Although the body plan of a developing embryo is ultimately determined by its genetic program, the proximate cause of morphogenesis is the generation and regulation of intercellular forces. Genetic approaches to development have been hugely successful, particularly in regard to the genes that determine patterns of cell-fate determination. However, additional genes then determine the mechanical consequences of cell-fate decisions. To unambiguously determine the role of these genes from mutant morphological phenotypes, it is crucial to quantitatively understand the forces underlying morphogenesis. In that spirit, this project involves experimental and computational investigations of the forces underlying the morphogenetic event of germ-band retraction in the fruit fly, Drosophila melanogaster. A working model for germ-band retraction will be experimentally challenged through laser-microsurgery and computational modeling. The working model is based on distinct roles for each of three tissues. These roles are embodied in the following hypotheses: (i) germ-band retraction is driven by spatially and temporally regulated contraction of the amnioserosa; (ii) the germ band itself responds passively to tension in the amnioserosa; and (iii) the distribution of tension in the amnioserosa is determined by contact between the amnioserosa and yolk sac. In light of these hypotheses, the specific goals of this project are:
1. To delineate the physical role of the amnioserosa in germ-band retraction (GBR), including the spatial and temporal limitations of this role. 2. To determine if there is an active component to the cell shape changes observed in the retracting germ band. 3. To quantitatively map and model the forces underlying GBR with high spatial and temporal resolution. 4. To link mutant GBR-failure phenotypes to defects in the underlying forces.
Note that these goals complement traditional genetic approaches to Drosophila embryogenesis. By focusing on a model organism for which a vast array of genetic techniques are available, the results of this research will provide much-needed leverage, enabling this and future investigations to better connect morphogenesis to the genetic program of development.
BROADER IMPACTS
Integral to success in the research goals stated above, the PI's educational plan will enable and encourage students from both physics and biology to work across the disciplinary divide. The four main thrusts of this plan are: (i) to improve the physics education of undergraduate life-science majors by implementing best practices from physics-education research; (ii) to provide interdisciplinary research opportunities for undergraduates; (iii) to recruit under-represented minorities into biophysical research through a partnership with Fisk University, a local HBCU, and (iv) to develop an interdisciplinary graduate course for physical scientists that stresses the ability to communicate complex ideas across disciplinary lines. The major broader impact of these integrated educational activities will be to strengthen interdisciplinary research, both by preparing students to work across the physics/biology interface and by increasing the appreciation of biophysics within the physics and biology mainstreams. Furthermore, the research project provides a new perspective on an exceedingly well-studied system. By defining the mechanical aspects of a major step in Drosophila embryogenesis, this research will build new intellectual infrastructure. It will enable the large community of researchers working on this problem to ask an entirely new set of questions. In addition, the software tools developed in this project (both for image processing and for analyzing the intrinsic forces using laser hole-drilling techniques) will be disseminated broadly to the Drosophila research community.
Although the body plan of a developing embryo is ultimately determined by its genetic program, the proximate cause of morphogenesis is the generation and regulation of intercellular forces. To unambiguously determine the developmental role of specific genes, it is crucial to quantitatively understand the forces underlying morphogenesis. In that spirit, this project used laser-microsurgery and computational modeling to investigate the forces underlying the morphogenetic event of germ band retraction in the fruit fly, Drosophila melanogaster. The primary research outcomes of this project were as follows: the development of laser-microsurgery as a truly quantitative tool for measuring mechanical forces in vivo – i.e. in actively developing fruit fly embryos; the development of techniques for holographic laser-microsurgery – i.e. the ability to ablate multiple locations simultaneously using a single laser pulse; and a deeper understanding of how the morphogenesis of one tissue (the germ band) can be driven by mechanical forces generated in an adjacent tissue (the amnioserosa). Our results show that the amnioserosa generates a contractile force that produces anisotropic mechanical stress in the germ band and helps this latter tissue retract and uncurl. This understanding comes from the results of laser-microsurgery: ablation of the amnioserosa halts germ band retraction; and incisions in the amnioserosa and germ band expand in a manner that maps out the anisotropic mechanical stress. To further our understanding, these laser-microsurgery results have been reproduced by computational cell-level finite element models. In addition, this project has contributed to the generation of software for high-throughput, manually assisted segmentation of confocal microscopy images (Seedwater Segmenter, http://code.google.com/p/seedwater/). The primary broader impact of this project has been the implementation of active learning strategies in Vanderbilt University’s introductory physics course for life science majors. With these strategies, we were able to demonstrably improve student learning. Our students made an average normalized knowledge gain on the Force Concept Inventory that was nearly twice that observed in typical lecture-based courses. These changes were also well received by students, with one anonymous end-of-semester response noting: "At the beginning of the semester I was not a fan of the clicker questions, wanting a more traditional lecture. Alas, I have changed my mind. Physics is such a practical science, a skill much like carpentry. You can't read how to hammer a nail." With these improvements, this project has contributed to strengthening our nation’s future scientific workforce by better preparing students to solve problems using approaches from multiple scientific disciplines (physics and biology in this case). This project also contributed to the future scientific workforce by expansion of a partnership between Vanderbilt and Fisk University (a local HBCU) to attract under-represented minority students into graduate study in biophysics and by providing research experience at the interface between physics and biology for eight undergraduate students and one high school student. These students should be well prepared to cross disciplinary boundaries in their futures when doing so is key to solving important and complex problems.
