Marine environments harbor by far the greatest range of biodiversity on Earth and this diversity of life provides untapped opportunities to understand how organisms "work" and how they interact with their natural environment. Recent developments in molecular technology in including next-generation DNA sequencing and functional genomics provide opportunities to build a new level of understanding of the molecular basis of cellular regulation during development, neurogenesis, cancer, adaptive resiliency, and regenerative biology. Marine animals, particularly invertebrates, are ideal experimental systems for a variety of investigations into fundamental properties of animals. This proposal uses delicate marine organisms, collected and cultured at the Whitney Lab for Marine Bioscience (www.whitney.ufl.edu/), to leverage these new technologies for an unprecedented molecular understanding of a variety of cellular behaviors related to the interrogation of gene expression at the resolution of identified cells. This grant provides instrumentation for the isolation of pure populations of identified single cells that can be used for downstream molecular characterization (e.g. the construction of gene regulatory networks involved in the establishment of stem cell or neural identity). This equipment will be housed on site, and can be deployed for a wide array of ongoing research projects of resident and visiting researchers. All research groups at the Whitney Lab remain actively involved in the Lab's 32 year-old NSF REU program, participate in K-12 STEM outreach programs (e.g. Scientist for a Day and Whitney Lab's Traveling Zoo), summer camps for 3rd- 4th graders, and a free public lecture series ("Evenings at Whitney"). Whitney recently opened a Sea Turtle Rehabilitation Hospital that focuses on the surgical removal and cure of debilitating fibropapilloma (FP) disease that affects all species of sea turtle worldwide. Whitney faculty are periodically consulted on social and land-use management issues, and share knowledge and expertise with the local community.
This project builds on previous expertise to utilize cutting edge, single cell omics techniques to understand the molecular regulation of distinct cell types. A cell labeling facility (microinjection rig) that can fluorescently label individual cells via a variety of molecular techniques, and an efficient multiuser friendly small volume fluorescent cell sorter (BD FACSMelody) will be deployed to address a wide array of ongoing research projects of resident and visiting researchers. Fluorescently labeled cells, whether it is a dextran lineage marker, nuclear markers of DNA content, mRNAs for specific genes injected in to living cells, reporter genes driven by endogenous enhancers, or CRSPR/Cas9 reporter gene knock-ins can be dissociated into single cells and sorted using a FACS (Fluorescently Activated Cell Sorter) machine. FACS machines were once large prohibitively expensive instruments needing large numbers of cells (1 x 106-9 ) and only able to sort limited wavelength channels. Newer generation FACS machines have become much more powerful with the development of multiple solid state lasers and small volume sorters (96 or 384 well formats). The capability to isolate single cells goes hand-in-hand with the ability to sequence transcriptomes and genomes of single or small numbers of molecularly identified cells, using Next Gen sequencing technology.
