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Browsing by Author "Marrs, James A."
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Item Another Swim in the Extensive Pool of Zebrafish Research(MDPI, 2024-02-29) Marrs, James A.; Sarmah, Swapnalee; Biology, School of ScienceItem Behavioral Effects of the Mixture and the Single Compounds Carbendazim, Fipronil, and Sulfentrazone on Zebrafish (Danio rerio) Larvae(MDPI, 2024-05-25) Gomes, Samara da Silva; da Silva, Jadson Freitas; Padilha, Renata Meireles Oliveira; de Vasconcelos, João Victor Alves; Neto, Luís Gomes de Negreiros; Marrs, James A.; Cadena, Pabyton Gonçalves; Biology, School of SciencePesticides are often detected in freshwater, but their impact on the aquatic environment is commonly studied based on single compounds, underestimating the potential additive effects of these mixtures. Even at low concentrations, pesticides can negatively affect organisms, altering important behaviors that can have repercussions at the population level. This study used a multi-behavioral approach to evaluate the effects of zebrafish larvae exposure to carbendazim (C), fipronil (F), and sulfentrazone (S), individually and mixed. Five behavioral tests, thigmotaxis, touch sensitivity, optomotor response, bouncing ball test, and larval exploratory behavior, were performed to assess potential effects on anxiety, fear, and spatial and social interaction. Significant changes were observed in the performance of larvae exposed to all compounds and their mixtures. Among the single pesticides, exposure to S produced the most behavioral alterations, followed by F and C, respectively. A synergistic effect between the compounds was observed in the C + F group, which showed more behavioral effects than the groups exposed to pesticides individually. The use of behavioral tests to evaluate pesticide mixtures is important to standardize methods and associate behavioral changes with ecologically relevant events, thus creating a more realistic scenario for investigating the potential environmental impacts of these compounds.Item Cadherin-7 function in zebrafish development(Springer, 2008-10) Liu, Qin; Marrs, James A.; Londraville, Richard L.; Wilson, Amy L.; Department of Medicine, IU School of MedicineCadherin cell adhesion molecules play crucial roles in vertebrate development. Most studies have focused on examining the functions of classical type I cadherins (e.g., cadherin-2) in the development of vertebrates. Little information is available concerning the function of classical type II cadherins (e.g., cadherin-7) in vertebrate development. We have previously shown that cadherin-7 mRNA exhibits a dynamic expression pattern in the central nervous system and notochord in embryonic zebrafish. To gain insight into the role of cadherin-7 in the formation of these structures, we analyzed their formation in zebrafish embryos injected with cadherin-7-specific antisense morpholino oligonucleotides (MO). Notochord development was severely disrupted in MO-injected embryos, whereas gross defects in the development of the central nervous system were not detected in MO-injected embryos. Our results thus demonstrate that cadherin-7 plays an important role in the normal development of the zebrafish notochord.Item Characterization of Ethanol-induced Effects on Zebrafish Retinal Development: Mechanistic Perspective and Therapeutic Strategies(2016) Muralidharan, Pooja; Marrs, James A.; Leung, Yuk Fai; Belecky-Adams, Teri; Meyer, Jason; Anderson, Ryan M.; Randall, Stephen K.Fetal alcohol spectrum disorder (FASD) is a result of prenatal alcohol exposure, producing a wide range of defects including craniofacial, sensory, motor and cognitive deficits. Many ocular abnormalities are frequently associated with FASD including microphthalmia, optic nerve hypoplasia, and cataracts. FASD is highly prevalent in low socioeconomic populations, where it is also accompanied by higher rates of malnutrition and alcoholism. Using zebrafish as a model to study FASD retinal defects has been extremely insightful in understanding the ethanol-induced retinal defects at the cellular level. Zebrafish embryos treated with ethanol from mid-blastula transition through somitogenesis (2-24 hours post fertilization; hpf) showed defects similar to human ocular deficits including microphthalmia, optic nerve hypoplasia, and photoreceptor differentiation defects. Ethanol exposure altered critical transcription factor expression involved in retinal cell differentiation. Retinoic acid (RA) and folic acid (FA) nutrient co-supplementation rescued optic nerve and photoreceptor differentiation defects. Ethanol exposure during retinal morphogenesis stages (16-24 hpf), produced retinal defects like those seen with ethanol exposure between 2-24 hpf. Significantly, during ethanol-sensitive time window (16-24 hpf), RA co-supplementation moderately rescued these defects, whereas, FA cosupplementation showed significant rescue of optic nerve and photoreceptor differentiation. RA, but not FA, supplementation after ethanol exposure could restore ethanol-induced optic nerve and photoreceptor differentiation defects. Ethanol exposure did not affect timing of retinal cell differentiation induction, but later increased retinal cell death and proliferation. Ethanol-treated embryos showed increased retinal proliferation in the outer nuclear layer (ONL), inner nuclear layer (INL), and ciliary marginal zone (CMZ) at 48 hpf and 72 hpf. In order to identify the genesis of ethanol-induced persistent retinal defects, ethanol effects on retinal stem cell populations in the CMZ and the Müller glial cells (MGCs) were examined. Ethanol treated retinas had an expanded CMZ indicated by histology and Alcama, a retinal stem cell marker, immunolabeling, but reduced expression of rx1 and the cell cycle exit marker, cdkn1c. Ethanol treated retinas also showed reduced MGCs. At 72 hpf, ONL of ethanol exposed fish showed fewer photoreceptors expressing terminal differentiation markers. Importantly, these poorly differentiated photoreceptors co-expressed the basic helix-loop-helix (bHLH) proneural differentiation factor, neurod, indicating that ethanol exposure produced immature and undifferentiated photoreceptors. Reduced differentiation along with increased progenitor marker expression and proliferation suggest cell cycle exit failure due to ethanol exposure. These results suggested that ethanol exposure disrupted stem cell differentiation progression. Wnt, Notch and proneural gene expression regulate retinal stem cell proliferation and transition into progenitor cells. Ethanol exposure disrupted Wnt activity in the CMZ as well as Notch activity and neurod gene expression in the retina. RA and FA co-supplementation were able to rescue Wnt activity in the CMZ and rescue downstream Notch activity. To test whether the rescue of these Wnt-active cells could restore the retinal cell differentiation pathways, ethanol treated embryos were treated with Wnt agonist. This treatment could restore Wnt-active cells in the CMZ, Notch-active cells in the retina, proliferation, and photoreceptor terminal differentiation. We conclude that ethanol exposure produced persistent defects in the stem cell Wnt signaling, a critical pathway in retinal cell differentiation. Further analysis of underlying molecular mechanisms will provide insight into the embryonic origins of ethanol-induced retinal defects and potential therapeutic targets to cure this disorder.Item Clarifying the Role of the CST Complex in DNA Replication and Repair(2021-12) Wysong, Brandon Carter; Balakrishnan, Lata; Marrs, James A.; Perrin, Benjamin J.Ends of linear chromosomes are maintained by specialized structures known as telomeres. These structures are protected by a number of essential protein complexes including the shelterin complex and CST (CTC1 – STN1 – TEN1) complex. CST is an RPA-like ssDNA binding protein that is vital for telomere length maintenance via inhibition of telomerase and stimulation of DNA polymerase α -primase during C-strand fill-in synthesis. CST is also known to possess additional genome-wide roles in regulating DNA replication and repair including helping facilitate replication re-start at stalled forks, activating checkpoint signaling at double-strand breaks, and promoting replication origin firing. Proper and efficient repair of DNA is critical in order to protect the integrity of the genome and prevent extreme mutagenesis. Telomeres have a strong predisposition to oxidative DNA damage in the form of 8-oxoguanine caused by exposure to reactive oxygen species and free radicals. These oxidative lesions are repaired by the base-excision repair (BER) pathway. Previous work has implicated telomeric proteins such as the shelterin complex in mediating BER. Here we show for the first time that the CST complex and individual subunits robustly stimulate a myriad of proteins involved in the BER pathway including Pol β, APE1, FEN1, and LIGI. CST’s ability to augment these BER-associated proteins could be instrumental in promoting efficient DNA repair. Additionally, we find that CTC1 and STN1 are able to significantly enhance the polymerase activity of Pol δ and Pol α on both random-sequence and telomeric-sequence DNA substrates in vitro. What is more, we establish the ability of CST to resolve G4 structure and promote Pol δ synthesis, which we predict is a key feature of CST’s involvement in DNA replication at telomeres, which are known to form replication-inhibiting G4’s. Our results define important mechanistic insight into CST’s role in DNA replication and repair, and provide a strong foundation for future studies relating defective telomere maintenance to aging disorders and cancers which impact human health.Item Complex cardiac defects after ethanol exposure during discrete cardiogenic events in zebrafish: prevention with folic acid(Wiley, 2013-10) Swapnalee, Sarmah; Marrs, James A.; Biology, School of ScienceFetal alcohol spectrum disorder (FASD) describes a range of birth defects including various congenital heart defects (CHDs). Mechanisms of FASD-associated CHDs are not understood. Whether alcohol interferes with a single critical event or with multiple events in heart formation is not known. RESULTS: Our zebrafish embryo experiments showed that ethanol interrupts different cardiac regulatory networks and perturbs multiple steps of cardiogenesis (specification, myocardial migration, looping, chamber morphogenesis, and endocardial cushion formation). Ethanol exposure during gastrulation until cardiac specification or during myocardial midline migration did not produce severe or persistent heart development defects. However, exposure comprising gastrulation until myocardial precursor midline fusion or during heart patterning stages produced aberrant heart looping and defective endocardial cushions. Continuous exposure during entire cardiogenesis produced complex cardiac defects leading to severely defective myocardium, endocardium, and endocardial cushions. Supplementation of retinoic acid with ethanol partially rescued early heart developmental defects, but the endocardial cushions did not form correctly. In contrast, supplementation of folic acid rescued normal heart development, including the endocardial cushions. CONCLUSIONS: Our results indicate that ethanol exposure interrupted divergent cardiac morphogenetic events causing heart defects. Folic acid supplementation was effective in preventing a wide spectrum of ethanol-induced heart developmental defects.Item Dual Functions of the Protein MgtE in Pseudomonas aeruginosa(2012-07-03) Coffey, Barbara M.; Anderson, Gregory G.; Marrs, James A.; Randall, Stephen K.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen which readily establishes itself in the lungs of people with cystic fibrosis (CF). Most CF patients have life-long P. aeruginosa infections. By modulating its own virulence and forming biofilms, P. aeruginosa is able to evade both host immune responses and antibiotic treatments. Previous studies have shown that the magnesium transporter MgtE plays a role in virulence modulation by inhibiting transcription of the type III secretion system, a mechanism by which bacteria inject toxins directly into the eukaryotic host cell. MgtE had already been identified as a magnesium transporter, and thus its role in regulating cytotoxicity was indicative of dual functions for this protein. This research focused on a structure-function analysis of MgtE, with the hypothesis that the magnesium transport and cytotoxicity functions could be exerted independently. Cytotoxicity assays were conducted using a co-culture model system of cystic fibrosis bronchial epithelial cells and a ∆mgtE strain of P. aeruginosa transformed with plasmids carrying wild type or mutated mgtE. Magnesium transport was assessed using the same mgtE plasmids in a Salmonella strain deficient in all magnesium transporters. Through analysis of a number of mgtE mutants, we found two constructs – a mutation in a putative magnesium binding site, and an N-terminal truncation – which demonstrated a separation of functions. We further demonstrated the uncoupling of functions by showing that different mgtE mutants vary widely in their ability to regulate cytotoxicity, whether or not they are able to transport magnesium. Overall, these results support the hypothesis of MgtE as a dual function protein and may lead to a better understanding of the mechanisms underlying P. aeruginosa virulence. By understanding virulence mechanisms, we may be able to develop treatments to reduce infections and pave the way to better health for people with cystic fibrosis.Item Elucidating Cellular Mechanisms Underlying Retinal Ganglion Cell Neurodegeneration in a Human Pluripotent Stem Cell-Derived Model(2022-12) Huang, Kang-Chieh; Cummins, Theodore R.; Meyer, Jason S.; Marrs, James A.; Perrin, Benjamin J.; Lasagna Reeves, Cristian A.Glaucoma is a leading cause of blindness characterized by the progressive loss of retinal ganglion cells (RGCs), essentially severing the connection between the eye and the brain. Among many underlying causes of the disease, mutations in the Optineurin (OPTN) gene result in severe RGC neurodegeneration in the absence of elevated intraocular pressure, providing a novel opportunity to study molecular mechanisms that lead to RGC neurodegeneration associated with glaucoma. Efforts of this study establishing a human pluripotent stem cell (hPSC)-derived in vitro disease model by inserting OPTN(E50K) mutation via CRISPR/Cas9 genome editing and investigate the cellular mechanisms of RGC neurodegeneration associated with glaucoma. OPTN(E50K) RGCs revealed neurodegeneration phenotypes, including downregulation of RGCs transcription factors, neurite retraction, and hyperexcitability, suggesting that OPTN(E50K) RGCs can serve as an appropriate disease model to study glaucoma-associated neurodegeneration. Since OPTN serves a primary role as an autophagy receptor, we further hypothesized that the OPTN(E50K) mutation disrupts autophagy in RGCs, and modulation of autophagy by mammalian target of rapamycin (mTOR)-independent pathways can preserve RGC phenotypes by maintaining mTOR signaling. OPTN(E50K) RGCs exhibited a higher number of OPTN puncta along with an overall reduced expression of OPTN protein, indicating a gain of toxic protein accumulation or loss of protein function. Furthermore, OPTN(E50K) RGCs revealed an accumulation of the autophagosome protein LC3 in a punctal manner as well as increased expression of lysosomal proteins, suggesting a disruption of degradation pathway in autophagosome and lysosome fusion. As mTOR complex 1 (mTORC1) signaling serves as a negative regulator of autophagy, a downregulation of mTORC1 signaling via activation of stress sensor adenosine monophosphate-activated protein kinase (AMPK) was observed as a possible compensatory mechanism for autophagy deficits in OPTN(E50K) RGCs. Pharmacological inhibition of mTOR in wild-type hRGCs resulted in similar disease-related phenotypes, while preservation of the mTOR pathway in OPTN(E50K) RGCs by treatment with the mTOR-independent autophagy modulator trehalose cleared OPTN accumulated puncta, preserving mTORC1 signaling, as well as rescuing neurodegenerative phenotypes. To further validate these associations in an animal model, the microbead occlusion mouse model was established by injection of magnetic microbeads in the anterior chamber to block aqueous outflow resulting ocular hypertension. In agreement with our findings in hRGCs, a decrease in mTOR signaling associated with an increase in the expression of autophagy-associated proteins was observed in RGCs in the microbead occlusion model. Additionally, these disease-related phenotypes were observed specifically within RGCs but not cortical neurons with an underlying OPTN(E50K) mutation, demonstrating that autophagy represents an essential pathway in RGCs to maintain homeostasis, and selective disrupt of autophagy in RGCs leads to neurodegeneration. Taken together, the results of this study highlight an essential balance between autophagy and mTORC1 signaling that is essential for the homeostasis of RGCs, while disruption to these signaling pathways contributes to neurodegenerative features in glaucoma. These results also demonstrated the ability to pharmacologically intervene to experimentally manipulate these pathways and rescue neurodegenerative phenotypes, providing a potential therapeutic target to prevent glaucoma-associated neurodegeneration.Item Embryonic Ethanol Exposure Affects Early- and Late-Added Cardiac Precursors and Produces Long-Lasting Heart Chamber Defects in Zebrafish(MDPI, 2017-12-01) Sarmah, Swapnalee; Marrs, James A.; Biology, School of ScienceDrinking mothers expose their fetuses to ethanol, which produces birth defects: craniofacial defects, cognitive impairment, sensorimotor disabilities and organ deformities, collectively termed as fetal alcohol spectrum disorder (FASD). Various congenital heart defects (CHDs) are present in FASD patients, but the mechanisms of alcohol-induced cardiogenesis defects are not completely understood. This study utilized zebrafish embryos and older larvae to understand FASD-associated CHDs. Ethanol-induced cardiac chamber defects initiated during embryonic cardiogenesis persisted in later zebrafish life. In addition, myocardial damage was recognizable in the ventricle of the larvae that were exposed to ethanol during embryogenesis. Our studies of the pathogenesis revealed that ethanol exposure delayed differentiation of first and second heart fields and reduced the number of early- and late-added cardiomyocytes in the heart. Ethanol exposure also reduced the number of endocardial cells. Together, this study showed that ethanol-induced heart defects were present in late-stage zebrafish larvae. Reduced numbers of cardiomyocytes partly accounts for the ethanol-induced zebrafish heart defects.Item Embryonic ethanol exposure alters expression of sox2 and other early transcripts in zebrafish, producing gastrulation defects(Springer Nature, 2020-03-03) Sarmah, Swapnalee; Srivastava, Rajneesh; McClintick, Jeanette N.; Janga, Sarath C.; Edenberg, Howard J.; Marrs, James A.; Biology, School of ScienceEthanol exposure during prenatal development causes fetal alcohol spectrum disorder (FASD), the most frequent preventable birth defect and neurodevelopmental disability syndrome. The molecular targets of ethanol toxicity during development are poorly understood. Developmental stages surrounding gastrulation are very sensitive to ethanol exposure. To understand the effects of ethanol on early transcripts during embryogenesis, we treated zebrafish embryos with ethanol during pre-gastrulation period and examined the transcripts by Affymetrix GeneChip microarray before gastrulation. We identified 521 significantly dysregulated genes, including 61 transcription factors in ethanol-exposed embryos. Sox2, the key regulator of pluripotency and early development was significantly reduced. Functional annotation analysis showed enrichment in transcription regulation, embryonic axes patterning, and signaling pathways, including Wnt, Notch and retinoic acid. We identified all potential genomic targets of 25 dysregulated transcription factors and compared their interactions with the ethanol-dysregulated genes. This analysis predicted that Sox2 targeted a large number of ethanol-dysregulated genes. A gene regulatory network analysis showed that many of the dysregulated genes are targeted by multiple transcription factors. Injection of sox2 mRNA partially rescued ethanol-induced gene expression, epiboly and gastrulation defects. Additional studies of this ethanol dysregulated network may identify therapeutic targets that coordinately regulate early development.