Almost all of our tangible understanding of the world is mediated by the skin, a highly innervated sensory organ. However, many injuries to the skin result in the severance of somatosensory axons, causing temporary or permanent loss of feeling. To reestablish innervation, epidermal and neuronal cells launch wound healing responses. Neuronal cells undergo a transition to a more juvenile state to promote new axon growth for reinnervation. Surrounding keratinocytes and immune cells migrate to the affected area to clear cellular debris and promote axon regeneration. Although we know these cellular processes occur, it is unclear how changes in gene expression direct these responses. My project aims to transcriptomically characterize healing responses using zebrafish (Danio rerio) as a model organism. Zebrafish are widely used in regeneration studies and have a fully sequenced genome available for reference. Unlike in human skin where complete regeneration is not observed, zebrafish have almost perfect regenerative abilities. To address our question, we plucked fish scales to induce a rapid regenerative response of both somatosensory neurons and skin. We then collected and purified RNA from somatosensory neurons and skin at zero, one, and three days post pluck for sequencing to characterize gene expression over time. With high-throughput RNA sequencing, we can uncover expression patterns and ontologies of differentially expressed genes in neurons and skin after injury. Thus far, we have found that several previously characterized regeneration associated genes (RAGs) are transiently upregulated in neurons following injury. Transcription factors involved in somatosensory development show a drop in gene expression following injury and are upregulated as the regenerating axon develops. We hope that by understanding skin reinnervation in successfully regenerating organisms, we can develop strategies to improve tissue recovery in humans through manipulation of conserved mechanisms.

Kaposi's Sarcoma associated Herpesvirus (KSHV) is an oncogenic human herpesvirus and the etiological agent of Kaposi's Sarcoma (KS), a cancer that afflicts HIV-positive individuals worldwide and is endemic in sub-Saharan Africa. KS tumor cells, known as spindle cells, originate from latent KSHV infection of endothelial cells. We have previously used a phosphor-proteomics approach to identify changes in the phosphorylation state of proteins during KSHV infection. We identified Platelet/Endothelial Cell Adhesion Molecule-1 (PECAM-1) to be significantly phosphorylated during KSHV infection as compared to mock infected cells. PECAM-1 phosphorylation leads to a cascade of signaling that promotes cell adhesion, migration, and cell survival, however, the role PECAM-1 plays to aid KSHV infection is currently unknown. To determine whether PECAM-1 expression and phosphorylation is important for KS pathogenesis, I propose to assess differences in gene expression levels with RT-qPCR, validate phosphorylation levels in vitro via western blot, and generate CRISPR-lentiviral constructs to knock out PECAM-1 and express PECAM-1 isoform variants in endothelial cells. These experiments will paint a more complete picture of how PECAM-1 interacts with endothelial cellular processes during KSHV infection. Many rounds of RT-qPCR have been conducted to asses gene expression levels with highly variable results. This indicates that some uncontrolled factor in the cell culture process is affected PECAM-1 expression levels. I hypothesize that cell density is the cause behind this variability and plan to use western blotting to elucidate differences in PECAM-1 protein levels in low and highly confluent cells.

Honeybees, Apis mellifera, demonstrate an ability to perform visual learning to such an extraordinary extent that it is not typically associated with smaller brains. Those learning abilities may be linked to their degree of sociality as they aggregate into colonies and synergistically work together on tasks. The Social Brain Hypothesis suggests that an organism's level of intelligence is correlated to how social their environment is. Some bees like the leaf cutter bee, Megachile rotundata, are solitary bees who do not live in a hive but instead reside in individual nests. The purpose of this research is to explore the visual learning capability of leaf cutter bees and compare them to the learning capability of the well documented honeybee. The methods used for this study include placing a tethered bee on a free rotating ball that is placed in front of a screen. Two colors are projected onto the screen and the bee controls the movement of the shapes. A positive and negative reinforcement are assigned to each shape. By analyzing how many times the bee picked each color, how fast it responded to the shapes, and how far from a distance it walked to fixate on that color, we can make an informed statement about the solitary bee’s ability to visually learn. We anticipate one of three outcomes, the leaf cutter bee will either have a greater learning rate, less learning rate, or equal learning rate to that of the honeybee. These results will help supplement more data for the Social Brain Hypothesis and allow a deeper understanding of how sociality is related to cognition. This study also has major relevance to pollination practices as leaf cutter bees are used in agriculture, therefore understanding the neural basis of cognition can open the door for new reforms in crop pollination.

It’s known that plant-pollinator relationships are central to the proper functioning of agricultural and ecological systems. Of the many navigation pathways pollinators use, floral scent signaling for insects is the most complex yet also the most at-risk from atmospheric human activity. Oenothera pallida, a primrose, interacts with the pollinators Hyles lineata, a hawk moth, and Megachile rotundata, a leaf-cutter bee, via this scent pathway. Because of their reactivity with floral scent, human-released ozone and NO₂ are the main perpetrators of scent degradation. To understand this relationship being damaged, I exposed the moth and bee species to a normal Oenothera scent versus a degraded one, recording the antennal response as well as the behavioral, expecting a poorer response to the degraded scent. Moth antennae act as the site of odor reception, bearing sensory hairs that detect odors, allowing the moths to navigate to scent sources. I conducted electroantennographic experiments (EAG) to record the electric signal from the insect antennae in response to each scent blend, with the degraded scent representing the impact of NO_x interactions. Following the EAG, I conducted Proboscis Extension Reflex (PER) experiments with Megachile to show the relationship between insect behavior and antennal physiology when in the presence of the scent blends. I expect that the EAG experiments show that the antennae respond worse to NOx degraded scents in comparison to the normal, unaltered scent blend. Likewise, Megachile has a worse PER when exposed to the degraded scent, linking the chemical biology of the scent interaction to the feeding and pollination behavior. This work has broader implications regarding the importance of plant-pollinator relationships, especially when considering environmental and agricultural health as well as the issue of food security in our changing climate.

The somatosensory system mediates our ability to perceive touch and temperature by relaying information from our periphery to our central nervous system. Peripheral axons of somatosensory neurons densely innervate tissues, such as skin to form intricate patterns that are maintained throughout adulthood. Multiple events that occur during the dramatic remodeling events of skin organogenesis guide such pattern formation. To study these processes, we use zebrafish (Danio rerio), a powerful model organism that develops ex utero, allowing direct observation and in vivo manipulation of the somatosensory system. However, current transgenic tools to label somatosensory neurons in zebrafish have several limitations, most notably, they do not allow permanent genetic labeling or inducible transgene expression. To address these limitations, we have taken a tool building approach using CRISPR/Cas9 to generate a knock-in line of zebrafish expressing tamoxifen-inducible Cre recombinase in somatosensory neurons. This will facilitate permanent, cell-specific labeling of somatosensory neurons in zebrafish, allowing researchers to, for example, perform temporal tracking of somatosensory neurons throughout development. Previous studies have uncovered several genes specifically expressed in zebrafish somatosensory neurons. These genes serve as potential targets for CRISPR/Cas9 mediated knock-in of CreERT2 such that when the selected somatosensory gene is expressed, CreERT2 is also expressed. In preliminary single guide RNA injection experiments, the somatosensory gene, trpv1, which encodes a thermosensitive ion channel, showed promising results as a knock-in target. Ultimately, the tools we are building will aid in advancing our understanding of the mechanisms behind the development of the skin sensory system, which will likely have implications in how scientists and health professionals approach a number of neuropathic diseases.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS), a vascularized tumor composed of spindle cells. While spindle cells express markers of blood and lymphatic endothelial cells (BECs and LECs) and KSHV can infect BECs and LECs in cell culture, it is not definitively known which cell type leads to KS development. Previous research suggests that LECs may be an important reservoir for KSHV infection: LECs are more susceptible to KSHV infection than BECs and maintain the KSHV episome over a greater number of passages compared to BECs. Importantly, KSHV-infected LECs, but not BECs, proliferate beyond normal replicative senescence, the first step in tumor formation. To further explore the hypothesis that LECs play an important role in KS development, I propose to overexpress Prox1 (the master gene controlling LEC differentiation) in BECs, knock out Prox1 in LECs, and assess the impact of KSHV infection on Prox1-expressing-BECs and ΔProx1-LECs. First, I will infect Prox1-expressing-BECs and ΔProx1-LECs with KSHV and measure infection rates 48 hours post infection. I expect the infection rate to be higher in the Prox1-expressing-BECs relative to ΔProx1-LECs. Next, I will infect Prox1-expressing-BECs and ΔProx1-LECs with KSHV and harvest cells every two days over the course of two weeks for immunofluorescence for latent gene expression. I expect the Prox1-expressing-BECs to maintain a higher rate of infection throughout this duration compared to the ΔProx1-LECs. Finally, I will infect Prox1-expressing-BECs and ΔProx1-LECs with KSHV at an early passage (before senescence), harvest cells at a later passage (when senescence is observed), and measure the percentage of senescent cells by staining cells for beta-galactosidase activity. I expect Prox1-expressing-BECs to have bypassed senescence at the later time point and ΔProx1-LECs to senesce. Results from these experiments will further our understanding of how KSHV alters human endothelial cells to induce KS tumorigenesis.

Skin is an organ system with a diverse cell ecosystem that plays important barrier and sensory functions, which are critical to organism survival. Because of its superficial location, the skin, and its composite cells, are easily damaged and need to be repaired to maintain homeostasis. Zebrafish are an excellent model to study skin repair because they regenerate tissue efficiently and share conserved skin architecture with other vertebrates. I am addressing two related questions that examine how the sensory axons that innervate the skin and resident skin cells interact during development and tissue repair. First, how do skin-resident immune cells called Langerhans cells contribute to skin repair? Previous work in the lab found that Langerhans cells phagocytose degenerating cutaneous sensory axons, leading me to hypothesize that Langerhans cells play a broad, and previously unappreciated role, in skin repair. As a first step, I have been examining the effects of genetic mutations on Langerhans cell development. I have compiled a data set from several mutants proposed to regulate Langerhans cell development using image analysis of transgenic zebrafish. Second, how do peripheral nerves in the skin influence tissue regeneration? To answer this, I have created week-long time-lapses of skin regeneration in mutants lacking peripheral neurons compared with wild type sibling fish. To induce regeneration, I removed a concentrated patch of scales, a type of skin appendage, on the lateral side centered above the pelvic fin. I then visualize and quantify scale regeneration using a fluorescent imaging of a stain that labels scales. By comparing the genetic groups, I hope to determine whether nerves influence the rate and amount of regrown scales. Together these projects will provide insights into how interactions between the complex cell types of the skin regulate tissue repair, which we hope will ultimately have implications for wound repair in humans.