Panlilio Jennifer M.
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ArticleGrxcr1 promotes hair bundle development by destabilizing the physical interaction between Harmonin and Sans usher syndrome proteins(Cell Press, 2018-10-30) Blanco-Sánchez, Bernardo ; Clément, Aurélie ; Fierro, Javier ; Stednitz, Sarah ; Phillips, Jennifer B. ; Wegner, Jeremy ; Panlilio, Jennifer M. ; Peirce, Judy L. ; Washbourne, Philip ; Westerfield, MonteMorphogenesis and mechanoelectrical transduction of the hair cell mechanoreceptor depend on the correct assembly of Usher syndrome (USH) proteins into highly organized macromolecular complexes. Defects in these proteins lead to deafness and vestibular areflexia in USH patients. Mutations in a non-USH protein, glutaredoxin domain-containing cysteine-rich 1 (GRXCR1), cause non-syndromic sensorineural deafness. To understand the deglutathionylating enzyme function of GRXCR1 in deafness, we generated two grxcr1 zebrafish mutant alleles. We found that hair bundles are thinner in homozygous grxcr1 mutants, similar to the USH1 mutants ush1c (Harmonin) and ush1ga (Sans). In vitro assays showed that glutathionylation promotes the interaction between Ush1c and Ush1ga and that Grxcr1 regulates mechanoreceptor development by preventing physical interaction between these proteins without affecting the assembly of another USH1 protein complex, the Ush1c- Cadherin23-Myosin7aa tripartite complex. By elucidating the molecular mechanism through which Grxcr1 functions, we also identify a mechanism that dynamically regulates the formation of Usher protein complexes.
PreprintDevelopmental exposure to domoic acid disrupts startle response behavior and circuitry in zebrafish(Oxford University Press, 2021-06-07) Panlilio, Jennifer M. ; Jones, Ian T. ; Salanga, Matthew C. ; Aluru, Neelakanteswar ; Hahn, Mark E.Harmful algal blooms produce potent neurotoxins that accumulate in seafood and are hazardous to human health. Developmental exposure to the harmful algal bloom toxin, domoic acid (DomA), has behavioral consequences well into adulthood, but the cellular and molecular mechanisms of DomA developmental neurotoxicity are largely unknown. To assess these, we exposed zebrafish embryos to DomA during the previously identified window of susceptibility and used the well-known startle response circuit as a tool to identify specific neuronal components that are targeted by exposure to DomA. Exposure to DomA reduced startle responsiveness to both auditory/vibrational and electrical stimuli, and even at the highest stimulus intensities tested, led to a dramatic reduction of one type of startle (short-latency c-starts). Furthermore, DomA-exposed larvae had altered kinematics for both types of startle responses tested, exhibiting shallower bend angles and slower maximal angular velocities. Using vital dye staining, immunolabeling, and live imaging of transgenic lines, we determined that although the sensory inputs were intact, the reticulospinal neurons required for short-latency c-starts were absent in most DomA-exposed larvae. Furthermore, axon tracing revealed that DomA-treated larvae also showed significantly reduced primary motor neuron axon collaterals. Overall, these results show that developmental exposure to DomA targets large reticulospinal neurons and motor neuron axon collaterals, resulting in measurable deficits in startle behavior. They further provide a framework for using the startle response circuit to identify specific neural populations disrupted by toxins or toxicants and to link these disruptions to functional consequences for neural circuit function and behavior.
ThesisImpacts of developmental exposures to the harmful algal bloom toxin domoic acid on neural development and behavior(Massachusetts Institute of Technology and Woods Hole Oceanographic Institution, 2019-06) Panlilio, Jennifer M.Harmful algal blooms (HABs) can produce potent neurotoxins that accumulate in seafood and affect human health. One HAB toxin of concern is domoic acid (DomA), a glutamate analog produced by the marine diatom Pseudo-nitzschia spp. Current regulatory limits are designed to prevent acute neurotoxicity in adult humans. However, research shows that low-level exposure during early life can lead to long-term changes in behavior, neural connectivity, and brain morphology. To determine the underlying mechanisms of developmental toxicity, this dissertation used zebrafish as a tool to: i) Establish the developmental window of susceptibility for DomA toxicity, ii) Characterize the behavioral consequences of exposures, and iii) Identify the cellular targets and processes perturbed by DomA. I found that DomA exposure particularly at 2 days post fertilization (dpf) led to altered startle response behavior, myelination defects, and the downregulation of axonal and myelin structural genes. Using vital dyes and immunolabeling, I assessed DomA-induced alterations in cells required for the startle response. I found no differences in the number of sensory neuromasts or in the sensory cranial ganglia structures that detect the acoustic stimuli. However, the majority of DomA-treated larvae lacked one or both Mauthner cells – hindbrain neurons critical for fast startle responses. DomA-treated larvae also had oligodendrocytes with fewer and shorter myelin sheaths, and appeared to aberrantly myelinate neuronal cell bodies. The loss of the Mauthner neurons and their axons may lead to a cellular environment where oligodendrocytes myelinate neuronal cell bodies in the absence of adequate axonal targets. Indeed, pharmacological treatment that reduced the oligodendrocyte number also led to the reduction in the number of these aberrant, myelinated cell bodies. These results indicate that exposure to DomA at a particular period in neural development targets specific cell types, disrupts myelination in the spinal cord, and leads to prolonged behavioral deficits. These mechanistic insights support hazard assessments of DomA exposures in humans during critical periods in early development.
ArticleDevelopmental neurotoxicity of the harmful algal bloom toxin domoic acid: Cellular and molecular mechanisms underlying altered behavior in the zebrafish model(National Institute of Environmental Health Sciences, 2020-11-04) Panlilio, Jennifer M. ; Aluru, Neelakanteswar ; Hahn, Mark E.Background: Harmful algal blooms (HABs) produce potent neurotoxins that threaten human health, but current regulations may not be protective of sensitive populations. Early life exposure to low levels of the HAB toxin domoic acid (DomA) produces long-lasting behavioral deficits in rodent and primate models; however, the mechanisms involved are unknown. The zebrafish is a powerful in vivo vertebrate model system for exploring cellular processes during development and thus may help to elucidate mechanisms of DomA developmental neurotoxicity. Objectives: We used the zebrafish model to investigate how low doses of DomA affect the developing nervous system, including windows of susceptibility to DomA exposure, structural and molecular changes in the nervous system, and the link to behavioral alterations. Methods: To identify potential windows of susceptibility, DomA (0.09–0.18 ng) was delivered to zebrafish through caudal vein microinjection during distinct periods in early neurodevelopment. Following exposure, structural and molecular targets were identified using live imaging of transgenic fish and RNA sequencing. To assess the functional consequences of exposures, we quantified startle behavior in response to acoustic/vibrational stimuli. Results: Larvae exposed to DomA at 2 d postfertilization (dpf), but not at 1 or 4 dpf, showed consistent deficits in startle behavior at 7 dpf, including lower responsiveness and altered kinematics. Similarly, myelination in the spinal cord was disorganized after exposure at 2 dpf but not 1 or 4 dpf. Time-lapse imaging revealed disruption of the initial stages of myelination. DomA exposure at 2 dpf down-regulated genes required for maintaining myelin structure and the axonal cytoskeleton. Discussion: These results in zebrafish reveal a developmental window of susceptibility to DomA-induced behavioral deficits and identify altered gene expression and disrupted myelin structure as possible mechanisms. The results establish a zebrafish model for investigating the mechanisms of developmental DomA toxicity, including effects with potential relevance to exposed sensitive human populations. https://doi.org/10.1289/EHP6652
ArticleDevelopmental exposure to non-dioxin-like polychlorinated biphenyls promotes sensory deficits and disrupts dopaminergic and GABAergic signaling in zebrafish(Nature Research, 2021-09-24) Brun, Nadja R. ; Panlilio, Jennifer M. ; Zhang, Kun ; Zhao, Yanbin ; Ivashkin, Evgeny ; Stegeman, John J. ; Goldstone, Jared V.The most abundant polychlorinated biphenyl (PCB) congeners found in the environment and in humans are neurotoxic. This is of particular concern for early life stages because the exposure of the more vulnerable developing nervous system to neurotoxic chemicals can result in neurobehavioral disorders. In this study, we uncover currently unknown links between PCB target mechanisms and neurobehavioral deficits using zebrafish as a vertebrate model. We investigated the effects of the abundant non-dioxin-like (NDL) congener PCB153 on neuronal morphology and synaptic transmission linked to the proper execution of a sensorimotor response. Zebrafish that were exposed during development to concentrations similar to those found in human cord blood and PCB contaminated sites showed a delay in startle response. Morphological and biochemical data demonstrate that even though PCB153-induced swelling of afferent sensory neurons, the disruption of dopaminergic and GABAergic signaling appears to contribute to PCB-induced motor deficits. A similar delay was observed for other NDL congeners but not for the potent dioxin-like congener PCB126. The effects on important and broadly conserved signaling mechanisms in vertebrates suggest that NDL PCBs may contribute to neurodevelopmental abnormalities in humans and increased selection pressures in vertebrate wildlife.