Dr. Alisa L. Katzen
- PhD, University of California, San Francisco
- Basil O'Connor Starter Scholar Research Award
Genetic analysis of cell cycle regulation during development; the role of a proto-oncogene.
During organismal development, control of the cell cycle is complex and is a critical component for determining whether a cell should divide, terminally differentiate, or die. Because it provides a powerful genetic and developmental system in which to dissect cellular and biochemical processes, Drosophila melanogaster is an attractive model system for studying the highly conserved biochemical pathways that regulate cellular division and differentiation during development.As a tool for understanding these processes, we are studying the function of Drosophila myb (Dm myb), a gene related to the proto-oncogene c-myb, which is a member of a small gene family in vertebrates. c-myb is thought to normally play an important role in controlling cell proliferation, and mutant versions of the gene have been implicated in causing cancer in chickens, mice, and humans. myb genes encode sequence-specific DNA-binding proteins that regulate transcription, but a great deal remains unknown about how this activity is governed and which genes are under myb control.
Elucidating the function of Drosophila myb and uncovering the signal transduction pathway(s) in which it participates will help us to understand regulation of the cell cycle during development and provide insights into how mutant versions of the gene cause uncontrolled cell growth. Using classical genetic screens, we generated two mutant alleles of Dm myb. Each contains a single base pair substitution resulting in the change of an amino acid perfectly conserved between Dm myb and its vertebrate counterparts. Examination of mutant phenotypes showed that Dm myb is important for both embryonic and adult development and that myb serves a role in the development of many tissues. Detailed analysis of the wing phenotype revealed that mutant wings contain approximately half the number of cells in wild type, and that the defect occurs during the final cell cycle. Mutant wing cells enter S-phase but are blocked before mitosis, with some of the cells beginning to endoreplicate their DNA. These results indicate that Dm myb is required for progression through the G2/M transition and for maintenance of diploidy. Recent studies of the abdominal phenotype have revealed additional roles for Dm myb in the cell cycle. The mutant cells proliferate slowly and abnormal mitoses associated with multiple functional centrosomes, unequal chromosome segregation, formation of micronuclei, and/or failure to complete cell division are observed (see Figure 1). These findings demonstrate that in abdominal epidermal cells, Dm myb is required to sustain the appropriate rate of proliferation, to suppress formation of supernumerary centrosomes, and to maintain genomic integrity.
Figure 1. Abnormal mitoses are observed in abdominal epidermal cells that are mutant for myb.
Abdominal epidermal samples from wild type (panel A) and myb2 mutants were triply stained to visualize nuclei (blue), microtubules (green), and red (centrosomes).
We have also generated transgenic animals in which we can induce ectopic expression of wild type and mutant versions of Dm myb. We have found that ectopic expression of Dm myb within the developing animal can have potent consequences, including inducing pattern disruption and even lethality. Ectopic expression of Dm myb can have opposing effects on cell cycle regulation, promoting proliferation in diploid cells (Figure 2), but suppressing endoreduplication in polyploid tissues (Figure 3). Therefore, we conclude that DMyb functions in multiple aspects of the cell division cycle to promote proliferation and maintain the integrity of the genome. A schematic is shown in Figure 4.
Figure 2. Ectopic expression of DMyb promotes S-phase in the ZNC of larval wing discs. Wing discs that were doubly stained to visualize nuclei (left panels) and for DNA synthesis by BrdU incorporation (right panels). (A) In wild type control discs, BrdU incorporation was not detected in the zone of non-proliferating cells (ZNC), which is composed of cells at the dorsoventral boundary (see arrowheads). (B) However, when DMyb is ectopically expressed in the posterior compartment of the disc (right side of the disc), BrdU incorporation could be detected in the posterior ZNC (indicated by arrow).
Figure 3. Ectopic DMyb activity inhibits endoreduplication and growth in salivary glands. Salivary glands were stained with DAPI to visualize nuclei. Salivary glands dissected from wild type larvae (A) were considerably larger than those dissected from larvae in which an activated form of DMyb had been ectopically expressed (B). In the right panels, a representative nucleus from each salivary gland is shown at higher magnification.
Figure 4. Schematic representation of the multiple roles DMyb plays in regulating the mitotic and endoreplicative cell cycles. DMyb is a positive regulator of both progression from G1 into S-phase and G2 into mitosis, but is a negative regulator of endoreplication, indicating that it plays an important role maintaining diploidy and preserving genomic integrity.
Our laboratory continues to pursue research that will help us to: 1) gain further understanding of myb function in regulating the cell cycle and differentiation during Drosophila development; 2) identify genes that participate in the same signal transduction pathway(s) as myb through genetic approaches; and 3) address the functional relationship between Drosophila myb and its vertebrate counterparts.