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.

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.

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.

Multiplexed CRISPR/Cas9 Targeting of Genes Implicated in Retinal Regeneration and Degeneration.

Thousands of genes have been implicated in retinal regeneration, but only a few have been shown to impact the regenerative capacity of Müller glia-an adult retinal stem cell with untapped therapeutic potential. Similarly, among nearly 300 genetic loci associated with human retinal disease, the majority remain untested in animal models. To address the large-scale nature of these problems, we are applying CRISPR/Cas9-based genome modification strategies in zebrafish to target over 300 genes implicated in retinal regeneration or degeneration. Our intent is to enable large-scale reverse genetic screens by applying a multiplexed gene disruption strategy that markedly increases the efficiency of the screening process. To facilitate large-scale phenotyping, we incorporate an automated reporter quantification-based assay to identify cellular degeneration and regeneration-deficient phenotypes in transgenic fish. Multiplexed gene targeting strategies can address mismatches in scale between "big data" bioinformatics and wet lab experimental capacities, a critical shortfall limiting comprehensive functional analyses of factors implicated in ever-expanding multiomics datasets. This report details the progress we have made to date with a multiplexed CRISPR/Cas9-based gene targeting strategy and discusses how the methodologies applied can further our understanding of the genes that predispose to retinal degenerative disease and which control the regenerative capacity of retinal Müller glia cells.

mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay.

As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, a low frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of adaptations that can occur may inform the strategies of mutant generation.

Development of the larval lymphatic system in zebrafish.

The lymphatic vascular system is a hierarchically organized complex network essential for tissue fluid homeostasis, immune trafficking and absorption of dietary fats in the human body. Despite its importance, the assembly of the lymphatic network is still not fully understood. The zebrafish is a powerful model organism that enables study of lymphatic vessel development using high-resolution imaging and sophisticated genetic and experimental manipulation. Although several studies have described early lymphatic development in the fish, lymphatic development at later stages has not been completely elucidated. In this study, we generated a new Tg(mrc1a:egfp)y251 transgenic zebrafish that uses a mannose receptor, C type 1 (mrc1a) promoter to drive strong EGFP expression in lymphatic vessels at all stages of development and in adult zebrafish. We used this line to describe the assembly of the major vessels of the trunk lymphatic vascular network, including the later-developing collateral cardinal, spinal, superficial lateral and superficial intersegmental lymphatics. Our results show that major trunk lymphatic vessels are conserved in the zebrafish, and provide a thorough and complete description of trunk lymphatic vessel assembly.

T Cell Immune Deficiency in zap70 Mutant Zebrafish.

ZAP70 [zeta-chain (TCR)-associated protein kinase, 70-kDa], is required for T cell activation. ZAP70 deficiencies in humans and null mutations in mice lead to severe combined immune deficiency. Here, we describe a zap70 loss-of-function mutation in zebrafish (zap70 y442 ) that was created using transcription activator-like effector nucleases (TALENs). In contrast to what has been reported for morphant zebrafish, zap70 y442 homozygous mutant zebrafish displayed normal development of blood and lymphatic vasculature. Hematopoietic cell development was also largely unaffected in mutant larvae. However, mutant fish had reduced lck:GFP + thymic T cells by 5 days postfertilization that persisted into adult stages. Morphological analysis, RNA sequencing, and single-cell gene expression profiling of whole kidney marrow cells of adult fish revealed complete loss of mature T cells in zap70 y442 mutant animals. T cell immune deficiency was confirmed through transplantation of unmatched normal and malignant donor cells into zap70 y442 mutant zebrafish, with T cell loss being sufficient for robust allogeneic cell engraftment. zap70 mutant zebrafish show remarkable conservation of immune cell dysfunction as found in mice and humans and will serve as a valuable model to study zap70 immune deficiency.

Emerging from the PAC: studying zebrafish lymphatic development.

Recently the zebrafish has emerged as a promising vertebrate model of lymphatic vasculature development. The establishment of numerous transgenic lines that label the lymphatic endothelium in the zebrafish has allowed the fine examination of the developmental timing and the anatomy of their lymphatic vasculature. Although many questions remain, studying lymphatic development in the zebrafish has resulted in the identification and characterization of novel and established mediators of lymphatic development and lymphangiogenesis. Here, we review the main stages involved in the development of the lymphatic vasculature in the zebrafish from its origins in the embryonic veins to the formation of the primary lymphatic vessels and highlight some of the key molecules necessary for these stages.

Central and C-terminal domains of heterotrimeric G protein gamma subunits differentially influence the signaling necessary for primordial germ cell migration.

Heterotrimeric G protein signaling is involved in many pathways essential to development including those controlling cell migration, proliferation, differentiation and apoptosis. One key developmental event known to rely on proper heterotrimeric G protein signaling is primordial germ cell (PGC) migration. We previously developed an in vivo PGC migration assay that identified differences in the signaling capacity of G protein gamma subunits. In this study we developed Gγ subunit chimeras to determine the regions of Gγ isoforms that are responsible for these differences. The central section of the Gγ subunit was found to be necessary for the ability of a Gγ subunit to mediate signaling involved in PGC migration. Residues found in the carboxy-terminal segment of Gγ transducin (gngt1) were found to be responsible for the ability of this subunit to disrupt PGC migration. The type of prenylation did not affect the ability of a Gγ subunit to reverse prenylation-deficient-Gγ-induced PGC migration defects. However, a version of gng2, engineered to be farnesylated instead of geranylgeranylated, still lacks the ability to reverse PGC migration defects known to result from treatment of zebrafish with geranylgeranyl transferase inhibitors (GGTI), supporting the notion that Gγ subunits are one of several protein targets that need to be geranylgeranylated to orchestrate the proper long-range migration of PGCs.

Prenylation-deficient G protein gamma subunits disrupt GPCR signaling in the zebrafish.

Prenylation of G protein gamma (gamma) subunits is necessary for the membrane localization of heterotrimeric G proteins and for functional heterotrimeric G protein coupled receptor (GPCR) signaling. To evaluate GPCR signaling pathways during development, we injected zebrafish embryos with mRNAs encoding Ggamma subunits mutated so that they can no longer be prenylated. Low-level expression of these prenylation-deficient Ggamma subunits driven either ubiquitously or specifically in the primordial germ cells (PGCs) disrupts GPCR signaling and manifests as a PGC migration defect. This disruption results in a reduction of calcium accumulation in the protrusions of migrating PGCs and a failure of PGCs to directionally migrate. When co-expressed with a prenylation-deficient Ggamma, 8 of the 17 wildtype Ggamma isoforms individually confer the ability to restore calcium accumulation and directional migration. These results suggest that while the Ggamma subunits possess the ability to interact with G Beta (beta) proteins, only a subset of wildtype Ggamma proteins are stable within PGCs and can interact with key signaling components necessary for PGC migration. This in vivo study highlights the functional redundancy of these signaling components and demonstrates that prenylation-deficient Ggamma subunits are an effective tool to investigate the roles of GPCR signaling events during vertebrate development.

Disruptions and satisfaction in internal medicine resident continuity clinic differ between inpatient and outpatient rotations.

BACKGROUND: Little is known about whether assignment to simultaneous inpatient and outpatient clinical duties causes disruptions during internal medicine resident continuity clinic and impacts trainee satisfaction. PURPOSE: Our purpose was to determine whether dual inpatient and continuity clinic responsibilities impact resident stress and document the number, type, and immediacy of interruptions in continuity clinics. METHODS: Methods included a prospective 2-residency survey of 70 internal medicine residents performing 240 half-day continuity clinic sessions. RESULTS: More than half (52%) of trainees on inpatient rotations felt pressured to return to their ward duties. Half (50%) of residents thought clinic increased work hours, and the majority (70%) did not think continuity clinic detracted from their education on inpatient or elective rotations. Disturbances were more likely to occur on inpatient rotations (odds ratio 4.52, 95% confidence interval = 2.298.92) than on outpatient rotations. The time required to address an interruption was 3.9 +/- 4.51 min. Residents thought many (46%) problems addressed during clinic could have waited until clinic completion. CONCLUSIONS: Residents on inpatient rotations who were commonly interrupted in clinic felt pressured to return to ward duties and unable to focus on their clinic patients. Internal medicine faculty should modify curriculum to minimize the interference of other duties in resident clinics.

A "block and rescue" pharmacogenetic approach to dissecting a biochemical pathway controlling germ cell migration.

Specific small molecule inhibitors of the de novo cholesterol synthesis pathway (statins) and the protein prenylation pathway were used to study their effect on germ cell migration. Hydroxymethylglutaryl-Coenzyme A reductase (HMGCoAR) catalyzes the rate-limiting step in the mevalonate pathway that produces isoprenoids and cholesterol. Pharmacological HMGCoAR inhibition by statins alters zebrafish development and germ cell migration. These effects were completely blocked by prior injection of mevalonate, the product of HMGCoAR activity, or the prenylation precursors farnesol and geranylgeraniol. Finally, pharmacological inhibition of geranylgeranyl transferase I activity, an enzyme downstream from mevalonate synthesis and responsible for the transfer of a lipid to target proteins, resulted in abnormal germ cell migration. These data together with new data from Drosophila demonstrate that protein prenylation is an evolutionarily conserved pathway mediating germ cell migration. Further, pharmacological block-and-rescue approach provided detailed information about the elements of isoprenoid biosynthesis that contribute to germ cell migration. A key question raised by this work is the identity of the prenylated protein which facilitates proper germ cell migration. Work from other laboratories suggests that germ cell migration might be a general model for the long-range migration of other cell types including cancer metastasis.