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.

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.

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.

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.

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.

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.

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.

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.

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.

Intrinsic epigenetic control of angiogenesis in induced pluripotent stem cell-derived endothelium regulates vascular regeneration.

Human-induced pluripotent stem cell-derived endothelial cells (iECs) provide opportunities to study vascular development and regeneration, develop cardiovascular therapeutics, and engineer model systems for drug screening. The differentiation and characterization of iECs are well established; however, the mechanisms governing their angiogenic phenotype remain unknown. Here, we aimed to determine the angiogenic phenotype of iECs and the regulatory mechanism controlling their regenerative capacity. In a comparative study with HUVECs, we show that iECs increased expression of vascular endothelial growth factor receptor 2 (VEGFR2) mediates their highly angiogenic phenotype via regulation of glycolysis enzymes, filopodia formation, VEGF mediated migration, and robust sprouting. We find that the elevated expression of VEGFR2 is epigenetically regulated via intrinsic acetylation of histone 3 at lysine 27 by histone acetyltransferase P300. Utilizing a zebrafish xenograft model, we demonstrate that the ability of iECs to promote the regeneration of the amputated fin can be modulated by P300 activity. These findings demonstrate how the innate epigenetic status of iECs regulates their phenotype with implications for their therapeutic potential.

A New Transgenic Line for Rapid and Complete Neutrophil Ablation.

Zebrafish lines expressing nitroreductase (NTR) in specific cell compartments, which sensitizes those cells to metronidazole (MTZ)-mediated ablation, have proven extremely useful for studying tissue regeneration and investigating cell function. In contrast to many cells, neutrophils are comparatively resistant to the NTR/MTZ targeted ablation strategy. Recently, a rationally engineered variant of NTR (NTR 2.0) has been described that exhibits greatly improved MTZ-mediated ablation efficacy in zebrafish. We show that a transgenic line with neutrophil-restricted expression of NTR 2.0 demonstrates complete neutrophil ablation, with an MTZ dose 100-fold less than current treatment regimens, and with treatment durations as short as 5 h.

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.

Mesoscopic oblique plane microscopy with a diffractive light-sheet for large-scale 4D cellular resolution imaging.

Fundamental understanding of large-scale dynamic connectivity within a living organism requires volumetric imaging over a large field of view (FOV) at biologically relevant speed and resolution. However, most microscopy methods make trade-offs between FOV and axial resolution, making it challenging to observe highly dynamic processes at cellular resolution in 3D across mesoscopic scales (e.g., whole zebrafish larva). To overcome this limitation, we have developed mesoscopic oblique plane microscopy (Meso-OPM) with a diffractive light sheet. By augmenting the illumination angle of the light sheet with a transmission grating, we improved the axial resolution approximately sixfold over existing methods and approximately twofold beyond the diffraction limitation of the primary objective lens. We demonstrated a FOV up to 5.4 mm × 3.3 mm with resolution of 2.5 µm × 3 µm × 6 µm, allowing volumetric imaging of 3D cellular structures with a single scan. Applying Meso-OPM for in vivo imaging of zebrafish larvae, we report here in toto whole-body volumetric recordings of neuronal activity at 2 Hz volume rate and whole-body volumetric recordings of blood flow dynamics at 5 Hz with 3D cellular resolution.

Large-scale phenotypic drug screen identifies neuroprotectants in zebrafish and mouse models of retinitis pigmentosa.

Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in multiple zebrafish and mouse RP models, reasoning drugs effective across species and/or independent of disease mutation may translate better clinically. We first performed a large-scale phenotypic drug screen for compounds promoting rod cell survival in a larval zebrafish model of inducible RP. We tested 2934 compounds, mostly human-approved drugs, across six concentrations, resulting in 113 compounds being identified as hits. Secondary tests of 42 high-priority hits confirmed eleven lead candidates. Leads were then evaluated in a series of mouse RP models in an effort to identify compounds effective across species and RP models, that is, potential pan-disease therapeutics. Nine of 11 leads exhibited neuroprotective effects in mouse primary photoreceptor cultures, and three promoted photoreceptor survival in mouse rd1 retinal explants. Both shared and complementary mechanisms of action were implicated across leads. Shared target tests implicated parp1-dependent cell death in our zebrafish RP model. Complementation tests revealed enhanced and additive/synergistic neuroprotective effects of paired drug combinations in mouse photoreceptor cultures and zebrafish, respectively. These results highlight the value of cross-species/multi-model phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for RP patients.

Photoreceptors are the cells responsible for vision. They are part of the retina: the light-sensing tissue at the back of the eye. They come in two types: rods and cones. Rods specialise in night vision, while cones specialise in daytime colour vision. The death of these cells can cause a disease, called retinitis pigmentosa, that leads to vision loss. Symptoms often start in childhood with a gradual loss of night vision. Later on, loss of cone photoreceptors can lead to total blindness. Unfortunately, there are no treatments available that protect photoreceptor cells from dying. Research has identified drugs that can protect photoreceptors in animal models, but these drugs have failed in humans. The classic way to look for new treatments is to find drugs that target molecules implicated in a disease, and then test them to see if they are effective. Unfortunately, many drugs identified in this way fail in later stages of testing, either because they are ineffective, or because they have unacceptable side effects. One way to reverse this trend is to first test whether a drug is effective at curing a disease in animals, and later determining what it does at a molecular level. This could reveal whether drugs can protect photoreceptors before research to discover their molecular targets begins. Tests like this across different species could maximise the chances of finding a drug that works in humans, because if a drug works in several species, it is more likely to have shared target molecules across species. Applying this reasoning, Zhang et al. tested around 3,000 drug candidates for treating retinitis pigmentosa in a strain of zebrafish that undergoes photoreceptor degeneration similar to the human disease. Most of these drug candidates already have approval for use in humans, meaning that if they were found to be effective for treating retinitis pigmentosa, they could be fast-tracked for use in people. Zhang et al. found three compounds that helped photoreceptors survive both in zebrafish and in retinas grown in the laboratory derived from a mouse strain with degeneration similar to retinitis pigmentosa. Tests to find out how these three compounds worked at the molecular level revealed that they interfered with a protein that can trigger cell death. The tests also found other promising compounds, many of which offered increased protection when combined in pairs. Worldwide there are between 1.5 and 2.5 million people with retinitis pigmentosa. With this disease, loss of vision happens slowly, so identifying drugs that could slow or stop the process could help many people. These results suggest that placing animal testing earlier in the drug discovery process could complement traditional target-based methods. The compounds identified here, and the information about how they work, could expand potential treatment research. The next step in this research is to test whether the drugs identified by Zhang et al. protect mammals other than mice from the degeneration seen in retinitis pigmentosa.

Engineering the Escherichia coli Nitroreductase NfsA to Create a Flexible Enzyme-Prodrug Activation System.

Bacterial nitroreductase enzymes that can efficiently convert nitroaromatic prodrugs to a cytotoxic form have numerous applications in targeted cellular ablation. For example, the generation of cytotoxic metabolites that have low bystander potential (i.e., are largely confined to the activating cell) has been exploited for precise ablation of specific cell types in animal and cell-culture models; while enzyme-prodrug combinations that generate high levels of bystander cell killing are useful for anti-cancer strategies such as gene-directed enzyme-prodrug therapy (GDEPT). Despite receiving substantial attention for such applications, the canonical nitroreductase NfsB from Escherichia coli has flaws that limit its utility, in particular a low efficiency of conversion of most prodrugs. Here, we sought to engineer a superior broad-range nitroreductase, E. coli NfsA, for improved activity with three therapeutically-relevant prodrugs: the duocarmycin analogue nitro-CBI-DEI, the dinitrobenzamide aziridine CB1954 and the 5-nitroimidazole metronidazole. The former two prodrugs have applications in GDEPT, while the latter has been employed for targeted ablation experiments and as a precise 'off-switch' in GDEPT models to eliminate nitroreductase-expressing cells. Our lead engineered NfsA (variant 11_78, with the residue substitutions S41Y, L103M, K222E and R225A) generated reduced metabolites of CB1954 and nitro-CBI-DEI that exhibited high bystander efficiencies in both bacterial and 2D HEK-293 cell culture models, while no cell-to-cell transfer was evident for the reduced metronidazole metabolite. We showed that the high bystander efficiency for CB1954 could be attributed to near-exclusive generation of the 2-hydroxylamine reduction product, which has been shown in 3D cell culture to cause significantly greater bystander killing than the 4-hydroxylamine species that is also produced by NfsB. We similarly observed a high bystander effect for nitro-CBI-DEI in HCT-116 tumor spheroids in which only a small proportion of cells were expressing variant 11_78. Collectively, our data identify variant 11_78 as a broadly improved prodrug-activating nitroreductase that offers advantages for both targeted cellular ablation and suicide gene therapy applications.

Drug screening with zebrafish visual behavior identifies carvedilol as a potential treatment for an autosomal dominant form of retinitis pigmentosa.

Retinitis Pigmentosa (RP) is a mostly incurable inherited retinal degeneration affecting approximately 1 in 4000 individuals globally. The goal of this work was to identify drugs that can help patients suffering from the disease. To accomplish this, we screened drugs on a zebrafish autosomal dominant RP model. This model expresses a truncated human rhodopsin transgene (Q344X) causing significant rod degeneration by 7 days post-fertilization (dpf). Consequently, the larvae displayed a deficit in visual motor response (VMR) under scotopic condition. The diminished VMR was leveraged to screen an ENZO SCREEN-WELL REDOX library since oxidative stress is postulated to play a role in RP progression. Our screening identified a beta-blocker, carvedilol, that ameliorated the deficient VMR of the RP larvae and increased their rod number. Carvedilol may directly on rods as it affected the adrenergic pathway in the photoreceptor-like human Y79 cell line. Since carvedilol is an FDA-approved drug, our findings suggest that carvedilol can potentially be repurposed to treat autosomal dominant RP patients.

Conditional reprogramming culture conditions facilitate growth of lower-grade glioma models.

BACKGROUND: The conditional reprogramming cell culture method was developed to facilitate growth of senescence-prone normal and neoplastic epithelial cells, and involves co-culture with irradiated fibroblasts and the addition of a small molecule Rho kinase (ROCK) inhibitor. The aim of this study was to determine whether this approach would facilitate the culture of compact low-grade gliomas. METHODS: We attempted to culture 4 pilocytic astrocytomas, 2 gangliogliomas, 2 myxopapillary ependymomas, 2 anaplastic gliomas, 2 difficult-to-classify low-grade neuroepithelial tumors, a desmoplastic infantile ganglioglioma, and an anaplastic pleomorphic xanthoastrocytoma using a modified conditional reprogramming cell culture approach. RESULTS: Conditional reprogramming resulted in robust increases in growth for a majority of these tumors, with fibroblast conditioned media and ROCK inhibition both required. Switching cultures to standard serum containing media, or serum-free neurosphere conditions, with or without ROCK inhibition, resulted in decreased proliferation and induction of senescence markers. Rho kinase inhibition and conditioned media both promoted Akt and Erk1/2 activation. Several cultures, including one derived from a NF1-associated pilocytic astrocytoma (JHH-NF1-PA1) and one from a BRAF p.V600E mutant anaplastic pleomorphic xanthoastrocytoma (JHH-PXA1), exhibited growth sufficient for preclinical testing in vitro. In addition, JHH-NF1-PA1 cells survived and migrated in larval zebrafish orthotopic xenografts, while JHH-PXA1 formed orthotopic xenografts in mice histopathologically similar to the tumor from which it was derived. CONCLUSIONS: These studies highlight the potential for the conditional reprogramming cell culture method to promote the growth of glial and glioneuronal tumors in vitro, in some cases enabling the establishment of long-term culture and in vivo models.

Downregulation of Nodal inhibits metastatic progression in retinoblastoma.

Retinoblastoma is the most common intraocular malignancy in children. We previously found that the ACVR1C/SMAD2 pathway is significantly upregulated in invasive retinoblastoma samples from patients. Here we studied the role of an ACVR1C ligand, Nodal, in regulating growth and metastatic dissemination in retinoblastoma. Inhibition of Nodal using multiple short hairpin (shRNAs) in WERI Rb1 and Y79 retinoblastoma cell cultures reduced growth by more than 90%, as determined by CCK-8 growth assay. Proliferation was also significantly inhibited, as found by Ki67 assay. These effects were paralleled by inhibition in the phosphorylation of the downstream effector SMAD2, as well as induction of apoptosis, as we observed more than three-fold increase in the percentage of cells positive for cleaved-caspase-3 or expressing cleaved-PARP1. Importantly, we found that downregulation of Nodal potently suppressed invasion in vitro, by 50 to 80%, as determined by transwell invasion assay (p = 0.02). Using an orthotopic model of retinoblastoma in zebrafish, we found 34% reduction in the ability of the cells to disseminate outside the eye, when Nodal was knocked down by shRNA (p = 0.0003). These data suggest that Nodal plays an important role in promoting growth, proliferation and invasion in retinoblastoma, and can be considered a new therapeutic target for both primary tumor growth and metastatic progression.

Let's get small (and smaller): Combining zebrafish and nanomedicine to advance neuroregenerative therapeutics.

Several key attributes of zebrafish make them an ideal model system for the discovery and development of regeneration promoting therapeutics; most notably their robust capacity for self-repair which extends to the central nervous system. Further, by enabling large-scale drug discovery directly in living vertebrate disease models, zebrafish circumvent critical bottlenecks which have driven drug development costs up. This review summarizes currently available zebrafish phenotypic screening platforms, HTS-ready neurodegenerative disease modeling strategies, zebrafish small molecule screens which have succeeded in identifying regeneration promoting compounds and explores how intravital imaging in zebrafish can facilitate comprehensive analysis of nanocarrier biodistribution and pharmacokinetics. Finally, we discuss the benefits and challenges attending the combination of zebrafish and nanoparticle-based drug optimization, highlighting inspiring proof-of-concept studies and looking toward implementation across the drug development community.

ACVR1C/SMAD2 signaling promotes invasion and growth in retinoblastoma.

Retinoblastoma is the most common intraocular cancer in children. While the primary tumor can often be treated by local or systemic chemotherapy, metastatic dissemination is generally resistant to therapy and remains a leading cause of pediatric cancer death in much of the world. In order to identify new therapeutic targets in aggressive tumors, we sequenced RNA transcripts in five snap frozen retinoblastomas which invaded the optic nerve and five which did not. A three-fold increase was noted in mRNA levels of ACVR1C/ALK7, a type I receptor of the TGF-ß family, in invasive retinoblastomas, while downregulation of DACT2 and LEFTY2, negative modulators of the ACVR1C signaling, was observed in most invasive tumors. A two- to three-fold increase in ACVR1C mRNA was also found in invasive WERI Rb1 and Y79 cells as compared to non-invasive cells in vitro. Transcripts of ACVR1C receptor and its ligands (Nodal, Activin A/B, and GDF3) were expressed in six retinoblastoma lines, and evidence of downstream SMAD2 signaling was present in all these lines. Pharmacological inhibition of ACVR1C signaling using SB505124, or genetic downregulation of the receptor using shRNA potently suppressed invasion, growth, survival, and reduced the protein levels of the mesenchymal markers ZEB1 and Snail. The inhibitory effects on invasion, growth, and proliferation were recapitulated by knocking down SMAD2, but not SMAD3. Finally, in an orthotopic zebrafish model of retinoblastoma, a 55% decrease in tumor spread was noted (p = 0.0026) when larvae were treated with 3 µM of SB505124, as compared to DMSO. Similarly, knockdown of ACVR1C in injected tumor cells using shRNA also resulted in a 54% reduction in tumor dissemination in the zebrafish eye as compared to scrambled shRNA control (p = 0.0005). Our data support a role for the ACVR1C/SMAD2 pathway in promoting invasion and growth of retinoblastoma.