Characterization of whole-organism and cultured epithelial cell stress responses to nickel toxicity in a marine tunicate.

Abstract

Botryllus schlosseri is a globally invasive colonial marine tunicate that relative to other chordates, possesses a remarkably high regenerative capacity through the maintenance of lifelong multipotent adult stem cells. B. schlosseri are comprised of several individual zooids that each contribute to the expansion of the colony through a form of weekly asexual budding termed blastogenesis, by which new clonal zooids arise as primary buds from the epithelial layers of the body walls of the prior zooid generation. As such, somatic cells of B. schlosseri, including those in the epithelia, have a significant potential to serve as powerful in vitro tools for revealing the cellular programs that drive regeneration and aging in one of our most closely related invertebrate species. Despite more than 40 years of research efforts, a continuous immortal cell line has not been established for B. schlosseri. For epithelial cells, the constrained cellular proliferation period for primary epithelial cells in culture presents a significant limitation in immortalization, with all documented cases entering a state of arrested cellular division after 24- to 72-hours. As such, characterizing the molecular networks regulating cell growth and arrest in this species is a critical step in developing strategies to overcome limited proliferation and facilitate the development of an immortalized epithelial cell line. To address this, this thesis evaluated organismal and in vitro cellular responses of B. schlosseri to elevated concentrations of nickel, an oxidative stressor and clastogenic compound with the potential to serve as an immortalizing agent. In chapter 1, an in vivo study evaluated the phenotypic and molecular nickel-induced stress responses of whole B. schlosseri colonies. This study was the first to establish estimates of median lethal nickel concentrations (LC50) for B. schlosseri at both 24- and 96-hours of exposure in full strength artificial seawater. B. schlosseri exhibited remarkably high nickel tolerance (24- hour LC50 = 177 mg/L and 96-hour LC50 =159 mg/L, nickel) where mortality was only observed at the highest dose administered (1000 mg/L nominal nickel (II) chloride [NiCl2]). Although B. schlosseri persisted in the second-to-highest dose evaluated (100 mg/L nominal NiCl2), colonies exhibited blastogenic arrest whereby primary buds suspended further growth and senescent zooids did not fully resorb. Differences in superoxide dismutase 1 (SOD1) activity for colonies in control, 1, or 100 mg/L nominal NiCl2 were evaluated at both 24- and 96-hours post-exposure. B. schlosseri exhibited a moderate non-monotonic SOD1 activity response in which elevated activity was observed only in colonies exposed to the intermediate 1 mg/L nominal NiCl2 dose. We then evaluated the transcriptomes of whole colonies exposed to control artificial seawater or 100 mg/L nominal NiCl2. B. schlosseri colonies exhibited a tight transcriptional response with only 54 differentially expressed genes out of 13,970 reliably expressed gene transcripts. Significantly differentially expressed pathways included gene transcripts associated with redox balance, extracellular matrix maintenance, vasculature remodeling, cellular differentiation, and apoptosis. In chapter 2, a series of in vitro experiments updated the methodology for culturing B. schlosseri epithelial cells, assessed primary epithelial cell phenotypic responses to NiCl2 exposure, and uncovered limitations for total RNA isolation from primary epithelial cells with proposed approaches for improved RNA extraction outcomes. Primary epithelial cells were cultured under an explant tissue method that partially desiccated intact zooids and buds for improved attachment to the plastic culture-ware substrate. The media formulation was simplified from prior reported attempts, with a final media composition of a 1:1 mixture of supplemented media and sterile artificial seawater (~750 mOsmo/kg; pH 8.1). Under these culturing conditions, both zooids and buds in late blastogenesis produced a comparable number of epithelial cells and proliferated for up to 24-hours in culture. Cells displayed typical epithelial morphological characteristics as reported previously for this species, including the generation of tunic-like secretions in culture. In an acute nickel toxicity assessment, cells displayed no changes in phenotype or cell number, suggesting an overall tolerance to the chemical. Assessment of transcriptional responses to elevated nickel exposure for primary epithelial cells was attempted, however RNA quality and yield remained below the threshold required for bulk-RNA sequencing. In a systematic evaluation to improve RNA isolation, these cells were not conducive to typical methods of animal cell lysing including, needle shearing, chemical detergents, and bead beating. Proposed improvements for molecular phenotyping of epithelial cells will involve the evaluation of the protective role of tunic-like secretions as well as alternative enzymatic digestion for improved RNA isolation. Together, these findings provide valuable insights into the molecular networks that regulate nickel-induced cellular stress responses and identified key methodological barriers that remain to be addressed for working towards immortalization of primary epithelial cell cultures of B. schlosseri.