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.

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.

Comparison of Hyperangulated and Standard Geometry Video Laryngoscopy Tracheal Intubation for Prehospital Care in a Manikin: A Randomized Controlled Crossover Trial.

OBJECTIVE: The purpose of this study was to investigate the efficacy of hyperangulated video laryngoscopy (HAVL) versus standard geometry video laryngoscopy (SGVL) in the simulated prehospital environment using a manikin. There is consensus that video laryngoscopy (VL) can be very useful in the emergency department when difficult intubations are predicted. Emergency medical service (EMS) providers are also often faced with difficult, rapidly deteriorating airway management situations that not only involve patient and operator factors but also include challenging unique environmental factors, such as nonoptimized positions in transport vehicles (eg, helicopters and ambulances) or at ground level or entrapped positions. To our knowledge, there has never been a study purposefully investigating the efficacy of hyperangulated geometry versus standard geometry VL techniques in the prehospital environment. METHODS: A single-center, randomized controlled crossover trial was performed using attending physician helicopter EMS providers. Physicians were randomized to perform 5 HAVL or SGVL intubations followed by the subsequent technique. Intubations were performed on ground level and then repeated in the helicopter with the first location also randomized. A manikin airway management trainer was used to simulate intubation in each environment. The time to intubation (primary outcome) as well as first-pass success and the Cormack-Lehane view were recorded for each attempt. Qualitative data were also obtained for physician preference and perceived difficulty. RESULTS: There was no statistically significant difference in the time to intubation with HAVL versus SGVL (ground: 15.02 vs. 14.88 seconds, P = .86; helicopter: 16.11 vs. 16.14 seconds, P = .93). First-pass success was 100% for both techniques in both scenarios. More Grade 1 views were obtained with HAVL (147/150 vs. 134/150). Moreover, most physicians preferred HAVL overall and felt that HAVL required less force (9/15 grounded manikin and 10/15 helicopter manikin) and led to the best chance for first-pass success (11/15 grounded manikin and 10/15 helicopter manikin). CONCLUSION: The results of this study are limited because of the static and highly favorable anatomy of a manikin versus the variability and often difficult anatomy of individual patients. Our results suggest that both techniques are efficacious when the patient is both on the ground or in the helicopter, although provider preference does seem to vary.

Assessment of dynamic changes in cardiac function during resuscitation of patients with suspected septic shock: A prospective, observational, cohort study.

STUDY OBJECTIVE: To describe changes in cardiac function throughout the course of resuscitation of patients with suspected septic shock. METHODS: Prospective observational cohort study of Point-of-Care Transthoracic Echocardiograms (TTE) obtained in Emergency Department (ED) patients with a presumed infectious cause of hypotension within one hour of initiating IV fluid resuscitation. Findings of this pre-resuscitation TTE were compared to mid-resuscitation TTE (obtained upon disposition from the ED), and post-resuscitation TTE (obtained after admission to hospital). RESULTS: 22 enrolled patients had a second TTE available for comparison to the initial, pre-resuscitation TTE. 12 patients had a mid-resuscitation TTE and 16 patients had a post-resuscitation TTE. We observed a high incidence of changes on TTE during the clinical course of resuscitation (14/22 [64%]). Patients who developed LV or RV dysfunction during resuscitation were more likely to require vasopressor infusion and ICU admission (Spearman's coefficients [95% CI] of 0.68 [0.36-0.86] and 0.47 [0.04;0.75] respectively). Development of RV dysfunction alone was associated with increased use of positive pressure ventilation and vasopressor infusion (Spearman's coefficients [95% CI] of 0.43 [0;0.72] and 0.47 [0.05,0.75] respectively). CONCLUSIONS: Cardiac function changes assessed by TTE are common during the resuscitation of patients with septic shock. These changes likely reflect the underlying physiology of patients with septic shock and correlate with need for interventions and higher level of care. Further work is required to characterize these changes and to elucidate how to use these physiologic data to guide management.

Protocol for differentiation of monocytes and macrophages from human induced pluripotent stem cells.

Study of disease-relevant immune cells, namely monocytes and macrophages, is limited based on availability of primary tissue, a limitation that can be remedied using human induced pluripotent stem cell (hiPSC) technology. Here, we present a protocol for differentiation of monocytes and macrophages from hiPSCs. We describe steps for hiPSC maintenance, mesoderm lineage induction, hematopoietic progenitor cells (HPCs) commitment and expansion, and myeloid lineage induction. We then detail procedures for monocyte formation and functional macrophage formation and polarization. For complete details on the use and execution of this protocol, please refer to Chen et al.1.

Establishment of a human induced pluripotent stem cell line (NIHTVBi031-A) derived from a COPA syndrome patient with a heterozygous p.Ala239Pro mutation.

We have successfully generated human induced pluripotent stem cells (hiPSC) from peripheral blood mononuclear cells (PBMCs) of a patient with COPA Syndrome. The patient, a 6 year old Caucasian male, has a spontaneous de novo missense mutation that replaced alanine with proline in the COPA gene. This paper confirms the differentiation potential of the hiPSC line, the presence of the p.Ala239Pro mutation, and the expression of typical pluripotency markers within the hiPSC line. The hiPSC line is ready for use as a cellular model of COPA Syndrome.

Human induced pluripotent stem cells (NIHTVBi029-A and NIHTVBi030-A) generated from two patients with a heterozygous mutation in the CDC42 gene.

Induced pluripotent stem cells (iPSCs) were successfully generated from peripheral blood mononuclear cells obtained from two patients with a heterozygous mutation in the CDC42 gene. Both iPSC lines expressed pluripotency markers, differentiated into the three germ layers in vitro, showed normal karyotypes, and retained the disease-causing mutation. Created iPSC lines and their differentiated derivatives may be of interest in the study of the physiology of disease mechanisms and therapy.

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.

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.

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.

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.

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.

Automated In Vivo Phenotypic Screening Platform for Identifying Factors that Affect Cell Regeneration Kinetics.

Various strategies for replacing retinal neurons lost in degenerative diseases are under investigation, including stimulating the endogenous regenerative capacity of Müller Glia (MG) as injury-inducible retinal stem cells. Inherently regenerative species, such as zebrafish, have provided key insights into mechanisms regulating MG dedifferentiation to a stem-like state and the proliferation of MG and MG-derived progenitor cells (MGPCs). Interestingly, promoting MG/MGPC proliferation is not sufficient for regeneration, yet mechanistic studies are often focused on this measure. To fully account for the regenerative process, and facilitate screens for factors regulating cell regeneration, an assay for quantifying cell replacement is required. Accordingly, we adapted an automated reporter-assisted phenotypic screening platform to quantify the pace of cellular regeneration kinetics following selective cell ablation in larval zebrafish. Here, we detail a method for using this approach to identify chemicals and genes that control the rate of retinal cell regeneration following selective retinal cell ablation.