cdc2
The Drosophila homologs of S. pombe cell division control (cdc) genes have been expressed in S. pombe as cDNAs from an SP6 promoter. For propagation in fission yeast, successful recovery of complementing plasmids requires adaptation of pooled plasmids from a Drosophila embryonic cDNA library. This is achieved by introducing an ars1-LEU2 DNA fragment into the vector. This library was introduced into S. pombe cdc2 and cdc25 mutants; plasmids were then isolated carrying cDNAs that complement these mutations. The gene that encodes the Drosophila cdc2 homolog maps to a single locus in the Drosophila genome at 31E on chromosome 2. It is expressed maternally to provide mRNA in syncytial embryos, and appears to be zygotically expressed in mitotically active regions of the cellularized embryo (Jimenez, 1990).
The spatial and temporal expression of CDC2 and CDC2C mRNAs are similar and are correlated with developmental periods of cell proliferation. Relatively high levels of transcripts are detected in the extremely rapid mitotic cycles preceding cellularization and during the post-blastodermal divisions following cellularization [Images]. In contrast, mRNA levels are somewhat lower during the short quiescent interval accompanying cellularization, and still much lower in late embryos, when only a few cells continue to divide. The transcripts present in early embryos are maternal. Signals become gradually restricted to the developing nervous system after germband retraction. Proliferation is restricted primarily to the nervous system at these later stages (Lehner, 1990).
Cells commit to mitosis by abruptly activating the mitotic cyclin-Cdk complexes. During Drosophila gastrulation, mitosis is associated with the transcriptional activation of cdc25(string), a phosphatase that activates Cdk1. This study demonstrated that the switch-like entry into mitosis observed in the Drosophila embryo during the 14th mitotic cycle is timed by the dynamics of Cdc25(String) accumulation. The switch operates as a short-term integrator, a property that can improve the reliable control of timing of mitosis. The switch is independent of the positive feedback between Cdk1 and Cdc25(String) and of the double negative feedback between Cdk1 and Wee1. It is proposed that the properties of the mitotic switch are established by the out-of-equilibrium properties of the covalent modification cycle controlling Cdk1 activity. Such covalent modification cycles, triggered by transcriptional expression of the activating enzymes, might be a widespread strategy to obtain reliable and switch-like control of cell decisions (Di Talia, 2012).
During Drosophila gastrulation, transcriptional activation of
string is associated with entry into mitosis. This study investigated how
String accumulation results in abrupt switch-like activation
of Cdk1 and entry into mitosis. The time interval between
String transcriptional activation and entry into mitosis is
controlled by the rate of string expression. The concentration
of String integrated over 2 min is the quantity that
best correlates with the decision of entering mitosis and such
integration time might be determined by the response time of
Cdk1 activity to changes in String concentration (Di Talia, 2012).
Two ultrasensitive steps (activation of Cdk1 by String and
entry into mitosis by Cdk1) control mitosis. Such a cascade can
provide high ultrasensitivity from two moderately ultrasensitive
steps. Positive feedback does not play an important role in the
control of entry into mitosis in the Drosophila gastrula and it is
proposed that the ultrasensitivity is rather due to the out-of-equilibrium
properties of the covalent modification cycle controlling Cdk1. Feedback mechanisms are conserved in Drosophila: it can be shown that mutants
(string9A and Wee19A) that disable feedbacks have the
expected effects on cell cycle control when overexpressed (Di Talia, 2012).
This raises the question of why feedbacks do not play a role in
WT cells. It is proposed that activation of mitosis in Drosophila is
too rapid for feedback to make a significant contribution to
Cdk1 activation. In Xenopus egg extract, positive feedback introduces
a 10 min delay between the accumulation of cyclin to
a critical threshold concentration and the activation of Cdk1. This delay has been interpreted as the time required to activate the feedback mechanism. Controlling entry into mitosis through rapid accumulation of String is, therefore,
likely to make the contribution of feedback irrelevant. When
string9A and Wee19A (Wee1) are overexpressed,
the time between String activation and mitosis can become significantly longer providing enough time for feedback to contribute to activation of Cdk1. It is speculated that the molecular network controlling entry into mitosis is highly
flexible and can operate as cyclin-driven switch dependent on feedback or as Cdc25-driven switch with the properties described in this article. These two different
strategies to control the cell cycle might reflect different
selective pressures on the control of mitosis and might be
utilized at different stages during development (Di Talia, 2012).
The control of cell division during Drosophila gastrulation
provides an extraordinary example of the temporal precision
with which cell behaviors can be timed during embryonic development. This precision might be required to avoid incompatible cell behaviors that might easily arise due to the fast timescales of Drosophila embryonic development. It is suggested that controlling entry into mitosis by transcriptional expression of string rather than accumulation of cyclins avoids the possible delay due to activation of
the positive feedback. Such a delay might be incompatible with the precise control of cell divisions observed during Drosophila gastrulation. Positive feedback
also has the potential of amplifying noise in the expression of
string and could in principle deteriorate the precision of
cdc25string transcriptional control. It is speculated that
String-driven switches similar to the one that this study has described might be a preferred solution for the precise temporal control of mitosis. Such switches, when operating as short-term integrators, have the ability to filter out the probably unavoidable fast fluctuations in the expression of string (Di Talia, 2012).
Covalent modification cycles are widespread signaling modules
that can generate ultrasensitivity when operating in the zero-order
regime. Theoretical work shows that transcriptionally driven
covalent modification cycle can effectively generate an ultrasensitive
response when they operate in the first-order regime as
long as they are not close to steady state. In this regime, these
cycles also display interesting signaling properties. They act as low-pass filters dampening fluctuations that happen on time scales faster
than the response time. Effectively, they resemble short-term integrators with an integration time that is determined by the response time (Di Talia, 2012).
Because the response time depends on the concentration of
the two opposing enzymes, the filtering properties of the cycle
can be easily tuned to the desired frequency. It is proposed that by driving the cycle with string expression, Drosophila is able to
achieve switch-like behavior while maintaining the robust
filtering properties of covalent modification cycles. Positive
feedback driven circuits that have similar integration properties
and therefore achieve both ultrasensitive and precise control
of cell decision might be much harder to design. It is speculated
that covalent modification cycles, triggered by transcriptional
expression of the activating enzymes, might be a widespread strategy to obtain reliable and switch-like control of cell decisions (Di Talia, 2012).
Embryos of most metazoans undergo rapid and synchronous cell cycles following fertilization. Using biosensors of Cdk1 and Chk1 activities, this study dissected the regulation of Cdk1 waves in the Drosophila syncytial blastoderm. Cdk1 waves were shown not to be controlled by the mitotic switch but by a double-negative feedback between Cdk1 and Chk1. S phase Cdk1 waves were shown to be fundamentally distinct and propagate as active trigger waves in an excitable medium, while mitotic Cdk1 waves propagate as passive phase waves. These findings show that in Drosophila embryos, Cdk1 positive feedback serves primarily to ensure the rapid onset of mitosis, while wave propagation is regulated by S phase events (Deneke, 2016).
The Drosophila midblastula transition (MBT), a major event in embryogenesis, remodels and slows the cell cycle. In the pre-MBT cycles, all genomic regions replicate simultaneously in rapid S phases that alternate with mitosis, skipping gap phases. At the MBT, down-regulation of Cdc25 phosphatase and the resulting inhibitory phosphorylation of the mitotic kinase Cdk1 create a G2 pause in interphase 14. However, an earlier change in interphase 14 is the prolongation of S phase. While the signals modifying S phase are unknown, the onset of late replication (where replication of constitutively heterochromatic satellite sequences is delayed), extends S-phase 14. Cdc25 mRNA was injected to bypass the developmentally programmed down-regulation of Cdc25 at the MBT. Introduction of either Cdc25 isoform (String or Twine) or enhanced Cdk1 activity triggered premature replication of late-replicating sequences, even after their specification, and thereby shortened S phase. Reciprocally, reduction of Cdk1 activity by knockdown of mitotic cyclins extended pre-MBT S phase. These findings suggest that high Cdc25 and Cdk1 contribute to the speed of the rapid, pre-MBT S phases and that down-regulation of these activities plays a broader role in MBT-associated changes than was previously suspected (Farrell, 2012 full text of article).
The experiments overrode the normal down-regulation of Cdc25 and Cdk1 activity at the MBT and showed that this developmental down-regulation is required for the introduction of late replication at S-phase 14 (see A model of how declining Cdc25 and Cdk1 activity results in S-phase 14 prolongation.). Increased Cdc25 or Cdk1 activity during cycle 14 abbreviates the late replication program normally active at the time. Conversely, it was found that decreasing Cdk1 activity during cycle 13 lengthens the rapid replication program that is active before the MBT. This suggests that Cdc25 and Cdk1 activity are regulating the length of S phase. Moreover, the pre-MBT S phases are unusual in having a rapid replication program and also in having high Cdc25 and Cdk1 activity during S phase. Then, cycle 14, when late replication begins in earnest, is the first cycle in which Cdc25 is effectively down-regulated and Cdk1 is inhibited by phosphorylation. Given these findings, it is proposed that this high Cdc25 and Cdk1 activity is actually the reason the pre-MBT S phases are rapid and the removal of these activities by down-regulation at the MBT is the developmental switch that lengthens S phase (Farrell, 2012).
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 Cdc25stg. 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 Cdk1ts, 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 Cdc25stg 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 Cdk1ts allows centriole multiplication without a deficit in daughter-centriole growth\. Another possibility is that Cdc25stg removes an inhibitor of daughter-centriole maturation. Indeed, tyrosine phosphorylated Cdc28 of S. cerevisiae inhibits spindle-pole-body duplication, and Cdc25Mih1 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 Cdk1ts 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).
Sister chromatid cohesion, mediated by the cohesin complex, is essential for faithful mitosis. Nevertheless, evidence suggests that the surveillance mechanism that governs mitotic fidelity, the spindle assembly checkpoint (SAC), is not robust enough to halt cell division when cohesion loss occurs prematurely. The mechanism behind this poor response is not properly understood. Using developing Drosophila brains, this study shows that full sister chromatid separation elicits a weak checkpoint response resulting in abnormal mitotic exit after a short delay. Quantitative live-cell imaging approaches combined with mathematical modeling indicate that weak SAC activation upon cohesion loss is caused by weak signal generation. This is further attenuated by several feedback loops in the mitotic signaling network. The study proposes that multiple feedback loops involving cyclin-dependent kinase 1 (Cdk1) gradually impair error-correction efficiency and accelerate mitotic exit upon premature loss of cohesion. These findings explain how cohesion defects may escape SAC surveillance (Mirkovic, 2017).
Mutations have been identified in the Drosophila cdc2 gene. The recessive lethality of these mutant alleles is rescued after P-element-mediated transformation with a genomic cdc2 fragment. Sequence analysis of amorphic alleles reveals non-conservative exchanges in evolutionary conserved positions. These alleles cause lethality at the larval-pupal interphase due to the absence of imaginal tissues. Embryonic lethality results when the maternal cdc2 contribution is reduced through the use of a temperature-sensitive allele. cdc2 function, therefore, is essential for cell proliferation throughout development. cdc2 function is clearly required for mitosis, but no evidence for a requirement in S-phase has been obtained. The reversible block of the mitotic proliferation observed in the peripheral nervous system of mutant embryos occurs exclusively in the G2-phase. While the mitotic proliferation of imaginal cells is blocked in the amorphic
mutant larvae, non-imaginal larval cells continued to grow and endoreplicate their DNA. The cdc2 mutant phenotype can neither be rescued with cdc2c (encoding a cdc2-like kinase) nor enhanced by a reduction of the cdc2c gene dose. These results indicate that the Cdc2- and Cdc2c-kinases control different processes (Stern, 1993).
In fission yeast, the product of the cdc2 gene is required both for entry into S phase and mitosis. Homologs of cdc2 have been isolated from a number of metazoans, but in general they have not been amenable to genetic analysis. P element transposon tagging of Cdc2 in Drosophila melanogaster and the analysis of 10 Cdc2 mutants is described. The recessive lethality of Cdc2P is associated with a P element located in the 5' untranslated region of the gene. Seven other alleles have unique single base pair substitutions in the coding region of Cdc2. One allele, Cdc2B47, is mutated in the splice donor site of exon 1. Most mutations in Cdc2, including the presumptive null allele Cdc2B47, die at the pupal stage, suggesting that the maternally supplied Cdc2 gene product drives earlier cell divisions. The phenotypes of these mutants are consistent with a role for Cdc2 in cell proliferation; however, no perturbation of the endoreduplication cycle associated with the acquisition of polyteny is observed (Clegg, 1993).
During larval development, Drosophila imaginal discs increase in size about 1000-fold and cells are instructed to acquire distinct fates as a function of their position. The secreted signaling molecules Wingless and Decapentaplegic have been implicated as sources of positional information that globally control growth and patterning. Local cell interactions play an important role in controlling cell proliferation in imaginal discs. As a first step to understanding how patterning cues influence growth, the effects of blocking cell division at different times were investigated in a spatially controlled manner by inactivation of the mitotic kinase Cdc2 in developing imaginal discs. Cell growth continues after inactivation of Cdc2, with little effect on overall patterning. The mechanisms that regulate the size of the disc therefore do not function by regulating cell
division, but appear to act primarily by regulating size in terms of physical distance or tissue volume (Weigmann, 1997).
The requirement for Drosophila cdc2 kinase has been analyzed during spermatogenesis after generating temperature-sensitive mutant lines (Dmcdc2ts) by re-constructing mutations in fission yeast cdc2+ known to result in temperature sensitivity. While meiotic spindles and metaphase plates are never formed in Dmcdc2ts mutants at high temperature, chromosomes still condense in late spermatocytes, and spermatid differentiation (sperm head and tail formation) continues. The same phenotype was 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. Confirming this notion, it is found that ectopic expression of the String/cdc25 phosphatase, which is known to activate the cdc2 kinase before mitosis, results in a partial rescue of meiotic divisions in twine mutant testis (Sigrist, 1995).
Asymmetric cell divisions can be mediated by the preferential segregation of cell-fate determinants into one of two sibling daughters. In Drosophila neural progenitors, Inscuteable, Partner of Inscuteable and Bazooka localize as an apical cortical complex at interphase, which directs the apical-basal orientation of the mitotic spindle as well as the basal/cortical localization of the cell-fate determinants Numb during mitosis. Although localization of these proteins shows dependence on the cell cycle, the involvement of cell-cycle components in asymmetric divisions has not been demonstrated. Neural progenitor asymmetric divisions require the cell-cycle regulator cdc2. By attenuating Drosophila cdc2 function without blocking mitosis, normally asymmetric progenitor divisions become defective, failing to correctly localize asymmetric components during mitosis and/or to resolve distinct sibling fates. cdc2 is not necessary for initiating apical complex formation during interphase; however, maintaining the asymmetric localization of the apical components during mitosis requires Cdc2/B-type cyclin complexes. These findings link cdc2 with asymmetric divisions, and explain why the asymmetric localization of molecules like Inscuteable show cell-cycle dependence (Tio, 2001).
The embryonic central nervous system (CNS) of Drosophila is derived from progenitors called neuroblasts (NBs). NBs undergo repeated asymmetric divisions, budding off a series of ganglion mother cells (GMCs) from their basal/lateral surfaces; GMCs can divide asymmetrically to produce progeny with distinct neuronal fates. Both the NB and GMC asymmetric divisions are mediated, in part, by a protein localization machinery that directs the preferential segregation of Prospero (Pros) or Numb to the more basally located daughter. Mitosis is driven by activation of the Cdc2 protein kinase, which, during the first 13 embryonic divisions, depends on dephosphorylation by the product of maternal string (cdc25). NB divisions occur after depletion of maternal string and depend on zygotic string. However, NBs, although arrested at G2 of cycle 14, do form in embryos lacking zygotic string. In contrast, loss of zygotic cdc2 does not substantially affect embryonic development, and lethality occurs during postembryonic development (Tio, 2001).
From a mutant screen two lines were identified that exhibit defective localization of Pros and Inscuteable (Insc) in NBs. Genetic mapping, complementation and DNA sequencing reveal that the phenotypes associated with both mutants were caused by the same mutation in cdc2, resulting in a glutamic acid to glutamine change at amino-acid 51. Embryos homozygous for cdc2E51Q show late embryonic lethality and exhibit various abnormalities that are also seen in insc and partner of inscuteable (pins) mutants, which can be explained by defects in asymmetric divisions (Tio, 2001).
To illustrate these defects, a focus was placed on the first GMC produced from NB4-2, GMC4-2a, which divides to generate two daughter neurons, RP2 and its sibling RP2sib. The Even-skipped (Eve) protein is expressed in the GMC4-2a sublineages. Eve is initially expressed in both RP2 and RP2sib; however, its expression is extinguished in RP2sib, such that late in embryonic development only one Eve+ neuron can be seen at the RP2 position in each wild-type hemineuromere. Two types of defects are seen in cdc2E51Q homozygotes. Fourteen per cent of the mutant hemisegments exhibit near the RP2 position a single Eve+ cell that has all of the characteristics of the RP2 neuron but is larger than the wild-type RP2 neuron. In cell-division mutants that prevent GMC4-2a division, GMC4-2a differentiates into an RP2-like cell that is larger than normal. More notably, 33% of the mutant hemisegments possess two cells near the RP2 position, both of which show characteristics of the RP2 neuron as judged by marker expression (Tio, 2001).
Duplication of the RP2 neuron is caused by an RP2sib -> RP2 transformation, indicating that the normally asymmetric division of GMC4-2a (GMC4-2a -> RP2 + RP2sib), has been converted to a symmetric division (GMC4-2a -> RP2 + RP2) in the mutant. This defect in asymmetric division is not restricted to the CNS; the normally asymmetric division of muscle progenitor, P15, which in wild type produces a single Eve-expressing muscle DA1 and a second daughter of unknown fate, can also become symmetric in mutant embryos, leading to the duplication of muscle DA1 (Tio, 2001).
The localization of Insc, Partner of Numb (Pon, which colocalizes with Numb) and Miranda (which colocalizes with Pros) was assessed in mutant neural progenitors which are clearly undergoing mitosis. Dividing wild-type GMC4-2a always localizes Insc as an apical crescent and Pon as a basal crescent. In dividing cdc2E51Q GMC4-2a, defective localization is observed of Insc (39%) and Pon (37), in the form of cortical distribution or misplaced crescents, and misorientation of the mitotic spindle (Tio, 2001).
These defects are not restricted to GMCs. Mislocalization of Insc (25%), Bazooka (38%), Pon and Miranda, and defective spindle orientation are also seen in mutant mitotic NBs. These data suggest that the underlying cause of the abnormal progenitor divisions may be the failure to localize the apical components during mitosis, and consequently localization of the basal determinants is also defective. Consistent with this notion, the duplicated RP2 neurons show identical nuclear size -- a phenotype characteristic of insc and pins mutants (Tio, 2001).
Embryonic lethality and defects in asymmetric division are seen in cdc2E51Q embryos but embryos totally lacking zygotic cdc2 function develop essentially normally, owing to the maternal contribution of cdc2. Several observations indicate that cdc2E51Q acts as a maternal effect dominant-negative allele that can antagonize the maternally inherited wild-type cdc2. Strong mutant phenotypes are seen in genotypically hemizygous (cdc2E51Q/deficiency) embryos only if the cdc2E51Q allele is inherited from the (cdc2E51Q/CyO) mother, and not if it comes from the father. Moreover an earlier arrest of cell divisions, resulting in a marked increase in the frequency of undivided GMC4-2a cells, is seen by overexpressing cdc2E51Q (from a uas-cdc2E51Q transgene) (Tio, 2001).
To show that defects in asymmetric cell divisions and protein localization are not peculiar to cdc2E51Q, a stock was used that is homozygous for an amorphic allele, cdc2B47, and in addition contains four copies of a transgene carrying a temperature-sensitive allele, cdc2A171T, of cdc2 (referred to as cdc2ts4X). Immunoprecipitates from cdc2ts4X embryonic extracts exhibit kinase activity that is highly temperature sensitive. Under appropriate temperature-shift conditions, these embryos can exhibit all of the phenotypes seen in cdc2E51Q including RP2 and muscle DA1 duplications, and defective protein localization in dividing progenitors (Tio, 2001).
These results suggest that the levels of Cdc2 activity determine whether and how a progenitor divides. When the level of Cdc2 is low, neural progenitors fail to divide; at intermediate levels they can divide, but mitotic NBs often fail to correctly localize asymmetric components, and GMC divisions can become symmetric with respect to segregation of cell-fate determinants and the size and fate of their daughters; only at higher levels of cdc2 do normal asymmetric divisions take place (Tio, 2001).
A binary expression system was used to express wild-type and mutated forms of cdc2 in the neural progenitors of cdc2E51Q embryos. Expression of a wild-type cdc2 transgene rescues the cdc2E51Q phenotypes, whereas the expression of enzymatically dead versions of cdc2, cdcT161A and cdc2K33R/T161A, which do not exhibit dominant-negative properties, did not rescue those phenotypes, suggesting that kinase activity is required for asymmetric divisions. Since Cdc2 kinase activity appears to be required to maintain apical Insc localization during mitosis, might it also be required for the apical localization of Insc during interphase? In embryos lacking zygotic string, NBs form but arrest at G2 during interphase of cell-cycle stage 14 and fail to enter mitosis because mitotic kinase activation does not occur. Insc, Pins and Bazooka form normal apical crescents, indicating that the initial apical localization of the apical components during interphase does not require mitotic kinase activation. Moreover, temperature upshifts can induce similar string-like phenotypes in cdc2ts4X embryos, and in these embryos the NBs arrested at G2 show apical localization of the apical components. These data suggest that Cdc2 kinase activity is required to maintain the apical complex proteins during mitosis but not for their establishment during interphase (Tio, 2001).
If Cdc2 activity is responsible for maintaining the apical components, premature inactivation/reduction of its activity -- at a point in mitosis when its activity is normally high -- should lead to premature delocalization of apical components like Insc. cdc2ts4X and wild-type control embryos were arrested at metaphase using colcemid treatment at 21°C for 30 min; half of the embryos were shifted to 31°C for 45 min, while the other half were kept at 21°C for 45 min, then both groups were fixed and stained for Insc. cdc2ts4X NBs arrested at metaphase and maintained at 21°C showed normal apical crescents of Insc (98%); similarly, colcemid-treated wild-type controls that were shifted to 31°C show 100% apical localization. cdc2ts4X NBs arrested at metaphase and upshifted to 31°C showed defective localization of Insc (only 7% have normal apical crescents) and Miranda. These results show that downregulating Cdc2 activity in NBs arrested at metaphase can cause delocalization of the apical (and basal) component proteins and provide direct evidence that elevated Cdc2 activity is required to maintain the localization of apical components during mitosis (Tio, 2001).
There are three known mitotic cyclins in Drosophila -- cyclin A, B and B3; these need to be destroyed for mitotic exit to occur. Is a subset of these cyclins preferentially required for maintaining the apical components? The temporal profile of Insc localization was followed with respect to the time of cyclin A (metaphase), cyclin B (early anaphase) and cyclin B3 (late anaphase) destruction. The results from anti-Insc/anti-cyclin-A/DNA-stain triple-labelling experiments show that Insc remains apically localized at metaphase/anaphase after destruction of cyclin A. Supporting the idea that cyclin A is dispensable, no evidence of defective Insc localization was detected in cyclin A single mutants and cyclin A;cyclin B3 or cyclin A;cyclin B double mutants. In wild-type NBs, Insc delocalization occurs after chromosome separation, coinciding with the time when cyclin B3 becomes undetectable. Single mutants in cyclin B and cyclin B3 do not show defects in Insc localization; however, in cyclin B;cyclin B3 double mutants, mislocalization of Insc can be seen in most mitotic NBs (72% for prophase; 71% for metaphase). These results indicate that whereas cyclin A appears dispensable, the B-type cyclins are required to maintain asymmetric localization of Insc during mitosis (Tio, 2001).
These data show that a key cell-cycle regulator is involved in mediating asymmetric cell divisions. Phosphorylation mediated by Cdc2 is likely to be important in maintaining the correct localization of the apical complex of asymmetry proteins during mitosis; however, Cdc2 probably does not act directly on Insc as the putative Cdc2 phosphorylation sites of Insc can be removed without affecting its function in overexpression paradigms. These observations provide an explanation for the normal temporal profile of the localization of molecules like Insc. Since long as there is Cdc2 kinase activity during mitosis, apical/cortical localization of molecules such as Insc is maintained; however, when kinase (and B-type cyclins) is destroyed towards the end of mitosis, the apical components become delocalized. These findings indicate that the tight temporal correlation of asymmetrically localized components important for mediating asymmetric divisions to the cell cycle may be because the two processes share key regulator(s) like cdc2 (Tio, 2001).
The centriole, organizer of the centrosome, duplicates by assembling a unique daughter identical to itself in overall organization and length. The centriole is a cylindrical structure composed of nine sets of microtubules and is thus predicted to have nine-fold symmetry. During duplication, a daughter lacking discrete microtubular organization first appears off the wall of the mother centriole. It increases in length perpendicularly away from the mother and terminates growth when it matches the length of the mother. How a unique daughter of the correct length and overall organization is assembled is unknown. Three types of unusual centriole configurations are observed in wing imaginal discs of Drosophila following inactivation of Cdk1. (1) Centriole triplets are observed consisting of one mother and two daughters; this suggests that centrioles have more than one potential site for the assembly of daughters. (2) Centriole triplets are observed comprising a grandmother, mother and daughter; this suggests that subsequent centriole duplication cycles do not require separation of mother and daughter centrioles. (3) Centriole pairs in which the daughter is longer than its mother have been observed. These findings suggest that regulatory events rather than rigid structural constraints dictate features of the stereotyped duplication program of centrioles (Vidwans, 2003).
Centrioles from wild-type (wt) third instar wing discs were examined. EM analysis revealed that 20% of centrioles in interphase cells were singlets, whereas the rest were pairs with immature daughters. Wing disc cells double in about 8 hours and spend approximately equal time in G1, S and G2 phases. It follows that about one-third of interphase cells from wt wing discs are in G1. In Drosophila embryos, G1 centrioles exist as singlets, daughter initiation accompanies S phase and maturation of daughter centrioles occurs in mitosis. If the timing of steps in the centriole cycle are conserved between Drosophila embryos and wing imaginal discs, then about two-thirds of interphase centriole pairs should contain immature daughters (corresponding to cells in S and G2) and the rest should be singlets. The observed preponderance of centriole pairs with immature daughters agrees with this expectation (Vidwans, 2003).
Inactivation of Cdk1 converts mitotic wing disc cells into endoreplicating cells. Consistent with this, wing imaginal discs in which Cdk1 had been inactivated (ts discs) are composed of fewer cells that are bigger and more brightly stained with a DNA stain (presumably due to suppression of mitosis and polyploidy, respectively). In contrast to naturally endoreplicating cells in which centriole duplication is arrested and the centrioles retain normal morphology, endoreplicating cells of ts discs exhibit a variety of centriole forms that suggests continued and anomalous duplication cycles. Triplet forms provide insights into the control of new daughter assembly while centriole pairs with unusually long daughters provide insights into the processes that terminate replicative growth of daughter centrioles (Vidwans, 2003).
Measurement of the mother centriole in control discs defines the average length of the mature centriole as 115 nm. The daughter centriole of a pair is generally shorter than (70 nm on the average) or occasionally comparable in length to its mother. This result is consistent with observations made in the embryos where daughter centrioles reach their mature length only upon entry into mitosis whereupon they soon separate from their mothers. In contrast, 15% of the centriole pairs from ts discs were longer than their mothers. The average daughter centriole length was observed to be 114 nm (a 63% increase over normal), while mother centriole length averaged 134 nm, a 17% increase. These data suggest that the observed aberrant centriole pairs are attributable to excess growth of the daughter and not shrinkage of the mother (Vidwans, 2003).
Centrioles duplicate precisely once in each cell cycle to generate daughters that are identical to themselves. Unlike the other replicative structure, DNA, the structure of the centriole does not provide insight into the mechanism of duplication. Rather, the structural relationships between mother and daughter centrioles highlight the mysterious features of centriole duplication. For example, while a centriole is purported to have ninefold symmetry along its long axis, it assembles only one daughter at a time. Additionally, the perpendicular arrangement of mother and daughter centrioles minimizes opportunities for structural templating; yet at the end of the duplication process, the daughter is as long as its mother. And finally, a daughter centriole fails to initiate a new centriole until it has dissociated from its mother, even thought the associated mother does not occlude the future site of centriole initiation on the daughter. The absence of an obvious structural basis for these observed 'rules' of centriole duplication suggests that these behaviors are guided by regulatory interactions rather than rigid structural constraints. If this were the case, one would expect that disruptions in the regulatory circuit might result in violations of the normal 'rules' of centriole duplication. Insight is gained into these issues when aberrant centriole structures are observed in Drosophila wing imaginal discs lacking Cdk1 (Vidwans, 2003).
One of the observed aberrant centriole morphologies consists of centriole pairs in which the daughter centriole is longer than its mother. These observations differ from previous descriptions of centriole elongation in that the extra growth appears to have occurred predominantly in the daughter centrioles. In contrast, the dramatic (20x) elongation of centrioles in Drosophila spermatogonial cells influences both mother and daughter centrioles (A. D. Tates, Cytodifferentiation during spermatogenesis in Drosophila melanogaster: an electron microscope study, PhD thesis, Rijksuniversiteit, Leiden, 1971 cited in Vidwans, 2003). Furthermore the production of a pseudocillia, which occurs in several species, involves elongation of the mother centriole. The growth of the mother centrioles by themselves or concomitantly with daughter centrioles in these cases indicates that the excess elongation is not connected with duplication (Vidwans, 2003).
While the inequity in length between mother and daughter centrioles upon inactivation of Cdk1 suggests that mis-specification of centriole length occurs during duplication, it should be noted that mother centrioles are also unusually long. The possibility exists that extra centriole growth outside the context of duplication produces these longer mothers. However, measurements of centriole length shows that daughters are more dramatically affected than mothers. This is consistent with the possibility that the long mother centrioles are secondary and represent long daughters seen in a subsequent round of duplication (Vidwans, 2003).
If daughter centriole length is structurally constrained by the mother centriole, daughter length would be at most that of the mother. The observation is emphasized that daughter centrioles within pairs can exceed the length of their mothers. Consequently, it is proposed that maximum centriole length is not a direct function of the length of the parent centriole (Vidwans, 2003).
A second type of aberrance observed consists of a mother centriole sporting two daughters simultaneously. While it is possible that these triplet centrioles are the result of reassociation of previously dissociated centrioles, it appears implausible that any random mechanism would produce the highly stereotyped and close association that is observed between the centrioles of such triplets. Therefore, it is suggested that these triplet centrioles result from two rounds of centriole initiation. It is emphasized that this observation demonstrates that more than one site on a mother centriole can be used for daughter centriole assembly in this experimental setting (Vidwans, 2003).
Drosophila centrioles at the developmental stages that were studied are unique: they are comprised of singlet microtubules and lack some of the sophisticated appendages normally associated with more 'mature' centrioles. It is possible that 'immature' Drosophila centrioles may be able to assemble non-canonical replicative structures unlike 'mature' centrioles that may be more constrained. Thus these observations might be unique to 'immature centrioles'. However, cancerous cells are often associated with aberrant centriole morphologies, which suggests that these observations might be generalized to other systems and that such anomalous centriole duplication might be one of the defects underlying the mitotic instability of cancer cells. The possible involvement in genome destabilization emphasizes the importance of understanding the basis of the regulation of daughter centriole assembly (Vidwans, 2003).
The steps between inactivation of Cdk1 and the final anomalies are unknown and could involve a complex cascade and multiple defects. Nonetheless, the observations are consistent with alteration of the centriole cycle at a single step. As described above, the two daughters in a triplet configuration differ in maturity, suggesting that they arise as a result of two duplication cycles without intervening disengagement of the products of the first cycle. Centriole pairs with excessively long daughters could also be due to continued (or renewed) replicative growth without disengagement. The simplest explanation for the observed anomalies is that coordination between disengagement and progression of duplication is lost because disengagement is defective or retarded. Alternatively, centriole maturation and duplication might lack normal constraints and occasionally get a step ahead of disengagement. In either case, it appears that disengagement is not totally blocked as centriole pairs continue to predominate (Vidwans, 2003).
Cell cycle regulators have been implicated in coordinating centriole duplication with cell cycle progression. In S. cerevisiae, mutation of Y19 of CDC28 (Cdk1) to E (cdc28-E19), a change that is presumed to mimic inhibitory phosphorylation at this position, or overproduction of the kinase phosphorylating this site, blocks duplication of the centrosome analog [the spindle pole body (SPB)] at the stage of mother-daughter separation. Importantly, the block is specific in that other aspects of the cycle continue. These results have led to the conclusion that Cdk1 dephosphorylation is required for SPB duplication. In Drosophila, maturation of daughter centrioles requires Cdc25string, a phosphatase that dephosphorylates Y-15 of Cdk1 (analogous to Y19 of Cdc28). Dephosphorylation of Cdk1 might be an activating change or Cdk1-YPO4 might impose a constraint on centriole duplication that is removed upon dephosphorylation. Two types of observation favor the interpretation that phosphorylated Cdk1 inhibits progress of the centriole cycle. (1) In S. cerevisiae the cdc28-E19 mutant has high kinase activity that is sufficient to support cell cycle events other than SPB duplication, hence the block to SPB duplication is not likely to be due to a lack of activity, but could be due to the ability of Cdc28-E19 kinase to mimic a phosphorylated form that can inhibit SPB duplication. (2) Findings in S. cerevisiae and S. pombe, as well as those reported here for Drosophila, suggest that loss of function of Cdk1 releases constraints on centriole/SPB duplication, arguing against a stimulatory role of cyclin/Cdk1. A plausible synthesis of these results suggests that cyclin/Cdk1-YPO4 inhibits progress of the SPB/centriole duplication cycle and its elimination leads to miscoordination of the duplication program. However, because the results are based on genetic tests, the connection between Cdk1 and the centriole cycle might be indirect and complex, and diverse interactions might contribute to the results in the different systems. More studies examining the role of the cyclin/Cdk1 complex in regulating progress of the centriole duplication cycle are needed (Vidwans, 2003).
In summary, evidence is provided that regulatory rather than structural constraints limit the number of daughters assembled by a centriole, and argue that the 'yardstick' that defines centriole length is independent of the mother centriole. The duplication program of the centriole remains puzzling, but there is hope that identification of regulatory contributions to this duplication will shed light on the process (Vidwans, 2003).
The role of genetic variants that affect cell size and proliferation in the determination of organ size has been investigated. Genetic mosaics of loss or gain of function were used in six different loci, which promoted smaller or larger than normal cells, associated with either smaller or larger than normal territories. These variants have autonomous effects on patterning and growth in mutant territories. However, there is no correlation between cell size or rate of proliferation on the size of the mutant territory. In addition, these mosaics show non-autonomous effects on surrounding wild-type cells, consisting always in a reduction in the number of non-mutant cells. In all mutant conditions the final size (and shape) of the wing is different from normal. The phenotypes of the same variants include higher density of chaetae in the notum. These autonomous and non-autonomous effects suggest that the control of size in the wing is the result of local cell communication defining canonic distances between cells in a positional-values landscape (Resino, 2004).
Size of insect organs is sex- and species-specific. In the
Drosophila wing, where most of the studies on size control
have been carried out, the determination of the size of
imaginal disc is disc-autonomous. Young imaginal discs
transplanted to the abdomens of adult flies grow after
several days of culture, irrespective of hormonal and
nutritional conditions, to a maximal size that corresponds
to that of mature imaginal discs. Minute mosaics and regeneration experiments reveal that
a final normal size is attained irrespective of the rate of cell proliferation. Clonal analysis of cell proliferation in wild-type wings show regional differences related to specification or differentiation, indicative of local as opposed to global control of organ size. Size
of the growing imaginal disc depends on the allocation of
postmitotic cells along the main axes of the wing in regimes
that change with developmental time. There is no indication that cell proliferation or cell allocation relates to the position of cells with respect to distances to compartments boundaries, where postulated diffusible morphogens are at maximal concentration (Resino, 2004).
If control of cell proliferation is local, the question arises as to how this is achieved. Can variations in cell size affect
the final size of the organ or its proliferation parameters?
These variations can be produced using mutations, usually
lethal in organisms, and have to be studied in genetic
mosaics. Mosaics of haploid territories (with half the cell
size of diploid cells) led to bigger territories with more
cells than diploid territories. Male wings have less and smaller cells than females, characteristics that are locally autonomous in gynandromorphs. For
mutations that affect cell size, it has to be considered that they cause different perturbations that may affect other cellular parameters in addition, such as cell viability, proliferation
rate or cell adhesion, which make difficult the interpretation
of the phenotype. Thus, the insufficient function of genes
involved in cell cycle progression, such as string (stg), cdc2 and cyclins or E2F (cycE positive regulator),
may retard the cell cycle and cause cell mortality, an
increase in cell size and smaller mosaic territories in
otherwise apparently normal sized discs. Mutant
cells in these mosaics do not differentiate properly. On the
contrary, over-expression of the same cell cycle genes (i.e.
stg, cycE, cycD-cdk4) or of their activators (i.e., E2F) in imaginal disc clones cause acceleration of their characteristic phases of the cell cycle, as well as a reduction of cell
size (except cycD-cdk4 combination) and an increase in
number of cells of the mutant territory compared with
control cells in apparently normal sized mosaic wing discs. These effects are more extreme in some genetic combinations (e.g., cycE-stg) because they
cause an acceleration of the whole cell cycle. These studies conclude that cell size
reduction/increase is 'compensated' by increment/decrement
in cell number in the mutant territory, as if the organ
would compute a global normal size, because the mutant
wing disc territories have an apparent wild-tupe size. This interpretation is biased by the
fact that those mosaics show high cell mortality. When this
is prevented with the coexpression of P35, the extra growth
of the mutant territories in discs and clones is even higher,
leading to abnormally shaped mutant territories. The over-expression of the cycD-cdk4 combination in the eye reaches the adult stage and causes larger and abnormally shaped mutant territories. These studies have not analyzed non-autonomous effects in non-mutant territories of the same discs (Resino, 2004 and references therein).
Less drastic mutant effects associated with cell viability are obtained with mutant perturbations in the signal transduction and reception of the insulin pathway. As a rule, loss of function of Drosophila Insulin Receptor (Inr), chico or Dp110 causes
reduction in both cell size and cell number of mutant
territories. This is similar to what happens in wild-tupe flies exposed to malnutrition or premature metamorphosis. This
holds for each member of the insulin receptor pathway
except for Drosophila S6 kinase (S6K), because S6K loss
of function only reduces cell size but not cell number. On the contrary, the gain of
function of genes of this pathway causes larger cells and an
increase in the number of cells of the mutant territory in
mosaics. The
loss of function of myc in diminutive mutants leads to
smaller flies, with smaller cells, in addition to poor cell
viability. Its overexpression causes
larger cells but not larger territories, suggesting that in this latter condition (but not the former)
the wing size in globally controlled by a normalizing
compensating mechanism (Resino, 2004 and references therein).
The results show a great heterogeneity
in the response of regional size to genetic perturbations that cause variations in cell size during cell proliferation. In fact, both smaller or larger than normal cell size may accompany normal, larger or smaller mutant territories. In the present paper, the effects on cell proliferation of
mutant conditions in six loci that cause smaller and larger
cell sizes have been studied. Of these, one corresponds to a new gene and five
to previously studied genes that affect cell size. They were
chosen as examples of the cell behavior variants, as
representatives of mutant effects on cell size (larger and
smaller than normal) and rate of proliferation (slower and
faster than normal). The choice was made without
considering the genetic/molecular bases of the corresponding wild-tupe alleles, in any case mechanistically far separated from the analyzed phenotype. Their
autonomous effects in mutant territories and in the mosaic
wing as a whole were studied: nonautonomous
effects were documented as well (Resino, 2004).
Adult cell size is measured by the exposed planar surface
of the cuticle cells. In principle, this may not reflect the size of the proliferating cells, when organ size is determined. However, in some of the cases examined in this study, cell dissociation
has revealed by direct estimation the larger or reduced cell
size in the proliferating wing disc cells. In others, cell size during growth is inferred by the
mutant effects on pattern formation, a process that precedes
final cell differentiation, as in the notum pattern of
microchaetae. This pattern results from the singularization
of sensory organ mother cells (SOMC) in a field of
epidermal cells through a process of lateral inhibition in
a field of proneural clusters. Thus, the final pattern reveals cell-cell interactions or communication, as observed in the form of cell projections emanating from epidermal cells. It holds for all mutant and genetic combinations examined in this study that the pattern, number
and density of chaetae are all altered in the notum (in the mutant
Dmcdc2E1-24 cells fail to differentiate chaetae). In all cases,
chaetae appear more densely spaced (separated by less
epidermal cells) associated with an increase in the total
number of chaetae. These variations to the
wild-tupe condition suggest that mutant cells have impaired
the capacity to signal among themselves to define spaced
SOMC singularization. Whether this is or is not associated
with cell size in individual cases is not known. These pattern
effects reveal abnormal cell communication between cells
during cell proliferation (Resino, 2004).
Although less easy to measure in mosaic nota, there
is a phenotypic association of variable cell size with a
reduction (in l(3)Me10, gigMe109, Dp110D945A) or an
increase (EP(3)3622, fta13, Dp110-CAAX) in notum sizes.
But there is no apparent causal relation between both
parameters of cell size and number of cells making the adult
notum. Perhaps cell viability associated with the mutation, as
in l(3)Me10 and gigMe109, may account for the observed lack
of correlation between both parameters. However, these
effects on notum size in other cases may also reflect failures
in cell-cell communication leading to more or less cell
proliferation (Resino, 2004).
The relationship between cell size and growth can be
more readily measured in the wing. The studied genetic
variants can be grouped, based on variations in these
parameters, as follows:
The autonomous effects on reduced clone size can result
from the poor viability of mutant cells (l(3)Me10 or
Dmcdc2E1-24), as shown in twin clonal analysis and cell
death monitoring. The increased clone size
of EP(3)3622, fta13 or Dp110-CAAX reflects higher than
normal cell proliferation, however there are no correlations
between cell size and clone size. Despite this lack of
correlation it holds for all mutants examined in this study that,
concerning the non-autonomous effects on growth in the
mosaic wing sector: the non-mutant cells of the sector are
always reduced in number. No cases were found in
which the reduction or increase in sector size by the
presence of mutant territories is compensated by wild-tupe
cells to obtain a normal sized sector (Resino, 2004).
The mosaic wings show, in addition to autonomous
effects within mutant sectors, non-autonomous effects in the
rest of the wing. It holds for all cases studied that wings with
entire or mosaic wing sectors show a reduction in the total
area of the wing or more in particular in non-mosaic areas
(sectors or compartments) of the wing. This
phenomenon is designated as 'positive' or 'negative' accommodation,
depending on its correlation with the size of the mutant
region. This phenomenon could be easily trivialized for
mutations that cause size reduction and 'positive accommodation'. It is
arguable that there are not enough cells in the
mutant territories to confront with normal growing cells
abutting the clone, the sector or the mutant compartment.
'Positive accommodation' could result from adjustment
between poorly growing cells and normal ones. However
this large effect hardly explain 'negative accommodation'
for the whole wing. 'Negative accommodation' occurs in
mosaic wings with mutant territories with more cells than
normal, such as EP(3)3622, fta13 or Dp110-CAAX (Resino, 2004).
Reduction in the size of non-mutant territories in mosaic
wings cannot be explained either by delay in development
(mosaic flies hatch at the same time as sib controls) or age of
clone initiation. It cannot be explained either by cell death,
because there is enough time for extraproliferation to reach
normal sized wings, since it occurs in mosaics where cell death
has been massively induced in Gal4 territories. 'Negative accommodation' is surprising because one
would expect that larger than normal mutant territories
should provide adjacent wild-tupe cells with more growth
signals (Resino, 2004).
To account for this 'negative accommodation' it is
postulated that mutant cells do not convey among themselves
and to wild-tupe cells sufficient signals necessary for them to
proliferate. These signals may depend on cell-cell communication. In the notum it has been seen that failures in cell-cell communication may account for abnormal chaetae
patterning and notum size. The same may apply to the wing
blade, although there are not enough pattern elements to
support this inference (Resino, 2004).
A model has been proposed to explain controlled cell
proliferation, based on local cell-cell signalling, as opposite
to reception of graded amounts of morphogens emanating
from compartment boundaries, such as Dpp and Hedgehog or
Wingless.
The Entelechia model (Interactive Fly editor's note: 'Entelechia' is a Greek term coined by Aristotle for the complete reality or perfection of a thing, and refers to the process of coming into being) states that cell proliferation results
from local interactions between neighboring cells. In these
interactions, cells compute positional values, presumably
expressed in the cell membrane. Positional value
discrepancies elicit cell division and readjustment of
positional values of daughter cells to those of neighboring
cells. These values differ along the two main axes of the
wing, A/P and Pr/Ds. Cell proliferation occurs within clonal
boundaries; those of compartments in the early disc and
other boundaries, such as veins, later. In these boundaries
the interchange of some type of signals help to increase
positional values at the border, eliciting cell division,
cascading down to intermediate regions with minimal
values. Cell proliferation is intercalar and driven by
differences in positional values between cells with lower
and higher values. These minimal differences may reflect
canonic efficiencies ('increments') in transduction of signals
(ligands/receptors) between neighboring cells. Cell division
ceases in the anlage when cells in the boundaries reach
maximal values and their increments, between all the cells
of a region become minimal. The anlage has then
reached the Entelechia condition of growth, characteristic of
the organ, the sex and species (Resino, 2004).
An organ such as the wing, grows co-ordinately through
compartments and clonal boundaries because maximal
positional values result from cell interactions at both sides
of the boundaries. In this respect compartments or wing
sectors are not independent units of cell proliferation. This
was first seen in bithorax-Complex (bx-C) mutants, where
either the A or P compartments of the haltere were transformed
to A or P compartments of the wing. The untransformed A or P haltere compartments contain now more cells, and the transformed ones less than a
wild-tupe A or P wing compartment. This
accommodation is explained as due to the reduced extent of the
compartment boundary between apposed mutant and nonmutant
compartments. Similar accommodation effects have
been already reported in other mutant conditions, such as mutants of the EGFR pathway in
extramacrochaetae (emc) and in
nubbin (nub). In the
latter case, the presence of proximal wing mutant territories
causes a distal reduction in growth in all the wing
compartments (Resino, 2004).
The Entelechia model helps to understand the behavior
of mosaic wings for the mutants examined in this study. In all cases,
clones or regions with smaller or larger cells and with less or
more cells than normal, cause autonomous effects on growth
in mutant territories but also a non-autonomous 'accommodation'
in the rest of the wing formed by wild-tupe cells. It should be emphasized that the effects on
proliferation between mutant and non-mutant territories are
reciprocal; the non-mutant territories rescuing proliferation
in the mutant territories and vice versa. It is hypothesized that failures in cell communication of positional values to/from
neighboring mutant or non-mutant cells affect the 'increment'
values of the model. This leads to reduced
proliferation in both genetic territories between cells
because cells cannot generate higher positional values and
thus promote intercalar proliferation. This
finding indicates that the size of territories does not depend
on distances from diffusible morphogen sources, measured
either in physical terms or in number of cells, or on other
postulated parameters such as measuring global cell mass
or wing length. How would
these global dimensions be defined, and how would they be
computed by individual cells? How would one explain that
mosaic territories separated from compartment boundaries
(or morphogen sources) can affect the growth of wild-tupe
territories far away all over the wing? It seems rather that
cell proliferation control depends on local cell interactions
(cell-cell communication) that define positional values
throughout the whole growing organ (Resino, 2004).
Stem cells must proliferate while maintaining 'stemness'; however, much remains to be learned about how factors that control the division of stem cells influence their identity. Multiple stem cell types display cell cycles with short G1 phases, thought to minimize susceptibility to differentiation factors. Drosophila female germline stem cells (GSCs) have short G1 and long G2 phases, and diet-dependent systemic factors often modulate G2. Previous studies have observed that Cyclin E (CycE), a known G1/S regulator, is atypically expressed in GSCs during G2/M; however, it has remained unclear whether CycE has cell cycle-independent roles in GSCs or whether it acts exclusively by modulating the cell cycle. In this study, CycE activity was detected during G2/M, reflecting its altered expression pattern, and it was shown that CycE and its canonical partner, Cyclin-dependent kinase 2 (Cdk2), are required not only for GSC proliferation, but also for GSC maintenance. In genetic mosaics, CycE- and Cdk2-deficient GSCs are rapidly lost from the niche, remain arrested in a G1-like state, and undergo excessive growth and incomplete differentiation. However, it was found that CycE controls GSC maintenance independently of its role in the cell cycle; GSCs harboring specific hypomorphic CycE mutations are not efficiently maintained despite normal proliferation rates. Finally, CycE-deficient GSCs have an impaired response to niche bone morphogenetic protein signals that are required for GSC self-renewal, suggesting that CycE modulates niche-GSC communication. Taken together, these results show unequivocally that the roles of CycE/Cdk2 in GSC division cycle regulation and GSC maintenance are separable, and thus potentially involve distinct sets of phosphorylation targets (Ables, 2013).
cdc2:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| References
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