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Poly- and perfluoroalkyl substances (PFAS) are a class of bioaccumulative toxicants used in numerous industrial and commercial products. Perfluorooctanesulfonic acid (PFOS) alters pancreatic organogenesis during development, and perfluorohexanesulfonic acid (PFHxS) has been suggested as a replacement for PFOS due to its shorter carbon chain, but they are often found together in surfactants, such as legacy aqueous film-forming foam. This study investigates how developmental exposures to a PFAS mixture (PFHxS + PFOS) impact the developing exocrine pancreas. Zebrafish embryos (Tg(ptf1a:GFP)) were exposed to 0.01% DMSO, or 8, 16, 32 μM PFHxS alone, 16 μM PFOS alone, and 8, 16, and 32 μM PFHxS plus 16 μM PFOS. Embryos underwent refreshing exposures (3 hours post fertilization (hpf) - 96 hpf) or static exposures (3, 24, 48, or 72 hpf - 96 hpf) and then live imaging to quantify the truncated exocrine pancreas phenotype that occurred, and at what point in development it became apparent. PFAS mixtures significantly impacted growth parameters and exocrine pancreas length. The truncated pancreas phenotype was seen most often in the 16 μM PFHxS + 16 μM PFOS treatment group, so this concentration was used for subsequent experiments. Time lapse imaging (58 - 72 hpf, 80 - 96 hpf) and cellular proliferation assays (3 - 96 hpf) were used to ascertain the cause of the truncated phenotype as an issue of cellular migration or proliferation within the pancreas. Cell migration and proliferation were decreased in response to toxicant exposure. This study offers insights to how developmental exposures to toxicants may impact the pancreas.

Tissue Factor Pathway Inhibitor (TFPI) is an anticoagulant protein containing three Kunitz domains, K1, K2 and K3. K1 inhibits Factor VIIa, K2 inhibits Factor Xa, and K3 enhances the Factor Xa inhibition by its interaction with Protein S. Since zebrafish is an excellent genetic model, we hypothesized that TFPI regulation could be studied using this model. As a first step, we confirmed the presence of tfpia in zebrafish. Subsequently, we performed knockdown of tfpia, and knockout of tfpia in K3 domain using CRISPR/Cas9. Both the tfpia knockdown and tfpia homozygous deletion mutants showed increased coagulation activities. Our data suggest that zebrafish tfpia is an orthologue for human TFPIα, and silencing it results in a thrombotic phenotype. We then optimized the piggyback knockdown method, where we could simultaneously piggyback 3 or 6 ASOs corresponding to 3 or 6 genes, respectively, using one VMO. These multiple gene knockdowns will increase the efficiency of genome-wide knockdowns. Since there are no studies on chromatin remodeling that control TFPI expression, we hypothesized that the genome-wide knockdowns of the Chromatin Binding and Regulatory Proteins (CBRPs) in zebrafish could help identify novel tfpia gene regulators. We chose 69 CBRPs and subjected them to simultaneous gene knockdowns. Our results have identified 5 novel regulators for tfpia. We exploited this information to discover UNC6852, a drug that enhances tfpia mRNA levels. This could be used as an antithrombotic drug. The approach developed here could be used to study the regulation of other coagulant and anticoagulant factors.

<p>Bioelectricity, or endogenous electrical signaling mediated by the dynamic distribution of charged molecules, is an ancient signaling mechanism conserved across living organisms. Increasing evidence has revealed that bioelectric signals play a critical role in many diverse aspects of biology such as embryonic development, cell migration, regeneration, cancer, and other diseases. However, direct visualization and manipulation of bioelectricity during development are lacking. Neuroscience has developed tools such as GEVIs (genetically encoded voltage indicators) and chemogenetics like DREADDs (designer receptor exclusively activated by designer drugs) which allow for real–time voltage monitoring and activation of mutated receptors by inert molecules for perturbing membrane potential (Vm). To uncover bioelectric activity during development, we generated a whole-zebrafish transgenic GEVI reporter line and characterized the electrical signaling during early embryogenesis using light sheet microscopy (LSM). Additionally, we generated tissue-specific transgenic lines that combined GEVIs and chemogenetic DREADD tools to manipulate Vm. We found zebrafish embryos display stage-specific characteristic bioelectric signals during the cleavage, blastula, gastrula, and segmentation periods. Furthermore, activation of DREADDs was able to alter cell-specific GEVI fluorescence intensity and could cause a melanophore hyperpigmentation phenotype. Ultimately, these results provide the first real-time systematic analysis of endogenous bioelectricity during vertebrate embryonic development. Additionally, we generated and tested zebrafish transgenic lines for simultaneous visualization and chemogenetic manipulation of Vm during development. These results provide a better understanding of developmental bioelectricity and new tools for future studies, which could eventually help uncover the cellular electric mechanisms behind tissue patterning and disease.</p>

Toll-like receptor (TLR) ligands and phytochemical feed additives (PFAs) were evaluated in this study to determine the effects of immune stimulation on gut immunity in the zebrafish, Danio rerio, and the channel catfish, Ictalurus punctatus. Rag1-/- (MT) zebrafish were used to study how the TLR ligands β-glucan and resiquimod (R848) affect the innate immune system in the gut of MT zebrafish. Enhanced expression of marker genes (NITR9, NCCRP-1 and MPEG-1) indicated stimulation of Natural Killer (NK) cells, non-specific cytotoxic cells (NCCs) and macrophages. After challenge with Edwardsiella ictaluri, MT zebrafish stimulated with β-glucan demonstrated higher survival and the presence of more macrophages/monocytes in the gut than control MT zebrafish. A PFA test diet containing a blend of prebiotic fiber, oregano, thyme, cinnamon essential oils, and Yucca schidigera (ONE Current™, OC) was fed to channel catfish for 3 months in ponds to determine the effect on channel catfish fingerling growth. Fish were fed in ponds and a tank bacterial challenge followed to test the efficacy of the product. Catfish fed OC demonstrated greater weight gain and feed conversion ratios, higher survival after challenge with E. ictaluri, greater phagocytosis or binding by macrophages and cytotoxic cells. Catfish fed OC also demonstrated greater gut surface area after 2 months feeding OC. To elucidate the effect(s) of each of the compounds in the OC diet on gut immune responses and to determine if PFAs can decrease bacterial colonization and replication within gut tissues, WT and MT zebrafish were fed diets containing different compounds included in OC. Quantification of live bacteria from gut and kidney tissue was determined after challenge with E. ictaluri. Expression levels of immune response genes were evaluated after ingestion of PFAs. Actifibe, Essential oil 25 ppm (EO 25) and Actifibe + EO demonstrated the lowest infection and colonization rate, upregulation of immune response genes, and significantly higher survival when challenged with E. ictaluri. This study demonstrates the potential for application of TLR ligand and feed administered PFAs to improve fish health. Our findings provide a more comprehensive understanding of host gut/pathogen interactions as well as suggestions for novel disease control measures.

Perturbation of cellular redox homeostasis to a more oxidized state has been linked to adverse human health effects such as diabetes and cancer. However, the impact of altering the regulation of redox homeostasis during development is not fully understood. Specifically, this project investigates the role of the Nrf2 antioxidant response pathway and its effect on glutathione (GSH; cellular redox buffer) at critical windows of development. To explore this, we used zebrafish embryos (Danio rerio) as a model due to the function of GSH and the Nrf2 being conserved among vertebrates. We exposed zebrafish embryos to three Nrf2 activators: two antioxidant enhancing molecules: sulforaphane (SFN; 40 µM) and Dimethyl fumarate (DFM; 7µM) as well as the pro-oxidant tert-Butylhydroquinone (tBHQ; 1µM) for 6 hours at critical windows development: 24, 48, 72 hours post fertilization (hpf). Following exposure, we visualized Nrf2 protein levels and glutathionylation rates using immunohistochemistry and confocal imaging. We found that changes in Nrf2 expression were dependent on the tissue type with there being significant changes in Nrf2 when looking at the pancreatic beta cells. Also in the beta cells, exposure to SFN, tBHQ, and DMF were found to increase Nrf2 translocation into the nucleus. Most notably, all three Nrf2 activators significantly altered glutathionylation levels depending on the time-point the zebrafish were exposed. SFN and tBHQ were also found to significantly increase glutathionylation at 48 and 72hpf, but led to a significant decrease at 96hpf while DMF increased glutathionylation at all three time-points. Interestingly, there was little correlation between Nrf2 protein levels and glutathionylation, but zebrafish with a mutated Nrf2 did have significantly different glutathionylation rates than the wild type fish. This suggests that oxidative stress is not the sole regulator of glutathionylation and instead Nrf2 may also be regulating glutathionylation through GSH storage. My data indicate that the effects of Nrf2 activation on Nrf2 levels and glutathionylation depend on the timing of exposure to the perturbing chemicals and the tissue type. Finding these windows of development where redox homeostasis is most sensitive in humans can allow for possible preventative and/or protective measures to oxidative stress during development.

Riboflavin transport is mediated, in part, by riboflavin transporter proteins 2 and 3, encoded by SLC52A2 and SLC52A3, respectively. Biallelic mutations in SLC52A2 and SLC52A3 impair riboflavin transporter protein function and riboflavin transport, causing disruptions to mitochondrial metabolism which result in sensory and motor neurodegeneration and give rise to riboflavin transporter deficiency (RTD) in humans. RTD is a rare neurodegenerative disease characterised by respiratory compromise, muscle and limb weakness, and vision and hearing impairments. RTD patients are treated with high-dose riboflavin supplementation which is effective in over 70% of cases but can be ineffective due to rapid excretion of riboflavin when its plasma concentration exceeds 0.5 μM. To address the need for alternative or supplemental RTD treatment, this study generated morpholino-mediated knockdown and CRISPR/Cas9 models of RTD in zebrafish. An RTD-like phenotype is observed in these RTD models including hearing loss, decreased motor axon length, and impaired locomotor activity. The slc52a3 morphant phenotype was found to be specific via coinjection of slc52a3 morpholino/human SLC52A3 mRNA, which achieved effective rescue of the morphant phenotype, as well as slc52a3 morpholino/p53 morpholino coinjection, which maintains the slc52a3 morphant phenotype. In line with clinical findings, riboflavin supplementation resulted in some improvement of the morphant phenotype. Probenecid was selected as a candidate drug due to its inhibitory effect on OAT-3, which mediates riboflavin excretion. However, supplementing riboflavin treatment with probenecid provided no additional benefit to the slc52a3 knockdown model. Further development of CRISPR/Cas9-knockout lines of slc52a2 and slc52a3, as well continued therapeutic screening of riboflavin and probenecid and consideration of alternative therapeutics will provide more opportunities to uncover novel therapeutic strategies to improve RTD treatment.

Thesis advisor: Sarah McMenamin / Pectoral fins are evolutionarily homologous to tetrapod limbs and can serve as useful models for studying the genetic and hormonal factors regulating appendage development. To test the roles of thyroid hormone (TH) in the development of these appendages, we examined the morphogenesis of zebrafish pectoral fins under euthyroid (EuTH) or hypothyroid (hypoTH) conditions. TH plays an important role in modulating the development of the pectoral fin endoskeleton, as well as the proximo-distal patterning of the fin rays. Additionally, in HypoTH fish, shh was expressed in the same domains, but at ~50% of WT expression levels. Nuclear TH acts by binding to dual-action receptors, including Thrab, which represses or activates expression depending on interaction with the TH ligand. When Thrab was absent in HypoTH fish, we found that many elements of the HypoTH phenotype were rescued, suggesting TH relieves Thrab- mediated repression. We also found that TH modulates the development of the musculature surrounding the pectoral fin. Lastly, we generated CRISPR-Mediated knockouts of dio1 and dio3b, to learn how the deiodination of TH may be affecting the development of the pectoral fin. / Thesis (BS) — Boston College, 2023. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Sex steroids are important hormones produced in the body to regulate an individual's physiology and behavior in preparation for reproduction. Aromatase (Cyp19a1) is the steroidogenic enzyme that converts aromatizable androgens into bioactive estrogens, and hence is in a pivotal position to mediate reproductive processes and sexual behavior. In mice, evidence from whole body aromatase knockout and estrogen receptor knockout lines has revealed the critical roles of estrogen signalling in ovarian development, gonadotropin signalling, ovulation, and sexual behavior. While the ovaries have high aromatase activity levels and are a major source of circulating estrogens in the female body, they can also be locally produced in tissues via tissue-specific aromatase expression. Of particular interest, the importance of brain-derived estrogens for reproductive processes and sexual behavior is still under study. Teleosts are an amenable model system for understanding the role of brain-derived estrogens in reproduction as they have two aromatase paralogs, cyp19a1a and cyp19a1b, that are highly expressed in the ovary and brain, respectively. In this thesis, I sought to identify the importance of brain-derived estrogens for sexual behaviors and reproductive health in zebrafish using cyp19a1⁻ᐟ⁻ mutant lines and a transgenic cyp19a1b-GFP line. In dyadic sexual behavior assays, female cyp19a1b⁻ᐟ⁻ mutants took 4.1 times longer to initiate spawning behavior with wildtype (WT) males compared to WT females. This suggested a potential impairment in mate identification and assessment in the female cyp19a1b⁻ᐟ⁻ mutants. The importance of the cyp19a1 paralogs for early larval development was revealed by 4 times higher progeny mortality from cyp19a1b⁻ᐟ⁻ mutant female and cyp19a1a⁻ᐟ⁻;cyp19a1b⁻ᐟ⁻ mutant male pairings compared to WT pairings. Gene expression analyses identified significantly lower levels of important neuroendocrine genes including the estrogen receptors and the nonapeptides, arginine vasopressin (avp) and oxytocin, in the telencephalon and hypothalamus of cyp19a1b⁻ᐟ⁻ mutant female compared to WT female brains. I performed acute intraperitoneal injections of Avp and Oxytocin, as well as mixtures of their respective receptor antagonists, in cyp19a1b⁻ᐟ⁻ mutant females to determine if the behavioral impairments could be rescued in adult fish. Arginine vasopressin was found to uniquely recover the delayed oviposition in female cyp19a1b⁻ᐟ⁻ mutants. Immunohistochemistry experiments using the transgenic cyp19a1b-GFP zebrafish line with a zebrafish-validated Avp antibody revealed a close neuroanatomical proximity and contact points between radial glial cell Cyp19a1b-expressing fibres and Avp-immunopositive neurons in preoptic areas. These findings suggest that brain-derived estrogens, via Cyp19a1b activity, might affect female sexual behavior by diffusing to nearby neurons to regulate Avp signalling levels in preoptic areas. Together, these findings establish the importance of cyp19a1b for female zebrafish sexual behavior and identify a positive link between Cyp19a1b and Avp. Future study can help to characterize the estrogen-dependent pathways involved in regulating Avp signalling in the female brain and the extent of evolutionary conservation of this regulation pathway for female sexual behavior across vertebrates.

No description available.