Cancer is characterized by uncontrolled cell proliferation, and its potential to affect almost anyone as they age poses a significant threat. Extracellular vesicles (EVs) are lipid-bilayer membrane-enclosed structures that cancer cells produce and use for intercellular communication. EVs are typically loaded with a variety of proteins, nucleic acids, and other cargo that can be delivered to recipient cells. Tumor-derived EVs aid in the progression of various cancers by enhancing malignant cell survival, proliferation, and invasion. Working with our graduate mentor, we conducted an 866 chemical screen and found kinase inhibitors that altered EV production by cancer cells via luminescence assay. From the hits, we chose to study kinases from the JNK and p38 MAPK pathways, which both promote cancer progression. Reactive oxygen species (ROS), which damage cells through oxidative stress, can activate both of these pathways. Based on this, we proposed the question: what role do ROS play in EV biogenesis and cancer development in living organisms? To answer this question, our research utilizes Drosophila melanogaster, an ideal in vivo model due to its vast genetic toolbox and brief generation times. We used Drosophila with the Ras^V12, scrib^-/- tumor model to study EV biogenesis, and crossed them with flies that have knocked down homologs of JNK and p38 MAPK. We then selected specific progenies and dissected the imaginal discs and placed them in media to allow for EV biogenesis, and quantification was done by live imaging EV production from tumor discs, fluorescence assays, and qPCR. Our preliminary results show that imaginal discs from Ras^V12, scrib^-/- flies produce a large amount of EVs. We anticipate that in organisms, both JNK and p38 MAPK knockdowns will lead to a decrease in EV production. Future work could be done to implement our findings in humans to potentially develop novel cancer therapeutics.

The notochord is a rod-like embryonic structure that plays a critical role in transmitting spatial cues to surrounding tissues, serving as the defining feature of chordates and a vital component of vertebrate embryonic development. Understanding the molecular and cellular mechanisms that govern notochord formation and maintenance is a fundamental knowledge gap in developmental biology. Our lab has previously identified wnt16 to be the second wnt gene expressed in the developing notochord in vertebrates and have found that wnt16^w1001/w1001 zebrafish mutant embryos exhibit significant reductions in notochord height, length, and notochordal vacuolated cell size. The fluid-filled vacuolated cells are osmotically active structures that experience osmotic pressure and rapidly inflate along the confines of the notochord sheath, contributing to embryonic body axis development. Glycosaminoglycans (GAGs) are a family of complex polysaccharides that are predicted to play an important role in vacuolated cell and notochord development due to their regulation of cellular tonic conditions and generation of hypotonic/hypertonic stress. Our study aims to establish potential mechanisms for the maintenance of vacuole cells and notochord development, which we hypothesize to act through the crosstalk between the Wnt16 signaling and GAG biosynthetic pathways. To test this, I performed single-cell RNA sequencing analysis on a zebrafish notochord-specific cell population, identifying five GAG biosynthetic genes expressed in clusters alongside wnt16. By determining expression profiles of these GAG genes in wild-type and wnt16^w1001/w1001 mutants, I aim to uncover the underlying mechanisms of the previously observed notochord phenotype. Furthermore, the notochord directly contributes to spine development, persisting within the nucleus pulposus, a structure entrapped in developing intervertebral discs (IVD) and predicted to be important in maintaining IVD health. As such, the implications of our study extend beyond the bench and may contribute to advancements in treating spinal diseases, particularly intervertebral disc degeneration (IVDD).

Understanding genetic risk factors for osteoporosis, a common chronic bone disease that increases fracture risk, is essential for developing new therapies. Genetic variants near SLC8A1, a member of the SLC8 gene family of sodium/calcium exchangers, have been associated with bone mineral density and fracture risk. More recently, I have shown that zebrafish slc8a4b, an ortholog of human SLC8A1, is highly expressed in osteoblasts through scRNA sequencing analysis. However, animal studies examining the expression pattern and necessity of slc8a4b in developing bone have yet to be conducted. Here, I tested the hypothesis that slc8a4b is highly expressed in osteoblasts and required for early bone formation in zebrafish. To evaluate the expression of slc8a4b, we performed whole-mount in situ hybridization chain reaction (HCR) RNA FISH of zebrafish embryos at 3dpf and 5dpf. To assess the function of slc8a4b, I utilized slc8a4b mutant allele sa34209, generated through large-scale zebrafish mutagenesis efforts. Structural modeling revealed that sa34209 results in severe protein truncation. To determine whether slc8a4b is necessary for skeletal development, I will incross adult slc8a4b^+/sa34209 heterozygous mutants to generate slc8a4b^{sa34209/sa34209} homozygous mutants and perform calcein staining at 5dpf and 13dpf to assess craniofacial and vertebral morphology in zebrafish. My preliminary data shows that slc8a4b is highly expressed in early craniofacial structures such as the opercle. This study will be the first to examine the necessity of slc8a4b in vivo and thus could uncover the role of solute carrier family 8 genes in skeletal development, which could lead to new therapies for osteoporosis.