A large-scale CRISPR screen reveals context-specific genetic regulation of retinal ganglion cell regeneration.

Many genes are known to regulate retinal regeneration after widespread tissue damage. Conversely, genes controlling regeneration after limited cell loss, as per degenerative diseases, are undefined. As stem/progenitor cell responses scale to injury levels, understanding how the extent and specificity of cell loss impact regenerative processes is important. Here, transgenic zebrafish enabling selective retinal ganglion cell (RGC) ablation were used to identify genes that regulate RGC regeneration. A single cell multiomics-informed screen of 100 genes identified seven knockouts that inhibited and 11 that promoted RGC regeneration. Surprisingly, 35 out of 36 genes known and/or implicated as being required for regeneration after widespread retinal damage were not required for RGC regeneration. The loss of seven even enhanced regeneration kinetics, including the proneural factors neurog1, olig2 and ascl1a. Mechanistic analyses revealed that ascl1a disruption increased the propensity of progenitor cells to produce RGCs, i.e. increased 'fate bias'. These data demonstrate plasticity in the mechanism through which Müller glia convert to a stem-like state and context specificity in how genes function during regeneration. Increased understanding of how the regeneration of disease-relevant cell types is specifically controlled will support the development of disease-tailored regenerative therapeutics.

Light wavelength modulates search behavior performance in zebrafish.

Visual systems have evolved to discriminate between different wavelengths of light. The ability to perceive color, or specific light wavelengths, is important as color conveys crucial information about both biotic and abiotic features in the environment. Indeed, different wavelengths of light can drive distinct patterns of activity in the vertebrate brain, yet what remains incompletely understood is whether distinct wavelengths can invoke etiologically relevant behavioral changes. To address how specific wavelengths in the visible spectrum modulate behavioral performance, we use larval zebrafish and a stereotypic light-search behavior. Prior work has shown that the cessation of light triggers a transitional light-search behavior, which we use to interrogate wavelength-dependent behavioral modulation. Using 8 narrow spectrum light sources in the visible range, we demonstrate that all wavelengths induce motor parameters consistent with search behavior, yet the magnitude of search behavior is spectrum sensitive and the underlying motor parameters are modulated in distinct patterns across short, medium, and long wavelengths. However, our data also establishes that not all motor features of search are impacted by wavelength. To define how wavelength modulates search performance, we performed additional assays with alternative wavelengths, dual wavelengths, and variable intensity. Last, we also tested blind larvae to resolve which components of wavelength dependent behavioral changes potentially include signaling from non-retinal photoreception. These findings have important implications as organisms can be exposed to varying wavelengths in laboratory and natural settings and therefore impose unique behavioral outputs.

Thalamic regulation of a visual critical period and motor behavior.

During the visual critical period (CP), sensory experience refines the structure and function of visual circuits. The basis of this plasticity was long thought to be limited to cortical circuits, but recently described thalamic plasticity challenges this dogma and demonstrates greater complexity underlying visual plasticity. Yet how visual experience modulates thalamic neurons or how the thalamus modulates CP timing is incompletely understood. Using a larval zebrafish, thalamus-centric ocular dominance model, we show functional changes in the thalamus and a role of inhibitory signaling to establish CP timing using a combination of functional imaging, optogenetics, and pharmacology. Hemisphere-specific changes in genetically defined thalamic neurons correlate with changes in visuomotor behavior, establishing a role of thalamic plasticity in modulating motor performance. Our work demonstrates that visual plasticity is broadly conserved and that visual experience leads to neuron-level functional changes in the thalamus that require inhibitory signaling to establish critical period timing.

Protocol for controlling visual experience during zebrafish development and modulation of motor behavior.

Sensory experience instructs neurodevelopment and refines sensory processing. Here, we describe a minimally invasive protocol to immobilize zebrafish during early development to control visual experience. We describe how to prepare larvae for embedding in agarose at two separate timepoints in development. Then we describe how to build a behavior rig and use software to track zebrafish behaviors. Finally, we detail analyzing behavioral data to validate the protocol and determine outcomes of sensory dependent plasticity. For complete details on the use and execution of this protocol, please refer to Hageter et al. (2023).1.

Thalamic neurons drive distinct forms of motor asymmetry that are conserved in teleost and dependent on visual evolution.

Brain laterality is a prominent feature in Bilateria, where neural functions are favored in a single brain hemisphere. These hemispheric specializations are thought to improve behavioral performance and are commonly observed as sensory or motor asymmetries, such as handedness in humans. Despite its prevalence, our understanding of the neural and molecular substrates instructing functional lateralization is limited. Moreover, how functional lateralization is selected for or modulated throughout evolution is poorly understood. While comparative approaches offer a powerful tool for addressing this question, a major obstacle has been the lack of a conserved asymmetric behavior in genetically tractable organisms. Previously, we described a robust motor asymmetry in larval zebrafish. Following the loss of illumination, individuals show a persistent turning bias that is associated with search pattern behavior with underlying functional lateralization in the thalamus. This behavior permits a simple yet robust assay that can be used to address fundamental principles underlying lateralization in the brain across taxa. Here, we take a comparative approach and show that motor asymmetry is conserved across diverse larval teleost species, which have diverged over the past 200 million years. Using a combination of transgenic tools, ablation, and enucleation, we show that teleosts exhibit two distinct forms of motor asymmetry, vision-dependent and - independent. These asymmetries are directionally uncorrelated, yet dependent on the same subset of thalamic neurons. Lastly, we leverage Astyanax sighted and blind morphs, which show that fish with evolutionarily derived blindness lack both retinal-dependent and -independent motor asymmetries, while their sighted surface conspecifics retained both forms. Our data implicate that overlapping sensory systems and neuronal substrates drive functional lateralization in a vertebrate brain that are likely targets for selective modulation during evolution.

Molecular regulation of retinal regeneration is context specific.

Many genes are known to regulate retinal regeneration following widespread tissue damage. Conversely, genes controlling regeneration following limited retinal cell loss, akin to disease conditions, are undefined. Combining a novel retinal ganglion cell (RGC) ablation-based glaucoma model, single cell omics, and rapid CRISPR/Cas9-based knockout methods to screen 100 genes, we identified 18 effectors of RGC regeneration kinetics. Surprisingly, 32 of 33 previously known/implicated regulators of retinal tissue regeneration were not required for RGC replacement; 7 knockouts accelerated regeneration, including sox2, olig2, and ascl1a . Mechanistic analyses revealed loss of ascl1a increased "fate bias", the propensity of progenitors to produce RGCs. These data demonstrate plasticity and context-specificity in how genes function to control regeneration, insights that could help to advance disease-tailored therapeutics for replacing lost retinal cells. One sentence summary: We discovered eighteen genes that regulate the regeneration of retinal ganglion cells in zebrafish.

Environmental and Molecular Modulation of Motor Individuality in Larval Zebrafish.

Innate behavioral biases such as human handedness are a ubiquitous form of inter-individual variation that are not strictly hardwired into the genome and are influenced by diverse internal and external cues. Yet, genetic and environmental factors modulating behavioral variation remain poorly understood, especially in vertebrates. To identify genetic and environmental factors that influence behavioral variation, we take advantage of larval zebrafish light-search behavior. During light-search, individuals preferentially turn in leftward or rightward loops, in which directional bias is sustained and non-heritable. Our previous work has shown that bias is maintained by a habenula-rostral PT circuit and genes associated with Notch signaling. Here we use a medium-throughput recording strategy and unbiased analysis to show that significant individual to individual variation exists in wildtype larval zebrafish turning preference. We classify stable left, right, and unbiased turning types, with most individuals exhibiting a directional preference. We show unbiased behavior is not due to a loss of photo-responsiveness but reduced persistence in same-direction turning. Raising larvae at elevated temperature selectively reduces the leftward turning type and impacts rostral PT neurons, specifically. Exposure to conspecifics, variable salinity, environmental enrichment, and physical disturbance does not significantly impact inter-individual turning bias. Pharmacological manipulation of Notch signaling disrupts habenula development and turn bias individuality in a dose dependent manner, establishing a direct role of Notch signaling. Last, a mutant allele of a known Notch pathway affecter gene, gsx2, disrupts turn bias individuality, implicating that brain regions independent of the previously established habenula-rostral PT likely contribute to inter-individual variation. These results establish that larval zebrafish is a powerful vertebrate model for inter-individual variation with established neural targets showing sensitivity to specific environmental and gene signaling disruptions. Our results provide new insight into how variation is generated in the vertebrate nervous system.