Cyclin A

DEVELOPMENTAL BIOLOGY

Effects of Mutation or Deletion

In eukaryotes, mitotic cyclins localize differently in the cell and regulate different aspects of the cell cycle. The relationship between subcellular localization of Cyclins A and B and their functions in syncytial preblastoderm Drosophila embryos has been investigated. During early embryonic cycles, Cyclin A is always concentrated in the nucleus and present at a low level in the cytoplasm. Cyclin B is predominantly cytoplasmic, and localized within nuclei only during late prophase. Also, Cyclin B colocalizes with metaphase but not anaphase spindle microtubules. Maternal gene doses of Cyclins A and B were manipulated to test their functions in preblastoderm embryos. Increasing doses of Cyclin B increases Cyclin B-Cdk1 activity, which correlates with shorter microtubules and slower microtubule-dependent nuclear movements. This provides in vivo evidence that Cyclin B-Cdk1 regulates microtubule dynamics. In addition, the overall duration of the early nuclear cycles is affected by Cyclin A but not Cyclin B levels. Taken together, these observations support the hypothesis that Cyclin B regulates cytoskeletal changes while Cyclin A regulates the nuclear cycles. Varying the relative levels of Cyclins A and B uncouples the cytoskeletal and nuclear events; therefore it is speculated that a balance of cyclins is necessary for proper coordination during these embryonic cycles (Stiffler, 1999).

Axial expansion occurs during cycles 4-6 when nuclei move along the anterior/posterior axis of the embryo. In cycles 7-10, nuclei migrate to the embryonic cortex. The microfilament breakdown during axial expansion was investigated. It has been proposed that microtubules regulate the stability of the microfilaments and direct their breakdown, thus controlling the movement of the nuclei. The network of astral microtubules pushes nuclei to the cortex of the embryo. These movements occur periodically and correlate with dynamic changes of the network. Microtubules undergo dramatic phase-specific morphological changes during cycles 4-7. Interphase astral microtubules radiate from centrosomes and form a network that generates the positioning of evenly spaced nuclei. This network breaks down in prophase and microtubules reorganize into mitotic spindles in metaphase. In anaphase the microtubules bring chromatids to the poles and the mitotic spindle breaks down; a midbody remains between the separating chromatids. Finally, microtubules reform asters as division is completed in telophase, and the midbody persists through interphase (Stiffler, 1999).

Cyclin B has been shown to regulate microtubule dynamics in Xenopus extracts, and cyclin B localizes to microtubules in blastoderm stage Drosophila embryos. Microtubular dynamics affects nuclear migration in the preblastoderm embryos. Whether or not cyclin B levels affect axial expansion and cortical migration during preblastoderm cycles was tested. To do this, the volume of interphase asters was calculated and microtubule configurations were observed in embryos possessing different doses of Cyclin B (1-, 2- and 4-cyclin B embryos). Astral microtubule volume is inversely proportional to Cyclin B dose. Confocal images reveal a similar correlation between microtubule length and cyclin B dose during interphase and metaphase. In interphase, microtubules are longer in 1- and 2-cyclin B embryos, when compared to those of 4-cyclin B embryos. During prophase in 1-cyclin B embryos, asters do not break down as completely as they do in 4-cyclin B embryos. During metaphase, mitotic spindles appear larger in 1-cyclin B embryos and smaller in 4-cyclin B embryos, as compared to 2-cyclin B embryos. In addition, metaphase asters are longer and clearly visible in 1-cyclin B compared to those in 2-cyclin B, but they are not detectable in 4-cyclin B embryos. During anaphase and telophase, larger microtubule asters are observed in 1-cyclin B embryos compared to those in 2- and 4-cyclin B embryos. To amplify the effects of cyclin B on microtubule morphology, 0- and 10-cyclin B embryos were examined. In 0-cyclin B embryos, microtubules are often elongated and organized into thick bundles throughout the embryo. Astral microtubules are often observed without nuclei; centrosomes appear to divide and migrate without associated nuclei. In 10-cyclin B embryos, microtubules are severely reduced in all phases of the cycle and nuclei remain close together. Microtubule morphology in 0- and 10-cyclin B embryos is more severely altered than that of 1- and 4-cyclin B embryos. Thus cyclin B levels correlate with microtubule abundance in different cycle phases (Stiffler, 1999).

Because cyclin A localized in nuclei, a test was performed to see whether reducing cyclin A affects cell-cycle duration. Decreased Cyclin A correlates with a significant increase in cell-cycle time during cycles 4-6, which is independent of cyclin B levels.There is a slight increase in the length of time from egg deposition to cycle 10 in 1-cyclin A embryos. In contrast, increased cyclin B does not have a linear effect on cycle time. These observations show that overall cycle length is sensitive to cyclin A levels. To analyze cell-cycle progression, the mitotic index for embryos was calculated at varying doses of cyclins A and B. During these early cycles, divisions are synchronous. Therefore, the mitotic index is the fraction of embryos in a specific cell-cycle phase, and represents the relative duration that embryos spend in that phase. During cycles 2 to 8, 4-cyclin B embryos have a longer metaphase and shorter interphase, compared to 1- or 2-cyclin B embryos, but other phases are not different. This shows that metaphase is longer in 4-cyclin B embryos while interphase is shorter; therefore the overall cell-cycle duration is the same. 1-cyclin B embryos spend less time in metaphase and more in interphase; thus the overall cell-cycle duration is again unaffected. However, less cyclin A affects the overall cycle duration, but the mitotic index is not different from controls. It is concluded that cyclin B dose affects metaphase and interphase duration while cyclin A regulates the overall cycle duration (Stiffler, 1999).

Cyclin A has a mitotic function: it acts synergistically with Cyclin B during the G2-M transition. In double mutant embryos that express neither Cyclin A nor Cyclin B zygotically, cell cycle progression is blocked just before the exhaustion of the maternally contributed Cyclin A and B stores. BrdU-labeling experiments indicate that cell cycle progression is blocked in G2 before entry into the fifteenth round of mitosis. Expression of either Cyclin A or B from heat-inducible transgenes is sufficient to overcome such a cell cycle block. This block is not observed in single mutant embryos deficient for either Cyclin A or B. In Cyclin B deficient embryos, cell cycle progression continues after the apparent exhaustion of the maternal contribution, suggesting that Cyclin B might not be essential for mitosis. However, mitotic spindles are clearly abnormal and progression through mitosis is delayed in these Cyclin B deficient embryos (Knoblich, 1993).

No functional overlap is observed between the cdc2 and the cdc2c kinases. The phenotype resulting from mutations in cdc2 is not affected by altering the level of cdc2c. Cyclin A and Cyclin B have largely overlapping functions. Cell proliferation is observed in the absence of either Cyclin A or Cyclin B, but not if both cyclins are absent. Cyclin A also has essential functions that cannot be taken over by Cyclin B, but these functions appear to be required at defined developmental stages in specific tissues only (Lehner, 1992).

The onset of pattern formation in the developing Drosophila eye is marked by the simultaneous synchronization of all cells in the G1 phase of the cell cycle. These cells will then either commit to another round of cell division or differentiate into neurons. Although cell cycle synchronization occurs in roughex (rux) mutants, cells circumvent G1 and all cells enter S phase, including cells that would normally differentiate. This leads to defects in early steps of pattern formation and cell fate determination. rux is suppressed by mutations in genes that promote cell cycle progression (i.e., Cyclin A and string) and enhanced by mutations in genes that promote differentiation (i.e., Ras1 and Star). A deficiency involving CycB does not suppress rux, a gene that encodes a novel protein of 335 amino acids. rux functions as a negative regulator of G1 progression in the developing eye (Thomas, 1994).

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

Cyclin B3 has been conserved during higher eukaryote evolution as evidenced by its identification in chicken, nematodes, and insects. Except for the destruction box, similarities are restricted to the cyclin box where the B3 types share between 39% and 46% identity. Cyclin B3 is present in addition to Cyclins A and B in mitotically proliferating cells and is not detectable in endoreduplicating tissues of Drosophila embryos. Cyclin B3 is coimmunoprecipitated with Cdk1(Cdc2) but not with Cdk2(Cdc2c). It is degraded abruptly during mitosis like Cyclins A and B. In contrast to these latter cyclins, which accumulate predominantly in the cytoplasm during interphase, Cyclin B3 is a nuclear protein. Genetic analyses indicate functional redundancies. Double and triple mutant analyses demonstrate that Cyclins A, B, and B3 cooperate to regulate mitosis, but surprisingly single mutants reveal that neither Cyclin B3 nor Cyclin B is required for mitosis. However, both are required for female fertility and Cyclin B also for male fertility (Jacobs, 1998).

In addition to cooperation of Cyclins B and B3, synergy of Cyclins A and B3 can be demonstrated. Although chromatin condensation is particularly slow, spindle formation appears to be less affected in Cyclin A;Cyclin B3 double mutants compared to Cyclin B:Cyclin B3 double mutants. On the basis the double mutant analyses it has been concluded that all of the mitotic cyclins cooperate to bring about progression through mitosis. These results are consistent with the idea that Cyclin A is most potent in triggering mitosis, whereas Cyclin B has an intermediate and Cyclin B3 the lowest potency. The results also confirm the suggestion that Cyclin B is particularly important for the organization of mitotic spindles. In addition, they suggest that Cyclin A, which translocates into the nucleus very early during prophase, and Cyclin B3, which accumulates in the nucleus already during interphase, are particularly important for chromosome condensation (Jacobs, 1998).

It has been proposed that different mitotic processes that occur after the metaphase-anaphase transition might be temporally arranged by the ordered disappearance of Cyclins A, B, and B3, such disappearances resulting from proteolytic degradation. This proposal is based not only on the ordered disappearance of the different mitotic cyclins, but also on the specific mitotic defects resulting after the expression of mitotic cyclins with amino-terminal deletions, which also remove the destruction box. These mutant nondestructible Cyclins A, B, and B3 are found to delay mitotic progression before sister chromatid separation, sister chromatid segregation to the poles, and telophase including cytokinesis, respectively. On the basis of this proposal, all these processes are expected to start simultaneously in Cyclin B;Cyclin B3 double mutants after the disappearance of Cyclin A during mitosis. In fact, chromosome segregation to the poles, chromosome decondensation, and cytokinesis are all attempted simultaneously in these double mutant embryos. However, the interpretation of these phenotypes is complicated by the fact that mitotic progression until metaphase is already severely affected, and not just exit from mitosis. This functional overlap between the cyclins is less pronounced in the germ line and in particular during oogenesis where both Cyclin B and Cyclin B3 are essential. Therefore, a future detailed characterization of the role of different mitotic cyclins in oogenesis should be especially informative with regard to cyclin type-specific functions and regulatory differences between mitosis and meiosis (Jacobs, 1998).

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).

In the central nervous system (CNS) of Drosophila embryos lacking either cyclin A or regulator of cyclin A (rca1) several ganglion mother cells (GMCs) fail to divide. Rca1 is novel 412 amino acid protein required for both mitotic and meiotic cell cycle progression, Whereas GMCs normally produce two sibling neurons that acquire different fates ('A/B'), non-dividing GMCs differentiate exclusively in the manner of one of their progeny ('B'). The rca1 mutation was initially identified and characterized from a screen for aberrant expression patterns of Even- skipped (Eve) protein in the embryonic CNS (I. Orlov, R. Saint, N. Patel, unpublished results cited by Lear, 1999). Eve is normally expressed in the nuclei of several cells in the CNS; these include GMC 1-1a and its progeny, aCC and pCC; GMC 4-2a and one of its progeny, RP2, as well as the EL, U, and CQ neurons. In cycA and rca1 mutants, Eve is expressed in fewer cells per hemisegment than wild-type. In the position where the siblings aCC and pCC normally sit, a single Eve-positive nucleus that is larger than the wild-type aCC or pCC is observed. In the position of RP2, there is still one Eve-positive nucleus, but again it often appears larger than normal. A loss of Eve expression is also observed where the U and CQ neurons normally sit and a decrease in the number of Eve-positive EL neurons (Lear, 1999).

The GMC 4-2a and GMC 1-1a lineages recieved the closest focus because of their well-characterized development and because various molecular markers exist that label these GMCs and their progeny. In wild-type embryos, GMC 4-2a divides early in stage 11, and two Eve-expressing nuclei are initially observed upon this division. Eve expression is quickly shut off in the smaller RP2 sibling nucleus but remains on in RP2. In cycA or rca1 mutants, Eve expression turns on normally in GMC 4-2a; however, two nuclei are rarely observed during stage 12, and the single Eve-expressing nucleus remains large. Likewise, GMC 1-1a normally divides during stage 10 in wild-type embryos to generate the Eve-positive neurons aCC and pCC. In cycA or rca1 mutants, GMC 1-1a expresses Eve as in wild-type but rarely divides. Instead, this GMC comes to reside in the same dorsal plane and posterior position where aCC and pCC sit in wild-type embryos. Other Eve-expressing lineages, including the U/CQ neurons and the EL neurons, appear to be affected as well in cycA and rca1 mutants. Notably, even the most severe alleles of cycA and rca1 examined do not show complete expressivity of CNS phenotypes in all lineages (Lear, 1999).

Having observed that GMCs acquire the fate of the 'B' sibling neuron in cycA or rca1 mutants, it was next determined whether GMCs could acquire the 'A' fate through activation of the Notch pathway. If Delta signal must be provided from a sibling neuron, then GMCs, which lack a true 'sibling', may not have the potential to acquire the 'A' fate through extracellular signaling. The rca1 mutation was combined with either a zygotic numb mutation or an activated form of Notch in order to examine this question. In zygotic numb mutants, sibling neuron fate alterations ('A/B' to 'A/A') occur infrequently or do not occur in some sibling pairs; depletion of both maternal and zygotic numb causes sibling neurons to acquire equalized fates ('A/A') with near-complete expressivity. In rca1;numb double mutant embryos, binary cell fate changes ('B' to 'A') in several GMCs as well. GMC 4-2a frequently adopts the 'A' fate of RP2 sibling in rca1;numb or hs-N intra;rca1 embryos. In contrast, GMC 4-2a always acquires the 'B' fate of RP2 in rca1 mutants alone. Notably, it was observed that the 'B' to 'A' fate change (RP2 to RP2 sibling) occurs with greater frequency in rca1;numb double mutants than the RP2/sib ('A/B') to sib/sib ('A/A') fate change that occurs in numb mutants alone (Lear, 1999).

Thus GMCs in cycA and rca1 mutants differentiate as neurons: they assume the 'B' fate normally taken by one of their sibling progeny. These GMC fate decisions correspond to Notch pathway mutants ('B/B'), and they oppose the fate changes observed in embryos lacking numb ('A/A'). The loss of zygotic numb or constitutive activation of Notch in a rca1 background allows for a binary fate switch in GMCs: GMCs often differentiate as the 'A' sibling in the context of these mutations. These results indicate that activation of the Notch pathway causes GMCs to adopt the 'A' neuronal fate. Thus, fate choice in non-dividing GMCs appears to occur in much the same way that binary fate decisions occur in sibling neurons. In some models of asymmetric division, a specific factor required to attain one of the sibling fates is produced only upon progression of the cell cycle. The observation that GMCs can attain the fate of either sibling neuron indicates that gene products dependent upon GMC division are not required in this fate decision (Lear, 1999).

A genetic screen was initiated to identify mutants that enhance or suppress weak pan gu (png) mutations by using the deficiency collection available from the Bloomington Stock Center to survey about 50% of the genome using a minimum number of stocks and crosses. Deficiencies that when heterozygous would dominantly suppress or enhance weak png mutations were scored by examining the nuclear phenotype of embryos. Females transheterozygous for the weak png³³¹⁸ allele and the strong png¹⁰⁵⁸ allele produce embryos in which some mitotic divisions occur before the nuclei ultimately become polyploid. These embryos contain up to 16 giant, polyploid nuclei. In the same collections, there are embryos that have a single, multilobed nucleus that results from the female meiotic products and male pronucleus undergoing DNA replication but no nuclear division. As these nuclei become polyploid they can fuse together. Thus, in the absence of mitosis and nuclear division, between one and five giant polyploid nuclei are produced. Two classes of embryos were scored as those with five or fewer nuclei vs. those with greater than five (multinucleated). The relative percentages of these two classes produced by png³³¹⁸/png¹⁰⁵⁸ females is affected by genetic background and can vary between 30% and 55%. Thus suppression was defined as genotypes producing >60% multinucleated embryos and enhancement as <30% (Lee, 2001).

In addition to Cyclin B, the levels of Cyclin A protein are decreased in proportion to the strength of the png allele. Surprisingly, neither a deficiency that uncovers cyclin A nor mutations in the gene enhance the png mutant phenotype. A negative result in this test does not exclude the possibility of interaction because a twofold reduction in gene dosage may not decrease the level of gene product below a threshold required to detect an effect. However, loss of one copy of the cyclin A gene in the mother does affect cell cycle parameters and nuclear division in the embryo, so one copy of the gene does not produce sufficient levels of the protein for normal cell division. Whether overexpression of cyclin A could suppress the png mutant phenotype was tested. This experiment produced an unexpected result. When cyclin A was induced by heat shock, the png phenotype was enhanced. This may be due to the ability of Cyclin A when overexpressed to drive cells inappropriately into S phase. Thus, this result may be a consequence of artifactually high levels of Cyclin A and may not accurately reflect the function of Cyclin A in the early embryonic divisions (Lee, 2001).

The suppression of png, plu, and gnu by overexpressing cyclin B is not complete because ultimately nuclear divisions fail, and the nuclei continue to replicate and become polyploid. It is possible that Cyclin B is the sole target of the Png/Plu Complex and Gnu and that the levels of increased Cyclin B protein in the png, plu, and gnu mutants (via increased copies of the cyclin B gene) are not adequate for completion of all the S-M cycles. However, it seems more likely that, although Cyclin B is a key target, other targets of the Png/Plu complex and Gnu are also important. Cyclin A is a particularly good candidate for two reasons. Cyclin A protein levels are decreased in png, plu, and gnu mutants; for png, the decrease is in proportion to the strength of the allele. Decreasing the dosage of maternal cyclin A to one copy causes an increase in cycle time during the early embryonic divisions. In contrast, decreasing the dosage of cyclin B does not affect the timing of nuclear cycles, whereas it does affect microtubule dynamics. These observations led to the conclusion that Cyclin B controls cytoskeletal events during the S-M cycles, but Cyclin A controls the nuclear cycles. If this model is correct, Cyclin A may be a critical target for the influence of Png, Plu, and Gnu on the nuclear cycles (Lee, 2001).

No enhancement of the png phenotype by mutations in cyclin A was observed, and overexpression of cyclin A unexpectedly enhanced the phenotype. This latter result likely reflects the ability of excess Cyclin A to promote DNA replication, but it is not clear why a reduction in Cyclin A does not affect the png phenotype. Further delineation of the role of Cyclin A levels will likely emerge from identification of Png kinase substrates and elucidation of the mechanism by which Png influences Cyclin A and B protein levels (Lee, 2001).

There are several mechanisms by which Png, Plu, and Gnu could affect Cyclin A and B protein levels, including maternal transcription, mRNA stability or processing, translation, and cyclin protein stability. Mutations in the pathway that target mitotic cyclin proteins for destruction were examined. No suppression of the png mutant phenotype was observed by reducing the dosage of the two known activators of cyclin destruction, fzy or fzr. Similarly, mutation of an APC/C subunit or several genes affecting the ubiquitin pathway did not alter the png mutant phenotype. These negative results do not exclude a role for Png in controlling APC/C-mediated protein degradation, since the dosage reductions may not have reduced protein activity below a crucial threshold. Additional experiments will be required to evaluate how PNG affects Cyclin A and B protein levels (Lee, 2001).

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

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

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

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

Myogenic cells, asymmetric lineages, the Notch pathway and the effects of blocking cell division in heart development

During the formation of the Drosophila heart, a combinatorial network that integrates signaling pathways and tissue-specific transcription factors specifies cardiac progenitors, which then undergo symmetric or asymmetric cell divisions to generate the final population of diversified cardiac cell types. Much has been learned concerning the combinatorial genetic network that initiates cardiogenesis, whereas little is known about how exactly these cardiac progenitors divide and generate the diverse population of cardiac cells. In this study, the cell lineages and cell fate determination in the heart have been examined by using various cell cycle modifications. By arresting the cardiac progenitor cell divisions at different developing stages, the exact cell lineages for most cardiac cell types have been determined. Once cardiac progenitors are specified, they can differentiate without further divisions. Interestingly, the progenitors of asymmetric cell lineages adopt a myocardial cell fate as opposed to a pericardial fate when they are unable to divide. These progenitors adopt a pericardial cell fate, however, when cell division is blocked in numb mutants or in embryos with constitutive Notch activity. These results suggest that a numb/Notch-dependent cell fate decision can take place even in undivided progenitors of asymmetric cell divisions. By contrast, in symmetric lineages, which give rise to a single type of myocardial-only or pericardial-only progeny, repression or constitutive activation of the Notch pathway has no apparent effect on progenitor or progeny fate. Thus, inhibition of Notch activity is crucial for specifying a myogenic cell fate only in asymmetric lineages. In addition, evidence is provided that whether or not Suppressor-of-Hairless can become a transcriptional activator is the key switch for the Numb/Notch activity in determining a myocardial versus pericardial cell fate (Han, 2003).

All mesodermal cells go through three postblastoderm cell cycles (cycle 14-16). The mesodermal cells enter the first postblastoderm division (mitosis 14) at 210 minutes of development as domain 10 (see Mitotic domains). They are the first embryonic cells to go through the second postblastoderm division at about 250 minutes. The mesodermal cells are thought to divide in an approximately synchronous fashion in the first two postblastoderm cell divisions. The third division (mitosis 16) takes place during late stage 10 to early stage 11 between 280-300 minutes. During this division, a continuous longitudinal zone that may generate the heart precursors and part of the visceral mesoderm appears as a subdomain of mitosis 16. It is likely that tinman is involved in generating this subdomain because it is specifically expressed in such a continuous longitudinal zone at stage 10 (Han, 2003).

The asymmetric cell divisions of the Eve lineage that generate the Eve paracardial cell (EPC) progenitors and muscle founders DO2 are arrested in CycA or Rca1 mutants, in which cell cycle 16 is blocked, but not in twi>dap (dacapo driven by twiGAL4) embryos, in which the subsequent division of the EPC progenitor is inhibited (cycle 17). Based on the effects of these genes in the ectoderm, it is likely that CycA mutant blocks the progression of cell cycle also in the mesoderm, but ectopic dacapo induces early exit from the cell cycle. Therefore, in CycA mutants mitosis may be blocked at a certain time point during development (such as at G2/M transition of mitosis 16), but ectopic dap may induce skipping of the last division. It has been shown that the Numb crescent in the precursor P2, which generates the DO2 founder and the EPC progenitor, appears at late stage 10 and the division happens between late stage 10 and early stage 11, consistent with this being mitosis 16 of the mesodermal cells. Therefore, it seems that many cells of the cardiac mesoderm go through three postblastoderm cell divisions (mitosis 14-16), but some of them (such as the EPC lineage) undergo an additional division (mitosis 17) (Han, 2003).

Two different models have been proposed for the mesodermal Eve lineages. One model suggests that each EPC share a progenitor with a muscle founder. The other model suggests that the two EPCs per hemisegment share a progenitor, which in turn share a progenitor with one of the muscle founder cells (DO2), whereas the other DA1 muscle founder derives from the second progenitor. The data presented in this paper strongly support the latter model. The most direct evidence derives from pan-mesodermal dap overexpression, which results in the formation of a single EPC, probably because of a block in the last division. By contrast, the first model predicts formation of either no EPC or two EPCs, clearly not what is observed. These conclusions are also supported by recent lineage tracing experiments (Han, 2003).

Recent studies have suggested that cardiac specification along the anterior-posterior axis is under the control of homeotic genes. For example, Svp myocardial cells are only present in the abdominal segments of the heart, but not in the thoracic segments, and is probably under the control of Antennapedia, which is active in these segments. In this study it was found that in addition to cell identity differences between the anterior two and the posterior heart segments, the lineage of the T3-A1 myocardial cells is also distinct. In cycle 16, blocking CycA or Rca1 mutants, the last cycle of myocardial divisions in T3-A1 is not arrested unlike the case for the posterior myocardial cells. The anterior myocardial progenitors may either undergo the last division during cycle 15 or they may be less susceptible to a loss-of-CycA-function. The first possibility is consistent with the observation that embryos with overexpression of the last division inhibitor, dap (but not cycle 16 arrested CycA mutants), exhibit a reduction of Tinman myocardial cells (TMCs) in T3-A1. Alternatively, it is possible that all T3-A1 myocardial lineages are asymmetric (as are the more posterior Svp lineages), except they all express tinman, due to the lack of svp in these two segments. Thus, blocking division in CycA mutants would not alter the number of TMC in these two segments. Recent lineage tracing data are consistent with this view, but further experiments are needed to elucidate these lineages (Han, 2003).

The second postblastoderm cell division of the mesodermal cells (mitosis 15) seems to be arrested if both CycA and CycB functions are lost. In CycA;CycB double mutants, only two of the normally six myocardial cells are formed in A2-A7 segments, one exhibiting TMC and the other Seven up myocardial cell (SMC) characteristics. Therefore, it is proposed that in each hemisegment two myocardial superprogenitors are specified: the TMC superprogenitor (TSP) divides twice symmetrically, whereas the SMC superprogenitor (SSP) first divides symmetrically and then asymmetrically. Present and previous studies suggest there are probably five progenitors in each hemisegment that give rise to 14 heart-associated cells (six myocardial and eight pericardial): the TSP gives rise to four myocardial cells (two of which are Labial mycardial cells); the SSP generates two myocardial and two SOPC (Svp and Odd co-expressing pericardial cells); the Eve-expressing pericardial cell progenitor gives rise to two EPCs; the remaining four pericardial cells, to two OPC and two LPC, probably derived from two symmetrically dividing precursors, although their lineage is not as well understood (Han, 2003).

Asymmetric divisions have been studied in the context of cell cycle progression in the Drosophila PNS and CNS. In the PNS, Notch activity is required for specification of a type I versus type II neuronal fate. When sensory organ progenitor cell division is blocked in stg^- mutants, the undivided precursor adopts a type II neuronal fate, whereas in numb;stg double mutants, a type I fate is chosen. In the CNS, Notch is required for specification of the sib cell fate versus the RP2 cell fate of the GMC1 asymmetric cell division. In Rca1 mutants, the undivided GMC1 adopts a RP2 fate, whereas in numb;Rca1 double mutants, the undivided GMC1 often adopts the sib cell fate. Both experimental outcomes are analogous to what is observed in CycA mutants: the undivided P2 progenitor adopts a pericardial fate in the absence of numb function instead of a myogenic fate in a wild-type background. Taken together, these observations suggest that arrest of an asymmetric cell division leads the undivided progenitor to adopt the fate of the daughter cell that inherits Numb, and in the absence of Numb the alternative fate is chosen (Han, 2003).

Cyclin A appears to contribute independently of Pim to the inhibition of premature sister chromatid separation

Sister chromatid separation during exit from mitosis requires separase. Securin inhibits separase during the cell cycle until metaphase when it is degraded by the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, sister chromatid separation proceeds even in the presence of stabilized securin with mutations in its D-box, a motif known to mediate recruitment to the APC/C. Alternative pathways might therefore regulate separase and sister chromatid separation apart from proteolysis of the Drosophila securin Pimples (PIM). Consistent with this proposal and with results from yeast and vertebrates, it is shown in this study that the effects of stabilized securin with mutations in the D-box are enhanced in vivo by reduced Polo kinase function or by mitotically stabilized Cyclin A. However, PIM is shown to contain a KEN-box, which is required for mitotic degradation in addition to the D-box; sister chromatid separation is completely inhibited by PIM with mutations in both degradation signals (Leismann, 2003).

Embryos homozygous for pim null mutations but equipped with a maternal pim⁺ contribution from pim heterozygous mothers progress normally through the initial embryonic cycles. Entry into mitosis 15 and progression to metaphase are still normal. Moreover, the transition from metaphase to anaphase is triggered as well, as evidenced by the degradation of the mitotic Cyclins A, B and B3. However, sister chromatid separation during mitosis 15 is completely inhibited. This block of sister chromatid separation after the exhaustion of the maternal pim⁺ contribution is almost completely prevented when pim embryos inherit a gpim^dba-myc transgene. gpim^dba-myc drives expression of Pim with C-terminal myc epitopes and a mutant D-box (AKPAGNLDA instead of KKPLGNLDN). Pim^dba-myc is stable during mitosis according to confocal immunofluorescence microscopy, in contrast to Pim-myc with the wild-type D-box. gpim^dba-myc expression is controlled by the normal pim regulatory region, and the resulting level of Pim^dba-myc before mitosis 15 was found to be comparable to wild-type Pim levels. Because stabilized Pim^dba-myc protein promotes sister chromatid separation in pim mutants, it appears that sister chromatid separation is not dependent on degradation of the Drosophila securin Pim. Analogous experiments with a gpim^dba transgene driving expression of a D-box mutant Pim version without myc epitopes also revealed rescue of mitosis 15 in pim mutants, excluding the possibility that sister chromatid separation in the presence of stabilized Pim^dba-myc occurs simply because C-terminal myc epitopes specifically abolish the inhibitory Pim function (Leismann, 2003).

Instead of being required during each mitosis, Pim degradation might be important to keep protein levels below a critical threshold. Moderate overexpression of wild-type pim (about fivefold) is sufficient to block sister chromatid separation. Moreover, although gpim^dba rescues sister chromatid separation during mitosis 15 and 16 in pim mutants, it does not allow later divisions, perhaps because the levels of stabilized Pim^dba have built up beyond the critical threshold (Leismann, 2003).

If degradation of the securin Pim was not an obligatory process required during each mitosis, separase bound to securin would be expected to have sufficient basal activity to allow sister chromatid separation. In this case, premature sister chromatid separation during interphase and early mitosis would have to be prevented by securin-independent regulation. Since securin-independent regulation at the level of Scc1 phosphorylation by Cdc5/Polo kinase has been described in yeast, whether a reduction in polo function enhances the effects of stabilized Pim^dba was investigated. Within the CNS of polo-mutant embryos, many abnormal cells were observed with very large polyploid nuclei, when these embryos also carried gpim^dba. Similar abnormal cells are almost never observed in either polo⁺ sibling embryos with gpim^dba or in polo^- sibling embryos without gpim^dba. In the presence of stabilized Pim^dba, therefore, the remaining level of maternal polo⁺ contribution is no longer sufficient to mask phenotypic abnormalities in polo-mutant embryos. Moreover, reduced polo⁺ function enhances the effects of stabilized Pim^dba (Leismann, 2003).

In addition to Scc1 regulation by Cdc5/Polo kinase, vertebrate Cdk1 has been shown to regulate separase independently of securin (Stemmann, 2001). The effects of stabilized Cyclin A in Drosophila embryos are consistent with the finding that vertebrate Cdk1 phosphorylates and thereby inhibits separase. Mutant Cyclin A versions that cannot be degraded during mitosis delay progression through the embryonic cell divisions during metaphase before sister chromatid separation. Therefore, Drosophila Cyclin A-Cdk1 complexes might inhibit separase activity. Accordingly, the effects of stabilized Cyclin ADelta1-53 are expected to be enhanced by expression of stabilized Pim^dba. Labeling with antibodies against tubulin and a DNA stain clearly reveal an increased number of metaphase figures in epidermal regions of embryos expressing both Cyclin ADelta1-53 and Pim^dba, compared with embryos expressing only Cyclin ADelta1-53. The stabilized Cyclin ADelta1-53 therefore results in a more pronounced metaphase delay in the presence of the stabilized Pim^dba (Leismann, 2003).

In principle, stabilized Cyclin A might delay cells in metaphase because it results in an inhibition of Pim degradation during mitosis. However, cells delayed in metaphase by stabilized Cyclin ADelta1-170 no longer contain Pim-myc according to immunolabeling experiments, whereas metaphase cells that do not express Cyclin ADelta1-170 are always positive for Pim-myc. It is concluded, therefore, that the metaphase delay induced by stabilized Cyclin A does not result from delayed Pim degradation (Leismann, 2003).

The phenotypic interactions between stabilized Pim^dba and Polo or Cyclin A are consistent with the notion that separase complexed with non-degradable securin might have sufficient activity to allow sister chromatid separation and that the timing of this process is controlled by pathways other than securin degradation. However, the sister chromatid separation in Pim^dba-expressing cells might also be supported by residual mitotic Pim^dba degradation. A KEN motif, which is found close to the N-terminus in all of the securins, might allow some limited mitotic Pim^dba degradation, escaping detection by confocal microscopy as applied in the previous experiments (Leismann, 2003).

To determine whether the KEN motif of Pim functions as a degradation signal, the mitotic stability of a myc-tagged Pim version with a mutant KEN-box (Pim^kena-myc with AAA instead of KEN) was analyzed. Pim^kena-myc, and Pim-myc for control, were expressed in the anterior region of embryos during cycle 14. Immunolabeling at the stage of mitosis 14 indicate that Pim^kena-myc is largely stable throughout mitosis, in contrast to Pim-myc, which is detected before but not after the metaphase-to-anaphase transition. Progression beyond the metaphase-to-anaphase transition was monitored by the labeling of DNA and Cyclin B, which is rapidly degraded when cells enter anaphase. These results show that the KEN-box is required and that the variant D-box (KKPLGNLDN), which is still present in Pim^kena-myc, is not sufficient for normal mitotic Pim degradation (Leismann, 2003).

Overexpression of Pim^kena-myc results in mitotic defects. Normal anaphase and telophase figures are not observed in Pim^kena-myc-positive cells that have progressed beyond the metaphase-to-anaphase transition according to the absence of anti-Cyclin-B labeling. Instead of pairs of well-separated telophase daughter nuclei, which are readily observed in Cyclin-B-negative regions in the Pim-myc control experiments, Cyclin-B-negative regions of Pim^kena-myc-expressing embryos display decondensing metaphase plates or chromatin bridges between partially separated nuclei. These abnormalities caused by Pim^kena-myc are indistinguishable from those previously observed with Pim^dba-myc, which has been shown to inhibit sister chromatid separation (Leismann, 2003).

Sister chromatid separation is also inhibited by strong overexpression of wild-type Pim-myc. By contrast, at low physiological expression levels, Pim-myc and, remarkably, also the stabilized versions Pim^dba-myc and Pim^kena-myc, can promote sister chromatid separation in pim mutants (Leismann, 2003).

To analyze the function of Pim with mutations in both D- and KEN-box, additional transgenes (g>stop>pim^kenadba and g>stop>pim^kenadba-myc) were constructed, allowing the expression of Pim^kenadba or Pim^kenadba-myc under the control of the normal pim regulatory region. To establish chromosomal insertions of these potentially detrimental transgenes, a stop cassette flanked by FLP recombinase target sites (>stop>) was inserted into the 5' untranslated region. This stop cassette was eventually excised by transmitting the established insertions via males expressing FLP recombinase specifically in spermatocytes. Expression of the paternally recombined transgenes (g>pim^kenadba and g>pim^kenadba-myc) started at the onset of zygotic expression during cycle 14 of embryogenesis. Expression of g>pim^kenadba and g>pim^kenadba-myc in pim-mutant embryos did not allow sister chromatid separation during mitosis 15. Instead of normal mitotic figures, which were readily apparent in pim⁺ sibling embryos, only decondensing metaphase plates were observed during exit from mitosis. Thus, pim-mutant embryos expressing g>pim^kenadba and g>pim^kenadba-myc display the same phenotype as pim mutants without transgene or with the non-recombined g>stop>pim^kenadba transgene (Leismann, 2003).

Control experiments with g>stop>pim transgenes encoding wild-type Pim show that expression after stop-cassette removal is sufficient to promote normal sister chromatid separation in pim mutants. Moreover, additional control experiments show that the recombined g>pim^kenadba-myc transgene is expressed as expected. Anti-myc immunoblotting clearly show expression, and co-immunoprecipitation experiments indicate that the Pim^kenadba-myc protein associates efficiently with Separase (SSE) and Three rows (THR), a Drosophila protein known to form trimeric complexes with SSE and Pim. In addition, although g>pim^kenadba-myc expression in pim⁺ sibling embryos has little effect during the initial embryonic cell divisions (mitosis 14-16), it results in a severe mutant phenotype in the CNS where additional cell divisions occur. Wild-type Pim therefore appears to protect cells from the effects of Pim^kenadba-myc but only as long as the latter has not yet accumulated to high levels (Leismann, 2003).

In summary, the experiments with g>pim^kenadba and g>pim^kenadba-myc in pim mutants show that sister chromatid separation does not occur in the presence of physiological levels of the double mutants Pim^kenadba and Pim^kenadba-myc, in contrast to the findings with the single mutants Pim^dba, Pim^dba-myc and Pim^kena-myc (Leismann, 2003).

It is concluded that mutations in either the D- or the KEN-box result in significant stabilization of Pim protein during mitosis. Neither the D- nor the KEN-box, therefore, are sufficient for normal degradation during the embryonic cell divisions in Drosophila. Similar observations have been described for human securin (Hagting, 2002; Zur, 2001). However, in contrast to Drosophila, mitotic degradation of human securin still occurs quite effectively when either only the D- or the KEN-box is intact. The D- and KEN-boxes of Drosophila Pim, therefore, might function less independently than the corresponding motifs in human securin. Eventually, the understanding of D- and KEN-box function will require structural analyses of their interactions with Fizzy/Cdc20 and Fizzy-related/Cdh1, which recruit proteins with these degradation signals to the APC/C. Fizzy and Fizzy-related are clearly both involved in Pim degradation, at least indirectly, since Pim is stabilized in both fizzy and fizzy-related mutants (Leismann, 2003).

Under the assumption that Pim^kenadba and Pim^kenadba-myc are still capable of providing the positive Pim function, these results with these stabilized mutants suggest that Pim must be degraded during each and every mitosis to allow sister chromatid separation. Although not detectable by confocal microscopy, the single mutants Pim^dba and Pim^kena might not be completely stable in mitosis. After low-level expression in pim-mutant embryos, residual mitotic degradation of single-mutant proteins might free some separase activity sufficient for sister chromatid separation. Similar results have been observed with the fission yeast securin Cut2, which is completely stabilized in a Xenopus extract destruction assay by mutations in either of the two D-boxes, and yet, low-level expression of single-but not double-mutant proteins is able to complement growth of cut2-ts strains at the restrictive temperature (Funabiki, 1997). It is emphasized that even in wild-type cells, mitotic Pim degradation appears to be far from complete, and it can be speculated that it is the Pim protein of a special pool of separase complexes that is more efficiently degraded, perhaps on kinetochores or during transport on spindles towards kinetochores. At high expression levels of Pim with or without single mutations, free excess of this securin might rapidly re-associate and inhibit the activated separase, resulting in the observed block of sister chromatid separation (Leismann, 2003).

These results also point to alternative pathways that might regulate separase activity and sister chromatid separation independently of Pim degradation. As in yeast, the success of mitosis in cells with reduced separase function is dependent on Polo kinase in Drosophila embryos. Moreover, since expression of mitotically stabilized Cyclin A versions result in a metaphase delay without inhibiting Pim degradation, Cyclin A appears to contribute independently of Pim to the inhibition of premature sister chromatid separation. Even though it remains to be analyzed whether Polo kinase and Cyclin A-Cdk1 act during Drosophila divisions as proposed for Polo homologs (Alexandru, 2001) and vertebrate Cyclin B-Cdk1 (Stemmann, 2001), these results indicate that separase and sister chromatid separation are unlikely to be regulated exclusively by securin degradation (Leismann, 2003).

Control of DNA replication and chromosome ploidy by geminin and cyclin A

Alteration of the control of DNA replication and mitosis is considered to be a major cause of genome instability. To investigate the mechanism that controls DNA replication and genome stability, RNAi was used to eliminate the Drosophila geminin from Schneider D2 (SD2) cells. Silencing of geminin by RNAi in SD2 cells leads to the cessation of mitosis and asynchronous overreplication of the genome, with cells containing single giant nuclei and partial ploidy between 4N and 8N DNA content. The effect of geminin deficiency is completely suppressed by cosilencing of Double parked (Dup), the Drosophila homologue of Cdt1, a replication factor to which geminin binds. The geminin deficiency-induced phenotype is also partially suppressed by coablation of Chk1/Grapes, indicating the involvement of Chk1/Grapes in the checkpoint control in response to overreplication. The silencing of cyclin A, but not of cyclin B, also promotes the formation of a giant nucleus and overreplication. However, in contrast to the effect of geminin knockout, cyclin A deficiency leads to the complete duplication of the genome from 4N to 8N. The silencing of geminin causes rapid downregulation of Cdt1/Dup, which may contribute to the observed partial overreplication in geminin-deficient cells. Analysis of cyclin A and geminin double knockout suggests that the effect of cyclin A deficiency is dominant over that of geminin deficiency for cell cycle arrest and overreplication. Together, these studies indicate that both cyclin A and geminin are required for the suppression of overreplication and for genome stability in Drosophila cells (Mihaylov, 2002).

Although geminin has recently been implicated in replication licensing in Xenopus egg extract, previous studies have suggested that the depletion of geminin did not cause overreplication in the Xenopus egg extract. The current data for SD2 cells clearly indicate that geminin participates in overreplication control in high eukaryotic cells. One possible explanation for these discrepancies could be that the maternal levels of free Cdt1 in Xenopus egg extract are not significantly affected by geminin depletion. However, it is possible that the control of overreplication in egg extract, which undergoes alternating S phase and mitosis, might be somewhat different from that in cultured SD2 or other somatic cells which show well-defined G1 and G2 phases (Mihaylov, 2002 and references therein).

The phenotype of geminin deficiency is intriguing. The asynchronous and partial overreplication of the genome suggests that the elimination of geminin may result in only a limited capacity for replication of the entire genome and that this replication capacity might be consumed by the replication process itself. Alternatively, geminin may have other functions that limit genome duplication in its absence. For example, geminin may affect Cdt1/Dup localization within the cell or the stability of the Cdt1/Dup protein. Geminin deficiency caused rapid downregulation of its binding partner, Cdt1/Dup. This effect appears to occur at the level of Cdt1/Dup RNA, suggesting that geminin deficiency may cause the downregulation of a factor required for Cdt1/Dup expression. It is possible that Cdt1 transcription is regulated by a checkpoint in response to overreplication. Such a possibility is supported by the observation that the cosilencing of Chk1 had a partial rescue effect on the levels of Cdt1 in geminin-deficient cells. However, these observations do not rule out the possibility that the loss of geminin also affects Cdt1/Dup protein stability or localization in the cell. A recent study suggests that Cdt1 protein, but not RNA, is regulated in a cell cycle-dependent fashion. Cdt1 protein is stable in G1 but is degraded by the ubiquitin-dependent proteolysis upon the entry of S phase. This observation is consistent with the data showing that limited Cdt1 protein is available for each S phase. Geminin knockout may release a limited amount of Cdt1, which is in complex with geminin, promoting the partial overreplication. In addition to the downregulation of Cdt1 RNA, the loss of Cdt1 is partially sensitive to MG132, an inhibitor of 26S proteasome that degrades polyubiquitinated proteins. Thus, in this study, the S phase induced by geminin deficiency may also work to destabilize the Cdt1 protein (Mihaylov, 2002).

In contrast to the effect of geminin deficiency, the silencing of cyclin A caused an initial G2 block followed by duplication of the entire genome, as judged by flow cytometry and morphology studies. This effect is similar to those seen in previous observations of the fission yeast cdc13 mutant, which encodes a mitotic cyclin. However, it is surprising that the silencing of cyclin B did not cause overreplication in SD2 cells. This analysis further suggests that cyclin A deficiency leads to the downregulation of cyclin B, but not vice versa. It has been shown that mutation of the cyclin A gene in Drosophila causes thoracic epidermis cells to skip the mitosis between S phases 16 and 17 and to undergo endoreduplication. The current results are consistent with these observations. In addition, the data unequivocally show that deficiency of cyclin A, unlike that of geminin, causes duplication of the entire genome. Furthermore, these studies indicate that in cyclin A-deficient cells, cyclin B is downregulated. The downregulation of both cyclin A and cyclin B in the cyclin A-deficient cells might explain why overreplication is not observed in the cyclin B-deficient cells, since they still contain relatively normal levels of cyclin A (Mihaylov, 2002).

The data indicate that the silencing of cyclin A induces an overreplication that is quite different from the one caused by geminin deficiency under the assay conditions. The loss of geminin causes only partial overreplication, while the silencing of cyclin A induces the full duplication of the genome. In addition, it appears that geminin deficiency induces substantial cell death while cyclin A silencing does not. The geminin deficiency-induced cell death can be rescued by Cdt1 cosilencing. These observations suggest that mechanisms for suppressing overreplication might be different for geminin and cyclin A. This notion is supported by the finding that the overreplication induced by geminin deficiency may not require the downregulation of cyclin A or cyclin B. Geminin deficiency does not appear to induce a decrease in cyclin A or cyclin B protein levels. Instead, the loss of geminin causes a marked increase in cyclin A and cyclin B protein levels. The overall kinase activity of cyclin B is slightly enhanced with the in vitro histone H1 kinase assay. Conversely, these studies show that cyclin A deficiency does cause a substantial decrease in geminin levels on days 1 and 2 after silencing. However, the downregulation of geminin is still far from complete compared with that caused by geminin knockout. Furthermore, cyclin A deficiency induces the downregulation of Cdt1 at the same time points (days 1 and 2). It thus appears that the ratio of geminin to Cdt1/Dup is not significantly altered by cyclin A deficiency. Moreover, because the silencing of cyclin A causes only G2 arrest on day 1, the downregulation of geminin in cyclin A-deficient cells does not appear to be sufficient to induce overreplication. These studies suggest that other events, independent of or in addition to the downregulation of geminin, may be required for the overreplication induced by cyclin A deficiency (Mihaylov, 2002).

Analyses of cyclin A and geminin double-knockout cells suggest that the loss of cyclin A is dominant over geminin deficiency. In these experiments, even though geminin is completely silenced, coelimination of cyclin A caused only G2 cell cycle arrest on day 1. The cosilencing of cyclin A and geminin on day 2 induced overreplication of the genome which, unlike that induced by geminin deficiency, produced a discrete 8N peak similar to that caused by cyclin A single knockout. These data suggest either that cyclin A is required for subsequent geminin-mediated replication control or that the loss of cyclin A may cause the replication to proceed in a geminin-independent mechanism (Mihaylov, 2002).

Human geminin was originally isolated during the analysis of proteins that are associated with human Chk2 protein. While this interaction appeared to be relatively weak during later verification, attempts have been made to address its potential significance for SD2 cells. In SD2 cells, Chk2 knockout did not have a significant effect on geminin deficiency-induced overreplication or the formation of giant nuclei. However, Chk1/Grapes deficiency significantly suppressed the geminin knockout phenotype, suggesting that Chk1/Grapes possesses a checkpoint function for the overreplication induced by geminin deficiency. This result is consistent with those of previous studies indicating that Chk1/Grapes regulates the DNA replication checkpoint for Drosophila. These studies have shown that interference of Drosophila nuclear division cycles 12 and 13 by X-irradiation or the DNA replication inhibitor aphidicolin activates the Chk1/Grapes signaling pathway. It has been shown that the activated Chk1 kinase phosphorylates Cdc25, promoting its complex formation with 14-3-3 and its subsequent retention in cytoplasm. Consequently, the activated Chk1/Grapes promotes the inhibitory phosphorylation of Cdc2 at threonine14 and tyrosine15 in a Cdc25/String-dependent process. In the current studies, it is likely that overreplication caused by geminin deficiency induces the Chk1/Grapes-mediated checkpoint, leading to the inhibition of the Cdc2 kinase activity and mitosis. This effect may be reflected in part by the observation that cyclin B-associated kinase activity is not dramatically induced by geminin deficiency compared to the marked increase of cyclin B protein levels in these cells. Since the loss of Chk1/Grapes or Cdt1 can either partially or completely rescue the geminin deficiency-induced phenotypes, these studies indicate that the loss of Chk1/Grapes does not suppress geminin deficiency through downregulation of Cdt1. Instead, the loss of Chk1/Grapes partially restores the Cdt1 levels in geminin-deficient cells. In mouse cells, Chk1 deficiency causes an aberrant G2/M cell cycle checkpoint during development or in response to DNA damage, causing the formation of nuclei containing highly condensed and aggregated chromatin and, consequently, massive apoptotic cell death. Although no extensive cell death was observed in Chk1/Grapes knockout SD2 cells, it is possible that Chk1/Grapes silencing allows cells to undergo aberrant mitosis, even though they are overreplicating their genome. This would produce a pseudorescue effect on the geminin deficiency-induced phenotypes. It is unclear how Chk1/Grapes rescues Cdt1/Dup expression. It is possible that Chk1/Grapes may be involved in the suppression of Cdt1/Dup transcription during overreplication. Alternatively, since Cdt1/Dup protein levels are regulated in a cell cycle-dependent fashion, being high in G1 and low in S and G2/M phases, the aberrant mitosis and possibly subsequent G1 phase induced by Chk1/Grapes deficiency may allow Cdt1/Dup to be expressed in G1. Further work is required to clarify these issues. Although no significant effect of Chk2 is seen, Chk2 may play a regulatory role for geminin under certain conditions (Mihaylov, 2002).

Genome instability is often associated with cancer. It is still not clear how these processes are linked to the alteration of DNA replication, mitosis, or G1 cell cycle regulation. The present work suggests that dysregulation of geminin/Cdt1 and cyclin A contributes to genome instability in Drosophila cells. Further studies are necessary to link alterations in the activities of geminin/Cdt1 and mitotic cyclins to human cancer (Mihaylov, 2002).

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).

Spire, an actin nucleation factor, regulates cell division during Drosophila heart development

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, this study demonstrates that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, it was determined that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. It is concluded that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis (Xu, 2012).

Proper dorsal vessel morphogenesis is critically dependent upon intercellular signaling and the regulation of gene expression. Although great progress has been made in the study of heart development, it is not known exactly how many genes and pathways are involved in this cardiogenic process or how many of these factors cooperate together. Previous genetic screens have identified genes that play roles in the specification, morphogenesis, and differentiation of the heart, including mastermind and tup. The current sensitized screen has also proved to be an efficient method to find additional factors in this process, suggesting that much remains to be learned about the molecular components involved in correct dorsal vessel formation (Xu, 2012).

Spir is required for the proper timing of cytoplasmic streaming in Drosophila, and loss of spir leads to premature microtubule-dependent fast cytoplasmic streaming during oogenesis, the loss of oocyte polarity, and female sterility. Even though it is known that spir is an important actin filament nucleation factor, the findings are the first report to describe a function of spir for cell division. Through phenotypic analysis of the spir mutant phenotype, it was found that many cardioblast nuclei are partially or completely divided. However, the cytoplasm is not divided in the absence of spir, which is consistent with the function of spir in cytoplasmic movement. Thirteen rapid nuclear division cycles without cell division initiate Drosophila embryo development, followed by three waves of cell division. The first wave of cell division occurs in the mesoderm at cell cycle 14. After this initial division, cells migrate, spread dorsally and undergo a second round of cell division at cell cycle 15. The third wave of cell division in the mesoderm occurs at the end of germband extension during cell cycle 16. There are two different types of cardioblast precursor cells: one type divides into two identical Tin-positive cardioblasts (TC), and the other type divides into one Svp-positive cardioblast (SC) and one Svp-positive pericardial cell (SPC). Based on the comparison of CycA and spir mutant phenotypes, a tentative cell division model is proposed to demonstrate spir function in determining cardiac cell fate (see A cell division model of spir function during heart development). In a wild-type background, one Svp-positive super progenitor (SSP) divides into two Svp-positive progenitors (SP), then each of these cells divides into one SPC and one SC. For Tin-positive super progenitors (TSP), after each divides into two Tin-positive progenitors (TP), each TP further divides into two identical TCs. In the current model, division from the super progenitor to progenitors takes place at cell cycle 15, and division from progenitors to full differentiated heart cells occurs at cell cycle 16. In CycA mutants, mitosis 16 is blocked such that both SPs and TPs stop cell division. This results in the two SPs assuming a myocardial fate. Thus the number of SCs remains normal, but half of the TCs are missing in the CycA mutants. The data suggest that in spir mutant embryos, most of the SPs fail to undergo full cell division at cycle 15 resulting in a SPC fate with paired nuclei. A subset of these cells are able to undergo the 15th cell division but are arrested at cycle 16 as double-nucleated cells which exhibit both Svp and Mef2 staining, characteristic of the SCs seen in the CycA mutants. Similarly, for TPs, cycle 16 was also blocked such that it resulted in two double-nucleated cells. In summary, Spir affects mitosis 16 for Tin-positive cell division and both mitosis 15 and 16 for Svp-positive cell division (Xu, 2012).

Antibody staining suggests that Spir is expressed ubiquitously before stages 12-13 and is located in both nuclei and cytoplasm. After cell cycle 16 cell division stops, occurring during stage 10-11. The expression of Spir in the cytoplasm then decreases gradually. At stage 15, the staining pattern shows mostly nucleus expression with some cytoplasmic expression and by stage 16 the nuclei become distinct indicating nucleus staining only. It is hypothesized that expression of Spir decreases in the cytoplasm but remains constant in the nuclei when cell division halts (Xu, 2012).

The genetic analysis of spir, Doc, pnr and tin suggests that these factors may regulate each other during dorsal vessel formation, and especially significant is the interaction between spir and pnr. Pnr is a GATA class transcription factor expressed in both the dorsal ectoderm and dorsal mesoderm, where it is required for cardiac cell specification. Proper dorsal vessel formation is inhibited in pnr loss-of-function embryos due to failure in the specification of the cardiac progenitors. In spir mutants, the expression pattern of Pnr remains normal. However, Spir is over-expressed in the cardiac mesoderm in pnr mutants, suggesting that Pnr may repress the expression of the spir (Xu, 2012).

In conclusion, Spir is a newly-identified factor functioning in cell division during dorsal vessel formation. Tin-, Eve- and Svp-positive heart cells are all affected in the absence of spir. Also, spir expression depends on the transcription factors Doc, tin and pnr. Genetic interaction data also show that spir cooperates with CycA in heart cell division (Xu, 2012).

Induction of endocycles represses apoptosis independently of differentiation and predisposes cells to genome instability

The endocycle is a common developmental cell cycle variation wherein cells become polyploid through repeated genome duplication without mitosis. Previous studies have show that Drosophila endocycling cells repress the apoptotic cell death response to genotoxic stress. This study investigated whether it is differentiation or endocycle remodeling that promotes apoptotic repression. It was found that when nurse and follicle cells switch into endocycles during oogenesis they repress the apoptotic response to DNA damage caused by ionizing radiation; this repression has been conserved in the genus Drosophila over 40 million years of evolution. Follicle cells defective for Notch signaling fail to switch into endocycles or differentiate and remain apoptotic competent. However, genetic ablation of mitosis by knockdown of Cyclin A or overexpression of fzr/Cdh1 induces follicle cell endocycles and represses apoptosis independently of Notch signaling and differentiation. Cells recovering from these induced endocycles regained apoptotic competence, showing that repression is reversible. Recovery from fzr/Cdh1 overexpression also results in an error-prone mitosis with amplified centrosomes and high levels of chromosome loss and fragmentation. These results reveal an unanticipated link between endocycles and the repression of apoptosis, with broader implications for how endocycles may contribute to genome instability and oncogenesis (Hassel, 2014).

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