dally, a Drosophila member of the glypican family of integral membrane proteoglycans, affects cell cycle progression and morphogenesis via a Cyclin A-mediated process

Hiroshi Nakato, Bethany Fox and Scott B. Selleck*

Department of Molecular and Cellular Biology, and The Arizona Cancer Center, Salmon Building, Rm 0975, 1515 N. Campbell Avenue, University of Arizona, Tucson, AZ 85724, USA

*Author for correspondence (e-mail: selleck{at}u.arizona.edu)

Accepted October 4, 2001


    SUMMARY
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 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
division abnormally delayed (dally) encodes an integral membrane proteoglycan of the glypican family that affects a number of patterning events during both embryonic and larval development. Earlier studies demonstrated that Dally regulates cellular responses to Wingless (Wg) and Decapentaplegic (Dpp) in a tissue-specific manner, consistent with its proposed role as a growth factor co-receptor. dally mutants also display cell cycle progression defects in specific sets of dividing cells in the developing optic lobe and retina. The affected cells in the retina and lamina show delays in completion of the G2-M segment of the cell cycle. We have investigated the molecular basis of dally-mediated cell division defects by examining the genetic interactions between dally and known cell cycle regulators.

Reductions in cyclin A but not cyclin B or string expression, suppress dally cell division defects in the optic lobe. cycA mutations also dominantly rescue many dally adult morphological defects including lethality, phenotypes that are unaffected by reducing cycB function. dally mutants show abnormal Cyclin A expression in the dividing cells affected, with appreciable levels of Cyclin A remaining in late prophase and metaphase, stages where Cyclin A is normally absent. Given that Dally is known to regulate the activity of secreted growth factors our findings suggest that extracellular cues influence the degradation of Cyclin A in a manner that controls cell cycle progression and ultimately, cell division patterning.

Key words: Glypican, dally, Cell division, Cyclin A


    Introduction
 Top
 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
During development cell division takes place in highly ordered patterns, producing cells at specific places and times required for normal tissue assembly. Tissues that show significant cell turnover or are capable of regeneration also depend on the precise control of cell division to maintain the tissue architecture essential for organ function. Despite dramatic advances in the elucidation of cell division control mechanisms in recent years, our understanding of the molecular mechanisms affecting cell division patterning during tissue assembly is rudimentary.

We have taken advantage of the highly ordered divisions required for normal assembly of the Drosophila visual system to identity genes required for cell division patterning during development. Previously, we described a gene required for ordered division in the eye and larval brain, division abnormally delayed (dally) (Nakato et al., 1995). dally mutants show defects in progression through G2-M for specific sets of dividing cells. dally mutations are pleiotropic, also affecting the morphogenesis of several adult tissues, including the eye, antenna, wing and genitalia.

dally encodes a member of the glypican family of integral membrane proteoglycans (Nakato et al., 1995). Glypicans bear one or more chains of the glycosaminoglycan heparan sulfate, and are attached to the cell surface via a glycosylphosphatidylinositol (GPI) linkage (Lander et al., 1996). Like the vertebrate glypicans, Dally is GPI-linked and heparan sulfate modified (Tsuda et al., 1999). Recently, a number of studies have demonstrated that Dally serves as a regulator of growth factor signaling during development, affecting the activity of both Wingless (Wg), and Decapentaplegic (Dpp) (Jackson et al., 1997; Lin and Perrimon, 1999; Tsuda et al., 1999) in a tissue-specific manner. These findings are consistent with Dally serving as a molecule that promotes the assembly of discrete signaling complexes on the cell surface.

In recent years a myriad of genetic studies have established the importance of proteoglycans and their glycosaminoglycan modifications in patterning and growth factor signaling during development (Selleck, 2000). Both the core proteins and the biosynthetic machinery required for their glycosaminoglycan modifications are critical. For example, mutations affecting a human core protein, Glypican-3 (GPC3), cause pre- and postnatal overgrowth and a number of morphological abnormalities including kidney dysplasia (Pilia et al., 1996). Mutations affecting glycosaminoglycan biosynthesis have profound effects on signaling during Drosophila development, compromising Wg, FGF, Hh and Dpp-mediated patterning (Baeg and Perrimon, 2000). Specific modifications of glycosaminoglycans are also critical for discrete patterning events, and indeed the signaling of specific growth factors (Bellaiche et al., 1998; Kamimura et al., 2001; The et al., 1999). Loss of heparan sulfate 2-O sulfotransferase, an enzyme required for a specific heparan sulfate modification, produces renal aplasia in the mouse (Bullock et al., 1998), and heparan sulfate 6-O sulfotransferase is required for the branching morphogenesis mediated by the FGFR-related protein breathless in Drosophila (Kamimura et al., 2001). While proteoglycans are known to affect both cell division and patterning during development, the molecular basis of their effects on cell cycle are not understood. We therefore examined the cell division abnormalities produced by mutations in a Drosophila glypican, Dally.

Earlier characterization of dally mutants identified two sets of morphologically similar sets of dividing cells in the eye and optic lobe that are affected by this cell surface proteoglycan. Lamina precursor cells (LPCs) are derived from the outer proliferative center of the optic lobe and go through two cell division cycles before producing lamina neurons, the synaptic targets of photoreceptors R1-6 (Selleck and Steller, 1991). The second of these divisions is triggered by photoreceptor axons arriving in the brain, while the first proceeds normally in the absence of photoreceptor ingrowth (Selleck et al., 1992). Each of these division cycles is disrupted in dally mutant third instar larvae (Nakato et al., 1995). The first division cycle is ‘delayed’, with cells failing to enter M phase on schedule. The second division does not occur, presumably because the abnormal timing of the first division disrupts the ability of photoreceptor axons to trigger the second division cycle. In the developing retina there are likewise two coordinated division cycles and the first of these shows the same G2-M progression delay in dally mutants. However, unlike the dally phenotype in LPCs, the second division cycle in the retina does occur.

dally phenotypes are the result, at least in part, of compromised Dpp signaling. In the eye, for example, dally phenotypes are dominantly enhanced by dpp mutations, and dally eye abnormalities can be rescued by increasing dpp+ expression. It is also evident that the cell cycle abnormalities found in dally mutant eye disks can be phenocopied by loss of Dpp signaling components. For example, dally mutants show a delay in the loss of Cyclin B in the first coordinated division cycle in the eye disk and mutant clones for either saxophone, schnurri or thickvein produce the same defect (Penton et al., 1997).

We have investigated the molecular basis of the cell cycle abnormalities in dally mutants by evaluating changes in the expression and function of known cell cycle regulators. We find that dally cell division phenotypes are selectively rescued by reduction in cycA function. Consistent with this genetic finding, Cyclin A protein is not lost at the appropriate step in M phase in dally mutants, suggesting that the cell cycle delay is the result of a failure to degrade Cyclin A on schedule.


    Materials and Methods
 Top
 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
For the genetic interaction experiments two independently derived null alleles of cyclin A were used, cycA3 and cycA5 (Lehner and O’Farrell, 1989). cyclin B null alleles tested were Df(2R)59AB and Df(2R)59AD (Lehner and O’Farrell, 1990). Three stg alleles were examined for their interaction with dally mutants, stgAR2, stgRXT13 (Thomas et al., 1994) and stg7B (Edgar and Datar, 1996). Two independently derived dally alleles were tested, dallyP2 and dally{Delta}P–305 (Nakato et al., 1995). TM3 Sb, TM2 Ubx, and TM6B Tb are ‘balancer’ chromosomes bearing multiple inversions that prevent the generation of any progeny with meiotic recombination between parental chromosomes.

The effect of cycA mutations on dally adult phenotypes was tested using the following cross, dallyP2TM3 Sb x dallyP2 cycA3/TM2 Ubx and comparing the phenotypes observed in dallyP2, cycA3/dally progeny with dallyP2/dallyP2 flies from control crosses reared under identical conditions (food, temperature, number of parents/vial). A similar cross with dallyP2 cycA5/TM3 Sb tested the interaction between dallyP2 and cycA5. Adults were examined and scored for the following phenotypes: rough eye, reduced antenna, incomplete wing vein V and reduced genitalia.

For the analysis of cycA effects on cell division in the larval brain, third instar larvae were obtained from the cross: dallyP2/TM6B Tb X dallyP2 cycA3 (or cycA5)/TM6B Tb. Tb is a dominant larval marker that allows the identification of dally cycA/dally larvae. The larval brains were dissected and stained with either anti-Cyclin B or anti-Cyclin A antibodies (Whitfield et al., 1990), as well as the fluorescent DNA stain, propidium iodide, according to previously published methods (Nakato et al., 1995). Each larval brain was serially sectioned by confocal microscopy in order to assess existence of two discrete domains of expression of Cyclin A or Cyclin B. Confocal analysis of these preparations was conducted using a Biorad MRC600 or Nikon confocal scanning microscope.

The analysis of genetic interactions between dally and cycB was performed by comparing cycBDf(2R)59AB/+; dally/dally with Sco/+; dally/dally progeny from the following crosses: +/+; dally/TM3 Sb (virgin females) x cycBDf(2R)59AB/Sco; dally/Sb (males). Two dally alleles were tested using this type of cross, dallyP2 and dally{Delta}P–305.

Scanning electron microscopy of adult heads was performed on an ISI DS-130 scanning electron microscope. All flies were reared on medium consisting of a mixture of instant fly food, agar and oatmeal with added yeast at 25°C as previously described (Condie and Brower, 1989; Manseau et al., 1997).


    Results
 Top
 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Suppression of dally adult phenotypes by reductions in cycA function
dally was identified in a screen for mutations affecting the patterning of cell division in the nervous system, where it disrupts G2-M progression in specific sets of dividing cells in the larval brain and eye disc (Nakato et al., 1995). Progression through G2-M phases of the cell cycle is affected by the activity of several genes, including cyclins A, B (Knoblich and Lehner, 1993; Lehner and O’Farrell, 1989; Lehner and O’Farrell, 1990; Whitfield et al., 1990) and a regulator of cyclin-dependent kinases (CDK), the phosphatase encoded by string (stg) (Edgar and O’Farrell, 1990). To better understand the molecular basis for the defects in dally mutants we conducted a series of genetic interaction experiments with mutations affecting cyclin A (cycA), cyclin B (cycB) and stg. Genetic interactions have proven to be a powerful method for establishing functional relationships between genes, including those that affect cell division control (Dong et al., 1997; Simon et al., 1991; Thomas et al., 1994).

We began by determining whether removing one functional copy of cycA or cycB influenced the adult phenotypes of animals homozygous for partial loss-of-function dally mutations. Reducing the level of cycA function had a dramatic effect on the lethality and morphological defects of dally mutants (Table 1). Flies heterozygous for either of two independently derived null alleles of cycA showed reduced lethality and reductions in the penetrance (% of animals affected) of defects in the eye, antenna, and wing (Table 1; Table 2). A more detailed analysis of eye defects in a separate experiment showed that cycA mutations reduced both the penetrance and severity of dally eye phenotypes, where cycA dally/dally mutants showed threefold fewer animals with severe defects than dally homozygotes (Fig. 1). Decreasing cycA function does not affect all dally-associated phenotypes; however, the penetrance of genitalia defects is unaltered by the level of cycA function (data not shown).


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Table 1.
 

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Table 2.
 


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Fig. 1. Eye phenotypes of dally mutants are rescued by reducing cycA function. Each dallyP2 adult was scored for eye defects and classified into ‘mild’, ‘moderate’, or ‘severe’ based on the degree of reduction in the number of ommatidia. Animals heterozygous for cycA3 showed approximately a threefold decrease in the number of animals with the severe class of defect compared with dally mutants wild-type for cycA (n=215 for dally/dally; n=187 for dally cycA/dally +).

 
These findings demonstrated that lethality, as well as several adult morphological defects associated with reductions in dally function, could be suppressed by reductions in cycA function. To test the specificity of this interaction, we examined whether reducing the function of another cyclin, cycB, could have similar effects on dally phenotypes. In contrast to our findings with cycA, removing one functional copy of cycB had no effect on dally adult phenotypes or lethality (Table 2). These findings argue that dally is selectively affecting the function of a specific cyclin, Cyclin A, and that this regulation is critical for the normal morphogenesis of the eye, wing and antenna.

Rescue of cell division abnormalities of dally mutants with reductions in cycA but not cycB or stg
dally function affects the normal cell cycle progression of a specific set of dividing cells in the larval brain, lamina precursor cells (LPCs). LPCs complete two division cycles from their origin in the outer proliferative center (OPC) before differentiating into lamina neurons, the synaptic target cells for photoreceptors R1-6 (Selleck et al., 1992). As LPCs progress through their two division cycles, they occupy more posterior positions with time, eventually differentiating into lamina neurons that are added to the anterior face of the developing lamina (Selleck et al., 1992; Selleck and Steller, 1991). The overall organization of the LPC divisions is very similar to the coordinated cell cycles observed across the morphogenetic furrow of the eye (Dong et al., 1997; Thomas et al., 1994), and indeed dally mutations affect cell cycle progression in the eye as well. The second LPC division cycle is triggered by photoreceptor axon ingrowth as processes contact LPCs and promote entry into S phase from G1 (Selleck et al., 1992).

dally mutants show defects in both division cycles, with a delay in progression through the G2-M segment of the first division cycle, and the complete absence of the second division. Cyclin B protein expression peaks in late G2 and early M, and is rapidly degraded at the metaphase-anaphase transition (Whitfield et al., 1990). Cyclin B therefore provides a cell cycle-specific marker for the two LPC division cycles and readily shows the absence of the second division cycle found in dally mutant larval brains.

We assessed the ability of cycA mutations to suppress LPC division defects by determining whether the second division cycle is restored in cycA, dally/dally double mutants. Reducing the level of cycA function had a dramatic effect on LPC division, restoring in large measure the ability of LPCs able to enter the second division cycle (Fig. 2A). To evaluate the ability of cycA mutations to rescue LPC division defects in detail we stained dally/+, dally/dally and dally cycA/dally third instar larval brains with anti-Cyclin B antibody and examined these preparations by serial section confocal microscopy. Each larval brain was scored in a double-blind fashion for the percentage of LPCs that showed the second domain of Cyclin B expression. Reducing cycA function dramatically suppressed LPC division defects, reflected in the percentage of LPCs that can proceed into the second division cycle (Fig. 2B). Detailed analysis of the serial sections for each larval brain revealed that the restoration of Cyclin B expression in the second LPC division of dally cycA/dally animals occurred in a spatially and temporally precise manner. These findings document that reductions in cycA restore the normal patterning of LPC divisions in dally mutants. A second independently derived cycA allele, cycA5, showed a similar effect on LPC divisions in dally mutants (data not shown).



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Fig. 2. Cell division defects in LPCs of dally mutants are rescued by reducing cyclin A expression. (A) Lateral views of brain lobes from dallyP2/TM6B (top left), dallyP2/dallyP2 (top right) and dallyP2 cycA3/dallyP2 (bottom left). cycB/+; dallyP2/dallyP2 (bottom right) third instar larvae are shown stained with anti-cyclin B antibody. The two LPC divisions are seen as two stripes of immuno-positive cells (marked 1 and 2) at the anterior edge of the lamina (LA). Note that the second domain of Cyclin B-positive cells, absent in dallyP2/dallyP2, is observed in the dallyP2 cycA3/dallyP2 preparation. We confirmed that cycB/+ animals show reduced levels but normal patterns of Cyclin B protein expression (data not shown). (B) Quantitative analysis of LPC division rescue by cycA. Third instar larval brains were stained with anti-Cyclin B antibody and scored for the percentage of LPCs showing the second stripe of Cyclin B expression. Each closed circle represents a single larval brain serially sectioned by confocal microscopy, and scored without knowledge of the genotype. cycA5 showed similar effects to cycA3 on expression of LPC division phenotype in dally mutants (data not shown).

 
Cyclins A and B, and the regulatory phosphatase Stg, all promote entry into mitosis from G2 (Edgar and O’Farrell, 1990; Knoblich and Lehner, 1993; Kumagai and Dunphy, 1991; Lehner and O’Farrell, 1990; Whitfield et al., 1990). If the rescue of dally cell cycle defects by reductions in cycA function is a consequence of a general decrease in signals promoting G2-M progression, we would expect mutations in either stg or cycB to similarly suppress LPC division abnormalities. We therefore determined whether null mutations in either cycB or stg could affect LPC division in dally mutants. Reducing the function of cycB had no effect on LPC divisions of dally mutants visualized with anti-cyclin B antibody staining (Fig. 2A). The first LPC division cycle remains and no recovery of the second division cycle is apparent.

For the analysis of stg dally/dally mutants, we used an anti-cyclin A antibody. Cyclin A expression in LPCs, such as that of Cyclin B, reflects the two consecutive division cycles normally present (Fig. 3). The normal pattern of Cyclin A expression is observed in dally heterozygotes, but larvae homozygous for dally show only the first domain of Cyclin A expression, indicative of the failure of the second division cycle (Fig. 3). In contrast to the effects of cycA mutations on LPC division, reductions of stg function did not rescue the second division cycle (Fig. 3). In fact, the domain of Cyclin A expression in the first LPC division is expanded, suggesting that a reduction of stg expression prolongs the already abnormal G2-M segment of the first LPC division.



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Fig. 3. Reducing string expression does not rescue dally LPC division phenotypes. Anti-cyclin A staining patterns of LPCs and lamina (LA) in dallyP2/TM6B (A), dallyP2/dallyP2 (B) and dallyP2 stg7B/dallyP2 (C) larval brains. Deleting one functional copy of stg does not rescue the second LPC division, and appears to expand the first G2/M domain of LPC division in dally mutants.

 
dally mutants show defects in Cyclin A expression
The rescue of cell division defects in dally mutants by reductions in cycA expression suggested that Cyclin A activity or expression is abnormally elevated in dally mutants. We therefore analyzed the cell cycle-dependent changes in Cyclin A expression in detail. The sudden proteolytic destruction of Cyclin A following entry into metaphase of the first LPC division can be appreciated as patches of cells completely devoid of anti-Cyclin A staining (Fig. 4). This pattern of Cyclin A loss in the first division cycle of LPCs is not found in dally mutant brains. Indeed, cells with highly condensed chromosomes, indicative of entry into M phase, retain significant levels of Cyclin A expression in LPCs of dally mutants.



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Fig. 4. Abnormal expression of Cyclin A in larval brains of dally mutants. Lateral views of LPCs and lamina (LA) of dally heterozygous (dallyP2/TM6B, A-C) or homozygous (dallyP2/dallyP2, D-F) larval brains stained with anti-cyclin A antibody (red, A,D) and propidium iodide (gray tones, B,E). C and F show merged images of A and B, and D and E, respectively. The numbers 1 and 2 in A mark the two consecutive LPC divisions that occur in front of and immediately posterior to the lamina furrow in a wild-type brain, respectively. The arrow marks a dividing cell in the first LPC division where loss of Cyclin A immunoreactivity is associated with entry into M phase, as shown by the condensed state of the chromosomes (A,B). The second LPC division cycle is completely absent in the dally mutant brain lobe, as shown by the lack of Cyclin A expression posterior to the lamina furrow. Arrows in D-F indicate cells in dally mutant brains that show condensed chromosomes, yet retain high levels of Cyclin A immunoreactivity.

 
Earlier reports demonstrated that in ventral ganglion neuroblasts, Cyclin A protein is degraded before entry into metaphase and prior to the loss of Cyclin B (Whitfield et al., 1990). This timing of Cyclin A loss is also found in wild-type or dally/+ LPCs. Using propidium iodide staining of chromosomes to monitor their state of condensation and position, it was possible to show that in wild-type LPCs Cyclin A protein is degraded prior to the alignment of chromosomes on the metaphase plate (Fig. 5). By contrast, LPCs of dally mutants retain significant levels of Cyclin A even after the chromosomes have condensed and assembled on the metaphase plate. Approximately 80% of metaphase cells (26 out of 32 cells observed) from dally mutant LPCs showed abnormally high levels of Cyclin A protein, while anti-Cyclin A signal was lost in at least 85% of wild-type metaphase cells (14 out of 16 cells). This prolonged and abnormal expression of Cyclin A could well disrupt the normal exit from mitosis, which requires Cyclin A destruction (Jacobs et al., 2001; Kaspar et al., 2001; Parry and O’Farrell, 2001; Sigrist et al., 1995).



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Fig. 5. Pattern of Cyclin A expression in wild-type and dally LPCs. A series of wild-type and dally mutant LPCS in M phase are shown, stained with anti-Cyclin A antibody (red) and propidium iodide (gray scale) to visualize the condensation state of chromosomes. Left panels show Cyclin A staining, middle panels show propidium iodide staining of chromosomes, and the right panels are merges of Cyclin A and propodium iodide images. The top six panels show wild-type LPCs. The arrowheads mark cells where Cyclin A has been degraded and propidium iodide staining shows the cells are in prophase, where chromosome condensation has begun, but not yet achieved the alignment indicative of metaphase. The arrow marks a cell in prophase that retains some residual level of Cyclin A immunoreactivity. dally mutant LPCs show a very different pattern of Cyclin A immunoreactivity in M phase. Shown are several LPCs (arrows) with significant levels of Cyclin A and highly condensed chromosomes characteristic of metaphase. In wild-type Cyclin A immunoreactivity is never seen in LPCs with this degree of chromosome condensation.

 

    Discussion
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 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
dally phenotypes are rescued by reductions in cycA function
dally was identified in a screen for genes affecting cell division patterning during development of the nervous system (Nakato et al., 1995). dally mutants show a very specific type of cell cycle defect in two groups of dividing cells in the developing eye disc and larval brain. In both cases, dally affects the first of two coordinated divisions that precede the differentiation of cells and their assembly into the mature eye and lamina, respectively. The cell cycle defect is seen as a prolonged expression of the mitotic cyclin, Cyclin B, and a delay of the exit from mitosis into the subsequent G1. These defects suggested that dally influences the activity of cyclins, molecules known to regulate cell cycle progression. We therefore explored the interaction between dally and the mitotic cyclins, Cyclin A and B, and the regulatory phosphatase encoded by stg.

We found that the adult phenotypes in the eye, wing and antenna, as well as the lethality of dally mutants, are all rescued selectively by reducing the expression of cycA. This rather surprising finding suggests that either the primary cause of morphological abnormalities in dally mutants is a defect in cell cycle regulation, or cycA plays a role in events outside of cell cycle control. There is circumstantial evidence that cyclins and Cdks function in non-dividing cells. Both G1 and mitotic cyclins are expressed in differentiated cells and proposed functions for cyclins outside of the cell cycle include stabilizing the differentiated state, coordinating metabolism of differentiated cells with extracellular cues, and providing components of an apoptotic pathway that can be activated in post-mitotic cells (Gao and Zalenka, 1997).

We do have evidence that dally participates in differentiation processes during imaginal disc development. dally serves as a component of the Dpp signaling apparatus in some tissues, and dally mutants show reduced activation of Dpp target genes, spalt and optomotorblind in imaginal discs (Jackson et al., 1997). It is surprising that the morphological defects in adult tissues, such as the eye and antenna where dally clearly participates in Dpp signaling events, are rescued by reducing cycA function. Perhaps there is a wider role for cell division regulation in controlling differentiation than is readily apparent. The normal assembly of the wing margin is certainly one example of where Wg and Notch affect cell cycle progression as part of the morphogenesis program (Johnston and Edgar, 1998). Inhibition of cell division is critical for normal gastrulation and is mediated by tribbles, an inhibitor of stg function (Grosshans and Wieschaus, 2000; Mata et al., 2000; Seher and Leptin, 2000). In addition, known regulators of cell division and growth can affect patterning (Blaumueller and Mlodzik, 2000; Boedigheimer et al., 1997; McCartney et al., 2000). Altering the length of the cell cycle can have dramatic effects on gene expression and morphogenesis in the chick limb (Ohsugi et al., 1997). In contrast to these findings, there is evidence from the analysis of animals mosaic for cdc2 that overall patterning is unaffected in the Drosophila wing disc when cell division does not take place (Weigmann et al., 1997). The role of cell division cycle control in differentiation remains poorly understood but it is intriguing that dally, a gene with clear cell cycle effects and a role in growth factor signaling, influences morphogenesis via a Cyclin A-mediated process.

dally mutations show a completely penetrant cell division defect in lamina precursor cells. All LPCs along the dorsal-ventral axis go through two synchronous cell divisions, reflected as two bands of Cyclin B expression at the surface of the larval brain. In dally mutants the first division cycle is delayed along the G2-M transition, and the second division does not take place at all, with the loss of the corresponding domain of Cyclin B. We used this patterning of Cyclin B expression to evaluate the ability of cycA to affect the cell division abnormalities in dally mutants. Larvae heterozygous for two different cycA null mutations showed a remarkable restoration of the second division cycle in independently derived dally mutants. This result shows that there is a functional link between dally and events regulated by Cyclin A in LPCs.

If the rescue of dally cell cycle defects is strictly a function of reducing signals promoting G2-M progression, we would expect that mutations in other genes known to drive cells into M phase would similarly suppress dally mutant phenotypes. This is not the case. Reducing the levels of stg, the phosphatase regulator of several mitotic cyclin-Cdk complexes, or cycB does not suppress dally cell division defects. Likewise, reductions in cycB activity had no effects on any of the adult phenotypes of dally mutants, providing further evidence that dally specifically affects Cyclin A function.

The suppression of dally-associated cell division defects by reducing cycA expression suggested that dally normally serves to reduce Cyclin A levels. We tested this by examining the pattern of Cyclin A expression in dally mutants. Indeed, the normal patterning of Cyclin A expression is disrupted, the sudden loss of the protein normally associated with entry into mitosis does not occur. dally mutants also show cells with high levels of Cyclin A beyond the phase of the cell cycle where it is normally degraded. These findings provide an explanation for the rescue of dally mutants by removing one functional cycA gene; Cyclin A is inappropriately elevated in dally mutants.

Mechanism of Cyclin A-induced cell cycle delay
Our analysis of the interaction between dally and cycA indicates that elevated levels of Cyclin A are responsible for the cell division defects found in dally mutants. This defect is characterized by prolonged expression of the mitotic cyclin, Cyclin B, and a delayed entry into M phase, indicating that an event somewhere during the G2-M segment is disrupted. However, we do not know which exact step within the G2-M segments of the cell cycle is abnormal and if this is the only defect caused by dally mutations. How then could elevated Cyclin A delay cell cycle progression? Expression of a form of Cyclin A that cannot be degraded by the cell cycle-dependent proteolytic machinery produces a delay in metaphase (Jacobs et al., 2001; Kaspar et al., 2001; Parry and O’Farrell, 2001; Sigrist et al., 1995). Perhaps elevated levels of Cyclin A found in dally mutants produce a delay in exit from mitosis. It is also possible that disruption of cycA function in G2 could affect other events during G2-M progression in dally mutants.

We have now established several functional links between dally and other genes affecting cell division and morphogenesis. What is the picture that is emerging? First, in imaginal tissues, dally affects cellular responses to the TGF-ß/BMP-related growth factor, Dpp (Jackson et al., 1997). In addition, clones of cells defective for Dpp reception in the eye disc show the same cell cycle defect, in the very same division cycle that we have observed in dally mutants (Penton et al., 1997). These latter findings suggest that dally participates in the cell cycle control functions of Dpp. Now we find that the cell cycle defects of dally mutants are selectively rescued by removing one functional copy of cycA, and that dally mutants show inappropriately high levels of Cyclin A. Perhaps Dally and Dpp cooperate in signaling events that downregulate the levels of Cyclin A.

This model is supported by the activity of TGF-ß in vertebrate cells. TGF-ß signaling has been shown in several different cell types to decrease the levels of cyclin A mRNA, and recent studies indicate this regulation occurs at the transcriptional level (Djaborkhel et al., 2000; Satterwhite et al., 1994; Slingerland et al., 1994; Sugiyama et al., 1997; Yoshizumi et al., 1997). Recent studies of Drosophila myb provide further evidence for the conservation of molecular activities of TGF-ß and Dpp. TGF-ß, in addition to downregulating cyclin A mRNA, inhibits the levels of B-myb mRNA (Satterwhite et al., 1994). Analysis of temperature-sensitive mutations in D-myb show that, like cycA, D-myb promotes G2-M progression (Katzen et al., 1998). Thus, a conserved molecular cassette can be proposed: (1) TGF-ß/Dpp signaling is enhanced at the cell surface by integral membrane proteoglycans of the glypican family; and (2) TGF-ß/Dpp serves to affect cell cycle progression in part by downregulation of both Cyclin A and Myb expression. The difference between the fly and vertebrate systems could simply be the outcome of the TGF-ß/Dpp signaling. In Drosophila the output is to promote cell cycle progression, whereas in vertebrate cells the principal activity of TGF-ß is to arrest cells in G1. Developmentally regulated cell cycle arrest in G2 has now been documented extensively in Drosophila (Edgar and O’Farrell, 1990; Johnston and Edgar, 1998; Kylsten and Saint, 1997; Milan et al., 1996) and Dpp may provide an important means of relieving this cell cycle arrest and integrating cell division with differentiation.


    ACKNOWLEDGMENTS
 
We thank C. Lehner and B. Edgar for providing cycA, B, and stg mutant strains, and W. Whitfield for generously supplying us with anti-Cyclin A and B antibodies. We also wish to acknowledge Vivian Lo and Momoko Fujise for their contributions to the genetic analyses and Satomi Takeo for figure assembly. This work is supported by NIH RO1 GM54832 to S.B.S. and by Human Frontier Science Program to H.N. and S.B.S.


    REFERENCES
 Top
 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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