the isolated CUL4A or CUL4B immunocomplexes can polyubiquitinate the associated cyclin E in vitro. These observations raise the possibility that cyclin E may also be a direct ubiquitination target of CUL4 E3 ligases in vivo. These studies indicated that loss of CAND1, APPBP1, or CSN has differential effects on Armadillo/β-catenin and cyclin E. This effect could be partly explained by the observation that while Armadillo is regulated by CUL1-containing SCF ligase, cyclin E is controlled by both CUL1 and CUL4 E3 ligases. Evidence is also provided that the effects of CAND1, APPBP1 or CSN deficiency on the substrates of various cullin E3 ligases may be different. Further analysis is required to investigate the mechanisms for these observations (Higa, 2006).

These data reveal that CUL4 E3 ligase represents a novel and conserved pathway from Drosophila to human cells in regulating CDK inhibitors and cyclin E. In the G1 cell cycle, the CDK inhibitors Dacapo and p27 appear to be the primary targets of CUL4 E3 ligases, since loss of CUL4 in Drosophila or CUL4A in human leads to the G1 cell cycle arrest rather than enhanced S phase entry. Since the gene encoding CUL4A is amplified in many breast cancers and hepatocellular carcinomas and since low or absent expression of p27 is often associated with malignant cancers, these studies also highlight how altered regulation of CUL4 E3 ligase may contribute to the genesis and progression of human cancers (Higa, 2006).

The Cyclin-dependent kinase inhibitor Dacapo promotes genomic stability during premeiotic S phase

The proper execution of premeiotic S phase is essential to both the maintenance of genomic integrity and accurate chromosome segregation during the meiotic divisions. However, the regulation of premeiotic S phase remains poorly defined in metazoa. Here, this study identified the p21Cip1/p27Kip1/p57Kip2-like cyclin-dependent kinase inhibitor (CKI) Dacapo (Dap) as a key regulator of premeiotic S phase and genomic stability during Drosophila oogenesis. In dap-/- females, ovarian cysts enter the meiotic cycle with high levels of Cyclin E/cyclin-dependent kinase (Cdk)2 activity and accumulate DNA damage during the premeiotic S phase. High Cyclin E/Cdk2 activity inhibits the accumulation of the replication-licensing factor Doubleparked/Cdt1 (Dup/Cdt1). Accordingly, this study found that dap-/- ovarian cysts have low levels of Dup/Cdt1. Moreover, mutations in dup/cdt1 dominantly enhance the dap-/- DNA damage phenotype. Importantly, the DNA damage observed in dap-/- ovarian cysts is independent of the DNA double-strands breaks that initiate meiotic recombination. Together, these data suggest that the CKI Dap promotes the licensing of DNA replication origins for the premeiotic S phase by restricting Cdk activity in the early meiotic cycle. dap-/- ovarian cysts frequently undergo an extramitotic division before meiotic entry, indicating that Dap influences the timing of the mitotic/meiotic transition (Narbonne-Reveau, 2009).

During the meiotic cycle, germ cells complete two divisions to produce haploid gametes. Before the two meiotic divisions, the germ cells duplicate their genomes during the premeiotic S phase. Events unique to the premeiotic S phase, such as the expression of REC8, a member of the kleisin family of structural maintenance of chromosome proteins, are required for the full execution of the downstream meiotic program. How this specialized meiotic S phase is regulated, as well as how similar it is to the mitotic S phase, has long been a question of interest. Studies from yeast indicate that the mitotic cycle and the meiotic cycle use much of the same basic machinery to replicate their genomes. For example, the minichromosome maintenance complex (MCM2-7), which functions as a DNA replication helicase, is essential for the duplication of the genome during both the mitotic and premeiotic S phase. Additionally, both the mitotic and premeiotic S phase require the activity of cyclin-dependent kinases (Cdks). Yet, despite its fundamental importance to both the maintenance of genomic integrity and the downstream events of meiosis, little is known about the regulation of premeiotic S phase metazoa (Narbonne-Reveau, 2009).

Drosophila provides an excellent model to examine the early events of the meiotic cycle, because the entire process of oogenesis takes place continuously within the adult female. In Drosophila, each ovary is composed of 12-16 ovarioles containing linear strings of maturing follicles also called egg chambers. New egg chambers are generated at the anterior of the ovariole in a region called the germarium that contains both germline and somatic stem cells. The germarium is divided into four regions according to the developmental stage of the cyst. Oogenesis starts in region 1 when a cystoblast, the asymmetric daughter of the germline stem cell, undergoes precisely four round of mitosis with incomplete cytokinesis to produce a cyst of 16 interconnected germline cells with an invariant pattern of interconnections (individual cells in the cyst are referred to as cystocytes). Stable actin-rich intercellular bridges called ring canals connect individual cystocytes within the cyst. Germline cyst formation is accompanied by the growth of the fusome, a vesicular and membrane skeletal protein-rich organelle that forms a branched structure extending throughout all the cells of the cyst. After the completion of the mitotic cyst divisions, all 16 cystocytes complete a long premeiotic S phase in region 2a of the germarium. Subsequently, the two cystocytes with four ring canals form long synaptonemal complexes (SCs) and begin to condense their chromatin, suggesting that they are in pachytene of meiotic prophase I. Several of the cells with three ring canals also assemble short SCs, and even cells with only one or two ring canals are occasionally seen to contain traces of SCs. However, as oogenesis proceeds, the SC is restricted to the two pro-oocytes and finally to the single oocyte in region 2b. The other 15 cystocytes lose their meiotic characteristics, enter the endocycle and develop as polyploid nurse cells (Narbonne-Reveau, 2009).

During both the mitotic cycle and the meiotic cycle, it is essential that the entire genome is duplicated precisely once during the S phase. In the mitotic cycle, the licensing of the DNA occurs when Cdc6 and Cdt1/Double Parked (Dup) load the MCM2-7 complex onto the origin recognition complex (ORC) to form the prereplication complex (preRC). PreRC formation occurs in late mitosis and G1 when Cdk activity is low. At the onset of S phase, Cdk activity increases, and the preRC initiates bidirectional DNA replication. PreRC formation must be suppressed after the initiation of S phase to prevent rereplication and thus ensure that each segment of the DNA is replicated exactly once per cell cycle. Cdks play a critical role in this process by preventing reestablishment of the preRC through multiple redundant mechanisms. Thus, during the mitotic cycle the precise regulation of Cdk activity ensures that each segment of DNA is replicated once, and only once, per cell cycle (Narbonne-Reveau, 2009).

The p21cip1/p27kip1/p57kip2-like cyclin-dependent kinase inhibitor (CKI) Dacapo (Dap) specifically inhibits Cyclin E/Cdk2 complexes (de Nooij, 1996; Lane, 1996). In Drosophila, Cyclin E/Cdk2 activity is required for DNA replication during both mitotic cycles and endocycles (Knoblich, 1994; Lilly, 1996). Similar to what is observed with CKIs in other animals, Dap functions to coordinate exit from the cell cycle with terminal differentiation. Indeed, high levels of Dap are observed upon exit from the cell cycle in multiple tissues during both embryonic and larval development. Additionally, in the adult ovary, high levels of Dap prevent oocytes from entering the endocycle with the nurse cells as ovarian cysts exit the germarium in stage 1 of oogenesis (Hong, 2003). However, in addition to its well-established developmental function, recent work indicates that during developmentally programmed endocycles Dap facilitates the licensing of DNA replication origins by reinforcing low Cyclin E/Cdk2 kinase activity during the Gap phase (Hong, 2007). In dap-/- mutants, cells undergoing endocycles have reduced chromatin bound MCM2-7 complex, indicating a reduction in the density of preRCs along the chromatin. Additionally, dap-/- cells accumulate high levels of DNA damage due to the inability to complete genomic replication (Hong, 2007). Thus, during developmentally programmed endocycles Dap functions to reinforce low Cdk activity during the Gap phase (Narbonne-Reveau, 2009).

This study demonstrates that the CKI Dap promotes genomic stability during the premeiotic S phase of the Drosophila oocyte. The data indicate that Dap facilitates the licensing of DNA replication origins for the premeiotic S phase by restricting Cyclin E/Cdk2 activity during the early meiotic cycle. These studies represent the first example of a CKI regulating premeiotic S phase and genomic stability in a multicellular animal. Additionally, Dap was found to influence the timing of the mitotic/meiotic switch in ovarian cysts (Narbonne-Reveau, 2009)..

Cells in the mitotic cycle and the meiotic cycle face a similar challenge. To maintain the integrity of the genome, they must replicate their DNA once, and only once, during the S phase. In mitotic cells, this goal is accomplished, at least in part, through the precise regulation of Cdk activity throughout the cell cycle. During the mitotic cycle, Cdk activity inhibits preRC formation. This inhibitory relationship, restricts the assemble of preRCs to a short window from late mitosis to G1, when Cdk activity is low, and provides an important mechanism by which mitotic cells prevent DNA rereplication. However, the inhibitory effect of Cdk activity on preRC assembly necessitates that cells have a strictly defined period of low Cdk activity before S phase, to assemble preRCs for the next round of DNA replication. In mammals and yeast, compromising this period of low Cdk activity by overexpression G1 cyclins results in decreased replication licensing and genomic instability (Narbonne-Reveau, 2009).

One means by which cells inhibit Cdk activity is the expression of CKIs. In the mitotic cycle of budding yeast, the deletion of the CKI Sic1, which contains a Cdk inhibitor domain that is structurally conserved with the inhibitor domain present in the dap homologue p27Kip1, results in inadequate replication licensing and genomic instability due to the precocious activation of Cdks in G1. The current data strongly suggest that Dap plays a similar role in defining a critical period of low Cdk activity during the early meiotic cycle in Drosophila females (Narbonne-Reveau, 2009).

Based on these results, it is proposed that the Dap facilitates the licensing of DNA replication origins in ovarian cysts by restricting the inhibitory effects of Cyclin E/Cdk2 kinase activity on preRCs formation before premeiotic S phase. The data support the model that in the absence of Dap, ovarian cysts enter premeiotic S phase with a reduced number of licensed origins and thus fail to complete genomic replication. This hypothesis is supported by several observations. First, relative to wild-type, dap-/- ovarian cysts spend an increased proportion of their time in premeiotic S phase, as evidenced by the increased proportion of 16-cell cysts that incorporated EdU. The lengthening of premeiotic S phase is in line with the hypothesis that dap-/- ovarian cysts initiate DNA replication from a reduced number of licensed origins. Second, dap-/- ovarian cysts accumulate DNA damage during the premeiotic S phase. The accumulation of DNA damage during the premeiotic S phase is consistent with decreased preRC assembly resulting in intraorigin distances that are too large to be negotiated by DNA polymerase during a single S phase. Third, dap-/- meiotic cysts have decreased levels of the preRC component Dup/Cdt1. Moreover, genetic analysis indicates that Dup/Cdt1 levels are indeed limiting for premeiotic S phase in the dap-/- background. Specifically, it was found that reducing the dose of dup/cdt1 dramatically increases the levels of DNA damage observed in dap-/- ovarian cysts in region 2a and 2b of the germarium. In Drosophila, the levels of Dup/Cdt1 are negatively regulated by Cyclin E/Cdk2 activity (Narbonne-Reveau, 2009).

The use of the CKI Dap to restrict Cdk activity and thus promote the formation of preRCs before S phase is observed in multiple cell types beyond the oocyte. In previous work, it was found that in dap-/- mutants, cells in developmentally programmed endocycles also accumulate DNA damage and have dramatically reduced levels of Dup/Cdt1 (Hong, 2007). Thus, Dap functions to promote the accumulation of Dup/Cdt1 in multiple developmental and cell cycle contexts in Drosophila. Indeed, in select mitotic cycles removing one copy of dup/cdt1 in a dap-/- background results in DNA damage and cell death. However, in most mitotic cycles the requirement for Dap is redundant with other mechanisms that restrict Cyclin E/Cdk2 activity (Narbonne-Reveau, 2009).

Why Dap is required for preRC assembly in some cell types but not others remains unclear. However, it is interesting to note that DNA replication that occurs outside the confines of the canonical mitotic cycle, during the meiotic S phase and the S phase of developmentally programmed endocycles, is most dependent on Dap function (Hong, 2007). Thus, the increased reliance on the CKI Dap to establish a period of low Cdk activity before the onset of DNA replication may be explained by the absence of cell cycle programs that are specific to the mitotic cycle. For example, the tight transcriptional control of S phase regulators during the mitotic cycle may make the presence of Dap unnecessary for proper S phase execution. Alternatively, there may be differential regulation of the machinery that controls the regulated destruction of cyclins in the archetypical mitotic cycle versus the variant cell cycles of meiosis and the endocycle. In the future, determining why Dap plays a nonredundant role in the regulation of DNA replication during the meiotic cycle, but not the mitotic cycle, will be an important avenue of study (Narbonne-Reveau, 2009).

In addition to its role in the regulation of premeiotic S phase, this study found that dap influences the number of mitotic cyst divisions that occur before meiotic entry. In dap-/- mutants, ∼25% of ovarian cysts complete a fifth mitotic division to produce ovarian cysts with 32 cells. Similarly, mutations that compromise the degradation of the Cyclin E protein also result in production of 32-cell cysts. In line with these observations, females with reduced levels of Cyclin E produce ovarian cysts that undergo only three mitotic divisions and thus contain eight cells. Why Cyclin E/Cdk2 activity influences the timing of meiotic entry is not fully understood. However, the data suggest that the cyst division phenotype is not a direct result of reducing the number of preRCs assembled for the premeiotic S phase. Specifically, it was found that in dap-/- females reducing the dose of dup/cdt1 does not increase the number of ovarian cysts that undergo an extra division. In contrast, reducing the dose of dup/cdt1 in dap-/- females significantly enhances the meiotic DNA damage phenotype. These data strongly suggest that the extramitotic cyst division observed in dap-/- ovarian cyst is not the direct result of high CyclinE/Cdk2 activity inhibiting preRC formation (Narbonne-Reveau, 2009).

Intriguingly, Cdk2 is not the only Cdk that influences the number of ovarian cyst divisions in Drosophila females. Surprisingly, increasing the activity of the mitotic kinase Cdk1 results in the production of egg chambers with eight-cell cysts. Moreover, decreased Cdk1 activity results in ovarian cysts undergoing five mitotic divisions to produce egg chambers with 32 cells. Thus, Cdk1 and Cdk2 seem to have opposing roles in the regulation of the ovarian cysts divisions and/or meiotic entry. One of several possible explanations for these data, is that the number of ovarian cyst divisions is influenced by the amount of time cystocytes spend in a particular phase (G1, S, G2, and M) of the cell cycle. In the mitotic cycle of the Drosophila wing, there is a compensatory mechanism that ensures that changes in the length of one phase of the cell cycle result in alterations in the other phases of the cell cycle to ensure normal division rates. This compensatory mechanism is likely to be operating in multiple cell types and may account for why Cdk1 and Cdk2 activity have opposite effects on the number of ovarian cyst divisions. Alternatively, Cdk1 and Cdk2 may act on truly independent pathways that have opposing roles in regulating the number of mitotic cyst divisions and/or the timing of meiotic entry. Ultimately, why Cdk1 and Cdk2 activity have opposite effects on the number of ovarian cyst divisions that occur before meiotic entry awaits the identification of essential downstream targets of these kinases (Narbonne-Reveau, 2009).

In summary, this study has defined two novel functions for a p21Cip/p27Kip1/p57Kip2-like CKI during the meiotic cycle, the regulation of the mitotic/meiotic transition and the maintenance of genomic stability during the premeiotic S phase (Narbonne-Reveau, 2009).

dacapo: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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