Botryllus schlosseri is a non-indigenous species of colonial tunicate commonly found in many marinas of the Puget Sound. Colonies of Botryllus can grow asexually through the process of blastogenesis (also called ‘budding’) which involves the formation of buds which mature into new zooids on a weekly cycle. Apoptosis and oxidative stress are involved in the final stage of blastogenesis, a time when the system resorbs the zooids and the newly matured buds take over. Although nickel has been found to be genotoxic and can induce many aspects of the cellular stress response in animals, little research has characterized these effects on marine invertebrates. In this study, we performed acute exposures of nickel chloride at sublethal concentrations in B. schlosseri collected from the field to identify changes to blastogenic timing, DNA damage, apoptosis, and the oxidative stress response. These data will establish the in vivo tolerance and stress responses of B. schlosseri to nickel exposure. Furthermore, these data will inform future studies which seek to use nickel as an immortalization agent in cell culture with the goal of generating the first continuous cell line for any marine invertebrate species.

The Developmental Origins of Health and Disease (DOHaD) hypothesis evaluates how the prenatal environment affects health after birth. The placenta is a multi-faceted organ that sustains life during human development and is key to evaluating the DOHaD hypothesis. Glial Cells Missing Transcription Factor 1 (GCM1) is a transcription factor that plays a critical role in placental development. Our goal is to understand the downstream effects of GCM1 on various genes necessary for placental development by evaluating gene expression after GCM1 knockdown. The BeWo choriocarcinoma cell line is a model of placental syncytiotrophoblasts cells which undergo a cell fusion process called syncytialization in the placenta to form multinucleated cells that help exchange nutrients. Previously, we knocked down GCM1 in full-term primary placental cells that spontaneously syncytialize and assessed gene expression using RNA sequencing. We identified 10 differentially expressed genes. Based on those findings, we hypothesized that GCM1 plays a greater role during early pregnancy leading us to repeat the GCM1 knockdown in BeWo cells. BeWo cells were treated with 20µM, 50µM and 100µM forskolin (FSK) for 48hr to induce syncytialization which was confirmed via qPCR of syncytialization markers GCM1 and Syncytin-2 and through fluorescence microscopy. GCM1 expression increased 3.15, 1.3, and 1.2 fold respectively after treatment with 20µM, 50µM, and 100µM FSK, whereas Syncytin-2 increased 78.1 fold after 50µM FSK treatment. We then performed an siRNA knockdown of GCM1 in unsyncytialized BeWo cells with two concentrations of siRNA (25nM and 50nM) for 24hrs and observed a 70% and 80% reduction in GCM1 expression, respectively. Next steps include optimizing the siRNA procedure for syncytialized BeWo cells and comparing these results to our previously conducted experiment. Overall, this will improve understanding of how GCM1 coordinates gene expression in the placenta during pregnancy.

The placenta is a crucial fetal organ providing oxygen and nutrients to the developing infant. Researchers typically use placental cell models to study the placenta, which are derived from immortalized cells. The use of in-vitro placental cell models is important because human samples are difficult to obtain, and placental biology is highly species-specific. Despite this, our understanding of the characteristics of these cell lines and how they compare to placental tissue samples is limited. This project aims to determine which placental cell model most directly reflects the gene expression of the human placenta. RNA sequencing data from the placental cell models HTR-8/SVneo, JEG-3, BeWo as well as data from primary trophoblast cells was obtained using the Gene Expression Omnibus (GEO) database or through lab-generated datasets. Data for each cell line was combined into a single dataset of shared genes (n=6835) and individual datasets of genes unique to each cell model. For genes that were unique to each cell model, I performed KEGG pathway analysis and characterized placenta specific genes using the Human Protein Atlas (HPA). BeWo cells expressed the highest number of unique genes (n=355) and shared the highest number of genes with the primary trophoblast cells (n= 1,167). Pathway analysis showed that genes unique to primary trophoblast cells (n=2661) were overrepresented in 24 pathways, unique BeWo genes were overrepresented in 15 pathways, while unique HTR-8/SVneo genes (n=355) were overrepresented for a single pathway- the neuroactive ligand-receptor interaction. Placenta specific genes were expressed within the uniquely expressed genes for JEG-3 (n=1), BeWo (n=1), and primary trophoblast cells (n=17), but not HTR-8/SVneo. Ultimately, the results from this project will provide a tool to evaluate differences in placental cell models and aid the placental biology research community in understanding which cell line is most representative of human placental tissue samples.