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DEVELOPMENTAL BIOLOGY

Embryonic

Maternal STG mRNA is distributed uniformly throughout the yolk and cytoplasm, and persists through cycle 13. It is degraded to an undetectable level during the first 20-30 minutes of interphase 14. Zygotic STG mRNA begins to accumulate in the latter half of interphase 14, approximately 25-35 minutes before the first cells enter mitosis 14. Accumulation is limited to those cells that will divide, and occurs in a temporal sequence nearly identical to, but preceding, the sequence of mitoses. The earliest zygotic stg expression is in a wide ribbon of cells along the ventral surface of the blastoderm that will invaginate to form the mesoderm. Several minutes later, as the cephalic furrow begins to form, expression begins in a set of bilaterally symmetric stripes and spots in the head region, then in the dorsolateral regions, and then along the margin of the ventral furrow and in expanded regions of the head and tail. In each case the appearance of STG correlates with future mitotic domains (Edgar, 1989).

Differential cell cycle regulation during postblastoderm development (cell cycles 14-16) occurs in G2. STG mRNA expressed from a heat shock promotor triggers mitosis and an associated S phase in G2 cells only during these cycles. The G2/M transition is the only differentially regulated transition in the cell cycle at this stage of development. Once a G2/M transition is triggered, subsequent aspects of cycle progression appear to proceed automatically, in orderly sequence until the next G2 is achieved. Hence, differential cell cycle timing at this developmental stage is controlled by stg. Heat-induced stg expression can be used to alter the normal pattern of embryonic mitoses (Edgar, 1990).

The conserved regulators of cell cycle progression--cyclins, cdc2 kinase , and String phosphatase (cdc25)--accommodate multiple modes of regulation during Drosophila embryogenesis. During cell cycles 2-7, cdc2/cyclin complexes are continuously present and show little fluctuation in abundance, phosphomodification, or activity. This suggests that cycling of the mitotic apparatus does not require cytoplasmic oscillations of known regulatory activities. Levels of String protein, found to be quite low at fertilization, rise during the first 8 cycles and then gradually decline. The accumulation of String and its gradual disappearance occurs without changes in the level of maternal STR mRNA, which remains stable until interphase 14. This suggests translational or post-translational control of String. String is phosphorylated, and the degree of phosphorylation, which begins to fluctuate in cycle 5 or 6, peaks at mitosis. If these fluctuations in String phosphorylation control its activity, and thus influence cdc2 activity, the level of tyrosine 15-phospho-cdc2 should fluctuate during these early cycles. No detectable such phosphorylation of String occurs until the complete disappearance of String in cycle 14. Thus cdc2 phosphorylation does not regulate the early cell cycles. It appears that cyclin synthesis is limiting for mitoses 10-13 (Edgar, 1994b).

The germ cells of metazoans follow a program of proliferation that is distinct from proliferation programs of somatic cells. Despite their developmental importance, the cell proliferation program in the metazoan primordial germ cells is not well characterized and the regulatory controls are not understood. In Drosophila, germ cell precursors (called pole cells) proliferate early in embryogenesis and then enter a prolonged quiescence. Pole cell formation begins when nuclei at the posterior end of the embryo pinch off to form pole buds during cycle 9. Pole buds divide twice while somatic nuclei undergo nuclear cycles 9 and 10. The pole buds then cellularize at the end of cycle 10. The pole cells divide asynchronously, zero to two times as suggested by cell counts, while somatic nuclei divide three more times (somatic cycles 11-13). Thus, polar nuclear divisions are asynchronous and lag behind somatic nuclear divisions during syncytial cycles 9 and 10. The polar division program deviates from the somatic division program at a time when pole nuclei and somatic nuclei still share a common cytoplasm: this is earlier than had previously been thought to occur. The lag in polar nuclear divisions is independent of grapes, which is required for lengthening somatic cell cycles 10-13. Mapping of the last S phase in pole cells and measurement of their DNA content indicate that pole cells become quiescent in G2 phase of the cell cycle. Cyclin A accumulates in arrested pole cells, consistent with a G2 arrest. Quiescent pole cells can be driven into mitosis by induction of either an activator of Cdc2 (Cdc25 [string] phosphatase) or a mutant form of Cdc2 that cannot be inhibited by phosphorylation. In contrast, induction of wild-type Cdc2 with a mitotic cyclin does not induce mitosis in pole cells. It is proposed that inhibition of Cdc2 by phosphorylation contributes to G2 arrest in pole cells during embryogenesis. Furthermore, pole cells enter G1 following induced mitoses, indicating that entry into both mitosis and S phase is blocked in quiescent pole cells. Although the data suggest that expression of String is likely to contribute to the reentry of pole cells into the cell cycle, how this reentry occurs during normal development is not understood. It is not clear exactly when quiescence is terminated in pole cells; although pole cell division is known to occur in late embryogenesis. These studies represent the first molecular characterization of proliferation in embryonic germ cells of Drosophila (Su, 1998).

Studies in unicellular systems have established that DNA damage by irradiation invokes a checkpoint that acts to stall cell division. During metazoan development, the modulation of cell division by checkpoints must occur in the context of gastrulation, differential gene expression and changes in cell cycle regulation. To understand the effects of checkpoint activation in a developmental context, a study was performed of the effect of X-rays on post-blastoderm Drosophila embryos. In Drosophila, DNA damage delays anaphase chromosome separation during cleavage cycles that lack a G2 phase. In post-blastoderm cycles that include a G2 phase, irradiation delays the entry into mitosis. Gastrulation and the developmental program of string (Cdc25) gene expression, which normally regulates the timing of mitosis, occurs normally after irradiation. The radiation-induced delay of mitosis accompanies the exclusion of mitotic cyclins from the nucleus. Furthermore, a mutant form of the mitotic kinase Cdk1 that cannot be inhibited by phosphorylation drives a mitotic cyclin into the nucleus and overcomes the delay of mitosis induced by irradiation. It is concluded that developmental changes in the cell cycle, for example, the introduction of a G2 phase, dictate the response to checkpoint activation, for example, delaying mitosis instead of or in addition to delaying anaphase. This unprecedented finding suggests that different mechanisms are used at different points during metazoan development to stall cell division in response to checkpoint activation. The delay of mitosis in post-blastoderm embryos is due primarily to inhibitory phosphorylation of Cdk1, whereas nuclear exclusion of a cyclin-Cdk1 complex might play a secondary role. Delaying cell division has little effect on gastrulation and developmentally regulated string gene expression, supporting the view that development generally dictates cell proliferation and not vice versa (Su, 2000).

To examine the effect of DNA damage on the progression of the cell cycle during Drosophila embryogenesis, embryos 0-4.5 hours of age were exposed to 570 rads of X-rays. At this dose, 40%-60% of cellular embryos die and fail to hatch into larvae. This dose therefore corresponds to the half-maximal lethal dose (LD50). When syncytial embryos are exposed to X-rays: nuclei enter mitosis normally but chromosome segregation is delayed. The delay is transient such that nuclei enter the next interphase without completely separating sister chromosomes, resulting in polyploid nuclei (Su, 2000).

In cellularized embryos, changes in cell cycle indicators that are consistent with a delay in the entry into mitosis are observed. In untreated embryos at these stages, cells divide in stereotypical clusters termed 'mitotic domains'. Both the location of a mitotic domain within the embryo and the time at which it goes through mitosis are invariant from embryo to embryo. The timing of morphogenetic movements that comprise gastrulation is likewise invariant from embryo to embryo. Thus, the wild-type pattern of mitotic cells at any specific time during this period, as indicated by the extent of gastrulation, is easily recognizable. In irradiated samples, embryos were found in which expected mitotic domains were not in mitosis, as judged by the absence of condensed chromosomes and mitotic figures. Antibody staining for a mitotic-specific phospho-epitope on histone H3 (PH3), and staining with wheatgerm agglutinin (WGA) to detect nuclear envelope breakdown, has confirmed the absence of mitoses in these embryos. It is inferred that irradiation delays the entry into mitosis in cellularized embryos, whereas under identical conditions, chromosome segregation is delayed in syncytial embryos. Treatment of cellularized embryos with a DNA-damaging agent, methyl methane sulfonate, results in a similar delay of mitosis. Therefore, the observed effect of irradiation on mitosis is probably due to the DNA-damaging activity of X-rays (Su, 2000).

It is an unprecedented finding that irradiation leads to two different cell cycle responses in a single organism: either the delay of anaphase chromosome segregation or the delay of mitosis. Mitotic chromosome segregation and the initiation of mitosis are regulated by different mechanisms. The former requires the proteolysis of proteins, such as PDS1 in budding yeast and cyclin A in Drosophila, whereas the latter requires the activation of mitotic cyclin-Cdk complexes. It is suggested that checkpoint activation by the same dose of radiation under identical conditions must have used different downstream mechanisms in order to delay chromosome segregation in the syncytium and mitosis in the cellularized embryos. Although mechanisms that operate in the syncytium remain elusive, the mechanisms used by cellularized embryos were addressed in this study (Su, 2000).

Despite the finding that irradiation does not interfere with String expression, it might have antagonized String activity. Cdc25Stg activates Cdk1 by removing the inhibitory phosphates on Thr14 and Tyr15. A Cdk1 mutant in which these residues have been mutated (Cdk1AF) bypasses the requirement for String. If the mechanism by which radiation delays mitosis solely involves inhibitory phosphorylation of Cdk1, Cdk1AF should bypass the radiation-induced delay. To test this hypothesis, Cdk1 or Cdk1AF, in conjunction with a mitotic cyclin, was expressed from a heat-inducible (hs) promoter during interphase 14. It was then asked whether irradiation could delay the onset of mitosis 14 in embryos expressing these transgenes. It was found that many cells of heat-shocked embryos that carried hs-Cdk1AF and hs-mitotic cyclin transgenes fail to delay mitosis after irradiation. This effect was seen with mitotic cyclins A, B or Bs -- a truncated version of cyclin B that is resistant to proteolysis. In contrast, embryos carrying hs-Cdk1, in combination with the same cyclins, behave like wild-type embryos and delay mitosis. It is concluded that Cdk1AF, and not Cdk1, can overcome the radiation-induced delay in mitosis. It is inferred that inhibitory phosphorylation on Cdk1 is required to delay mitosis in response to DNA damage, in agreement with previous results from fission yeast and vertebrates (Su, 2000).

Interestingly, the ability of Cdk1AF and cyclins to overcome the delay of mitosis in Drosophila was seen only in certain cells of the embryos, and these cells represent mitotic domains, for example, domain 4. Cells of mitotic domains are distinguished from their neighbors by their accumulation of String protein. Although further experiments are required to demonstrate the importance of String, the perfect coincidence of clusters of irradiated cells that entered mitosis in the presence of Cdk1AF as well as accumulated String, has led to the following suggestion: although cyclin-Cdk1AF activity is not present in sufficient quantities to promote mitosis by itself under these experimental conditions, this activity can induce endogenous String to activate endogenous Cdk1 and induce mitosis. A similar feedback mechanism has been proposed for human Cdk1 and Cdc25. It follows then that endogenous String and Cdk1 might be inhibited by irradiation, but that this inhibition can be overcome by a small amount of Cdk1AF activity (Su, 2000).

The same amount of Cdk1AF activity overcomes another consequence of irradiation, namely, the nuclear exclusion of a mitotic cyclin. Nuclear cyclin/Cdk1 activity is a prerequisite to mitosis and the exclusion of cyclin B1 from the nucleus appears to contribute to the delay in mitosis after irradiation in human cells. In cellular-stage Drosophila embryos, cyclins A and B remain enriched in the cytoplasm in interphase. Cyclin A accumulates in the nucleus of cells that initiate mitosis, as does cyclin B. In irradiated embryos, both cyclins A and B are excluded from nuclei although their levels remain unchanged. In cells that express Cdk1AF (with a mitotic cyclin) that enter mitosis even after irradiation, nuclear accumulation of cyclin A is evident. Thus, a low level of Cdk1 activity, provided by Cdk1AF in these experiments, leads to both the nuclear accumulation of a cyclin and the entry into mitosis (Su, 2000).

Given these two observations -- that Cdk1AF drives the nuclear accumulation of cyclin A and that nuclear accumulation of mitotic cyclins coincides with the entry into mitosis in unperturbed cell cycles -- it has been proposed that Cdk1 activity normally drives the nuclear accumulation of cyclin-Cdk1 complexes. In support of this idea, Cyclin A remains excluded from nuclei in string mutants. In accordance with this, Cdk1AF, in conjunction with endogenous String, overcomes the radiation-induced delay of mitosis because Cdk1AF can start the feedback loop that activates endogenous Cdk1 by endogenous String and Cdk1 activity can drive the nuclear accumulation of cyclin-Cdk1. These ideas help explain previous observations in human cells. In the latter, although the exclusion of cyclin B1 from nuclei appears to be of some importance to regulating mitotic entry, Cdk1AF can overcome the checkpoint-induced delay of mitosis, regardless of whether cyclin B1 or NLS-cyclin B1, which is constitutively localized to the nucleus, is co-expressed. Thus, Cdk1AF in human cells, as in Drosophila, might also drive the nuclear accumulation of cyclin-Cdk complexes and the entry into mitosis by initiating a positive feedback loop for the activation of endogenous Cdk1. Whether a similar feedback loop of Cdk1, String and cyclin-localization operates to control mitosis in other tissues, such as larval imaginal discs, remains to be seen (Su, 2000).

Whereas the segmental nature of the insect head is well established, relatively little is known about the genetic and molecular mechanisms governing this process. The phenotypic analysis is reported of mutations in collier (col), which encodes the Drosophila member of the COE family of HLH transcription factors and is activated at the blastoderm stage in a region overlapping a parasegment (PS0: posterior intercalary and anterior mandibular segments) and a mitotic domain, MD2. col mutant embryos specifically lack intercalary ectodermal structures. col activity is required for intercalary-segment expression both of the segment polarity genes hedgehog, engrailed, and wingless, and of the segment identity gene cap and collar. The parasegmental register of col activation is controlled by the combined activities of the head-gap genes buttonhead and empty spiracles and the pair-rule gene even skipped; it therefore integrates inputs from both the head and trunk segmentation systems, which were previously considered as being essentially independent. During the interphase of cycle 14, the broad band of ectopic col activation resolves into a distinctive stripe, separated from the normal PS0 stripe by one to three cells going from ventral to dorsal. col expression precisely overlaps the expression of string (stg), in the region prefiguring mitotic domain 2. Expression of stg, which triggers the G2/M transition, is unchanged in embryos deficient for col and vice versa, arguing that MD2 cells undergo a concerted mitotic and differentiation program, set upstream of both col and stg. This led to an examination of whether eve was also involved in defining the position of MD2, using antibodies against the phosphorylated form of histone H3 as a marker of mitosis. Like col transcription, MD2 expands posteriorly in eve mutant embryos at early cycle 14 to form a second, ectopic, stripe of mitotic cells at the beginning of gastrulation. Together, these results show that col expression and MD2 position integrate inputs from both the head and trunk segmentation systems, which were previously considered as being essentially independent (Crozatier, 1999b).

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

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

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

RNAi of mitotic cyclins in Drosophila uncouples the nuclear and centrosome cycle

Successful cell duplication requires orderly progression through a succession of dramatic cell-cycle events. Disruption of this precise coupling can compromise genomic integrity. The coordination of cell-cycle events is thought to arise from control by a single master regulator, cyclin:Cdk, whose activity oscillates. However, very little is known of how individual cell-cycle events are coupled to this oscillator and how the timing of each event is controlled. An approach with RNA interference (RNAi) and real-time imaging was developed to study cyclin contributions to the rapid syncytial divisions of Drosophila embryos. Simultaneous knockdown of all three mitotic cyclins, Cyclin A, Cyclin B, and Cyclin B3, blocked nuclei from entering mitosis. Despite nuclear arrest, centrosomes and associated myosin cages continue to divide until the midblastula transition. Centrosome division is synchronous throughout the embryo and the period of the uncoupled duplication cycle increases over successive divisions. In contrast to its normal actions, injection of a competitive inhibitor of the anaphase-promoting complex/cyclosome (APC/C) after knockdown of the mitotic cyclins does not interfere with the centrosome-duplication cycles. Finally, how cyclin knockdown affects the onset of cellularization at the midblastula transition was studied and it was found that nuclear cell-cycle arrest did not advance or delay onset of cellularization. This study shows that knockdown of mitotic cyclins allows centrosomes to duplicate in a cycle that is uncoupled from other cell-cycle events. It is suggested that high mitotic cyclin normally ensures that the centrosome cycle remains entrained to the nuclear cycle (McCleland, 2008).

Interestingly, these data show that reduction in mitotic cyclin blocks mitosis without blocking centrosome duplication. It is suggested that the uncoupled centrosome cycles represent full duplication cycles, because three cycles of centrosome duplication were observed, which requires more than a division of previously duplicated centrosome components. Furthermore, the appearance and movements of centrosomes is similar to that seen in normal cycles. How might cyclin knockdown bypass the normally tight coordination between centrosome and nuclear division (McCleland, 2008)?

After cyclin knockdown, reduced accumulation is seen of a GFP-cyclin reporter and reduced cyclin A is seen on Western blots. The residual cyclin could retain some function, but it is not adequate to facilitate mitosis. Thus, the level of mitotic cyclin required for centrosome multiplication (if any) is less than the level required for mitosis. Accordingly, in a standard cell-cycle paradigm, cyclin accumulation would first satisfy the threshold for centrosome multiplication and only later reach the mitotic threshold, followed by the resetting of the cycle by mitotic cyclin destruction. A simple interpretation of these results is that cyclin accumulation has been arrested between the two thresholds so that mitosis is blocked but centrosome multiplication proceeds. However, if the centrosome cycle is mechanistically coupled to the mitotic cycle, one might expect that blocking mitosis would also block the centrosome cycle, unless mitotic cyclins are also required for the coupling mechanism. Indeed, there are a number of indications that mitotic cyclins influence the centrosome cycle. Moreover, there are also observations that suggest several points of coupling of the centrosome-cycle and cell-cycle progression (McCleland, 2008).

The centrosome-duplication cycle normally occurs in lockstep with progress through the cell cycle. During syncytial mitoses, the centrioles of a centriole pair disjoin at the transition to anaphase, daughter centriole assembly begins in anaphase, and centrosomes move apart during interphase. Subsequent shifts in this coordination occur in parallel with changes in cyclin:Cdk regulation. When a G2 phase appears in cycle 14, completion and maturation of daughter centrioles is held in abeyance until expression of Cdc25^stg. Furthermore, when a G1 appears in cycle 17, initiation of daughter centrioles is deferred because of cyclin E downregulation. Apparently, multiple steps of the centrosome cycle are coupled to the cell cycle, and previous work suggests various ways that cyclin:Cdk might couple the centrosome and mitotic cycles (McCleland, 2008).

Conceptually, the once-per-cell-cycle duplication of centrosomes is similar to the regulation of DNA replication. DNA replication is coupled to oscillations in cyclin:Cdk activity because cyclin:Cdk inhibits one step of replication but is required to promote another. However, centrosomes have been found to duplicate in experimental conditions apparently lacking oscillations of cyclin:Cdk1. In a Xenopus egg extract arrested with low mitotic cyclin:Cdk1 kinase activity by inhibition of DNA synthesis, centrosomes continued replicating in a cyclin E dependent fashion. Thus, cyclin E:Cdk2 makes a positive contribution to the centrosome cycle, but centrosomes multiplied in its continuous presence, indicating that this cyclin:Cdk does not block duplication. Similarly, upon deletion of S. cerevisiae Clb 1-4 (G2 cyclins), uncoupled duplication of the spindle-pole bodies occurred in the continuous presence of Cln2 (G1 cyclin) or Clb5 (S phase cyclin). Thus, G1 cyclins and/or S phase cyclins promote centrosome duplication without blocking it. Importantly, in normal cycles the G1 cyclins do not provoke multiple rounds of uncoupled centrosome division (McCleland, 2008).

How might such divisions be suppressed? Interestingly, as in the above experiments in Xenopus and yeast, treatments that eliminate or suppress mitotic cyclin:Cdk1 seem to uncouple centrosome replication. Centrioles multiplied without mitosis in Drosophila upon temperature inactivation of a Cdk1^ts, and centrosomes amplified in sea urchin and frog embryos arrested by inhibition of protein synthesis, which presumably blocks cyclin accumulation. Thus, the findings are in accord with previous observations in suggesting that mitotic cyclins are required to enforce coupling of the centrosome cycle to the mitotic cycle (McCleland, 2008).

Suppression of uncoupled centrosome cycles by mitotic cyclin:Cdk1 could be the result of inhibition of one or more steps of the centrosome cycle. Indeed, stabilized versions of the mitotic cyclins or inhibition of the APC/C blocks mitotic exit and blocks daughter-centriole production, showing that mitotic cyclins have either a direct or indirect inhibitory action on centrosome replication (McCleland, 2008).

Because several steps of the centrosome cycle appear coupled to the cell cycle, the cyclin inputs might be complex. For example, the finding that Cdc25^stg promotes daughter-centriole maturation in G2 of cycle 14 suggests that cyclin:Cdk1 activation is required for centrosome maturation. However, this is not easily consistent with the observation that inactivation of Cdk1^ts allows centriole multiplication without a deficit in daughter-centriole growth\. Another possibility is that Cdc25^stg removes an inhibitor of daughter-centriole maturation. Indeed, tyrosine phosphorylated Cdc28 of S. cerevisiae inhibits spindle-pole-body duplication, and Cdc25^Mih1 reverses this inhibition. In summary, present evidence is consistent with direct or indirect inhibition of centrosome duplication by mitotic cyclin (McCleland, 2008).

It is noted that the multiple centrioles produced after inactivation of Drosophila Cdk1^ts did not separate and that the separation of yeast spindle-pole bodies requires active Cdc28. It is suggested that there is also a positive contribution of mitotic cyclin:Cdk1 to centrosome multiplication but that this requirement is either absent in the early syncytial cycles or that it is satisfied by a low level of mitotic cyclin that persists following RNAi. If a residue of Cdk1 promotes the uncoupled centrosome cycle, why does it not also promote mitosis? Perhaps the cyclin level is too low, the residual Cdk1 activity is localized to the centrosome, or the nuclear cycle is prevented by an unappreciated checkpoint (McCleland, 2008).

If cyclin:Cdk1 provides both negative and positive contributions to the centrosome cycle, a simple model could explain coupling of the centrosome cycle to mitosis. G1 cyclins promote centrosome duplication but also trigger mitotic cyclin accumulation. If kinase inactive mitotic cyclin:Cdk1 inhibits a step in centrosome maturation, this would ensure centrosomes do not divide until mitotic entry, whereupon Cdk1 activation would allow completion of centrosome duplication. Furthermore, if active cyclin:Cdk1 kinase and metaphase activities suppressed centrosome separation, separation of the duplicated centrosome would await mitotic exit. Additional studies will be required to define this multistep coupling mechanism (McCleland, 2008).

The switch from maternal to zygotic regulation at the MBT involves a wholesale reorganization of many regulatory circuits. Although there has been great interest in the mechanisms that time and coordinate this transition, little is known about either the timer or the mechanism. Experiments in frog and fly have suggested that the MBT occurs when the exponential multiplication of nuclei increases the nuclear to cytoplasmic ratio to a threshold. But what provides the readout of the increasing nuclear density? In flies, the capacity to promote mitotic cyclin destruction correlates with an increase in the nuclear to cytoplasmic ratio, or, as emphasized by some authors, it also correlates with the increase in centrosomes and mitotic apparatuses. This relationship between cyclin degradation and nuclear concentration might explain the gradual prolongation of the blastoderm cycles and onset of the MBT. This interphase prolongation has been suggested to allow time for transcription of components necessary at the MBT. Accordingly, knockdown of cyclin synthesis should dramatically influence MBT timing, perhaps directly if cyclin:Cdk levels provide a regulatory input or indirectly if cell-cycle length or nuclear density provides an input (McCleland, 2008).

Knockdown of mitotic cyclins blocked mitosis at the injected pole, modestly extended the cell-cycle length in more distal regions, and usually left the cycle unaffected at the most distal pole. When the unaffected end of such chimeric embryos completed cycle 13, ingression of the cellularization membranes occurred in concert in regions of the embryo in cycle 14, cycle 13, and cycle 12. Thus, local knockdown of cyclins, prolongation of the cell cycle, and reduction of local nuclear density was not sufficient to forestall cellularization. Although other MBT parameters have yet to be characterized, the apparently normal gastrulation of embryos arrested in cycle 13 suggests concerted transition of the various MBT events. These findings are not easily consistent with earlier ideas because the experiment alters many parameters thought to contribute to triggering the MBT. It is noted that one parameter that is not changed by cyclin RNAi is the increasing centrosome density, which remains a viable candidate for triggering the MBT (McCleland, 2008).

A mis-expression study of factors affecting Drosophila PNS cell identity

Drosophila PNS sense organs arise from single sensory organ precursor (SOP) cells through a series of asymmetric divisions. In a mis-expression screen for factors affecting PNS development, string and dappled were identified as being important for the proper formation of adult external sensory (ES) organs. string is a G2 regulator. dappled has no described function but is implicated in tumorigenesis. The mis-expression effect from string was analysed using timed overexpression during adult ES-organ and, for comparison, embryonic Chordotonal (Ch) organ formation. Surprisingly, string mis-expression prior to SOP division gave the greatest effect in both systems. In adult ES-organs, this lead to cell fate transformations producing structural cells, whilst in the embryo organs were lost, hence differences within the lineages exist. Mis-expression of dappled, lead to loss and duplications of entire organs in both systems, potentially affecting SOP specification, in addition to affecting neuronal guidance (O'Farrell, 2008).

To identify novel alleles affecting ES-organ development the GeneSearch (GS) P-element was mobilized. This element, when coupled with a GAL4 source, promotes transcription of flanking genes. After crossing to sca-GAL4, expressed in proneural clusters, SOPs, and their progeny, resulting adult phenotypes of mature ES-organs were scored. Screening approximately 20,000 progeny, 21 strains with phenotypes were recovered. Of these, eight were selected for further studies based on phenotype consistency. By Southern blot only three strains were found to carry single GS-element insertions. These were mapped by plasmid rescue and were found to sit upstream of the genes stg (2 strains) and dpld (1 strain) respectively. Using the hsp70-GAL4 driver RNA was specifically overexpressed downstream of the GS-elements. After conversion of the RNA into DNA, sequencing using element-specific primers, revealed that GS-element dependent overexpression came from the immediate neighbour gene, dpld (1 strain) and stg (2 strains). Of the five chromosomes carrying more than one insertion, one chromosome produced two discrete transcripts (identified by PCR), making a total of nine transcripts identified in the screen (O'Farrell, 2008).

Dpld is a member of the Tripartite Motif (TRIM) superfamily of proteins originally isolated in a screen for tumour causing genes. Its molecular function remains unknown. Plasmid rescue experiments placed the P{GS}dpld^GS8193 insertion site 0.45 kb upstream of the dappled translational start site. sca-dpld^GS8193 had strongest effects on macrochaetae development. General mis-spacing of organs over the thorax was noted with some individuals lacking macrochaetae while other had excess. Occasionally, duplications of bristles (twinning) from individual organs was observed. The ectopic and mis-positioned organs suggest a role for dappled in SOP cell selection. However, a caveat to mis-expression screening is the relevance of the phenotype, i.e. whether the gene is normally expressed within the affected tissue. In the case of dappled, no expression in the thorax has been documented. Expression of dappled within the developing embryonic PNS has been documented. This, and the fact that embryo possesses adult ES analogous ES- and Ch-organs, which are easily studied, prompted a continued study of dappled within these PNS lineages. sca-dpld^GS8193 expression also gave rise to embryonic PNS phenotypes. Frequently, PNS axonal patterning was non-wildtype, with lateral sense organs, Lch, Les and Lda aberrantly positioned, including Lch5 organs that occasionally appeared fused to Lch5s of adjacent segments. In all cases, the fused appearance paralleled a loss of organ in the adjacent segment, discarding a simple duplication explanation. Instead, the positional defects are thought to arise potentially as a result of axonal guidance problems. Lch5 axons follow the tracheal spiracular branch (TSB) to reach the intersegmental nerve leading to the CNS. The LdaA sense organ marks the first turning point for these axons to seek contact with the tracheal spiracular branch (TSB). Since the PNS pattern deviates from wildtype in sca-dpld embryos, it is possible that missing guidance cues resulted in misrouting of Lch5 axons. Also, post-mitotic PNS neurons normally express dappled during the period of axonal outgrowth, indicating that the pathfinding defect may constitute a relevant cell autonomous effect, where misrouted axons subsequently pulled their corresponding organs into the neighbouring segment (O'Farrell, 2008).

Insertions into the string locus were isolated twice. Plasmid rescue placed P{GS}stg¹⁰¹⁵ within the string 5'UTR, 0.6 kb from the translational start site. The most prominent external phenotype on the dorsal thorax resulting from mis-expression of string in developing ES-organs was the duplication of microchaetal bristles (twinning) which accounted for 56% of the total aberrations observed. Macrochaetae were frequently lost from the thorax, a phenomenon that results from interference of string in the lateral inhibition process. Given that this effect potentially represents cell fate transformations resulting from mitotic timing, an element of asymmetric cell divisions little studied, the observation was investigated further, reasoning that if cell fate transformations caused this phenotype, it would most likely be a pIIb to pIIa transformation, duplicating bristle and socket cells at the expense of neuron and sheath cells. Using MAb22c10 staining as a marker for neurons and the nuclear β-galactosidase signal of the pros-lacZ enhancer trap to mark sheath cells, wildtype and sca-stg^GS1015 thoraxes were compared. In wildtype flies a ratio of 0.89 sheath cells per organ counted was observed, close to the expected ratio of 1, whereas sca-stg^GS1015 animals displayed only 0.48 sheath cells per organ. This ratio was further reduced to 0.28 when only ES-organs of aberrant morphology were scored, strongly suggesting that sheath cell numbers were affected. Examining five twin-bristled organs in greater detail a single MAb22C10-positive neuron was observed associated to each such organ while lacking Pros-positive nuclei in their vicinity. Neighbouring wildtype organs displayed single neuron and Pros-positive cells associated with the organ. The detection system (DAB) used for this analysis also generated a weak non-specific staining of bristle and socket cells, enabling visualisation of the structural cell types of the ES-organs. Thus, each twin bristle was observed to be associated with a distinct socket cell, i.e. two bristles and two socket cells. Interestingly, enervation of just one bristle per twin pair was detected. Potentially this represents a duplication of the pIIa cell type, generating double bristle and socket cell numbers. Typically one would expect this to occur as a result of a pIIb to pIIa transformation, which e.g. can occur at divisions of SOPs having lost pros expression. However, the presence of five cells, including a neuron, which stems from the pIIIb, deters this model. In an effort to understand this transformation, it was asked in which precursor cell division ectopic string expression affected. For this, the hs-stg³ construct was used and developing pupae were subjected to a single heat-shock at distinct time-points, during the 12–22 h post pupal formation period containing all notal microchaetae mitoses. This also generated twinning of bristles. Neither heat-shocked wildtype, nor non-heat-shocked hs-stg³ flies showed abnormal bristle phenotypes. After metamorphosis, flies were counted for the number of notal twin microchaetae present. On this basis subgroups of mild (<3 twin microchaetae per thorax), or severe (≥3) phenotypic effects were created. Surprisingly, induction of string prior to SOP cell division gave rise to the highest levels of twinning, indicating that a precocious SOP mitosis had a “carry-over” effect to daughter cells. Potentially, precursor cells born from this division were mis-specified. In wildtype PNS organ development, neurons may form at both pIIb and pIIIb divisions, depending on organ type. Furthermore, it has been demonstrated that modulation of Hamlet levels, a determinant normally present in the pIIIb and neuron only, can affect the normal pII/IIIB output to become pIIa-like, and vice versa. It was reasoned that precocious mitosis of the SOP leads to the formation of a pIIa and a non-wildtype pIIb precursor. The aberrant pIIb undergoes two consecutive mitoses, giving birth to the ectopic bristle and socket, plus one neuron. Potentially, insufficient time in G2 for the SOP interferes with build-up or segregation of such determinants (O'Farrell, 2008).

The effect was examined of hs-stg³ upon the embryonic Ch-organs. A 30 min heat-shock was applied to hs-stg³ embryos across the Ch-organ division time window (6–8 h). Two dominating Ch-organ phenotypes were found, Lch5 organs that lacked two Ch-organs, and partial dorsalisation of Lch5 organs. The former phenotype was predominant for induced mitoses preceding and during the period of SOP divisions. Dorsalised defects resulted from mitoses induced during and after SOP divisions. Since two SOPs of the Lch5 are sequentially recruited by the first three SOPs specified, it was hypothesise that ectopic string expression, causing early mitosis, interferes with this process, potentially not allowing time enough for it to occur before the pre-defined SOP cells divide. However, since ectopic string expression affects lateral inhibition in adult macrochaetal development, such an effect cannot be discounted in this study (O'Farrell, 2008).

Speculating that the partial dorsalisation phenotype could result from a lack of the ligament cells required to drag the Lch5 ventrally, ligament cells were examined using anti-REPO. This revealed a decrease in Lch5 ligament cells. Ch-ligament cells arise from the pIIb division and may be directly affected by precocious mitosis, or alternatively may suffer mis-specification as a result of a precocious SOP division. Additionally, completely dorsalised Lch5 were less frequently observed, that had failed to descend from their dorsal point of origin (O'Farrell, 2008).

The terminal fates of Lch5 organ cells were examined. Contrary to findings in the adult, ectopic string had no impact on Ch-organ structural cap and cap attachment cells (pIIa daughter cells). Nor was a change in the ratio of neuron and sheath cells within the organs (pIIIb daughter cells) observed in response to ectopic string expression at any given time-point. Therefore, while ectopic string affected development of the Lch5, these effects did not present themselves as cell fate transformations like those observed in the adult (O'Farrell, 2008).

In summary, both adult and embryonic SOP cells were the most susceptible PNS precursor cells to ectopic string, as judged by timed mis-expression studies. The SOP precursor potentially requires a minimal time in which to build fate determinants, which is certainly the case for the specification of its own daughter cells. But the findings suggest that this could be required to provide determinants to granddaughter cells also (O'Farrell, 2008).

While the SOP was the cell most sensitive to string-dependent precocious mitosis, phenotypic consequences were different for adult and embryonic PNS. Even adult macro- and microchaetal phenotypes differed. Thus, while these PNS lineages are analogous, they are not identical. The differences observed likely represent distinct mechanisms within each lineage, potentially contributing to the cellular diversity observed in the mature organs (O'Farrell, 2008).

Specification of differentiated adult progenitors via inhibition of endocycle entry in the Drosophila trachea

A population of Drosophila adult tracheal progenitor cells arises from differentiated cells of the larval main trachea that retain the ability to reenter the cell cycle and give rise to the multiple adult tracheal cell types. These progenitors are unique to the second tracheal metamere as homologous cells from other segments, express fizzy-related (fzr), the Drosophila homolog of CDH1 protein of the APC complex, and enter endocycle and do not contribute to adult trachea. This study examined the mechanisms for their quiescence and show that they reenter the cell cycle by expression of string/cdc25 through ecdysone. Furthermore, preventing endocycle entry is both necessary and sufficient for these tracheal cells to exhibit markers of adult progenitors, thus modifying their genetic program. Finally, Hox-mediated regulation of fzr expression was shown to be responsible for progenitor identity and thus specifies a group of differentiated cells with facultative stem cell features (Djabrayan, 2014: PubMed).

String and wing development

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

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

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

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

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

Notch-dependent String and Fizzy-related expression is required for the mitotic-to-endocycle transition in follicle cells

During Drosophila oogenesis, Notch function regulates the transition from mitotic cell cycle to endocycle in follicle cells at stage 6. Loss of either Notch function or its ligand Delta (Dl) disrupts the normal transition; this disruption causes mitotic cycling to continue and leads to an overproliferation phenotype. In this context, the only known cell cycle component that responds to the Notch pathway is String/Cdc25 (Stg), a G2/M cell cycle regulator. Prolonged expression of string is not sufficient to keep cells efficiently in mitotic cell cycle past stage 6, suggesting that Notch also regulates other cell cycle components in the transition. By using an expression screen, such a component was found: Fizzy-related/Hec1/Cdh1 (Fzr), a WD40 repeat protein. Fzr regulates the anaphase-promoting complex/cyclosome (APC/C) and is expressed at the mitotic-to-endocycle transition in a Notch-dependent manner. Mutant clones of Fzr have revealed that Fzr is dispensable for mitosis but essential for endocycles. Unlike in Notch clones, in Fzr mutant cells mitotic markers are absent past stage 6. Only a combined reduction of Fzr and ectopic Stg expression prolongs mitotic cycles in follicle cells, suggesting that these two cell cycle regulators, Fzr and Stg, are important mediators of the Notch pathway in the mitotic-to-endocycle transition (Schaeffer, 2004).

In Drosophila, nurse and follicle cells in the adult ovary endocycle in a regulated manner. It has been suggested that endocycling requires the loss of M-phase cyclin-dependent kinase (Cdk) activity and oscillations in the activity of S-phase Cdk. In Drosophila follicle cells, the function of the Notch pathway in the mitotic-to-endocycle transition has been well established. Lack of Notch activity in Drosophila follicle cells leads to prolonged mitosis at the expense of endocycles, suggesting that Notch functions in this context as a tumor suppressor. Because very few signaling pathways that stop the mitotic cell cycle have been identified, it is important to understand the relationship between the Notch pathway and known cell cycle regulators in more detail (Schaeffer, 2004).

String encodes the Drosophila homolog of the yeast cell cycle regulator Cdc25, a phosphatase whose role is to activate the Cdk-cyclin complex at the G2/M transition by dephosphorylating the inhibitory sites. As a consequence, cells are propelled into mitosis. Notch signaling downregulates string at stage 6 of oogenesis to allow the cells to transit into the endocycle. The 4.9 kb and 6.4 kb elements found in the 50 kb-long string promoter drive string expression in follicle cells from germarium to stage 3 and from stage 4 to stage 6, respectively. The Notch-Delta cascade achieves the tight downregulation of the 6.4 kb element at stage 6, when the mitotic-to-endocycle transition takes place. A string rescue construct that contains 15.3 kb of the string promoter restores only the early string expression pattern between germarium and stage 1-2 egg chambers (because of the 4.9 kb element) but does not contain the control element active between stages 3 and 6 (the 6.4 kb element). Although stg clones produce cells arrested in G2, the mutant nuclei were larger than in the wild-type cells when stg clones were produced in the background of the 15.3 kb rescue construct. Furthermore, the mutant clones are half the size of sister clones, suggesting that the mutant cells stop division and possibly enter endocycle too early. If downregulating String leads the follicle cells to enter an endocycle rather than to completely arrest, then the sole role of Notch, which downregulates string expression at the switch, is to act on string to promote endocycling. If this is the case, string expression is the only limiting factor in the mitotic-to-endocycle transition. Also, because ectopic expression of stg in Drosophila embryos and discs is capable of driving cells blocked in G2 into mitosis, continuous string expression should keep most cells in the mitotic phase (Schaeffer, 2004).

stg (either with a heat-shock-inducible promoter or with one or two copies of the UAS-stg transgene) was overexpressed via the flip-out Gal4 system to analyze whether String is sufficient to prolong division of follicle cells past stage 6. Overexpression of string with a transgene driven by a heat-shock promoter did not show any ectopic Cyclin B or Phospho-Histone 3 (PH3) expression. With one copy of the UAS string transgene, prolonged mitotic divisions were rarely observed in follicle cells past stage 6, except in the posterior region, where 10% of the clones that overexpressed string showed ectopic Cyclin B or PH3 expression. When two copies of the UAS-stg construct were present, leading to higher string expression levels, a higher incidence of Cyclin B and PH3 expression was seen in posterior clones. However, only in a few cases did the ectopic expression of string in lateral and anterior follicle cells prolong their mitotic state. In addition, in most cells, overexpression of string did not affect endocycling (Schaeffer, 2004).

Because the String protein is not enough to create extra cell divisions, except in the highly sensitized posterior area, it was proposed that the mitotic-to-endocycle transition is regulated by a combination of String and other Notch-controlled components yet to be uncovered. Lack of String generally arrests cells in G2, when high levels of mitotic cyclins can be found. Cyclin A and Cdc2 have been implicated in inhibiting the assembly of prereplication complexes in G2. Furthermore, when Cdc2 or Cyclin A activity is eliminated, mutant cells enter endocycles in Drosophila because the assembly of prereplication complexes is then allowed. The hypothesis here is that the Notch signaling pathway allows cells to bypass this inhibition by activating a specific gene/genes that would allow cells to continue to cycle without undergoing the mitotic phase. Expression of such a gene would be activated after/during the mitotic-to-endocycle transition and possibly act on mitotic cyclin regulation and/or the mitotic cyclin-associated kinase, Cdc2. In order to find these genes, an expression screen was performed for genes differentially expressed before and after the transition (Schaeffer, 2004).

400 lethal X chromosome P element enhancer trap lines were screened for changes in expression levels at stage 7 by using the β-gal reporter gene. Three interesting functional groups were obtained from this screen: adhesion molecules, transcriptional control proteins, and cell cycle regulators. Premature expression of fzr caused formation of enlarged nuclei, a potential indication of precocious endocycles. Therefore the cell cycle regulator Fzr was analyzed in more detail in the mitotic-to-endocycle transition (Schaeffer, 2004).

The lines fzrG0326 and fzrG0418 have the P{lacW} element inserted in the first intron and at the 5'-end of the Fizzy-related gene, respectively, and are hypomorphic alleles of fzr. These constructs drive expression of the reporter gene after the transition, from stage 6-7 onward. This expression is tightly correlated with the end of mitotic cycles; no fzr expression is observed in follicle cells that show PH3 staining. A similar expression pattern of Fzr was observed with the Fzr specific antibody, and the fzr mRNA pattern in follicle cells reflects this pattern as well (Schaeffer, 2004).

Fzr, also known as Retina aberrant in pattern, is a conserved WD domain protein that is required during G1 for proteolysis of mitotic regulators such as Aurora-A kinase and Cyclins A, B, and B3 in an APC/C-dependent manner. Loss of Fzr in Drosophila causes cells to progress through an extra division cycle in the epidermis and inhibits endoreduplication in the salivary gland cells, whereas fzr overexpression inhibits mitosis and transforms mitotic cycles into endoreduplication cycles. This finding suggests that, in at least some cell types, the Fzr protein is essential for the mitotic-to-endocycle transition. Because fzr expression is upregulated in follicle cells when the Notch cascade is activated, whether the fzr expression was responsive to Notch activity was tested by using the fzrG0326 (fzr-LacZ) enhancer trap line to analyze fzr expression levels in follicle cells that surround the Dl germline clones. A clear reduction of fzr expression was observed in all Dl germline clones past stage 6, demonstrating that fzr expression is dependent on Notch activity in the mitotic-to-endocycle transition (Schaeffer, 2004).

Mitotic-cyclin protein levels are downregulated at the mitotic-to-endocycle transition. Cyclin A protein levels are reduced at the end of mitotic cycles in the follicle cells. Similarly, Cyclin B is downregulated at the protein level at the mitotic-to-endocycle transition. In situ hybridization studies indicated that neither gene was regulated at the transcriptional level during or after the transition but showed mRNA expression in the follicle cells throughout oogenesis until stage 10. Thus, both the Cyclin A and the Cyclin B protein levels are regulated posttrancriptionally at the mitotic-to-endocycle transition. This regulation is critical for the mitotic-to-endocycle transition because continuous expression of cyclin A in posterior follicle cells results in small nuclei and a reduced DNA level, indicative of a defect in the transition to endocycles. This supports previous reports showing that overexpression of cyclin A inhibits the progression of endoreplication cycles in Drosophila salivary glands (Schaeffer, 2004).

Because the downregulation of Cyclin A and B expression coincides with the upregulation of Fzr and because Fzr is required for proteolysis of Cyclin A and Cyclin B in embryonic epidermal cells, clones for a fzr null allele (fzr^ie28) were generated to test whether Fzr might be responsible for the mitotic-to-endocycle transition by downregulating the mitotic cyclin levels. Initially, the ovaries were immunostained with antibodies against Cyclin B. The clonal cells lacking Fzr function showed a limited but consistent increase of Cyclin B level after stage 6, when Cyclin B is normally absent. In a similar manner, fzr^ie28 mutant cells strongly upregulated Cyclin A after the mitotic-to-endocycle transition. It is therefore concluded that, as seen in other systems, Fzr function in follicle cells is to degrade mitotic cyclins (Schaeffer, 2004).

Upregulation of the mitotic Cyclin A during endocycles has been shown to inhibit endoreplication. The ovaries bearing fzr^ie28 mutant cells were stained with DAPI to observe nuclei size and shape. In addition to showing a failure of Cyclin A and B removal, the fzr^ie28 mutant cells showed phenotypes that indicated endocycle inhibition. The small nuclei size and reduced DNA level seen in the fzr^ie28 mutant cells are reminiscent of the Notch phenotype and thereby show that Fzr is required for the mitotic-to-endocycle transition. Unlike the Notch clones in which Cyclin B and PH3 expression were detected after stage 6, the fzr^ie28 mutant cells do not shown signs of overproliferation. No PH3 staining is ever observed in mutant clones after the transition, suggesting that the fzr^ie28 mutant cells do not continue to divide past stage 6. The 6.4 kb Stg-LacZ transgene, abruptly downregulated by Notch at the mitotic-to-endocycle transition, did not show any prolonged expression in fzr^ie28 mutant cells after stage 6, indicating that cells are not in a mitotic phase. The number of cells in mutant and sister clones (two copies of GFP) were counted. The same number of cells was observed in mutant clones as in the associated twin spot; the ratio varied from 0.64 to 3, with a mean of 1.01). All these clues led to the idea that despite mitotic cyclins' upregulation in fzr^-/- mutant cells, those cells do not divide past stage 6 (Schaeffer, 2004).

Because the number of cells in the mutant follicle cell clones and the associated twin spots are the same and because the expression of the 6.4 kb stg-LacZ transgene is normal prior to the switch to endocycling, it is concluded that, even though Fzr is required for endocycles, it is dispensable for the mitotic stages in the Drosophila ovary. Similarly, it has been shown that completion of mitosis does not require Fzr in embryos (Schaeffer, 2004).

Although fzr^ie28 mutant cells show upregulation of the mitotic cyclins, these cells do not divide. In all dividing cells, the G2/M transition depends on String to activate the kinase activity of the mitotic cyclin-Cdc2 complexes. Because the 6.4 kb stg-LacZ transgene is downregulated by the Notch pathway after the transition, the fzr^ie28 mutant cells might require Stg to prolong mitosis. To test this, string was overexpressed by using a heat shock promoter in the fzr^ie28 follicle cell clones. The flies were heat shocked twice, once to promote the formation of fzr^ie28 mutant clones and again, 12 hr prior to dissection, in order to induce stg expression. In a wild-type fzr background, this low level of string expression alone is insufficient for prolonging mitosis past the mitotic-to-endocycle transition (no upregulation of Cyclin B and PH3 markers). However, this prolonged stg expression is enough to push the fzr^ie28 mutant cells into mitosis, as shown by the PH3 staining and mitotic figures in egg chambers at stage 9. More strikingly, PH3-positive cells as well as mitotic figures were seen in nonclonal areas heterozygous for fzr^ie28, which prompted a test to see whether reducing the level of Fzr to one copy while overexpressing stg by heat shock is sufficient to produce the PH3-positive cells. In order to demonstrate a direct stg effect, the flies were examined 2 hr after heat shock. The fzrG0326 enhancer trap line (fzr LacZ) was used to reduce the level of Fzr and to mark the stages precisely. It was found that 39% of ovarioles of the experimental group fzrG0326;;Hs Stg displayed PH3-positive cells and mitotic figures at stage 7-8, whereas 0%-2% did so in the control groups. In order to further determine whether the ratios of cells in a mitotic stage in mutant and control situations were similar, the number of PH3-positive cells observed in a single focal plan was quantified before and at stage 7-8, in the experimental group fzrG0326;;Hs Stg as well as in three control groups. On average, 12%-15% of the cells showed PH3 staining at mitotic stages before the transition (before stage 7), but none did so after the transition (stage 7-8). In contrast, ovaries with reduced fzr and prolonged stg expression showed PH3 staining after stage 7, whereas the control groups did not. In comparison, 8.5% of cells in the egg chamber did exhibit PH3 staining. It is possible that the percentage of mitotic cells observed in mutant egg chambers past the transition was somewhat lower than the percentage of mitotic cells observed in wild-type egg chambers before the transition because of the low level of string expression given by the heat shock construct or subtle effects of yet-unraveled components in the transition. However, these data strongly suggest that reducing the Fzr level in combination with prolonged stg expression can prolong the mitotic stage in follicle cells (Schaeffer, 2004).

In Drosophila, loss of Fzr causes progression through an extra division cycle in the epidermis and inhibition of endoreplication in the salivary glands, in addition to the upregulation of mitotic cyclins. In follicle cells loss of Fzr causes an inhibition of endoreplication as well as an upregulation of the mitotic cyclins, particularly Cyclin A, but no prolonged mitosis. This difference might be due to the lack of String in follicle cells. It is possible that in the epidermis, residual String might dephosphorylate and therefore activate the mitotic cyclin/Cdk complexes and allow an extra mitosis to proceed, whereas in follicle cells the absence of String might result in G2-arrest. This is supported by the fact that overexpressing a string transgene under the control of a heat shock promoter rescues cell division in a fzr mutant (Schaeffer, 2004).

Notch mutant cells are mitotic: in those cells, Stg is upregulated, and Fzr is not activated. Those two events (upregulation of String and downregulation of Fzr) are able to keep the cells in mitotic cycle in 39% of stage 7-8 egg chambers. It is therefore possible that Notch controls the mitotic to endocycle transition by repressing String to block mitosis and by activating Fzr to allow endocycle progression (Schaeffer, 2004).

Based on earlier studies, it has been proposed that endocycle is induced by lack of M-phase Cdk activity. However, the regulation and exact manifestation of this task has not been previously uncovered. This study shows that in Drosophila follicle cells the Notch pathway executes the task by first freezing the mitotic cyclin/Cdk complex in an inactive, phosphorylated form and thereafter inducing the degradation of the mitotic cyclins to allow progression to S phase. Further studies will reveal whether Notch action is also required for G1-to-S-phase transition or whether these two alterations, lack of String, and expression of Fzr are sufficient to transform mitotic cells to endocycling cells (Schaeffer, 2004).

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