Transcriptomic comparison of two selective retinal cell ablation paradigms in zebrafish reveals shared and cell-specific regenerative responses.

Retinal Müller glia (MG) can act as stem-like cells to generate new neurons in both zebrafish and mice. In zebrafish, retinal regeneration is innate and robust, resulting in the replacement of lost neurons and restoration of visual function. In mice, exogenous stimulation of MG is required to reveal a dormant and, to date, limited regenerative capacity. Zebrafish studies have been key in revealing factors that promote regenerative responses in the mammalian eye. Increased understanding of how the regenerative potential of MG is regulated in zebrafish may therefore aid efforts to promote retinal repair therapeutically. Developmental signaling pathways are known to coordinate regeneration following widespread retinal cell loss. In contrast, less is known about how regeneration is regulated in the context of retinal degenerative disease, i.e., following the loss of specific retinal cell types. To address this knowledge gap, we compared transcriptomic responses underlying regeneration following targeted loss of rod photoreceptors or bipolar cells. In total, 2,531 differentially expressed genes (DEGs) were identified, with the majority being paradigm specific, including during early MG activation phases, suggesting the nature of the injury/cell loss informs the regenerative process from initiation onward. For example, early modulation of Notch signaling was implicated in the rod but not bipolar cell ablation paradigm and components of JAK/STAT signaling were implicated in both paradigms. To examine candidate gene roles in rod cell regeneration, including several immune-related factors, CRISPR/Cas9 was used to create G0 mutant larvae (i.e., "crispants"). Rod cell regeneration was inhibited in stat3 crispants, while mutating stat5a/b, c7b and txn accelerated rod regeneration kinetics. These data support emerging evidence that discrete responses follow from selective retinal cell loss and that the immune system plays a key role in regulating "fate-biased" regenerative processes.

NTR 2.0: a rationally engineered prodrug-converting enzyme with substantially enhanced efficacy for targeted cell ablation.

Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell-ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here we used rational engineering and cross-species screening to develop an NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell-loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community.

Structural Evaluation of a Nitroreductase Engineered for Improved Activation of the 5-Nitroimidazole PET Probe SN33623.

Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase (E. coli NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole. To elucidate the mechanism behind these enhancements, we resolved the crystal structure of the mutant enzyme to 1.98 Å and compared it to the wild-type enzyme. Structural analysis revealed an expanded substrate access channel and new hydrogen bonding interactions. Additionally, computational modeling of SN33623, CB1954, and metronidazole binding in the active sites of both the mutant and wild-type enzymes revealed key differences in substrate orientations and interactions, with improvements in activity being mirrored by reduced distances between the N5-H of isoalloxazine and the substrate nitro group oxygen in the mutant models. These findings deepen our understanding of nitroreductase substrate specificity and catalytic mechanisms and have potential implications for developing more effective theranostic imaging strategies in cancer treatment.

Notch Inhibition Promotes Regeneration and Immunosuppression Supports Cone Survival in a Zebrafish Model of Inherited Retinal Dystrophy.

Photoreceptor degeneration leads to irreversible vision loss in humans with retinal dystrophies such as retinitis pigmentosa. Whereas photoreceptor loss is permanent in mammals, zebrafish possesses the ability to regenerate retinal neurons and restore visual function. Following acute damage, Müller glia (MG) re-enter the cell cycle and produce multipotent progenitors whose progeny differentiate into mature neurons. Both MG reprogramming and proliferation of retinal progenitor cells require reactive microglia and associated inflammatory signaling. Paradoxically, in zebrafish models of retinal degeneration, photoreceptor death does not induce the MG to reprogram and regenerate lost cells. Here, we used male and female zebrafish cep290 mutants to demonstrate that progressive cone degeneration generates an immune response but does not stimulate MG proliferation. Acute light damage triggered photoreceptor regeneration in cep290 mutants but cones were only restored to prelesion densities. Using irf8 mutant zebrafish, we found that the chronic absence of microglia reduced inflammation and rescued cone degeneration in cep290 mutants. Finally, single-cell RNA-sequencing revealed sustained expression of notch3 in MG of cep290 mutants and inhibition of Notch signaling induced MG to re-enter the cell cycle. Our findings provide new insights on the requirements for MG to proliferate and the potential for immunosuppression to prolong photoreceptor survival.SIGNIFICANCE STATEMENT Inherited retinal degenerations (IRDs) are genetic diseases that lead to the progressive loss of photoreceptors and the permanent loss of vision. Zebrafish can regenerate photoreceptors after acute injury by reprogramming Müller glia (MG) into stem-like cells that produce retinal progenitors, but this regenerative process fails to occur in zebrafish models of IRDs. Here, we show that Notch pathway inhibition can promote photoreceptor regeneration in models of progressive degeneration and that immunosuppression can prevent photoreceptor loss. These results offer insight into the pathways that promote MG-dependent regeneration and the role of inflammation in photoreceptor degeneration.

The Crystal Structure of Engineered Nitroreductase NTR 2.0 and Impact of F70A and F108Y Substitutions on Substrate Specificity.

Bacterial nitroreductase enzymes that convert prodrugs to cytotoxins are valuable tools for creating transgenic targeted ablation models to study cellular function and cell-specific regeneration paradigms. We recently engineered a nitroreductase ("NTR 2.0") for substantially enhanced reduction of the prodrug metronidazole, which permits faster cell ablation kinetics, cleaner interrogations of cell function, ablation of previously recalcitrant cell types, and extended ablation paradigms useful for modelling chronic diseases. To provide insight into the enhanced enzymatic mechanism of NTR 2.0, we have solved the X-ray crystal structure at 1.85 Angstroms resolution and compared it to the parental enzyme, NfsB from Vibrio vulnificus. We additionally present a survey of reductive activity with eight alternative nitroaromatic substrates, to provide access to alternative ablation prodrugs, and explore applications such as remediation of dinitrotoluene pollutants. The predicted binding modes of four key substrates were investigated using molecular modelling.

Three-dimensional automated reporter quantification (3D-ARQ) technology enables quantitative screening in retinal organoids.

The advent of stem cell-derived retinal organoids has brought forth unprecedented opportunities for developmental and physiological studies, while presenting new therapeutic promise for retinal degenerative diseases. From a translational perspective, organoid systems provide exciting new prospects for drug discovery, offering the possibility to perform compound screening in a three-dimensional (3D) human tissue context that resembles the native histoarchitecture and to some extent recapitulates cellular interactions. However, inherent variability issues and a general lack of robust quantitative technologies for analyzing organoids on a large scale pose severe limitations for their use in translational applications. To address this need, we have developed a screening platform that enables accurate quantification of fluorescent reporters in complex human iPSC-derived retinal organoids. This platform incorporates a fluorescence microplate reader that allows xyz-dimensional detection and fine-tuned wavelength selection. We have established optimal parameters for fluorescent reporter signal detection, devised methods to compensate for organoid size variability, evaluated performance and sensitivity parameters, and validated this technology for functional applications.

Immunomodulation-accelerated neuronal regeneration following selective rod photoreceptor cell ablation in the zebrafish retina.

Müller glia (MG) function as inducible retinal stem cells in zebrafish, completely repairing the eye after damage. The innate immune system has recently been shown to promote tissue regeneration in which classic wound-healing responses predominate. However, regulatory roles for leukocytes during cellular regeneration-i.e., selective cell-loss paradigms akin to degenerative disease-are less well defined. To investigate possible roles innate immune cells play during retinal cell regeneration, we used intravital microscopy to visualize neutrophil, macrophage, and retinal microglia responses to induced rod photoreceptor apoptosis. Neutrophils displayed no reactivity to rod cell loss. Peripheral macrophage cells responded to rod cell loss, as evidenced by morphological transitions and increased migration, but did not enter the retina. Retinal microglia displayed multiple hallmarks of immune cell activation: increased migration, translocation to the photoreceptor cell layer, proliferation, and phagocytosis of dying cells. To test function during rod cell regeneration, we coablated microglia and rod cells or applied immune suppression and quantified the kinetics of (i) rod cell clearance, (ii) MG/progenitor cell proliferation, and (iii) rod cell replacement. Coablation and immune suppressants applied before cell loss caused delays in MG/progenitor proliferation rates and slowed the rate of rod cell replacement. Conversely, immune suppressants applied after cell loss had been initiated led to accelerated photoreceptor regeneration kinetics, possibly by promoting rapid resolution of an acute immune response. Our findings suggest that microglia control MG responsiveness to photoreceptor loss and support the development of immune-targeted therapeutic strategies for reversing cell loss associated with degenerative retinal conditions.

Rapid adaptive optical recovery of optimal resolution over large volumes.

Using a descanned, laser-induced guide star and direct wavefront sensing, we demonstrate adaptive correction of complex optical aberrations at high numerical aperture (NA) and a 14-ms update rate. This correction permits us to compensate for the rapid spatial variation in aberration often encountered in biological specimens and to recover diffraction-limited imaging over large volumes (>240 mm per side). We applied this to image fine neuronal processes and subcellular dynamics within the zebrafish brain.

Gfap-positive radial glial cells are an essential progenitor population for later-born neurons and glia in the zebrafish spinal cord.

Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial-derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ subtype lacking this activity. Fate mapping studies in this line indicate that gfap+ radial glia generate later-born CoSA interneurons, secondary motorneurons, and oligodendroglia. In another transgenic line using the gfap promoter-driven expression of the nitroreductase enzyme, we induced cell autonomous ablation of gfap+ radial glia and observed a reduction in their specific derived lineages, but not Blbp+ and Sox2+/gfap-negative NSCs, which were retained and expanded at later larval stages. Moreover, we provide evidence supporting classical roles of radial glial in axon patterning, blood-brain barrier formation, and locomotion. Our results suggest that gfap+ radial glia represent the major NSC during late neurogenesis for specific lineages, and possess diverse roles to sustain the structure and function of the spinal cord. These new tools will both corroborate the predicted roles of astroglia and reveal novel roles related to development, physiology, and regeneration in the vertebrate nervous system. GLIA 2016;64:1170-1189.

Three-Dimensional Vascular Network Assembly From Diabetic Patient-Derived Induced Pluripotent Stem Cells.

OBJECTIVE: In diabetics, hyperglycemia results in deficient endothelial progenitors and cells, leading to cardiovascular complications. We aim to engineer 3-dimensional (3D) vascular networks in synthetic hydrogels from type 1 diabetes mellitus (T1D) patient-derived human-induced pluripotent stem cells (hiPSCs), to serve as a transformative autologous vascular therapy for diabetic patients. APPROACH AND RESULTS: We validated and optimized an adherent, feeder-free differentiation procedure to derive early vascular cells (EVCs) with high portions of vascular endothelial cadherin-positive cells from hiPSCs. We demonstrate similar differentiation efficiency from hiPSCs derived from healthy donor and patients with T1D. T1D-hiPSC-derived vascular endothelial cadherin-positive cells can mature to functional endothelial cells-expressing mature markers: von Willebrand factor and endothelial nitric oxide synthase are capable of lectin binding and acetylated low-density lipoprotein uptake, form cords in Matrigel and respond to tumor necrosis factor-α. When embedded in engineered hyaluronic acid hydrogels, T1D-EVCs undergo morphogenesis and assemble into 3D networks. When encapsulated in a novel hypoxia-inducible hydrogel, T1D-EVCs respond to low oxygen and form 3D networks. As xenografts, T1D-EVCs incorporate into developing zebrafish vasculature. CONCLUSIONS: Using our robust protocol, we can direct efficient differentiation of T1D-hiPSC to EVCs. Early endothelial cells derived from T1D-hiPSC are functional when mature. T1D-EVCs self-assembled into 3D networks when embedded in hyaluronic acid and hypoxia-inducible hydrogels. The capability of T1D-EVCs to assemble into 3D networks in engineered matrices and to respond to a hypoxic microenvironment is a significant advancement for autologous vascular therapy in diabetic patients and has broad importance for tissue engineering.

Zebrafish rest regulates developmental gene expression but not neurogenesis.

The transcriptional repressor Rest (Nrsf) recruits chromatin-modifying complexes to RE1 'silencer elements', which are associated with hundreds of neural genes. However, the requirement for Rest-mediated transcriptional regulation of embryonic development and cell fate is poorly understood. Conflicting views of the role of Rest in controlling cell fate have emerged from recent studies. To address these controversies, we examined the developmental requirement for Rest in zebrafish using zinc-finger nuclease-mediated gene targeting. We discovered that germ layer specification progresses normally in rest mutants despite derepression of target genes during embryogenesis. This analysis provides the first evidence that maternal rest is essential for repression of target genes during blastula stages. Surprisingly, neurogenesis proceeds largely normally in rest mutants, although abnormalities are observed within the nervous system, including defects in oligodendrocyte precursor cell development and a partial loss of facial branchiomotor neuron migration. Mutants progress normally through embryogenesis but many die as larvae (after 12 days). However, some homozygotes reach adulthood and are viable. We utilized an RE1/NRSE transgenic reporter system to dynamically monitor Rest activity. This analysis revealed that Rest is required to repress gene expression in mesodermal derivatives including muscle and notochord, as well as within the nervous system. Finally, we demonstrated that Rest is required for long-term repression of target genes in non-neural tissues in adult zebrafish. Our results point to a broad role for Rest in fine-tuning neural gene expression, rather than as a widespread regulator of neurogenesis or cell fate.

The nitroreductase system of inducible targeted ablation facilitates cell-specific regenerative studies in zebrafish.

At the turn of the 20th century, classical regenerative biology--the study of organismal/tissue/limb regeneration in animals such as crayfish, snails, and planaria--garnered much attention. However, scientific luminaries such as Thomas Hunt Morgan eventually turned to other fields after concluding that inquiries into regenerative mechanisms were largely intractable beyond observational intrigues. The field of regeneration has enjoyed a resurgence in research activity at the turn of the 21st century, in large part due to "the promise" of cultured stem cells regarding reparative therapeutic approaches. Additionally, genomics-based methods that allow sophisticated genetic/molecular manipulations to be carried out in nearly any species have extended organismal regenerative biology well beyond observational limits. Throughout its history, complex paradigms such as limb regeneration--involving multiple tissue/cell types, thus, potentially multiple stem cell subtypes--have predominated the regenerative biology field. Conversely, cellular regeneration--the replacement of specific cell types--has been studied from only a few perspectives (predominantly muscle and mechanosensory hair cells). Yet, many of the degenerative diseases that regenerative biology hopes to address involve the loss of individual cell types; thus, a primary emphasis of the embryonic/induced stem cell field is defining culture conditions which promote cell-specific differentiation. Here we will discuss recent methodological approaches that promote the study of cell-specific regeneration. Such paradigms can reveal how the differentiation of specific cell types and regenerative potential of discrete stem cell niches are regulated. In particular, we will focus on how the nitroreductase (NTR) system of inducible targeted cell ablation facilitates: (1) large-scale genetic and chemical screens for identifying factors that regulate regeneration and (2) in vivo time-lapse imaging experiments aimed at investigating regenerative processes more directly. Combining powerful screening and imaging technologies with targeted ablation systems can expand our understanding of how individual stem cell niches are regulated. The former approach promotes the development of therapies aimed at enhancing regenerative potentials in humans, the latter facilitates investigation of phenomena that are otherwise difficult to resolve, such as the role of cellular transdifferentiation or the innate immune system in regenerative paradigms.

Evaluating human cancer cell metastasis in zebrafish.

BACKGROUND: In vivo metastasis assays have traditionally been performed in mice, but the process is inefficient and costly. However, since zebrafish do not develop an adaptive immune system until 14 days post-fertilization, human cancer cells can survive and metastasize when transplanted into zebrafish larvae. Despite isolated reports, there has been no systematic evaluation of the robustness of this system to date. METHODS: Individual cell lines were stained with CM-Dil and injected into the perivitelline space of 2-day old zebrafish larvae. After 2-4 days fish were imaged using confocal microscopy and the number of metastatic cells was determined using Fiji software. RESULTS: To determine whether zebrafish can faithfully report metastatic potential in human cancer cells, we injected a series of cells with different metastatic potential into the perivitelline space of 2 day old embryos. Using cells from breast, prostate, colon and pancreas we demonstrated that the degree of cell metastasis in fish is proportional to their invasion potential in vitro. Highly metastatic cells such as MDA231, DU145, SW620 and ASPC-1 are seen in the vasculature and throughout the body of the fish after only 24-48 hours. Importantly, cells that are not invasive in vitro such as T47D, LNCaP and HT29 do not metastasize in fish. Inactivation of JAK1/2 in fibrosarcoma cells leads to loss of invasion in vitro and metastasis in vivo, and in zebrafish these cells show limited spread throughout the zebrafish body compared with the highly metastatic parental cells. Further, knockdown of WASF3 in DU145 cells which leads to loss of invasion in vitro and metastasis in vivo also results in suppression of metastasis in zebrafish. In a cancer progression model involving normal MCF10A breast epithelial cells, the degree of invasion/metastasis in vitro and in mice is mirrored in zebrafish. Using a modified version of Fiji software, it is possible to quantify individual metastatic cells in the transparent larvae to correlate with invasion potential. We also demonstrate, using lung cancers, that the zebrafish model can evaluate the metastatic ability of cancer cells isolated from primary tumors. CONCLUSIONS: The zebrafish model described here offers a rapid, robust, and inexpensive means of evaluating the metastatic potential of human cancer cells. Using this model it is possible to critically evaluate whether genetic manipulation of signaling pathways affects metastasis and whether primary tumors contain metastatic cells.

Silencer-delimited transgenesis: NRSE/RE1 sequences promote neural-specific transgene expression in a NRSF/REST-dependent manner.

BACKGROUND: We have investigated a simple strategy for enhancing transgene expression specificity by leveraging genetic silencer elements. The approach serves to restrict transgene expression to a tissue of interest - the nervous system in the example provided here - thereby promoting specific/exclusive targeting of discrete cellular subtypes. Recent innovations are bringing us closer to understanding how the brain is organized, how neural circuits function, and how neurons can be regenerated. Fluorescent proteins enable mapping of the 'connectome', optogenetic tools allow excitable cells to be short-circuited or hyperactivated, and targeted ablation of neuronal subtypes facilitates investigations of circuit function and neuronal regeneration. Optimally, such toolsets need to be expressed solely within the cell types of interest as off-site expression makes establishing causal relationships difficult. To address this, we have exploited a gene 'silencing' system that promotes neuronal specificity by repressing expression in non-neural tissues. This methodology solves non-specific background issues that plague large-scale enhancer trap efforts and may provide a means of leveraging promoters/enhancers that otherwise express too broadly to be of value for in vivo manipulations. RESULTS: We show that a conserved neuron-restrictive silencer element (NRSE) can function to restrict transgene expression to the nervous system. The neuron-restrictive silencing factor/repressor element 1 silencing transcription factor (NRSF/REST) transcriptional repressor binds NRSE/repressor element 1 (RE1) sites and silences gene expression in non-neuronal cells. Inserting NRSE sites into transgenes strongly biased expression to neural tissues. NRSE sequences were effective in restricting expression of bipartite Gal4-based 'driver' transgenes within the context of an enhancer trap and when associated with a defined promoter and enhancer. However, NRSE sequences did not serve to restrict expression of an upstream activating sequence (UAS)-based reporter/effector transgene when associated solely with the UAS element. Morpholino knockdown assays showed that NRSF/REST expression is required for NRSE-based transgene silencing. CONCLUSIONS: Our findings demonstrate that the addition of NRSE sequences to transgenes can provide useful new tools for functional studies of the nervous system. However, the general approach may be more broadly applicable; tissue-specific silencer elements are operable in tissues other than the nervous system, suggesting this approach can be similarly applied to other paradigms. Thus, creating synthetic associations between endogenous regulatory elements and tissue-specific silencers may facilitate targeting of cellular subtypes for which defined promoters/enhancers are lacking.

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.

Nanoparticle-based targeting of microglia improves the neural regeneration enhancing effects of immunosuppression in the zebrafish retina.

Retinal Müller glia function as injury-induced stem-like cells in zebrafish but not mammals. However, insights gleaned from zebrafish have been applied to stimulate nascent regenerative responses in the mammalian retina. For instance, microglia/macrophages regulate Müller glia stem cell activity in the chick, zebrafish, and mouse. We previously showed that post-injury immunosuppression by the glucocorticoid dexamethasone accelerated retinal regeneration kinetics in zebrafish. Similarly, microglia ablation enhances regenerative outcomes in the mouse retina. Targeted immunomodulation of microglia reactivity may therefore enhance the regenerative potential of Müller glia for therapeutic purposes. Here, we investigated potential mechanisms by which post-injury dexamethasone accelerates retinal regeneration kinetics, and the effects of dendrimer-based targeting of dexamethasone to reactive microglia. Intravital time-lapse imaging revealed that post-injury dexamethasone inhibited microglia reactivity. The dendrimer-conjugated formulation: (1) decreased dexamethasone-associated systemic toxicity, (2) targeted dexamethasone to reactive microglia, and (3) improved the regeneration enhancing effects of immunosuppression by increasing stem/progenitor proliferation rates. Lastly, we show that the gene rnf2 is required for the enhanced regeneration effect of D-Dex. These data support the use of dendrimer-based targeting of reactive immune cells to reduce toxicity and enhance the regeneration promoting effects of immunosuppressants in the retina.

An inducible model of chronic hyperglycemia.

Transgene driven expression of Escherichia coli nitroreductase (NTR1.0) renders animal cells susceptible to the antibiotic metronidazole (MTZ). Many NTR1.0/MTZ ablation tools have been reported in zebrafish, which have significantly impacted regeneration studies. However, NTR1.0-based tools are not appropriate for modeling chronic cell loss as prolonged application of the required MTZ dose (10 mM) is deleterious to zebrafish health. We established that this dose corresponds to the median lethal dose (LD50) of MTZ in larval and adult zebrafish and that it induced intestinal pathology. NTR2.0 is a more active nitroreductase engineered from Vibrio vulnificus NfsB that requires substantially less MTZ to induce cell ablation. Here, we report on the generation of two new NTR2.0-based zebrafish lines in which acute ß-cell ablation can be achieved without MTZ-associated intestinal pathology. For the first time, we were able to sustain ß-cell loss and maintain elevated glucose levels (chronic hyperglycemia) in larvae and adults. Adult fish showed significant weight loss, consistent with the induction of a diabetic state, indicating that this paradigm will allow the modeling of diabetes and associated pathologies.

A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening.

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ~5-fold more effective than the canonical nitroreductase NfsB.

A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening.

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ∼5-fold more effective than the canonical nitroreductase NfsB.

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.