College instructors who learn how to teach actively at the beginning of their careers do not have to modify deeply set habits that rely on lectures. Instead, they develop their teaching while they learn evidence-based practices. We developed a workshop for the Science Teaching Experience Program: Working in Science Education (STEP-WISE), a professional development opportunity for postdoctoral scholars who want to learn how to teach effectively and gain undergraduate teaching experience. The program’s mission is to “engage a diverse pool of postdoctoral scholars at the University of Washington and affiliate institutions in a closely mentored apprenticeship to learn how to teach scientifically with inclusive, demonstrably effective, student-centered pedagogies.” In this workshop, STEP-WISE participants learn how to implement active learning techniques they can use in their own classes by experiencing inclusive strategies that include individual quick writes, think-pair-share, gallery walk, and a jigsaw. Through these activities, workshop participants experience the different learning techniques as they explore different aspects of teaching. Afterwards, they reflect on how they can use these same strategies in teaching their own classes. This workshop was developed for postdoctoral scholars who are new to active learning, but it can also work for graduate students, faculty, and others interested in the topic. Primary image: Teaching active learning actively. Our workshop participants discuss and reflect on several active learning strategies including individual write, gallery walk, and jigsaw while experiencing these strategies themselves. ­­

STEP Forward is a partnership between two established programs that train postdoctoral fellows in science communication: one that focuses on formal communication within college classrooms, and another that focuses on informal science communication at Pacific Science Center, a science museum. By combining these programs, postdoctoral fellows developed broad and more cohesive science communication skills. Here, we describe the components of STEP Forward and report the participants’ experiences as captured by open-ended responses from surveys administered before, during, and after their training at the museum. We use content analysis to examine their perceptions of what they gained from the program. Participants indicated that they eagerly took part in public events at Pacific Science Center, gained confidence in speaking with the general public, and valued the opportunity to bring their scientific research to a broader audience. Collectively, these results indicate that STEP Forward has a positive impact on the postdocs, especially given that the postdoctoral stage is one at which scholars begin to explore different careers and skill sets. We conclude that the program is beneficial, noting that while it is currently novel, it is also a replicable and feasible model for holistic postdoctoral training and science outreach.

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.

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.

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.

Modulation of Ca(2+) within cells is tightly regulated through complex and dynamic interactions between the plasma membrane and internal compartments. In this study, we exploit in vivo imaging strategies based on genetically encoded Ca(2+) indicators to define changes in perikaryal Ca(2+) concentration of intact photoreceptors. We developed double-transgenic zebrafish larvae expressing GCaMP3 in all cones and tdTomato in long-wavelength cones to test the hypothesis that photoreceptor degeneration induced by mutations in the phosphodiesterase-6 (Pde6) gene is driven by excessive [Ca(2+)]i levels within the cell body. Arguing against Ca(2+) overload in Pde6 mutant photoreceptors, simultaneous analysis of cone photoreceptor morphology and Ca(2+) fluxes revealed that degeneration of pde6c(w59) mutant cones, which lack the cone-specific cGMP phosphodiesterase, is not associated with sustained increases in perikaryal [Ca(2+)]i. Analysis of [Ca(2+)]i in dissociated Pde6β(rd1)mouse rods shows conservation of this finding across vertebrates. In vivo, transient and Pde6-independent Ca(2+) elevations ('flashes') were detected throughout the inner segment and the synapse. As the mutant cells proceeded to degenerate, these Ca(2+) fluxes diminished. This study thus provides insight into Ca(2+) dynamics in a common form of inherited blindness and uncovers a dramatic, light-independent modulation of [Ca(2+)]i that occurs in normal cones.

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.

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.

Ascidian metamorphosis transforms a free-swimming larval chordate ascidian into a sessile adult through a distinct series of metamorphic events. Initially, larvae must become competent to respond to settlement cues. Settlement is then marked by dramatic body plan remodeling and may be accompanied by attachment to the substrate. Subtractive hybridization has revealed that many innate immunity transcripts are upregulated during metamorphosis in the ascidian Boltenia villosa. Several of these genes have well-known roles in the mannose-binding lectin (MBL)-complement pathway of innate immunity, including MBL and MBL-activated serine protease (MASP). MBL recognizes and binds to bacterial pathogens, activates MASP, and triggers the complement cascade. In B. villosa, larvae upregulate BvMASP at the time of competency to initiate settlement. We show that several bacterial strains can induce settlement and that the timing of BvMASP expression in the papillae-associated tissue (PAT) cells is tightly correlated with larval competency. We further demonstrate that serine protease inhibitors used to block the complement response also block metamorphosis, allowing tail resorption, but preventing further morphological changes. Based on these experiments, we propose that the MBL-complement pathway may be important for competency, bacterial substrate detection and body plan remodeling during metamorphosis.