Wayne Rickoll

Updated: September 2004

Abstract
The cell shape changes and cell movements of development are consequences of forces generated by sub-cellular structures, such as microfilaments and microtubules. I plan to identify the structures and the mechanisms involved in producing such forces. Genetic approaches in Drosophila will be coupled with precision UV-laser microsurgery and disruption of specific sub-cellular structures by drug treatment. Experimentally and genetically perturbed embryos will be monitored by high-resolution imaging of living embryos, and then analyzed by high-resolution transmission electron microscopy. These studies will help us understand the forces that direct epidermal reshaping during dorsal closure in Drosophila and during epidermal wound healing in humans.

Research interests
I complement and strengthen my efforts in teaching biology by doing research in the area of developmental biology. My research focuses on the process of morphogenesis. During morphogenesis the internal and external forms of an organism are produced. In multicellular organisms, morphogenesis is accomplished by complex changes in cell shape, cell migration, and/or cell rearrangements. I am interested in these cellular events that cause morphogenesis.

Dorsal closure is a morphogenetic process currently of great interest in developmental biology. This process occurs late in Drosophila embryogenesis when laterally located epidermal cells extend upward, without any cell proliferation or recruitment, until they fuse together at the dorsal midline to fully enclose the embryonic gut. Dorsal closure, which requires cytoskeletal-dependent epidermal cell extension, is currently being used as a model system to study wound healing in humans.

I have studied dorsal closure in Drosophila in collaboration with Dan Kiehart at Duke since 1993. So far our collaboration has produced the intellectual background and materials for a number of senior thesis students. Dissemination of our collaborative work has occurred in one co-authored review chapter (1), one co-authored paper (2), and two poster presentations (one with student co-author) and one invited platform presentation (with one student co-author) at National Drosophila Conferences. The study Dan, I and others recently published in the Journal of Cell Biology (2) demonstrated that Drosophila epidermal cells undergoing dorsal closure rapidly recover from laser wounds, establishing this as a valid experimental system for examining the cellular and molecular processes involved in human wound healing.

Working on ultrastructural changes during dorsal closure has been one of the most exciting and motivating projects I have ever done. Usually, the Drosophila embryo is very reluctant in allowing a researcher to morphologically visualize structures that seem very likely to be causally involved in morphogenetic changes. Dorsal closure is very much an exception. By using a relatively new specimen preparation technique called high pressure rapid freezing (HPRF), I have been able to obtain incredibly provoking images of cellular and extracellular structures that immediately suggest potentially informative experimental approaches.

The paper Dan, others and I published in the Journal of Cell Biology on wound healing repair in dorsal closure generated increased broad interest in dorsal closure. In this research we used a laser ablation system set up on a scanning laser confocal microscope (scanning microscope) that allows the optical destruction of areas as small as six cells in the epidermis of Drosophila embryos. Laser ablation of cells at the leading edge of the epidermis in living embryos whose cytoskeleton is labeled with green fluorescent protein (gfp) has demonstrated that multiple forces, not just the actin-myosin cytoskeleton, are required for dorsal closure, and that wound healing does occur in the Drosophila epidermis. These results have stimulated NIH-supported work in a number of labs now using dorsal closure in Drosophila as a model system to study wound healing, as it occurs in humans. Dorsal closure is a valid model because a comparable actin-myosin network is present in both the epidermal cells of Drosophila during dorsal closure and our epidermis during wound healing.

The laser ablation system has now been modified to produce a small enough spot to ablate a single cell. Another refinement added to the system is laser beam steering, in which a specific pattern on the tissue's surface can be traced using the computer's mouse, and the tracing subsequently precisely guides the laser cuts to ensure accuracy in ablation. Finally, genetic stocks of Drosophila have been constructed that permit labeling of a number of different proteins located within cells, such as actin or microtubules, with as many as three differently colored fluorescent labels--a yellow fluorescent protein (yfp), a cyan fluorescent protein (cfp) as well as the original gfp. One can also produce stocks in which specific tissues of the embryo we have shown to be critical in dorsal closure, for example amnioserosa and epidermis, express different fluorescent proteins, and thus are "color-coded" (3).

We have started to assay the wound healing process using both scanning microscopy and transmission electron microscopy (TEM). Live embryos, in which very specific cells have been ablated, are analyzed with time-lapse scanning microscopy, and at points during the wound healing process, we fix embryos using HPRF for the best possible preservation (4). The sub-cellular changes that occur during the wound healing process are evaluated by transmission electron microscopy.

This past summer, I spent a month at Duke using HPRF to prepare wounded embryos for TEM analysis here. We also prepared embryos mutant in six different genes, in which the mutant phenotype is defective dorsal closure. All material we have preserved can be sectioned and studied using the equipment we have here in our Electron Microscopy lab.

We plan to also investigate pharmaceutical perturbation of the wound healing response using drugs that specifically inhibit protein synthesis and actin and microtubule function, in order to determine the roles of these components in wound healing. The effects of these drugs on sub-cellular structures will again be monitored by both scanning microscopy, and HPRF followed by TEM analysis. In such experiments, we will be able to ask and answer questions such as is protein synthesis required for the timely and correct reestablishment of the actin-myosin cytoskeleton following ablation and subsequent wound healing? We have hypothesized that signaling via microtubules may be an essential first step in the establishment of the actin-myosin network. Thus another question of great interest to us is whether inhibiting microtubules early in the process will affect the establishment of the actin-myosin network? The only way we can ask and answer such questions is by using the experimental approaches and instrumentation described above. The only lab in the world that has a laser ablation system set up on a scanning microscope and has direct and immediate access to HPRF instrumentation is Dan Kiehart's lab at Duke. All experimental material prepared in using this unique and highly technical equipment will be sectioned and studied using the equipment we have here in our Electron Microscopy lab.

References

1. Kiehart, D.P. , R.A. Montague, W.L. Rickoll, D. Foard, and G.H. Thomas. (1994). High-resolution microscopic methods for the analysis of cellular movements in Drosophila embryos. In: Methods in Cell Biology, edited by R. Goldstein and R. Storti, pp. 507-532.

2. Kiehart, D.P., C.G. Galbraith, K.A. Edwards, W.L. Rickoll, and R.A. Montague. (2000) Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila. J Cell Biol. 149(2): 471-90.

3. Hutson, M. S. ,Y. Tokutake, M.-S. Chang, J. W. Bloor, S. Vanakides, D. P. Kiehart and G. S. Edwards. (2003) Laser dissection and quantitative analysis of dorsal closure in Drosophila morphogenesis. Science 300: 145-149.

4. McDonald, K., D.J. Sharp and W.L. Rickoll. (2000) Electron Microscope Techniques for Drosophila. In: Drosophila Protocols. Cold Spring Harbor Laboratory Press, Eds. W. Sullivan, M. Ashburner, and R.S.