BIOLOGICAL OVERVIEW

The Drosophila compound eye is well suited to the study of cell cycle regulation. The eye develops from a columnar epithelium called the eye imaginal disc. During the third and final larval stage in Drosophila development, a wave of differentiation sweeps across the eye disc, from posterior to anterior. The front of this wave is marked by a depression in the disc epithelium, the morphogenetic furrow (MF). Ahead of the MF, cells are unpatterned and undifferentiated; they progress through the cell cycle asynchronously. All cells become synchronized in G1 beginning just anterior to the MF, such that cells at the anterior edge of the MF are in early G1. Cells in G2 ahead of the furrow are driven through mitosis by a burst of string expression in a band of cells just anterior to the MF under the control of the patterning gene hedgehog. String drives G2 cells through mitosis and into G1 (Heberlein, 1995).

Cells proceeding through G1 are found more posterior (relative to the MF). Cells emerging from the posterior edge of the MF either become postmitotic, without entering S phase, and differentiate into neurons, or they up-regulate Cyclin E before entering a final synchronous S phase. As cells transit S and G2, Cyclin A and CycB proteins accumulate. G1 cells are prevented from entering S phase by the product of the roughex locus. In roughex null mutants, cells enter S phase precociously in early G1. By preventing early accumulation of Cyclin A, Roughex delays premature entry into S phase, one part of the cell cycle driven by Cyclin A. In addition, Cyclin E promotes down-regulation of Roughex, allowing accumulation of Cyclin A for its function in S and G2 (Thomas, 1997 and references).

The R8 neuron is likely to be the first cell fate to be established within the MF, and several lines of evidence suggest that cellular interactions play a role in restricting to one per cluster the number of R8 cells that form within the MF. Atonal acts as the proneural gene for photoreceptor neurons. Notch is required for restriction of atonal expression to a single R8 precursor. Rough functions to downregulate atonal in cells not selected for R8 fate. Boss is subsequently expressed in R8 cells and is required for the R7 photoreceptor fate (Dokucu, 1996 and Krämer, 1991).

Mutation of roughex perturbs cell fate determination. Many rux mutant clusters contain multiple boss-expressing cells. In some of these clusters, R8 cells are missing. There is also a reduction in the number of cells expressing bar and Seven-up. This may be due to errors in cell fate determination. Alternatively, the reduced number of cells expressing these markers may reflect cell death. Extensive cell death is seen in rux mutants beginning with the MF and extending to the posterior edge of the disc. In rux mutant discs, neuronal differentiation is delayed by approximately 6 hours of development (Thomas, 1994).

It is found that mutations in ras1 and Star enhance rux phenotypes. In rux mutations, the length of G1 may be reduced, making cellular interactions more sensitive to a reduction in the level of intercellular signaling molecules. A more intriguing notion is that the establishment of G1 itself may be promoted by intercellular signals. For example, Ras1 and Star may act in a signaling pathway to activate Rux function, or Rux may be part of a signaling cascade that negatively regulates cell cycle progression in the MF. MF movement may actually be driven by a signaling cascade that, among other processes, regulates cell cycle synchronization. hedgehog is thought to function as a secreted diffusible signal to induce progression of the MF (Thomas, 1994 and references).

The key to understanding the function of roughex comes from an analysis of the interactions of rux with cell cycle proteins. Rux acts as a negative regulator of Cyclin A. In screens for dominant suppressors of the roughex rough-eye phenotype, three regulators of cyclin dependent kinase (Cdk) activity were identified: cycA, string and Regulator of cyclin A1 (Rca1) (Thomas, 1994 and Dong, 1997). Because Stg activates CycA-Cdk complexes in vitro and Rca1 encodes an upstream positive regulator of cycA (Dong, 1997), a test was made as to whether rux suppresses entry in S phase by preventing ectopic activation (directly or indirectly) of a CycA-Cdk complex in the G1 domain within the MF. Consistent with this interpretation, overexpression of cycA mimics the rux mutant phenotype, showing extensive induction of S-phase cells just anterior to and within the MF. Coexpression of rux results in suppression of the ectopic S phases induced by cycA in all discs assayed. Therefore, ectopic CycA expression can drive G1 cells into S phase, and coexpression of Rux inhibits this phenotype. It is concluded that rux acts as a negative regulator of CycA. This is the first demonstration of a role for CycA in regulation of G1 or S phase in Drosophila (Thomas, 1997).

Striking defects in the level and subcellular distribution of CycA are observed in cells ectopically expressing high levels of Rux. In the eye, dividing cells lose their connection to the basal lamina and mitotic nuclei are found at the extreme apical surface of the disc epithelium. In wild type cells, CycA accumulates in the cytoplasm of cells with basally located nuclei. As nuclei rise apically on entry into mitosis, CycA localizes transiently to the nucleus and then disappears. In CycA overexpressors, no cytoplasmic CycA staining is detected in apical focal planes. Instead, CycA accumulates transiently in basally located nuclei of cells behind the MF. These cells do not show features of cells that are entering mitosis. It is thought that Rux controls CycA levels by promoting its nuclear localization and thereby its rapid degradation. Since Rux does not physically associate with CycA, exactly how Rux regulates CycA levels and subcellular distribution remains unknown (Thomas, 1997).

Rux can be shown to physically interact with CycE, but does not inhibit the kinase activity of Drosophila CycE-Cdk complexes. Also, Rux overexpression fails to inhibit CycE-induced S phase. Therefore, rux does not appear to inhibit CycE-dependent processes in vivo. Conversely, CycE inhibits Rux accumulation. Rux rapidly disappears from cycling cells at the posterior edge of the MF where the level of CycE increases. It can be shown that overexpression of CycE, but not of CycA, inhibits Rux protein accumulation. These results suggest that CycE targets Rux for destruction in cells that re-enter S phase behind the MF. Therefore, during normal eye development, accumulation of CycE in late G1 cells may down-regulate Rux protein. In turn, this facilitates the accumulation of CycA, which is required in subsequent cell cycle stages (Thomas, 1997).

The control of CycA activity in G1 cells in the MF shows striking similarity to the control of G2 cyclins during G1 in yeast. Degradation of the G2 cyclin, CLB2, in S. cerevisiae continues in early G1 prior to Start (the initiation of DNA synthesis). In addition, overexpression of CLB2 protein in G1 drives cells into S phase, and the CLB degradation pathway is inhibited by G1 cyclins in late G1 (Amon, 1994).

It is concluded that cell cycle arrest in G1 at the onset of patterning in the Drosophila eye is mediated by roughex. Rux inhibits entry into S phase by preventing accumulation of CycA protein during G1, and CycE promotes down-regulation of Rux, allowing accumulation of CycA for its function in S and G2 (Thomas, 1997). The next several years should see renewed efforts to unravel the role of CycA in the G1-S transition in Drosophila. These future analyses should reveal the pathway by which Roughex regulates CycA.

GENE STRUCTURE

Bases in 5' UTR - 85

Bases in 3' UTR - 221

PROTEIN STRUCTURE

Amino Acids - 335

Structural Domains

Roughex is novel, with no homology to any other reported protein (Thomas, 1994). The interaction of a variety of proteins, including CKIs, with cyclins is mediated by RXL motifs. Rux contains three RXL motifs, starting at positions 30, 197 and 249, that could mediate the observed interaction of Rux with cyclins (Foley, 1999).

date revised: 3 June 97  

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