Effects of Mutation and Overexpression (part 1/2)

stg mutation cause a nonspecific reduction in the number of differentiated cuticular structures. stg mutants have a markedly reduced number of cells relative to wild-type embryos during the later stages of embryogenesis. Mutants undergo the first 13 nuclear divisions with normal timing and morphology but fail to initiate the 14th mitosis. Cells formed during interpahse 14 in stg mutants remain in interphase for the duration of embryogenesis (Edgar, 1989).

Roughex, a novel protein required for establishment of G1, is required to negatively regulate eitherstring, Cyclin A or both. roughex mutants, which circumvent G1 and enter S, are suppressed by reduced gene dosage of either CycA or string (Thomas, 1994).

A coordinate program of transcription of S-phase genes (DNA polymerase alpha, Proliferating cell nuclear antigen and the two ribonucleotide reductase subunits) can be induced in Drosophila embryos by the G1 cyclin, Cyclin E. This program drives an intricate spatial and temporal pattern of gene expression that perfectly parallels the embryonic program of S-phase control. string mutation does not disrupt this dynamic pattern of expression. Thus, the transcriptional program is not a secondary consequence of cell cycle progression. It seems that developmental signals control this transcriptional program and the activation of this program drives transition (either directly or indirectly) from G1 to S phase in the stereotyped embryonic pattern (Duronio, 1994).

What factors contribute to the maternal/zygotic transition, the point at which zygotic factors begin to control cell cycle in Drosophila? Proliferation of embryonic nuclei is the initial driving force for the maternal/zygotic transition. As nuclei multiply, they progressively deplete factors required for cell cycle progression, thus lengthening the interphases starting in cycle 10. The first critical factors to be titrated out are probably Cyclins A and B, but not Cdc25 phosphatase. Embryos lacking maternal string proceed normally through cell cycles 1 to 13, always transversing the maternal/zygotic transition during interphase 13. This result suggests that twine might play a role in early embryonic mitoses, since twine, along with string serves the cdc25 function in Drosophila. When string and twine are simultaneously deleted, germ cell proliferation is blocked and no ovarioles or eggs are produced. Lowering the maternal dose of both string and twine advances the maternal/zygotic transition. Premature cell cycle arrest in embryos from double mutants correlates with reduced cdc25 function. Increasing twine, but not string, can postpone the maternal/zygotic transition. Degradation of maternal cdc25 is the critical event that inactivates the maternal cell cycle oscillator. As a consequence of the loss of cdc25 phosphatase activity at the maternal/zygotic transition, inhibitory tyrosine phosphorylation of cdc2 kinase by Drosophila wee1 kinase can effect a G2 arrest, causing the prolonged cell cycle characteristic of the maternal/zygotic transition (Edgar, 1996).

Mesodermal progenitors arise in the Drosophila embryo from discrete clusters of lethal of scute (l'sc)-expressing cells. Individual progenitors are specified by the sequential deployment of unique combinations of intercellular signals. Initially, the intersection between the Wingless (Wg) and Decapentaplegic (Dpp) expression domains demarcate an ectodermal prepattern that is imprinted on the adjacent mesoderm in the form of L'sc preclusters. One precluster, preC1, is found in the ventral mesoderm, and the other, preC2, is localized to the dorsal mesoderm. PreC2 encompasses the territory in which dorsal L'sc clusters C2 and C14-C17, the subject of this paper, subsequently develop. All mesodermal cells within preC2 precluster are competent to respond to a subsequent instructive signal mediated by two receptor tyrosine kinases (RTKs), the Drosophila epidermal growth factor receptor (Egfr) and the Heartless (Htl) fibroblast growth factor receptor. By monitoring the expression of the diphosphorylated form of mitogen-associated protein kinase (MAPK), these RTKs are seen to be activated in small clusters of cells within the original competence domain (precluster). Each cluster represents an equivalence group because all members initially resemble progenitors in their expression of both L'sc and mesodermal identity genes. Thus, localized RTK activity induces the formation of mesodermal equivalence groups. The RTKs remain active in the single progenitor that emerges from each cluster under the subsequent inhibitory influence of the neurogenic genes. The singling out of progenitors from mesodermal equivalence groups depends on lateral inhibition mediated by the neurogenic genes. Moreover, Egfr and Htl are differentially involved in the specification of particular progenitors (Carmena, 1998).

Transduction of RTK signals occurs, at least in part, via the Ras/MAPK cascade. Constitutive activity of Egf receptor, Htl, or Ras1 is associated with the development of supernumerary Eve-expressing mesodermal cells. However, it has not been established whether this response is due to activated Ras1-induced proliferation of normal Eve cells or is due to the recruitment of additional cells to an Eve-positive fate. Activated Ras1 does not increase the sizes of L'sc clusters, suggesting that Ras1 is not simply stimulating the division of mesodermal cells. However, to definitively address this question, the effects of constitutive Ras1 activity were examined in string (stg) mutant embryos. Because strong alleles of stg prevent all post-blastoderm cell divisions, a potential cell-proliferation effect of activated Ras1 should be blocked in this genetic background. However, stg should not inhibit the overproduction of Eve-expressing cells if Ras1 promotes their determination. In a stg mutant, only one to two Eve-positive cells are present in each hemisegment. These cells correspond to the Eve progenitors that form in wild-type embryos. The presence of only one Eve progenitor in some segments of stg mutant embryos presumably is due to the smaller number of mesodermal cells that contribute to L'sc clusters when zygotic cell divisions do not occur. Significantly, activated Ras1 generates more Eve progenitors in a stg mutant than are seen in the mutant alone. It is concluded that Ras1 promotes the formation of additional Eve progenitors by inducing more mesodermal cells to assume this fate and not by stimulating the normal progenitors to divide. Next, an assessment was carried out to determine if the Ras1 pathway is sufficient to induce progenitor differentiation by examining the myogenic effects of Ras1 at later developmental stages in both wild-type and myoblast city (mbc) mutant embryos. In the absence of mbc function, muscle fusion does not occur and differentiated muscle founders appear as spindle-shaped myoblasts that express myosin and founder cell markers, such as Eve. In contrast to the mbc mutant in which one Eve-expressing muscle DA1 founder is present in each hemisegment, multiple occurrences of such cells form in mbc embryos under the influence of activated Ras1. All of the Eve plus myosin-positive myoblasts seen in these embryos have the elongated morphology of muscle founders, as opposed to the round shape of neighboring Eve-negative myoblasts. Large syncytia containing many Eve-positive nuclei are present when Ras1 is activated in a wild-type background. Although extra Eve pericardial cells initially appear under the influence of activated Ras1, Eve expression in such cells is lost by later stages. It is concluded that ectopic activation of the Ras1 pathway not only stimulates Eve expression in additional progenitors, but also promotes the formation and differentiation of supernumerary muscle founders (Carmena, 1998).

Arresting cell division using the string mutation or blocking DNA replication with aphidicolin fails to prevent ectopic expression of the homeotic gene Ultrabithorax in Polycomb mutants. Thus, even in the absence of DNA replication, Pc is required to maintain spatially restricted patterns of homeotic gene expression (Gould, 1990).

The requirement for Drosophila cdc2 kinase during spermatogenesis has been examined after generating temperature-sensitive mutant lines (Dmcdc2ts). While meiotic spindles and metaphase plates do not form in Dmcdc2ts mutants at high temperature, chromosomes still condense in late spermatocytes and spermatid differentiation (sperm head and tail formation) continues. The same phenotype are also observed in twine and twine/Dmcdc2ts double mutant testes, consistent with the idea that the cdc2 kinase activity required for meiotic divisions is activated by the Twine/cdc25 phosphatase. Ectopic expression of the String/cdc25 phosphatase, known to activate the cdc2 kinase before mitosis, results in a partial rescue of meiotic divisions in twine mutant testis (Sigrist, 1995).

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

In Drosophila, the sensory mother cells of macrochaetes are chosen from among the mitotically quiescent clusters of cells in wing imaginal discs, where other cells are proliferating. The pattern of cyclin A, one of the G2 cyclins, reveals that mitotically quiescent clusters of cells are arrested in G2. When precocious mitoses are induced during sensory mother cell determination by the ectopic expression of string, a known G2/M transition regulator, the formation of sensory mother cells is disturbed, resulting in the loss of macrochaetes in the adult notum. This suggests that G2 arrest of the cell cycle ensures the proper determination of sensory mother cells, and that G2 arrest in mitotically quiescent clusters of cells is controlled by the down-regulation of string transcription (Kimura, 1997).

In larval tissues polyteny results from the endo cell cycle, a modified cell cycle in which there is only an S (synthesis) phase and a G (gap) phase. A key regulator of the mitotic cell cycle, the product of the string gene (the Drosophila homologue of cdc25), is not required for the endo cell cycle (Smith, 1991)

stringis essential for the generation of imaginal tissue. Clones of a strong mutant allele were generated in growing imaginal tissue. Normal bristle formation is prevented by induction of string clones, indicating that string is essential for the generation of the adult cuticle, the tissue generated by division of histoblast nests. String's role in cell cycle regulation is illustrated by the observation that ectopic expression of string in the imaginal discs of the thorax induces mitoses in G2-arrested cells. Ectopic String also induces premature and extra mitoses in the developing eye-antennal imaginal disc. In contrast to the ability of String to induce mitosis in imaginal discs, imaginal histoblasts prove to be refractory to ectopic String. These experiments suggest that in wandering third istar larvae, a factor or factors other than String are limiting for entry of abdominal histoblasts into mitosis This is the first example of G2 arrest during fly development that is not mediated by string transcriptional regulation (Kylsten, 1997).

Cell intrinsic and cell extrinsic factors mediate asymmetric cell divisions during neurogenesis in the Drosophila embryo. In one of the well-studied neuronal lineages in the ventral nerve cord (the NB4-2->GMC-1->RP2/sib lineage), Notch (N) signaling interacts with asymmetrically localized Numb (Nb) to specify sibling neuronal fates to daughter cells of GMC-1. The NB4- 2 is delaminated in the second wave of NB delamination during mid stage 9 (~4.5 hours) of embryogenesis and is located in the 4th row along the anteroposterior axis and 2nd column along the mediolateral axis within a hemisegment. The NB4-2 generates its first GMC (GMC-1, also known as GMC4-2a) ~1.5 hours after formation. The GMC-1 divides ~1.5 hours later to generate two cells, the RP2 and the sib. The RP2 cell eventually occupies its position in the anterior commissure along with the other RP neurons (RP1, RP2, RP3 and RP4) and projects its anteroipsilateral axon to the intersegmental nerve bundle (ISN) and innervates muscle #2 on the dorsal musculature. The sib cell migrates to a position posterior and more dorsal to RP2. The DiI tracing of the NB4-2 lineage indicates that the sib has no axonal projection at mid stage 17 of embryogenesis; thus, its ultimate fate has not been determined. In this study, loss-of-function mutations in N and nb, cell division mutants cyclinA (cycA), Regulator of cyclin A1 (Rca1) and string/cdc25 phosphatase (stg/cdc25 phosphatase), and the microtubule destabilizing agent, nocodazole, were all used to investigate asymmetric cell fate specifications by N and Nb in the context of cell cycle. Mutation in rca1 gene was initially identified as a dominant suppressor of roughex (rux) eye phenotype. In rux, the cells enter S-phase precociously due to ectopic activation of a CycA/Cdk complex in early G1 (Dong, 1997). In embryos lacking the rca1 activity, the cells appear to arrest in G2 of the cell cycle (at stages 15- 16) similar to cycA mutants (Wai, 1999 and references).

The loss of cycA, rca1 or stg leads to a block in the division of GMC-1, however, this GMC-1 exclusively adopts an RP2 identity. The requirement of cycA or rca1 for cell division in the CNS is lineage specific. Anti-Eve staining of cycA or rca1 mutant embryos indicates that loss of these gene products does not affect all the Eve-positive lineages in the ventral nerve cord. Eve is expressed in other neuronal lineages such as the CQs, the Us and the ELs. The CQs are formed from NB7-1, an S1 neuroblast. The GMC for these neurons are formed at the same time as the GMC for the aCC/pCC neurons (generated from another S1 neuroblast, NB1-1) and divide at the same time as GMC for the aCC/pCC lineage. The NB7-1 in cycA or rca1 mutants does not divide to generate an Eve-positive GMC for the CQs. However, the effect on CQs is partially penetrant in both the mutants. Thus, ~75% of the hemisegments had missing CQs in cycA mutants; in rca1 mutants, this figure is ~50%. The effect on the generation of U neurons is as follows: in cycA mutants, the effect is fully penetrant; whereas, in rca1 mutants, 65% of the hemisegments were missing the Us. It must be pointed out that in those hemisegments where these neurons (Us and CQs) are formed, the number of these neurons is fewer than normal. Finally, the effect of the loss of cycA or rca1 on another Eve-positive lineage, the EL neurons, is minimal. The EL neurons are formed from NB3-3, an S4 neuroblast (the formation of this neuroblast extends between S3-S5). None of the hemisegments have missing EL neurons, in either the cycA mutants or the rca1 mutants. The above result indicates that the loss of rca1 or cycA does not affect the division of all neuroblasts. One possibility for this result is that the maternal deposition of these gene products is masking the zygotic loss of these gene products in these lineages. However, this seems unlikely since the GMCs for the aCC/pCC or the RP2/sib lineages are generated earlier than the GMCs for the EL neurons. Moreover, the maternal deposition of CycA, for example, is completely exhausted before stage 7 and none of the neuroblasts have delaminated from the neuroectoderm at this stage of development. Thus, these results indicate that the effect of loss of cycA or rca1 is lineage specific and every neuronal lineage is not sensitive to the loss of these cell division genes. It is most likely that some other cyclins (i.e., Cyclin B) complement the loss of CycA in these lineages (Wai, 1999).

While the loss of N leads to the specification of RP2 fates to both progeny of GMC-1 and loss of nb results in the specification of sib fates to these daughter cells, the GMC-1 in the double mutant between nb and cycA assumes a sib fate. While the GMC-1 fails to divide to generate two cells in these double mutants, the GMC-1 assumed a sib fate. About ~35% of the hemisegments show this phenotype. This penetrance of the phenotype is slightly higher than the phenotype observed in nb single mutants alone. This suggests that cycA mutation has an enhancing effect on the nb phenotype. This would argue that normally a small amount of the Nb protein segregates into a sib cell and that, in the absence of cell division, all of Nb is accumulated in one cell, and therefore, is much more effective in blocking the N signaling. Moreover, since the nb phenotype is epistatic to the cell division mutant phenotype, Nb must be acting downstream of these genes. This result is consistent with the finding that Nb becomes localized during metaphase and is not localized in stg mutants. Thus, in rca1 or cycA mutants, the absence of a localized Nb prevents the N signaling from specifying sib fate and, as a result, the GMC-1 assumes an RP2 fate. These epistasis results indicate that both N and nb function downstream of cell division genes and that progression through cell cycle is required for the asymmetric localization of Nb. In the absence of entry into metaphase, the Nb protein prevents the N signaling from specifying sib fate to the RP2/sib precursor. These results are also consistent with the finding that the sib cell is specified as RP2 in N;nb double mutants. Finally, these results show that nocodazole-arrested GMC-1 in wild-type embryos randomly assumes either an RP2 fate or a sib fate. This suggests that microtubules are involved in mediating the antagonistic interaction between Nb and N during RP2 and sib fate specification (Wai, 1999).

A screen was carried out in order to identify genes interacting with Armadillo, the Drosophila homolog of ß-catenin. Two viable fly stocks have been generated by altering the level of Armadillo available for signaling. Flies from one stock overexpress Armadillo (Armover) and, as a result, have increased vein material and bristles in the wings. Flies from the other stock have reduced cytoplasmic Armadillo following overexpression of the intracellular domain of DE-cadherin (Armunder). These flies display a wing-notching phenotype typical of wingless mutations. Both misexpression phenotypes can be dominantly modified by removing one copy of genes known to encode members of the wingless pathway. This paper identifies and describes further mutations that dominantly modify the Armadillo misexpression phenotypes. These mutations are in genes encoding three different functions: establishment and maintenance of adherens junctions, cell cycle control, and Egfr signaling (Greaves, 1999).

Mutations in 17 genes (26 deficiencies) were characterized that interact with Armover and/or Armunder. Interaction strength varies from deficiency to point mutation, suggesting that several genes in the original deficiencies could have contributed to, or modified, the interaction. Only for 7 of the 17 genes have interactions been identical between the point mutation and the corresponding starting deficiency. The 17 genes were sorted into four groups. Group 4 consisted of cell cycle genes: Four cell cycle genes, cdc2, string/cdc25, pebble (pbl), and cyclinA, were uncovered by the screen. string enhances Armover and weakly suppresses Armunder. The other three, cdc2, pbl, and cycA, all modify Armover. These modifications are weak and variable and more work is needed to assess their significance (Greaves, 1999).

Genes encoding cell cycle components also interact with Arm misexpression lines; this was unexpected. However, recent work by others has established a link between Wg and the cell cycle. At the wing margin of the anterior compartment, wg suppresses string (stg) transcription via induction of the proneural genes achete (ac) and scute (sc). This is followed by G2 arrest and the formation of specialized sensory bristles. In the posterior compartment no such G2 arrest takes place, and therefore there is no simple explanation as to why stg should enhance the number of ectopic noninnervated bristles induced by excess wg signaling. This and the finding that stg suppresses the Armunder phenotype implies that somehow loss of stg activity potentiates Wg signaling (Greaves, 1999).

The proper localization of Numb depends on its interaction with the adapter protein Partner of numb (Pon). In pon mutant embryos, the formation of Numb crescent is delayed in neuroblasts and is disrupted in muscle progenitor cells (Lu, 1998). Pon was isolated on the basis of its physical interaction with Numb. Pon is asymmetrically localized during mitosis and colocalizes with Numb. Ectopically expressed Pon responds to the apical-basal polarity of epithelial cells and is sufficient to localize Numb basally. It is proposed that PON is one component of a multimolecular machinery that localizes Numb by responding to polarity cues conserved in neural precursors and epithelial cells (Lu, 1998 and Lu, 1999).

In principle, the asymmetric localization of Numb/Pon can be accomplished by one or a combination of the following mechanisms: localization and local translation of their mRNAs; active transport of the proteins by motor molecules along the cytoplasmic or cortical cytoskeleton; passive diffusion (3D in the cytosol or 2D along the cortex) and trapping of the proteins by basally localized anchor molecules, or protein targeting to the membrane followed by selective degradation at one side of the cortex. The available method to detect protein localization by immunostaining of fixed embryos only provides static images of the proteins in different cells and is inadequate to distinguish among the above possibilities (Lu, 1999).

The mechanism of asymmetric Pon localization has been shown to operate at the protein level. The asymmetric localization domain of Pon has been mapped to its C-terminal region. Using a fusion between this localization domain and GFP, the entire process of Pon localization was monitored in neuroblasts of living embryos. This in vivo analysis reveals that the asymmetric localization of Pon is a dynamic, multistep process. The protein is first recruited from the cytosol to the cell cortex, a step that requires cell cycle progression into mitosis. Cortically recruited Pon then moves on the cortex and is later restricted to the basal side to form a crescent. The crescent disintegrates upon exit from mitosis. Photobleaching experiments reveal both apical and basal movements of Pon on the cortex. These movements can still occur when myosin motor activity is inhibited by drug treatment. Genetic and pharmacological analyses further reveal that the formation and anchoring of the Pon crescent at the basal cortex require actomyosin and Inscuteable (Lu, 1999).

The gradual recruitment of Pon from the cytosol to the cell cortex at early stages of the cell cycle appears to be coupled to cell cycle progression. To test whether entry into mitosis is a prerequisite for this cortical recruitment, Pon-GFP was introduced into cell cycle mutant backgrounds. In string mutants, postblastoderm cells arrest at the G2 phase of the cell cycle. In neuroblasts of string mutants, the GFP signal is diffuse in the cytoplasm, with some uniform cortical staining. Even after 1 hr of recording, the cytoplasmic signal is not cleared, although the uniform cortical signal seems to increase slightly over time. In contrast, in wild-type neuroblasts, the cytoplasmic signal is cleared within 5-6 min after its appearance. Thus, cortical recruitment of Pon-GFP depends on entry into mitosis (Lu, 1999).

In pebble mutants, cytokinesis of postblastoderm cell divisions is blocked, but other cell cycle events including the asymmetric localization of Numb and Prospero still occur. The initial cortical recruitment and formation of a basal Pon-GFP crescent are normal in pebble mutant neuroblasts. However, by continuously monitoring the Pon-GFP crescent, it was observed that within 10-15 min of its formation, the crescent starts to disintegrate and the protein is dispersed uniformly on the cortex. Gradually, the cortical signal is decreased to background levels, presumably due to degradation or release from the cortex. By comparing the time interval between crescent formation and disintegration in pebble mutants (10-15 minutes) with the interval between crescent formation and the later stages of the neuroblast cell cycle in wild-type embryos, it was determined that the timing of Pon-GFP crescent disintegration in pebble mutants coincides with the end of a normal neuroblast division. This result suggests that at the exit from mitosis certain cell cycle events disassemble or inactivate the localization machinery and that this can occur in the absence of cytokinesis (Lu, 1999).

The defect in the cortical recruitment of Pon-GFP in string mutants suggests that the assembly or proper functioning of the machinery that recruits Pon to the cortex depends on cell cycle progression into mitosis. It is also possible that the cortical recruitment of Pon may depend on its posttranslational modification such as phosphorylation by the p34cdc2 kinase, which is inactive in string mutants. Further biochemical characterization of Pon protein, such as analyzing its posttranslational modification during the cell cycle, should provide more insight into the mechanistic aspects of this regulation (Lu, 1999).

The disintegration of Pon-GFP crescent at the exit from mitosis in pebble mutants implicates a role for the cell cycle machinery in disabling the protein localization machinery. The absence of cytokinesis in pebble mutants allows this step to be observed in more detail. In wild-type neuroblasts, the cleavage furrow coincides with the border of the Pon-GFP crescent at cytokinesis, therefore the GFP signal is distributed all around the GMC cell membrane as soon as the GMC is formed. This makes the crescent disintegration step not observable in wild-type embryos. However, in both wild-type GMC cells and pebble mutant neuroblasts, the uniform cortical GFP signal is gradually decreased to background levels as the cell cycle progresses, suggesting that Pon-GFP is eventually released from the cortex and becomes degraded or delocalized in both wild-type and pebble mutant embryos. In this regard, it will be interesting to test whether the anaphase-promoting complex/cyclosome ubiquitin ligase or components of the mitosis exit signaling pathway are involved in the disintegration of Pon-GFP crescent and the subsequent release of the protein from the cortex (Lu, 1999).

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

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

In order to show that the CNS midline cells control proliferation of the ventral neuroectoderm by activation of stg, stg expression was analyzed in both wild-type and sim mutant embryos. The stg expression profile almost completely matches that of phosphohistone H3 expression. stg expression in the medial, intermediate, and lateral neuroectoderm of wild-type embryos is abolished in sim embryos at stage 10. This indicates that the CNS midline cells promote mitosis of the ventral neuroectoderm by activation of stg expression through cell signaling (Chang, 2000).

To separate cell cycle-independent regulation of the sim gene from the effect on proper cell division in the ventral neuroectoderm, the stg mutant was employed in order to block cell division. The stg mutant is arrested at the G2 phase of cycle 14 since zygotic stg controls the G2/M transition at cell cycle 14. Therefore, analysis of the ventral neuroectodermal marker gene expression in stg and sim;stg double mutants allows one to determine whether sim regulates the cell cycle-independent expression of the genes that determine the identity of the ventral neural and ectodermal cells. The expression of neural (ac, castor, en) and ectodermal (BP28, otd, pnt) markers was analyzed in sim, stg, and sim;stg double mutants. ac gene is expressed in four ventral neuroectodermal clusters in each hemisegment and is successively maintained only in a single NB that is selected from each cluster: MP2, 3-5, 7-1, and 7-4. The expression of ac in S1 NBs is absent in 90% of the examined hemisegments of the sim and of sim;stg double mutant embryos. It is not, however, affected in stg embryos. This observation suggests that the CNS midline provides the ventral neuroectodermal cells with the extrinsic signal(s) that is required for the initial establishment of the ventral neuroectodermal cell fate (Chang, 2000).

Castor is expressed in the S3-S5 NBs 1-2, 2-1, 3-2, 3-3, 3-4, 4-1, 5-1, 5-2, 5-3, 6-1, 7-1, 7-2, and 7-4 of the wild-type embryos at stage 11. In sim embryos, its expression is absent in the medial NBs 1-2, 2-1, 4-1, and 5-1. Castor expression in most of the intermediate and lateral NBs is more severely reduced in the stg mutant than in the sim mutant embryos. This indicates that mitosis is required for the proper expression of Castor in the individual divided NBs. It is maintained in more than 95% of the NBs 2-1, 3-4, 4-1, and 6-1 of the stg mutant embryos. In sim;stg double mutant embryos, the expression of Castor disappears in all the medial NBs 2-1 and 4-1. This result indicates that the CNS midline cells are required for the identity determination of the medial NBs 2-1 and 4-1. It is also demonstrated that mitotic cell division is essential for the proper expression of Castor in order to establish the identity of the NBs 1-2 and 5-1, which undergo several rounds of cell division before Castor expression (Chang, 2000).

The expression pattern of En was examined in the sim, stg, and sim;stg double mutants in order to elucidate the effect of the sim gene on the formation of lateral neurons. The number of the lateral En-positive 10-12 cells of the wild-type embryos is severely reduced to 2-3 cells in more than 88% of hemisegments of the sim;stg mutant, while it is reduced to 5-7 cells in the stg mutant. This result indicates that the CNS midline cells are also required for the proper generation of the En-positive neurons (Chang, 2000).

The enhancer trap line BP28 and otd and pnt genes were used as ventral ectodermal markers. The expression of the BP28 enhancer trap line and otd and pnt genes is abolished in the sim mutant. Beta-galactosidase expression of BP28 is missing in the ventral ectodermal cells of sim and sim:stg mutants. The cell number of the ventral ectoderm is reduced to half in the stg mutant since these cells cannot divide. otd is expressed in two stripes of longitudinal columns of ventral ectoderm in the wild-type embryos at stage 11. It is absent in the sim and sim;stg mutants except in a few NBs. However, it is reduced approximately by half in the stg mutant. The expression of another ectodermal marker, pnt, disappears completely in the ventral region of the sim, stg, and sim;stg mutants (Chang, 2000).

These results show that the CNS midline cells provide the ventral neuroectodermal cells with the extrinsic signal(s) that is required for their unique identity, which is established both by cell cycle progression and by cell cycle-independent determinants (Chang, 2000).

This analysis has demonstrated that the expression of neural (ac, castor/ming, en) and epidermal (BP28, otd) markers in the ventral neuroectodermal cells of the stg mutant disappears in the sim;stg double mutant. This indicates that the CNS midline cells also contribute to the establishment of NB identity by inducing the cell cycle-independent expression of NB, neural, and ectodermal marker genes by cell-cell interaction between the CNS midline and the ventral neuroectodermal (Chang, 2000).

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).

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).

Armadillo levels are sensitive to string dosage

The Armadillo protein of Drosophila is both a structural component of adherens junctions at apical cell membranes and also a key cytoplasmic transducer of the Wingless signalling pathway. The Gal4-UAS system has been used to over-express Armadillo in the wing: this hyperactivates the Wingless pathway and leads to the formation of ectopic, supernumerary wing bristles. This adult phenotype is dominantly enhanced by mutations in string and, conversely, is suppressed by co-expression of String. Furthermore, the steady state levels of Armadillo protein produced from the UAS transgene are also sensitive to string dosage in the cells of the larval imaginal wing disc. Consistent with the role of String in promoting mitosis and with the genetic interaction data, a strong correlation is found between progression through mitosis and a reduction in Armadillo levels. Significantly, this is true whether Armadillo is over-expressed or not, and both cytoplasmic (signalling) and membrane-associated (junctional) Armadillo appears to be affected. It is concluded that this phenomenon may reduce the efficacy of Wingless signalling and/or intercellular adhesion during cell division (Marygold, 2003).

Thus String has gene dosage-dependent effects on Wg-Arm signalling in the adult wing and on Arm protein stability in imaginal wing disc cells. The nature of these interactions suggest that Stg behaves as a negative regulator of Wg-Arm signalling: over-expression of Stg lowers Arm levels and inhibits Wg signal transduction, while a reduction of stg dosage has the opposite effect. Stg is the major mitotic inducer in Drosophila cells. Consistent with this role, it is found that inhibition of mitoses in imaginal wing discs increases the number of cells containing high Arm levels, while induction of mitoses across the disc reduces Arm levels. At the cellular level, high cytoplasmic Arm expression is only detected in cells which are in interphase, while cells undergoing mitosis show reduced levels of cytoplasmic Arm. Taken together, these data suggest that cytoplasmic Arm is destabilized or degraded significantly during Stg-mediated mitosis and that this effect can attenuate Wg signalling (Marygold, 2003).

At the present time, the precise molecular mechanism whereby progression through mitosis leads to a reduction in cytoplasmic Arm levels is not understood. Nonetheless, it is reasoned that the Stg-dependent effects observed must be caused through the ability of Stg to remove inhibitory phosphate from Cdk1 and thereby induce mitotic entry. This is because: (1) Cdk1 is the only reported target of Stg; (2) the sole function ascribed to Stg is to promote G2-M progression, and (3) passage through mitosis correlates with reduced Arm levels in these assays. It is therefore concluded that the decreased stability of cytoplasmic Arm during mitosis occurs as a result of increased Cdk1 activity, though it is not known how direct this effect may be. One attractive possibility is that Arm itself is targeted for proteolysis at the metaphase-anaphase transition of mitosis via the anaphase-promoting complex/cyclosome (APC/C), although the Arm protein lacks the canonical sequence recognition motifs that are found in known APC/C substrates. Alternatively, an upstream component of the canonical Wg pathway could be specifically regulated during mitosis such that cytoplasmic Arm is destabilized as a result (Marygold, 2003).

Regardless of the exact mechanism(s) by which passage through mitosis lowers cytoplasmic Arm, it is interesting to ask why varying the gene dosage of stg should modify the sensitized phenotypes of ectopic Arm? Genetic manipulations of Stg activity might feasibly affect the relative number of Cdk1 molecules that are dephosphorylated by Stg at mitosis. Over-expression of Stg, for example, could cause hyperactivation of Cdk1, leading to excessive phosphorylation of Cdk1 substrates (such as the APC/C) and, ultimately, a near-complete destruction of the over-expressed Arm. Alternatively, as Stg is the limiting inducer of mitosis in Drosophila cell cycles, manipulating stg dosage may have a more significant effect on the absolute or relative length of time cells spend in mitosis and/or interphase. Such effects could thus impinge on the amount of over-expressed Arm that is degraded during mitosis and the degree of productive Wg signalling during interphase. In this regard, it is interesting to note that mutations in genes encoding other, non-limiting promoters of the G2-M transition (including Cdk1, Cyclin A, Cyclin B and Cyclin B3) do not interact in the En>Arm-based assays (Marygold, 2003).

Mitotic G2-arrest is required for neural cell fate determination in Drosophila

In the wing discs of Drosophila, the mechanosensory precursor cells are singled out from clusters of cells blocked at the G2 phase of the cell cycle. This mitotic quiescence and the selection of the precursors are under strict spatio-temporal control. G2 cells were forced to enter mitosis by overexpression of string, the Drosophila homolog of the cdc25 gene. Premature entrance in the cell cycle is associated with a loss of precursor cells. Precursors are lost consecutively to a transcriptional down-regulation of the determinant proneural achaete/scute genes. This down-regulation results from an over-activation of the Enhancer of Split genes, known as effectors of the Notch signalling pathway. It is concluded that exit from the cell cycle is required for proper neural cell fate determination (Nègre, 2003).

Thus, forcing G2 arrested cells into mitosis results in a loss of adult sense organs. The corresponding precursors are also lost. This result was obtained by using two distinct transgenic systems to control the timing and spatial location of stg-overexpression. In both cases, precursors are not selected because ac/sc proneural expression is repressed. This repression occurs at a transcriptional level. Noteworthy is the fact that bristles are lost using either the sca-Gal4 driver to overexpress stg, or the klu-Gal4 driver; this demonstrates that overexpression of stg not only prevents the early accumulation of Ac/Sc (klu-Gal4 driver), but can also downregulate Ac/Sc after the levels of these proteins have started to rise (sca-Gal4 driver). Thus, it is concluded that the arrest in G2 is necessary for proper determination of precursor cells. The complexity of the 5' regulatory sequences of stg indicates that this mitotic regulator might itself integrate information from patterning genes. For instance, the regulatory regions of the stg gene possess putative recognition sites for Achaete and Scute transcription factors. Here, it has been shown that stg can itself control the expression of developmental genes. The effect of stg on cell determination is unlikely to be direct, however, since the only known function of stg is to dephosphorylate the CDK1- cyclin B mitotic kinase. Future genetic approaches may reveal whether or not String has other biochemical targets (Nègre, 2003).

After stg overexpression using the klu-Gal4 driver, it was observed that E(Spl) expression is maintained in proneural regions in absence of Ac/Sc. It was also observed that stg can cause accumulation of the E(Spl) bHLH genes outside of proneural clusters, in a cell-autonomous mode. Maintenance of expression of E(Spl), a transcriptional repressor of the ac/sc expression, is relevant. It can functionally justify the loss of precursor cells. Nevertheless, it has been reported that E(Spl) transcription is dependent on the ac-sc genes in the proneural clusters. One explanation could be that deregulation of the cell cycle directly or indirectly increases transcription of the E(Spl) genes by modifying activity of upstream activators of the E(Spl) expression. Considering this hypothesis, E(Spl) should sometimes be expressed in incorrect positions compared to its wild-type expression. On the contrary, because E(Spl) genes are expressed at the exact positions for proneural clusters, it is suggested that forcing cell cycle more likely affects E(Spl) expression at a post-translational level rather than at a transcriptional level. In the mutants, initial transcription of E(Spl) genes would still have been dependent on Ac/Sc, which begin to accumulate in proneural domains. But, it is known that at least E(Spl) m5, m7 and m8 isoforms contain a PEST-rich motif that harbors an invariant Serine residue, which is phosphorylated by the casein kinase II. Casein kinase II is a ubiquitous serine/threonine kinase whose activity fluctuates with cell cycle progression. Phosphorylation usually regulates protein stability via activation of PEST motifs. Modification in the phosphorylation status of some E(Spl) proteins could exhibit a longer half-life in vivo, thus leading to their predominance over the proneural proteins, and therefore to an inhibition of neurogenesis. In other words, premature entry in the cell cycle would introduce an external bias in the highly dynamic process that opposes the antagonistic E(Spl) and Ac/Sc proteins and which normally occurs in cells of proneural clusters. It would confer an advantage to E(Spl) over proneural activity and would explain persistence of E(Spl) proteins after proneural products have disappeared (Nègre, 2003).

The stg-overexpressing clones, which coexpress high levels of E(Spl), are of low frequency. In the wing primordium, E(Spl) mRNAs are normally detected in proneural clusters, but also in a broad pattern that has no direct relationship with the sensory organs. These Ac/Sc-independent forms of E(Spl) may have been stabilized outside of proneural domains (Nègre, 2003).

The Stg-overexpressing clones are systematically smaller that control clones. This size difference could be related to the capacity of the Notch pathway to negatively control cell proliferation, thus forming a negative feedback loop to precisely coordinate growth and patterning. Striking similarities with the Minute- clones induced in wild-type wing discs may be mentioned here. The Minute- clones display a cell-autonomous slow mitotic rate. Interestingly, when a wing disc is mosaic for two populations of cells, the one dividing more slowly tends to be rapidly eliminated. This phenomenon is termed cell competition. Another feature of the Minute- clones is their fragmentation into patches, which is a rare phenomenon. The idea is favored that clones coexpressing stg and E(Spl) could also be split clones. This dispersion may reflect a difference in surface affinities, which could result in a loss of cohesion (Nègre, 2003).

Altogether, these results suggest that proneural competence can only develop in mitotically arrested cells. The programmed incompatibility between cell cycling and proneural product accumulation may have several general, and not mutually exclusive, functional correlates. In proneural clusters, keeping cells together in a continuous group may be necessary. Indeed, cell interactions could be required to maintain Ac/Sc levels via indirect autoregulation through cell- cell signalling. Furthermore, a G2 arrest may be necessary to preserve a balance between the levels and/or activities of E(Spl) and Ac/Sc products that could directly or indirectly be dependent on post-transductional modifications. The relative strength of the signal impinging on a given cell determines whether products of the proneural genes or products of the E(Spl) become finally predominant. Changing the cell cycle phase could disrupt this equilibrium. Finally, divisions that underly normal cell proliferation and those involved in the fixed lineage of the precursor cell, make different demands on the cytoskeletal machinery. The asymmetric divisions of the precursor cell are strictly controlled in orientation and in time. These controls are presumably essential to realize a correct lineage. A period of mitotic quiescence may give the precursor cell the time and/or conditions required to reorganize its cytoskeleton in order to shift to an asymmetric mode of division. Although a quiescent period systematically precedes the emergence of neural precursors, re-entry into mitosis is independently controlled in the precursor and the surrounding epidermis. This suggests that quiescence is a necessary step preceding the lineage of the precursor. Moreover, the decision of the precursor to enter in its lineage is made independently of the mitotic state of its surrounding cells (Nègre, 2003).

In Drosophila embryos, clusters of synchronously dividing cells appear over the blastoderm surface shortly after cellularization. These mitotic domains correspond to distinct developmental fates. In the case of mitotic domain 10, which corresponds to the prospective mesoderm, it has been demonstrated that mitotic inhibition is essential for normal gastrulation. Mitotic domains have been also described in vertebrates. In zebrafish embryos, mitotic domains arise after the midblastula transition (MBT) with characteristic cell cycle lengths. In Xenopus laevis, axial mesodermal cells become non-mitotic after involution, while cells in the future cement gland cease completely proliferation as they begin to accumulate pigment. No cell division is observed in the presumptive notochord. A recent study in Xenopus shows that in the ciliary marginal zone of the retina, which generates neurons and glial cells, activation of the cell cycle by injection of cyclin E1 inhibits the function of the related proneural gene Xath5. Reciprocally, a forced cell cycle exit, caused by the cyclin kinase inbibitor p27Xic1, potentiates the Xath5 function. Nevertheless, in the same system, forced cell cycle exit by activation of the Notch pathway, either inhibits Xath5 induction of retina glial cells, or promotes the function of coexpressed proneural genes (Nègre, 2003 and references therein).

In this study, causal relationship has been demonstrated to exist between cell cycle and neural determination in an endogenous system: the Drosophila wing imaginal discs, in which E(Spl) effectors of the Notch pathway behave as integrative sensors of the cell cycle status (Nègre, 2003).

Multiple protein phosphatases are required for mitosis in Drosophila

Approximately one-third of the Drosophila kinome has been ascribed some cell-cycle function. However, little is known about which of its 117 protein phosphatases (PPs) or subunits have counteracting roles. This study investigated mitotic roles of PPs through systematic RNAi. It was found that G2-M progression requires Puckered, the JNK MAP-kinase inhibitory phosphatase and PP2C in addition to string (Cdc25). Strong mitotic arrest and chromosome congression failure occurs after Pp1-87B downregulation. Chromosome alignment and segregation defects also occurs after knockdown of PP1-Flapwing, not previously thought to have a mitotic role. Reduction of several nonreceptor tyrosine phosphatases produced spindle and chromosome behavior defects, and for corkscrew, premature chromatid separation. RNAi of the dual-specificity phosphatase, Myotubularin, or the related Sbf 'antiphosphatase' resulted in aberrant mitotic chromosome behavior. Finally, for PP2A, knockdown of the catalytic or A subunits led to bipolar monoastral spindles, knockdown of the Twins B subunit led to bridged and lagging chromosomes, and knockdown of the B' Widerborst subunit led to scattering of all mitotic chromosomes. Widerborst is associated with MEI-S332 (Shugoshin) and is required for its kinetochore localization. This study has identified cell-cycle roles for 22 of 117 Drosophila PPs. Involvement of several PPs in G2 suggests multiple points for its regulation. Major mitotic roles are played by PP1 with tyrosine PPs and Myotubularin-related PPs having significant roles in regulating chromosome behavior. Finally, depending upon its regulatory subunits, PP2A regulates spindle bipolarity, kinetochore function, and progression into anaphase. Discovery of several novel cell-cycle PPs identifies a need for further studies of protein dephosphorylation (Chen, 2007).

P2A is a heterotrimeric serine/threonine phosphatase composed of invariant catalytic (C) and structural (A) subunits together with one member of a family of B regulatory subunits thought to direct the AC core to different substrates. The Drosophila gene for the catalytic subunit of type 2A protein serine/threonine phosphatase (PP2A) is known as microtubule star (mts) because mutant embryos show multiple individual centrosomes with disorganized astral arrays of microtubules. In agreement with this mutant phenotype, it was found that S2 cells depleted for Mts (PP2A-C) displayed aberrant elongated arrays of microtubules with a high proportion (5- to 10-fold increase over the control) of bipolar monoastral spindles or monopolar spindles emanating from a single centrosomal mass. This phenotype is also consistent with the observations in Xenopus egg extracts where mitotic microtubules grow longer and bipolar spindles can not be assembled after inhibition of PP2A by low concentrations of okadaic acid (OA). It is speculated that the monopolar spindle phenotype after mts dsRNA treatment is a consequence of the spindle collapse rather than a failure in centrosome duplication or separation because most of the RNAi-treated cells showed well-separated centrosomes during prophase. In support of this view, spindle bipolarity can be rescued by restoration of microtubule dynamics in OA-treated Xenopus egg extracts (Chen, 2007).

In Drosophila, as in many other eukaryotes, mitosis-specific phosphorylation of histone H3 requires Aurora B activity, but the identity of the opposing phosphatase remains unclear. Because P-H3 (Ser 10) levels were used for monitoring the mitotic index in this analysis, it is possible that a high mitotic index observed after RNAi for PPs may also reflect a defect in dephosphorylating P-H3 in the absence of PPs upon mitotic exit. The phosphorylation state of this histone was therefore studied after RNAi for PPs that displayed a significant increase in the mitotic index in the screen. The immunostaining of control cells showed that P-H3 signals began to decrease at early telophase and then disappeared completely at late telophase. After RNAi knockdown of Mts (PP2A-C) or Pp1-87B, however, the majority of mitotic cells were arrested at prometaphase, but late telophase figures could occassionally be found showing an abnormal accumulation of P-H3 on decondensed chromosomes. To better assess the effect of depletion of these two PPs on P-H3 dephosphorylation, the spindle-assembly checkpoint was inactivated by simultaneously knocking down BubR1. It was found that this rescued the prometaphase arrest of cells simultaneously depleted for Mts or Pp1-87B; this allowed a study of telophase cells. P-H3 was present in the majority of such telophase cells compared to control cells, indicating that both PPs are required for P-H3 dephosphorylation. These results are in accordance with previous studies showing that reduction of PP1 activity can partially suppress defects in the mitotic histone H3 phosphorylation in yeast and C. elegans (Chen, 2007).

Downregulation of Pp2A-29B, the structural A subunit, revealed almost identical aberrant phenotypes to those observed after mts (PP2A-C) RNAi. Consistently, knockdown of Pp2A-29B (PP2A-A) led to a reduction of the protein level of Mts (PP2A-C) (Chen, 2007).

The Drosophila genome contains 4 B-type PP2A regulatory subunits, twins/tws/aar (B sub-type), widerborst/wdb (B' sub-type), Pp2A-B' (B' sub-type), and Pp2A-B" (B" sub-type), but mitotic defects have so far only been reported for mutants of tws. Consistent with the phenotype of tws mutants, it was observed that RNAi for this gene led to an increased proportion of anaphase figures showing lagging chromosomes and chromosome bridges (Chen, 2007).

In metazoans, the B' regulatory subunits of PP2A have evolved into two related subclasses with conserved central regions and diverged amino and carboxy termini. The protein encoded by widerborst (wdb) is more closely related to the human α and ɛ subunits (79%-80% identity) than to the β, γ, or δ subunits (69%-75% identity). Whereas RNAi for tws led to lagging chromosomes, wdb RNAi led to dramatic scattering of chromosomes throughout the spindle. Whether this dramatic effect of wdb RNAi on chromosome segregation reflected any particular subcellular localization of this regulatory subunit was examined. To this end, a GFP-tagged Wdb was expressed in S2 cells. During interphase and prophase, Wdb::GFP partially colocalized with the centromeric marker CID (CENP-A). After spindle formation, Wdb::GFP was found adjacent and external to the centromeres. Although less pronounced, this distribution remained during chromosome segregation at anaphase. Because MEI-S332 (Drosophila Shugoshin) is a dynamic centromeric marker, its distribution was examined in wdb RNAi cells. In control cells, MEI-S332 localized in a band between each pair of the centromeres at metaphase. After downregulation of wdb, however, greatly reduced MEI-S332 staining was found on the metaphase chromosome. In contrast, depletion of MEI-S332 by RNAi did not affect the normal localization of the Wdb B' PP2A subunit. Thus, it is concluded that the Wdb B' subunit is required for correct localization of MEI-S332 but not vice versa. Whether the two proteins existed in the same complex was examined. To address this, a Protein A (PrA)-tagged form of MEI-S332 was expressed in S2 cells to purify potential protein complexes and identify its components by mass spectrometry. The catalytic C (Mts), the structural A (PP2A-29B), and the regulatory B' (Wdb) and B (Tws) subunits of PP2A were identified associated with MEI-S332. Three recent studies also identified PP2A complexed to the B' subunit bound to Shugoshin (Sgo) in human and yeast cells, where they are thought to protect centromeric cohesin subunits from phosphorylation that would promote premature sister-chromatid separation. As with the archetypal family member, Drosophila MEI-S332, the Shugoshins function primarily to protect sister chromatids from separation in the first meiotic division but are also present in mitotic divisions. Consistent with these observations in Drosophila S2 cells, it has been found that depletion of PP2A in human cells led to premature dissociation of Shugoshin 1 (Sgo1) from the kinetochore and loss of mitotic centromere cohesion. The finding of Shugoshin complexed to PP2A/B' in yeast and human, and now in Drosophila, points to a highly evolutionally conserved role for this particular PP2A heterotrimer in regulating sister-chromatid cohesion. Interestingly, Tws B regulatory subunit was also recovered associated with MEI-S332. How this subunit of PP2A might function with MEI-S332 should be the subject of future investigations (Chen, 2007).

Only a moderatedly elevated mitotic index (by approximately 10%) was observed after downregulation of the second Drosophila B' regulatory subunit (Pp2A-B'/B56-1). However, when this second B' subunit was simultaneously knocked down with Wdb, this led to similar phenotypes seen in Mts (PP2A-C) or Pp2A-29B (PP2A-A)-depleted cells. Western-blot analysis showed that the Mts (PP2A-C) level decreased after simultaneous knockdown of both B' subunits, suggesting that this phenotype could be partially due to the loss of PP2A catalytic subunit, although the possibility that the two B' subunits share partially redundant mitotic functions cannot be excluded (Chen, 2007).

Cell-cycle kinases represent a large family of enzymes governing the cell division cycle. It is therefore not surprising that a considerable number of counteracting cell-cycle phosphatases (19% of the genes for tested) were identified in the current study. In addition to finding all the well-known PPs required for cell-cycle progression in Drosophila (Mts, Tws, String, Pp4-19C, and Pp1-87B), the Drosophila counterparts of some eight PPs implicated in cell-cycle functions were identified from studies on other organisms together with six PPs for which novel cell-cycle roles were ascribe. These results were validated by confirming the observed phenotypes with a second nonoverlapping dsRNA. In two cases (flw and csw), their mitotic roles were confirmed through the analysis of phenotypes in mutant larval neuroblasts. The RNAi phenotypes of catalytic subunits were evaluated by observing similar phenotypes after downregulation of the corresponding regulatory subunits (e.g., Pp4-19C and PPP4R2r, Mts/PP2A-C and Pp2A-29B/PP2A-A, and simultaneous RNAi of the two PP2A-B' regulatory subunits). Although a recent large-scale RNAi screen based solely on flow cytometry in Drosophila S2 cells identified many regulators of the cell cycle, cell size, and cell death, this study showed a very low degree of overlap with the cureent analysis (only six), reflecting the need for more sensitive small-scale screens that can examine the functional requirements of assayed proteins in greater detail. These results have provided novel insights into the cell-cycle functions of the Drosophila PPs, and it is likely that, in many cases, these functions have been conserved in other metazoans including humans. This study should guide future work aimed at elucidating the significance and mechanisms of the balanced activities of PKs and PPs in regulating the cell division cycle. The challenge ahead will be to match up the functions of the PPs that were identified with their corresponding counteracting PKs and to identify their common key substrates (Chen, 2007).

Cell cycle control of Wnt receptor activation

Low-density lipoprotein receptor related proteins 5 and 6 (LRP5/6; Drosophila Arrow) are transmembrane receptors that initiate Wnt/β-catenin signaling. Phosphorylation of PPPSP motifs in the LRP6 cytoplasmic domain is crucial for signal transduction. Using a kinome-wide RNAi screen, it was shown that PPPSP phosphorylation requires the Drosophila Cyclin-dependent kinase (CDK) L63. L63 and its vertebrate homolog PFTK are regulated by the membrane tethered G2/M Cyclin, Cyclin Y, which mediates binding to and phosphorylation of LRP6. As a consequence, LRP6 phosphorylation and Wnt/β-catenin signaling are under cell cycle control and peak at G2/M phase; knockdown of the mitotic regulator CDC25/string, which results in G2/M arrest, enhances Wnt signaling in a Cyclin Y-dependent manner. In Xenopus embryos, Cyclin Y is required in vivo for LRP6 phosphorylation, maternal Wnt signaling, and Wnt-dependent anteroposterior embryonic patterning. G2/M priming of LRP6 by a Cyclin/CDK complex introduces an unexpected new layer of regulation of Wnt signaling (Davidson, 2009).

Wnt/β-catenin signaling regulates patterning and cell proliferation throughout embryonic development and is widely implicated in human disease, notably cancer. Two principal classes of transmembrane (TM) receptors function to transduce Wnt/β-catenin signaling; the seven pass TM Frizzled (Fz) proteins and the single pass TM low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6; Drosophila Arrow). Frizzled receptors activate β-catenin-dependent (canonical) as well as β-catenin-independent (noncanonical, such as planar cell polarity) pathways, while LRP5/6 function more specifically in the Wnt/β-catenin pathway (Davidson, 2009).

LRP6 signaling requires Ser/Thr phosphorylation of its intracellular domain (ICD), which contains five PPPSPXS dual phosphorylation motifs comprising Pro-Pro-Pro-Ser-Pro (PPPSP) and directly adjacent casein kinase 1 (CK1) sites. Phosphorylation of the most N-terminal PPPSP (S1490) involves glycogen synthase kinase 3 (GSK3), while CK1g phosphorylates two Ser/Thr clusters near S1490. Phosphorylation of CK1 sites is downstream of, and requires, PPPSP phosphorylation; however, alternative epistasis models have also been proposed. Both PPPSP and CK1 site phosphorylation is necessary for Axin binding to LRP6 and Wnt/β-catenin pathway activation. Phosphorylated PPPSPXS motifs directly inhibit the ability of GSK3 to phosphorylate β-catenin, providing a potential mechanism linking LRP6 activation to β-catenin stabilization. Investigating how LRP6 phosphorylation is regulated is thus crucial for understanding Wnt receptor activation and downstream signaling. Constitutive, non-Wnt-induced S1490 phosphorylation has been observed, suggesting that additional proline-directed kinases may be involved, such as the ERK or Cyclin-dependent kinase (CDK) subgroups (Davidson, 2009).

CDKs are regulators of the cell cycle and require Cyclin partners, whose levels are precisely controlled during the cell cycle, endowing CDKs with both temporal activity and substrate specificity. Several less well-characterized CDK-like proteins exist, including the PFTAIRE kinase subfamily. This study reports on the identification of a Cyclin/PFTAIRE-CDK complex that phosphorylates LRP6 S1490 in a cell cycle-dependent manner, which brings Wnt/β-catenin signaling under G2/M control and introduces a surprising new principle in Wnt regulation (Davidson, 2009).

An important issue in the field of Wnt/β-catenin signaling concerns the regulation of LRP5/6/Arrow function via phosphorylation. This study has identified the unusual plasma membrane tethered Cyclin Y/PFTAIRE complex which functions predominantly at the G2/M phase of the cell cycle to phosphorylate the PPPSP motifs of LRP6. The results suggest a G2/M priming model of LRP5/6/Arrow phosphorylation, where the Cyclin Y/CDK complex phosphorylates LRP6 at PPPSP motifs, which then primes adjacent phosphorylation by CK1. However, PPPSP priming alone is not sufficient for phosphorylation by CK1, as Wnt-induced LRP6 aggregation is also required. Combined phosphorylation at PPPSP and CK1 sites then promotes Gsk3-Axin binding to LRP6 and signalosome formation. Since GSK3 and Cyclin Y/CDK are both essential for LRP6 priming they apparently act nonredundantly. So why is there a dual kinase input to PPPSP phosphorylation? The phosphorylation of LRP6 by GSK3 occurs in acute response to Wnt signaling and it was suggested that it serves to amplify receptor activation. Cyclin Y/CDK phosphorylates Wnt independently at G2/M, thereby gating signal transduction in proliferating cells. One possibility is that individually both kinases prime LRP6 substoichiometrically at the five PPPSP sites and that only their combined action is sufficient for full LRP6 signaling competence (Davidson, 2009).

These findings have important implications for the link between proliferation and Wnt signaling. It has been long known that there is cross talk between mitogenic growth factors and Wnt signaling. The current results may explain why mitogenic growth factors synergize with Wnt/β-catenin signaling, namely by G2/M priming of LRP6 through enhanced cell proliferation, which sensitizes LRP6 for incoming Wnt signals. Moreover, not only extracellular but also intracellular cell cycle check point regulators controlling G2/M entry are likely to affect Wnt signaling (Davidson, 2009).

Wnt/β-catenin signaling itself promotes G1 progression by inducing c-myc and cyclin D1. This suggests that Wnt/β-catenin signaling can entrain a positive feedback loop in proliferating cells by promoting cell cycle progression, which triggers LRP6 phosphorylation at G2/M. Simultaneous stimulation by Wnt and mitogenic growth factors could initiate such a loop. Indeed, the results may explain the previously noted G2/M enrichment of β-catenin and Wnt signaling. Likewise, protein levels of the direct Wnt target gene Axin2, considered a marker gene for Wnt/β-catenin signaling, also peak during mitosis (Davidson, 2009).

What may be the function of a Wnt positive feedback loop during the cell cycle? One of the many roles of Wnt/beta-catenin signaling is to promote cell proliferation and the positive feedback loop suggested by this study may enhance the systems' levels properties of the cell cycle. Specifically, the loop may promote synchrony of cell cycle regulated events or constitute a bistable switch between cell proliferation and cell cycle exit (Davidson, 2009).

One interesting question raised by this study concerns preferential transcription of Wnt target genes around G2/M. Most genes are transcriptionally silenced between late prophase and early telophase, yet TOPFLASH reporter and AXIN2 peak around G2/M. It will therefore be interesting to investigate whether Wnt target genes are transcribed during the more permissive stages G2, early prophase, or late telophase (Davidson, 2009).

Another important question raised by this study is whether G2/M priming is essential or only modulatory for Wnt/β-catenin signaling in general, in particular in light of Wnt signaling in nondividing cells. The fact that LRP6 signaling is promoted by G2/M phase does not exclude Wnt/β-catenin signaling in other cell cycle phases or in nondividing cells. Even though during interphase the levels of LRP6 signalosomes, Sp1490, β-catenin, and reporter activation are lower compared to G2/M, such Wnt/β-catenin signaling is likely physiologically relevant and may involve additional PPPSP kinases, such as GSK3. Surprisingly little is known about Wnt/β-catenin signaling in nondividing cells. In transgenic Wnt-reporter mice, Wnt activity is detected in apparently postmitotic cells in the adult brain, retina, and certain liver cells. In the adult liver, Wnt/β-catenin signaling controls perivenous gene expression. Furthermore, Wnts play a role in axon remodeling in postmitotic neurons and at least one study suggests that this can involve the β-catenin pathway. In light of the current results it will be interesting to examine more systematically Wnt/β-catenin signaling and in particular the LRP6 kinases involved in postmitotic cells (Davidson, 2009).

Traditionally it is thought that Wnt/β-catenin signaling acts to regulate gene expression of downstream targets. Why then should Wnt/β-catenin signaling peak at G2/M? One likely answer is that components of the Wnt/β-catenin pathway play a crucial role during mitosis beyond transcriptional activation. In C. elegans, Wnt signaling regulates the orientation of the mitotic spindle in early development. In mammalian cells, phosphorylated β-catenin itself binds to centrosomes and is involved in spindle separation during mitosis. Likewise, GSK3, Adenomatous polyposis coli protein (APC) and Axin2, which are components of the β-catenin destruction complex, also have direct functions in mitosis. Taken together these data suggest that Cyclin Y/CDK phosphorylates LRP6 at G2/M to induce Wnt/β-catenin signaling for orchestrating a mitotic program (Davidson, 2009).

String (Cdc25) regulates stem cell maintenance, proliferation and aging in Drosophila testis

Tight regulation of stem cell proliferation is fundamental to tissue homeostasis, aging and tumor suppression. Although stem cells are characterized by their high potential to proliferate throughout the life of the organism, the mechanisms that regulate the cell cycle of stem cells remain poorly understood. This study shows that the Cdc25 homolog String (Stg) is a crucial regulator of germline stem cells (GSCs) and cyst stem cells (CySCs) in Drosophila testis. Through knockdown and overexpression experiments, it was shown that Stg is required for stem cell maintenance and that a decline in its expression during aging is a critical determinant of age-associated decline in stem cell function. Furthermore, it was shown that restoration of Stg expression reverses the age-associated decline in stem cell function but leads to late-onset tumors. It is proposed that Stg/Cdc25 is a crucial regulator of stem cell function during tissue homeostasis and aging (Inaba, 2011).

The present study reveals the function of Stg in stem cell proliferation in the Drosophila testis. The highly specific expression pattern of Stg in GSCs and CySCs indicates its function in stem cells. Specifically, Stg contributes to the maintenance of stem cells, as its knockdown leads to a decrease in GSC and CySC numbers, whereas its overexpression causes an increase in CySC number. It is currently unknown whether stem cells that are defective in Stg (or with reduced Stg expression) are lost due to cell death or differentiation. It is possible that cells that do not 'qualify' as stem cells undergo differentiation, as shown for melanocyte stem cells. The reduced proliferation of GSCs and/or CySCs is unlikely to be a direct cause of decreased stem cell numbers: RNAi-mediated knockdown of Stg did not influence spindle orientation in GSCs or CySCs and thus GSCs are likely to be dividing asymmetrically, preserving GSC number. It is possible that GSCs with a reduced proliferation rate are somehow sensed by a quality control mechanism, leading to apoptosis or the differentiation of such GSCs and thus to a reduction in GSC number (Inaba, 2011).

Are the effects of Stg manipulation described in this study specific to Stg function, or are they general consequences of manipulating the stem cell cycle? The first possibility is favored for the following reasons. First, other cell cycle regulators, such as Cyclins A, B and E, do not show stem cell-specific high-level expression, implying that these cell cycle regulators are equally important in stem cell compartments and transit-amplifying cells. Second, overexpression of Cdc2 (Cdk1) or Cdk4 together with Cyclin D did not lead to overproliferation of the CySCs, in contrast to the effect of Stg overexpression in CySCs. Given the specific, high-level expression of Stg in the stem cell compartments, Stg might function as a rate-limiting factor of cell cycle progression in stem cells (Inaba, 2011).

The data also indicate that CySCs are likely to play a permissive (but not instructive) role in GSC division, whereas GSCs play a minor role in CySC division. This leads to a higher CySC:GSC division ratio (the CySC mitotic index is more than twice the GSC mitotic index) under certain conditions: when CySC divisions are induced by overexpression of Stg in these cells, and when GSC divisions are reduced in aging testis leaving the CySC division rate unchanged. The fact that the GSC:CySC division ratio can diverge from a strict 1:2 ratio suggests that the nature of the coordination is not so tight that any modulation in the division rate in one population is reflected in the division rate of the other population. Instead, it seems that the system only provides a 'cap' on GSC division (Inaba, 2011).

Stg expression specifically decreases in GSCs, but not in CySCs, with increasing age. Although re-expression of Stg in the germline suppressed the age-associated decline in GSC division and CySC number, it led to late-onset tumor development. This implies that, although the reduction in Stg expression contributes to tissue aging, it is an important tumor-suppressor mechanism; indeed, CDC25 has been shown to be overexpressed in many human cancers. Tge current work raises the possibility that the cells-of-origin of cancers that express CDC25 might be stem cell populations. It remains unclear whether the overexpression of Stg is sufficient to induce tumors or merely enhances a tumorigenic phenomenon inherent to Drosophila testes during aging. Indeed, wild-type and control flies also develop tumors at a lower frequency and Stg overexpression does not immediately lead to tumorigenesis, in contrast to the overexpression of 'stem cell factors' such as Upd and Zfh1. Therefore, the possibility is favored that the overexpression of Stg/Cdc25 enhances tumor formation when combined with other tumorigenic circumstances, such as the genetic background and age-associated imbalance of stem cell proliferation, as indicated in the present study (Inaba, 2011).

This study presents a potential hurdle in harnessing stem cells for therapeutics. Although it has been speculated that restoration of stem cell activity might help prevent aging or tissue-degenerative diseases, the data indicate that reversing the aging phenotype of one stem cell population does not necessarily lead to desirable consequences. To harness stem cell potential for therapeutic use, it will be crucial to understand how distinct stem cell populations decline with age and with unique kinetics, and how these distinct kinetics are coordinated among multiple stem cell populations over the course of aging to achieve tissue homeostasis and tumor suppression. In summary, the present study provides a cellular mechanism that links cell cycle regulation, stem cell identity, tissue aging and tumor-suppression mechanisms (Inaba, 2011).

String and eye development

Continued: see String: Effects of mutation part 2/2

string: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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