Marcus C. Davis, Ph.D. - US grants

Terrestrial (land living) vertebrates possess a specialized region of their appendages called the autopod (hands & feet; fingers & toes) which are not present in their aquatic fish ancestors. Variation in key genes that form the autopod generate the diversity of crawling, flying, and swimming adaptations we see in nature, but also explain common forms of congenital birth defects seen in human hands and feet. Previously, this laboratory discovered that genes involved in autopod formation, called Hox genes, are active in the fins of a primitive living fish, the North American paddlefish Polyodon spathula. This fish does not have an autopod region, nor did it evolve from an ancestor that had an autopod. Why then, do paddlefish have these genes and what is their function? To address these questions, these investigators are using modified versions of these genes designed to either shut down gene function or amplify it above normal levels within the developing fins of embryonic paddlefish. These results should reveal the normal function of these genes, and interpret the results of abnormal gene expression in comparison to data from human and mouse birth defects. Initial results demonstrate a pattern of loss of skeletal structures that links specific Hox genes to the formation of specific portions of the fin, in a manner similar to humans and mice. These results will provide significant insights into autopod formation that will answer both evolutionary questions and contribute to clinical approaches for addressing congenital autopod birth defects in humans. In addition, undergraduates at Kennesaw State will comprise the core of this research, providing numerous students with research experience to prepare them for careers in medicine and the biological sciences.

The Zeiss LSM 700 Laser Scanning Confocal Imaging System will be used in multidisciplinary research and education in the Departments of Biology & Physics and Chemistry and Biochemistry at Kennesaw State University (KSU) and will be the anchor instrument of KSU's microscopy core facility in the new Science Laboratory Building. The LSM 700 and integrated workstation with ZEN (Zeiss Efficient Navigation) software is capable of covering a broad range of imaging needs, from multiple fluorescence and co-localization analysis to live cell imaging to photoactivation of fluorophores. This flexibility will maximize the number of researchers, and types of research, that can make use of the microscope. The confocal imaging system will advance KSU research programs, foster new research directions, and generate data that will complement many other techniques employed by researchers at KSU, such as: fluorescent and standard microscopy, cell culture and in vivo assays, gel-shift, equilibrium saturation and competition assays. The LSM 700 system will enhance the ability of KSU researchers to generate and interpret data. The integration of confocal results with experiments already performed at KSU will provide crucial insights for on-going research in developmental biology, molecular genetics, neurobiology, botany, ecology, evolutionary biology, biochemistry, and related fields at KSU and our collaborator institutions. Although the primary use of the microscope will be for research, it will also be integrated into research training and course curricula.

Confocal microscopy is an increasingly common research tool for visualizing biological and biochemical systems. Acquisition of the LSM 700 confocal microscope will have a significant impact on the research environment at Kennesaw State University (KSU), giving researchers in the Departments of Biology & Physics and Chemistry & Biochemistry the ability to more fully describe interactions important to the systems they study. Publication of innovative research by KSU faculty incorporating confocal data will provide insights into such fields as cell and developmental biology, biochemistry, ecology, evolutionary biology, and anatomy. In addition, the instrument broadens the array of research tools available to researchers at KSU and makes them more attractive collaborators for scientists at more research-intensive institutions. Beyond primary research, teaching experiments will be designed to incorporate confocal microscopy into the classroom. Bringing a more quantitative approach to bear on research problems using confocal microscopy will also make faculty better teachers and afford invaluable research training to undergraduates, including members of underrepresented groups. Hands-on training in the technology and usage of confocal imaging will better prepare KSU graduates for STEM careers.

An award is made to Kennesaw State University (KSU) to fund an electrophoretic nucleic acids analyzer and accessories for the use in multidisciplinary research and research training. Electrophoresis is the primary means of analyzing nucleic acids, separating nucleic acids based on their size and, to a lesser extent, conformation. Through an electrophoretic analysis, critical qualitative and quantitative information regarding the composition of a nucleic acid sample can be obtained. The nucleic acid analyzer will be part of the existing massively parallel / next generation sequencing facility, including an Ion Torrent Personal Genome Machine semiconductor sequencer (PI Van Dyke), thereby significantly expanding facility capabilities. Primary benefits will be in research, including projects involving undergraduate and graduate (Master of Science in Integrative Biology, Master of Science in Chemical Sciences) students. Additionally, programs that enhance research access to under-represented populations, including KSU's NIH-funded Peach State Bridges to the Doctorate and NSF-funded Chemistry and Biochemistry Summer Undergraduate Research Experiences programs, will benefit from this instrumentation. Involvement of the facility with course curricula is also planned, especially given the increasing importance of next-generation sequencing and transcriptome analysis in both health-related and research fields. Hands-on training in the technology used for contemporary nucleic acid sample quality control will better prepare KSU trainees for a broad range of STEM careers.

An electrophoretic nucleic acids analyzer provides automated sample processing and scalable throughput for a variety of DNA and RNA quality control applications. For example, they are routinely used to determine the integrity of genomic DNA samples before their use in involved and costly procedures such as next generation sequencing, microarrays, and quantitative PCR. The nucleic acids analyzer will be used initially by a core group of five laboratories at KSU and their collaborators. Dr. Michael Van Dyke investigates orphan transcriptional regulators in a variety of organisms, including E. coli and Thermus thermophilus. Dr. Martin Hudson studies the role of heparan sulfate proteoglycans and anosmin-like proteins in nervous system development, using C. elegans and stem cell models of nervous system differentiation. Dr. Joel McNeal investigates photosynthetic gene evolution, host/parasite interactions, and microbial endophytes in the parasitic plant genus Cuscuta. Dr. Melanie Griffin studies the global targets of the leucine response protein in Pseudomonas aeruginosa. Dr. Marcus Davis explores the developmental mechanisms that pattern vertebrate appendages (fins and limbs) and the evolution of HOX gene regulation. Nucleic acids analysis will provide their materials, ranging from combinatorially selected fusion libraries to developmental stage-selected RNAs, with essential quality control assessments, thereby increasing the likelihood of success in their research and teaching endeavors.

The fins of fishes and the limbs of terrestrial (land living) vertebrates are anatomically distinct: Fins are 'capped' in a series of rays, while limbs terminate in an autopod (hands/feet). These differences support the view that fin rays and autopods are patterned by distinctly separate genes and genetic 'circuits'. If this were true, studies of fin ray growth and regeneration may not be directly transferable to studies of limb birth defects and regeneration research, including in humans. However, preliminary data from this project shows that the same genetic circuits do form the skeletons of both fins and limbs. This project will confirm this common fin/limb building program, first characterizing the activity of key genes in the developing fins of two fish ideally suited to address these questions, the American paddlefish and the small spotted catshark. These results will then guide experiments to test gene function, which will be compared to known data for developing limbs. Outcomes will show that a shared genetic program regulates formation of fins and limbs and demonstrate that fish fins are a powerful research tool for the study of limbs. Part of the success of this program will be the training of new researchers - a diverse group of graduate and undergraduates will bring their talents to this project and contribute to public outreach experiences in the Atlanta area public school system.


The developmental basis of the fin-to-limb transition remains a longstanding question in evolutionary biology. In current models, fins are patterned by distinct proximal and distal developmental modules, generating adult skeletal compartments containing endochondral or dermal elements respectively. Emphasis on skeletal type led to the hypothesis that fin-folds and autopods are not homologous, patterned by different modules, despite similar distal positions. However, new findings raise an intriguing alternative hypothesis: that autopods share a deep regulatory homology with dermal fin rays. This project will build a model for the evolutionary origin of the distal paired appendage gene regulatory network (GRN), expanding on discoveries in two phylogenetically well-positioned vertebrates, the American paddlefish (Polyodon spathula) and the small spotted catshark (Scyliorhinus canicula). To test this model, RNA-seq will be used to build a comprehensive transcriptional map in the fin compartments of paddlefish. These datasets will be compared to existing transcriptomic resources to identify candidate distal appendage GRN genes, which will be tested via pharmacological perturbation assays in paddlefish and complementary gene expression assays in catshark. CRISPR-Cas9 mediated gene knockdowns will test HoxA gene function in paddlefish fins. Together, the outcomes of this project will elucidate key components of the distal appendage GRN, catalyze new research directions that regulate early skeletogenesis, and reinvigorate interest in the use of fins for the study of limb birth defects and regeneration studies. The proposed work will train a diverse group of graduate and undergraduate students, including those recruited through minority participation programs. The PI and students will collaborate on outreach programs to Atlanta area public high schools through virtual access to the PI's lab, classroom visits, and summer intern programs.