CRISPR-Generated Nrf2a Loss- and Gain-of-Function Mutants Facilitate Mechanistic Analysis of Chemical Oxidative Stress-Mediated Toxicity in Zebrafish.

The transcription factor Nrf2a induces a cellular antioxidant response and provides protection against chemical-induced oxidative stress, as well as playing a critical role in development and disease. Zebrafish are a powerful model to study the role of Nrf2a in these processes but have been limited by reliance on transient gene knockdown techniques or mutants with only partial functional alteration. We developed several lines of zebrafish carrying different null (loss of function, LOF) or hyperactive (gain of function, GOF) mutations to facilitate our understanding of the Nrf2a pathway in protecting against oxidative stress. The mutants confirmed Nrf2a dependence for induction of the antioxidant genes gclc, gstp, prdx1, and gpx1a and identified a role for Nrf2a in the baseline expression of these genes, as well as for sod1. Specifically, the 4-fold induction of gstp by tert-butyl hydroperoxide (tBHP) in wild type fish was abolished in LOF mutants. In addition, baseline gstp expression in GOF mutants increased by 12.6-fold and in LOF mutants was 0.8-fold relative to wild type. Nrf2a LOF mutants showed increased sensitivity to the acute toxicity of cumene hydroperoxide (CHP) and tBHP throughout the first 4 days of development. Conversely, GOF mutants were less sensitive to CHP toxicity during the first 4 days of development and were protected against the toxicity of both hydroperoxides after 4 dpf. Neither gain nor loss of Nrf2a modulated the toxicity of R-(-)-carvone (CAR), despite the ability of this compound to potently induce Nrf2a-dependent antioxidant genes. Similar to other species, GOF zebrafish mutants exhibited significant growth and survival defects. In summary, these new genetic tools can be used to facilitate the identification of downstream gene targets of Nrf2a, better define the role of Nrf2a in the toxicity of environmental chemicals, and further the study of diseases involving altered Nrf2a function.

Notch signaling regulates the extent of hair cell regeneration in the zebrafish lateral line.

Mechanosensory hair cells within the zebrafish lateral line spontaneously regenerate after aminoglycoside-induced death. Exposure of 5-d-old larvae to 400 microM neomycin for 1 h results in death of almost all lateral line hair cells. Regeneration of new hair cells is observed by 24 h after neomycin treatment with nearly complete replacement by 72 h. Using bromodeoxyuridine incorporation, we show that the majority of new hair cells are generated from a transient increase in support cell proliferation that occurs between 12 and 21 h after neomycin damage. Additional observations reveal two distinct subsets of proliferating support cells within the neuromasts that differ in position, morphology, and temporal pattern of proliferation in response to neomycin exposure. We hypothesize that proliferative hair cell progenitors are located centrally within the neuromasts, whereas peripheral support cells may have a separate function. Expression of Notch signaling pathway members notch3, deltaA, and atoh1a transcripts are all upregulated within the first 24 h after neomycin treatment, during the time of maximum proliferation of support cells and hair cell progenitor formation. Treatment with a gamma-secretase inhibitor results in excess regenerated hair cells by 48 h after neomycin-induced death but has no effect without previous damage. Excess hair cells result from increased support cell proliferation. These results suggest a model where Notch signaling limits the number of hair cells produced during regeneration by regulating support cell proliferation.

Differential copper-induced death and regeneration of olfactory sensory neuron populations and neurobehavioral function in larval zebrafish.

Fish rely heavily on their sense of smell to maintain behaviors essential for survival, such as predator detection and avoidance, prey selection, social behavior, imprinting, and homing to natal streams and spawning sites. Due to its direct contact with the outside environment, the peripheral olfactory system of fish is particularly susceptible to dissolved contaminants. In particular, environmental exposures to copper (Cu) can cause a rapid loss of olfactory function. In this study, confocal imaging of double-transgenic zebrafish larvae with differentially labeled ciliated and microvillous olfactory sensory neurons (OSNs) were used to examine cell death and regeneration following Cu exposure. Changes in cell morphologies were observed at varying degrees within both ciliated and microvillous OSNs, including the presence of round dense cell bodies, cell loss and fragmentation, retraction or loss of axons, disorganized cell arrangements, and loss of cells and fluorescence signal intensity, which are all indicators of cell death after Cu exposure. A marked loss of ciliated OSNs relative to microvillous OSNs occurred after exposure to low Cu concentrations for 3 h, with some regeneration observed after 72 h. At higher Cu concentrations and 24-h exposures, ciliated and microvillous OSNs were damaged with increased severity of injury with longer Cu exposures. Interestingly, microvillous, but not ciliated OSNs, regenerated rapidly within the 72-h time period of recovery after death from Cu exposure, suggesting that microvillous OSNs may be replaced in lieu of ciliated OSNs. An increase in bromodeoxyuridine labeling was observed 24 h after Cu-induced OSN death, suggesting that increased proliferation of the olfactory stem cells replaced the damaged OSNs. Olfactory behavioral analyses supported our imaging studies and revealed both initial loss and restoration of olfactory function after Cu exposures. In summary, our studies indicate that following zebrafish OSN damage by Cu, regeneration of microvillous OSNs may occur exceeding ciliated OSNs, likely via increased proliferation of the cellular reservoir of neuronal OSC precursors. Transgenic zebrafish are a valuable tool to study metal olfactory injury and recovery and to characterize sensitive olfactory neuron populations in fish exposed to environmental pollutants.

Synaptojanin 1 is required for endolysosomal trafficking of synaptic proteins in cone photoreceptor inner segments.

Highly polarized cells such as photoreceptors require precise and efficient strategies for establishing and maintaining the proper subcellular distribution of proteins. The signals and molecular machinery that regulate trafficking and sorting of synaptic proteins within cone inner segments is mostly unknown. In this study, we show that the polyphosphoinositide phosphatase Synaptojanin 1 (SynJ1) is critical for this process. We used transgenic markers for trafficking pathways, electron microscopy, and immunocytochemistry to characterize trafficking defects in cones of the zebrafish mutant, nrc(a14) , which is deficient in phosphoinositide phosphatase, SynJ1. The outer segments and connecting cilia of nrc(a14) cone photoreceptors are normal, but RibeyeB and VAMP2/synaptobrevin, which normally localize to the synapse, accumulate in the nrc(a14) inner segment. The structure of the Endoplasmic Reticulum in nrc(a14) mutant cones is normal. Golgi develop normally, but later become disordered. Large vesicular structures accumulate within nrc(a14) cone photoreceptor inner segments, particularly after prolonged incubation in darkness. Cone inner segments of nrc (a14) mutants also have enlarged acidic vesicles, abnormal late endosomes, and a disruption in autophagy. This last pathway also appears exacerbated by darkness. Taken altogether, these findings show that SynJ1 is required in cones for normal endolysosomal trafficking of synaptic proteins.

Signaling pathways regulating zebrafish lateral line development.

The lateral line organ is a mechanosensory organ of fish and amphibians that detects changes in water flow. The lateral line organ of zebrafish has been used as a model for cell polarity and collective cell migration as well as hair cell loss and regeneration. A combination of genetic tools and live imaging has allowed dissection of signaling pathways that regulate these processes. Here, we summarize recent findings on the roles of the FGF, Wnt/beta-catenin, and Notch pathways in the initial formation of the posterior lateral line primordium, as well as during organ patterning, migration, cell fate specification and hair cell regeneration.