The morphogenetic furrow of the developing Drosophila eye provides a model system for studying the role of cell cycle genes and their control of cell proliferation and differentiation. A depression in the apical surface of the eye imaginal disc epithelium of the third instar larva, the morphogenetic furrow moves from the posterior region of the disc to the anterior. The passing of the furrow results in cell differentiation and in the induction of synchronous cell proliferation marked by the orderly expression of cell cycle genes.
Prior to the passing of the furrow there are many S phase and mitotic cells expressing Cyclin A and Cyclin B. Initially, a domain of string expression occurs, prior to the passing of the furrow, found in a narrow band of cell roughly 5- to 6-cell diameters wide. Cyclin D expression reaches high levels immediately after the induction of string. The string domain represents a transition point: S phase ceases at its anterior boundary, expression of Cyclin A and Cyclin B terminates, and an increase in cells in mitosis is seen within the string domain (Thomas 1994). Significantly, the band of Cyclin D expression occurs before cells reenter into the cell cycle. Cyclin E is expressed posterior to the band of Cyclin D expression, in a region partially overlapping the synchronous band of S-phase cells (Thomas 1994).
What is the function of string and Cyclin D expression ahead of the developing furrow? The expression of string is accompanied by an increase in the number of cells in mitosis as would be expected if the function of string were to drive cells in G2 into a round of mitosis. This increase in density of mitotic activity represents a two fold increase over the number seen in the unpatterned region anterior to the furrow (Thomas, 1994).
Cells in G1 are inhibited from entering S phase. Thus some aspect of cell cycle regulation inhibits passage into S for a certain period of time. The occasional S phase cells seen overlapping the anterior edge of STG mRNA suggests that cells already in S phase complete it before progressing into G2. One gene involved in regulation of the G1 phase is roughex. Mutations in roughex cause cells to circumvent G1, and all cells enter S phase, including cells that would normally differentiate. This leads to defects in early steps of pattern formation and cell fate determination.
The G1 period is accompanied by cell shape changes that define the furrow and marks the beginning of cluster differentiation. In the midst of the furrow, cellular interactions play a role in restricting the number of R8 cells that form within the morphogenetic furrow to one per cluster. At the posterior edge of the morphogenetic furrow, cells are formed into regularly spaced preclusters that contain postmitotic precursors serving as the first five photoreceptor cells to differentiate, R8 and R2-5. Following an extended G1 period, marked by Cyclin D and Cyclin E expression and the passing of the furrow, the remaining cells pass synchronously into S phase (following the passage of the furrow) and then into G2 and M, accompanied by the expression and degradation of Cyclins A and B. The expression of Cyclin D and E prior to the passing of the furrow set the stage for the cycle of synchronous division in the cells that do not immediately differentiate. Paradoxically, although all cells passing through the furrow express Cyclin D and Cyclin E, only a fraction of those cells undergo the synchronized division that follows the passing of the furrow (Thomas, 1994 and Finley, 1996).
The three mammalian D-type cyclins are thought to promote progression through the G1 phase of the cell cycle as regulatory subunits of cyclin-dependent kinase 4 and 6. In addition, they have been proposed to control the activity of various transcription factors without a partner kinase. This study describes phenotypic consequences of null mutations in Cyclin D, the single D-type cyclin gene in Drosophila. As previously observed with null mutations in the single Drosophila Cdk4 gene, these mutations do not primarily affect progression through the G1 phase. Moreover, the apparently indistinguishable phenotypes of double (CycD and Cdk4) and single mutants (CycD or Cdk4) argue against major independent functions of Cyclin D and Cdk4. The reduced cellular and organismal growth rates observed in both mutants indicate that Cyclin D-Cdk4 acts as a growth driver (Emmerich, 2004).
D-type cyclin-cdk complexes are of crucial importance in human tumorigenesis. Since these complexes have been conserved in evolution, it is readily possible to use model organisms like D. melanogaster for functional characterizations. This study extends previous characterization of Drosophila Cdk4 mutants by phenotypic comparisons with CycD mutants. As observed for Cdk4, Cyclin D is not required for progression through the G1 phase of the cell cycle. Some escapers develop to the adult stage even when both maternal and zygotic Cdk4+ or CycD+ function is abolished. Moreover, FACS analyses demonstrate that the cell-cycle profile of wing-imaginal disc cells homozygous for null mutations in Cdk4 or CycD is essentially indistinguishable from that of wild type. The evidence therefore is not consistent with the prevailing idea that D-type cyclin-cdk complexes primarily regulate progression through the G1 phase. In cultured mammalian cells, where the most support for this suggestion has accumulated, D-type cyclin-cdk complexes have been shown to act in part by titrating CIP/KIP inhibitors away from CycE/Cdk2 complexes, which are thus freed to stimulate cell-cycle progression. In contrast, binding of Dacapo, the single known Drosophila CIP/KIP family member, to Drosophila Cyclin D-Cdk4 has not been detectable. This provides a potential explanation for the apparent discrepancy. It should be noted, however, that the strong genetic interactions reported among CycD, CycE, and Cdk2, previous interaction tests performed with Rbf indicate that Drosophila CyclinD-Cdk4 complexes do play a significant, if redundant, activating role in the E2F/RBF network, just as described in mammals. In the CycD and Cdk4 mutants, CycE/Cdk2 complexes are presumably sufficient to perform this function (Emmerich, 2004).
While not revealing a specific role during G1, the Drosophila mutant phenotypes provide compelling evidence that Cyclin D-Cdk4 promotes cellular growth and thereby accelerates progression through all the cell-cycle phases proportionally. CycD and Cdk4 mutants develop into small but normally proportioned flies with an average weight of ~20% less than that of wild-type siblings. Conversely, overexpression of Cyclin D and Cdk4 has the opposite effect, causing increased growth in organs such as the eye, wing, and salivary glands. Moreover, growth regulation by Cyclin D and Cdk4 is also clearly apparent at the cellular level. Clones of wing-imaginal disc cells either lacking one of the Cyclin D-Cdk4 complex partners or overexpressing the complex grow slower or faster, respectively, than wild-type clones (Emmerich, 2004).
In Drosophila, the growth-promoting function of Cyclin D-Cdk4 can be interrogated using genetic approaches. Initial results have so far argued that Cyclin D-Cdk4 is not part of one of the other pathways (insulin/TOR, ras, myc, bantam), which are known to control cellular and organismal growth rates. However, the Hif-1 prolyl hydroxylase has been identified as a key growth stimulator downstream of Cyclin D-Cdk4. This raises the possibility that Cyclin D-Cdk4 is interconnected with metabolic pathways sensitive to oxygen levels. Future analyses might therefore reveal whether an involvement in oxygen-related metabolism represents the evolutionary conserved role of Cyclin D-Cdk4 in multicellular eukaryotes and throw a new light on its significance in human tumors, where oxygen limitation is a known and crucial challenge. Cyclin D-Cdk4 has also recently been implicated in the JAK-STAT pathway by an independent genetic approach in Drosophila (Emmerich, 2004 and references therein).
This comparison of CycD and Cdk4 mutant phenotypes is also of interest with regard to functions provided by these proteins independently. In particular, D-type cyclins have been proposed to regulate a number of transcription factors without a partner kinase. Moreover, overexpression of UAS-CycD alone or UAS-Cdk4 alone does often have phenotypic consequences that vary in extent with different GAL4 driver lines. For instance, ey-GAL4-driven UAS-CycD expression suppresses the inhibitory effects of simultaneous UAS-RBF1 expression dramatically. Similarly, da-GAL4-driven UAS-CycD expression during development of otherwise wild-type flies results in an increased adult fly weight. However, in these experiments in Drosophila, the overexpressed Cyclin D might execute its effect in combination with excess Cdk4 expressed from the endogenous Cdk4 gene, as suggested by the finding that da-GAL4-driven UAS-CycD expression in Cdk4 mutants increases adult fly weight at most marginally. The findings that loss of CycD+ or Cdk4+ function, as well as simultaneous loss of both CycD+ and Cdk4+ function, results in essentially indistinguishable phenotypes and does not necessarily prevent development to the adult stage, demonstrate that neither Cyclin D nor Cdk4 provides essential functions in Drosophila independently of each other (Emmerich, 2004).
Drosophila Cyclin D shows strongest similarity to cyclins in a 135 amino acid region, extending from residue 157 to residue 292. This region contains matches to the 20 most conserved residues of most cyclins. The first 97 amino acids contain PEST sequences involved in rapid protein turnover. Drosophila Cyclin D is 39% identical to human Cyclin D2 but is only 18 to 24% identical to the other classes of cyclins identified either in humans or Drosophila. The consensus Cyclin D sequence consists of 65 residues, of which 24 are unique to D cyclins (Finley, 1996).