Comparing robotic and manual injection methods in zebrafish embryos for high-throughput RNA silencing using CRISPR-RfxCas13d.

In this study, the authors compared the efficiency of automated robotic and manual injection methods for the CRISPR-RfxCas13d (CasRx) system for mRNA knockdown and Cas9-mediated DNA targeting in zebrafish embryos. They targeted the no tail (TBXTA) gene as a proof-of-principle, evaluating the induced embryonic phenotypes. Both Cas9 and CasRx systems caused loss of function phenotypes for TBXTA. Cas9 protein exhibited a higher percentage of severe phenotypes compared with mRNA, while CasRx protein and mRNA showed similar efficiency. Both robotic and manual injections demonstrated comparable phenotype percentages and mortality rates. The findings highlight the potential of RNA-targeting CRISPR effectors for precise gene knockdown and endorse automated microinjection at a speed of 1.0 s per embryo as a high-throughput alternative to manual methods.

Clinical validation of RCSMS: A rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva.

Peru's holds the highest COVID death rate per capita worldwide. Key to this outcome is the lack of robust, rapid, and accurate molecular tests to circumvent the elevated costs and logistics of SARS-CoV-2 detection via RT-qPCR. To facilitate massive and timely COVID-19 testing in rural and socioeconomically deprived contexts, we implemented and validated RCSMS, a rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva. RCSMS uses the power of CRISPR-Cas technology and lateral flow strips to easily visualize the presence of SARS-CoV-2 even in laboratories with limited equipment. We show that a low-cost thermochemical treatment with TCEP/EDTA is sufficient to inactivate viral particles and cellular nucleases in saliva, eliminating the need to extract viral RNA with commercial kits, as well as the cumbersome nasopharyngeal swab procedure and the requirement of biosafety level 2 laboratories for molecular analyses. Notably, RCSMS performed outstandingly in a clinical validation done with 352 patients from two hospitals in Lima, detecting as low as 50 viral copies per 10 µl reaction in 40 min, with sensitivity and specificity of 96.5% and 99.0%, respectively, relative to RT-qPCR. The negative and positive predicted values obtained from this field validation indicate that RCSMS can be confidently deployed in both high and low prevalence settings. Like other CRISPR-Cas-based biosensors, RCSMS can be easily reprogrammed for the detection of new SARS-CoV-2 variants. We conclude that RCSMS is a fast, efficient and inexpensive alternative to RT-qPCR for expanding COVID-19 testing capacity in Peru and other low- and middle-income countries with precarious healthcare systems.

Fish models in prion biology: underwater issues.

Transmissible spongiform encephalopathies (TSEs), otherwise known as prion disorders, are fatal diseases causing neurodegeneration in a wide range of mammalian hosts, including humans. The causative agents - prions - are thought to be composed of a rogue isoform of the endogenous prion protein (PrP). Beyond these and other basic concepts, fundamental questions in prion biology remain unanswered, such as the physiological function of PrP, the molecular mechanisms underlying prion pathogenesis, and the origin of prions. To date, the occurrence of TSEs in lower vertebrates like fish and birds has received only limited attention, despite the fact that these animals possess bona fide PrPs. Recent findings, however, have brought fish before the footlights of prion research. Fish models are beginning to provide useful insights into the roles of PrP in health and disease, as well as the potential risk of prion transmission between fish and mammals. Although still in its infancy, the use of fish models in TSE research could significantly improve our basic understanding of prion diseases, and also help anticipate risks to public health. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.

Origin of Nogo-A by domain shuffling in an early jawed vertebrate.

Unlike mammals, fish are able to regenerate axons in their central nervous system. This difference has been partly attributed to the loss/acquisition of inhibitory proteins during evolution. Nogo-A--the longest isoform of the reticulon4 (rtn4) gene product--is commonly found in mammalian myelin where it acts as a potent inhibitor of axonal regeneration. Interestingly, fish RTN4 isoforms were previously reported to lack the most inhibitory Nogo-A-specific region (NSR). Nevertheless, fish axons collapse on contact with mammalian NSR, suggesting that fish possess a functional Nogo-A receptor but not its ligand. To reconcile these findings, we revisited the early evolution of rtn4. Mining of current genome databases established the unequivocal presence of NSR-coding sequences in fish rtn4 paralogues. Further comparative analyses indicate that the common ancestor of fish and tetrapods had an NSR-coding rtn4 gene, which underwent duplication and divergent evolution in bony fish. Our genomic survey also revealed that the cephalochordate Branchiostoma floridae contains a single rtn gene lacking the NSR. Hence, Nogo-A most probably arose independently in the rtn4 gene of a gnathostome ancestor before the split of the fish and tetrapod lineages. Close examination of the NSR uncovered clusters of structural and sequential similarities with neurocan (NCAN), an inhibitory proteoglycan of the glial scar. Notably, the shared presence of transposable elements in ncan and rtn4 genes suggests that Nogo-A originated via insertion of an ncan-like sequence into the rtn4 gene of an early jawed vertebrate with myelinated axons.

Regulation of embryonic cell adhesion by the prion protein.

Prion proteins (PrPs) are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1) mediates Ca(+2)-independent homophilic cell adhesion and signaling; and (2) modulates Ca(+2)-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin-based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development.

Reggies/flotillins regulate retinal axon regeneration in the zebrafish optic nerve and differentiation of hippocampal and N2a neurons.

The reggies/flotillins--proteins upregulated during axon regeneration in retinal ganglion cells (RGCs)--are scaffolding proteins of microdomains and involved in neuronal differentiation. Here, we show that reggies regulate axon regeneration in zebrafish (ZF) after optic nerve section (ONS) in vivo as well as axon/neurite extension in hippocampal and N2a neurons in vitro through signal transduction molecules modulating actin dynamics. ZF reggie-1a, -2a, and -2b downregulation by reggie-specific morpholino (Mo) antisense oligonucleotides directly after ONS significantly reduced ZF RGC axon regeneration: RGC axons from reggie Mo retinas were markedly reduced. Moreover, the number of axon-regenerating RGCs, identified by insertion of A488-coupled dextran, decreased by 69% in retinas 7 d after Mo application. At 10 and 14 d, RGCs decreased by 53 and 33%, respectively, in correlation with the gradual inactivation of the Mos. siRNA-mediated knockdown of reggie-1 and -2 inhibited the differentiation and axon/neurite extension in hippocampal and N2a neurons. N2a cells had significantly shorter filopodia, more cells had lamellipodia and fewer neurites, defects which were rescued by a reggie-1 construct without siRNA-binding sites. Furthermore, reggie knockdown strongly perturbed the balanced activation of the Rho family GTPases Rac1, RhoA, and cdc42, influenced the phosphorylation of cortactin and cofilin, the formation of the N-WASP, cortactin and Arp3 complex, and affected p38, Ras, ERK1/2 (extracellular signal-regulated kinases 1 and 2), and focal adhesion kinase activation. Thus, as suggested by their prominent re-expression after lesion, the reggies represent neuron-intrinsic factors for axon outgrowth and regeneration, being crucial for the coordinated assembly of signaling complexes regulating cytoskeletal remodeling.

CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos.

Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to degrade RNA in yeast, plants, and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-RfxCas13d (CasRx) is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that zygotically expressed and maternally provided transcripts are efficiently targeted, resulting in a 76% average decrease in transcript levels and recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish, and mouse embryos. Altogether, our results demonstrate that CRISPR-RfxCas13d is an efficient knockdown platform to interrogate gene function in animal embryos.

Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains.

Reggie-1 and -2 proteins (flotillin-2 and -1 respectively) form their own type of non-caveolar membrane microdomains, which are involved in important cellular processes such as T-cell activation, phagocytosis and signalling mediated by the cellular prion protein and insulin; this is consistent with the notion that reggie microdomains promote protein assemblies and signalling. While it is generally known that membrane microdomains contain large multiprotein assemblies, the exact organization of reggie microdomains remains elusive. Using chemical cross-linking approaches, we have demonstrated that reggie complexes are composed of homo- and hetero-tetramers of reggie-1 and -2. Moreover, native reggie oligomers are indeed quite stable, since non-cross-linked tetramers are resistant to 8 M urea treatment. We also show that oligomerization requires the C-terminal but not the N-terminal halves of reggie-1 and -2. Using deletion constructs, we analysed the functional relevance of the three predicted coiled-coil stretches present in the C-terminus of reggie-1. We confirmed experimentally that reggie-1 tetramerization is dependent on the presence of coiled-coil 2 and, partially, of coiled-coil 1. Furthermore, since depletion of reggie-1 by siRNA (small interfering RNA) silencing induces proteasomal degradation of reggie-2, we conclude that the protein stability of reggie-2 depends on the presence of reggie-1. Our data indicate that the basic structural units of reggie microdomains are reggie homo- and hetero-tetramers, which are dependent on the presence of reggie-1.

An evolutionary basis for scrapie disease: identification of a fish prion mRNA.

Infectious prion proteins cause neurodegenerative disease in mammals owing to the acquisition of an aberrant conformation. We cloned a Fugu rubripes gene that encodes a structurally conserved prion protein, and found rapid rates of molecular divergence among prions from different vertebrate classes, along with molecular stasis within each class. We propose that a directional trend in the evolution of prion sequence motifs associated with pathogenesis and infectivity could account for the origin of scrapie in mammals.

Disparate evolution of prion protein domains and the distinct origin of Doppel- and prion-related loci revealed by fish-to-mammal comparisons.

Prions result from the misfolding and selective accumulation of the host-encoded prion protein (PrP) in the brain. Despite intensive research on mammalian models, basic questions about the biological role of PrP and the evolutionary origin of prion disease remain unanswered. Following our previous identification of novel fish PrP homologues, here we generated new fish PrP sequences and performed genomic analysis to demonstrate the existence of two homologous PrP loci in bony fish, which display extensive molecular variation and are highly expressed in adult and developing fish brains. The fish PrP genomic regions contain PrP-related loci directly downstream of each PrP locus, suggesting an independent origin of prion-related proteins in fish and mammals. Our structural prediction analysis uncovers a conserved molecular "bauplan" for all vertebrate PrPs. The C- and N-terminal protein domains have evolved independently from one another, the former having retained its basic globular structure despite high sequence divergence and the latter having undergone differential expansion-degeneration cycles in its repetitive domains. Our evolutionary analysis redefines fundamental concepts on the functional significance of PrP domains and opens up new possibilities for the experimental analysis of prion misfolding and neurodegeneration in a non-mammalian model like the zebrafish.

Uncontrolled SFK-mediated protein trafficking in prion and Alzheimer's disease.

Prions and Amyloid beta (Aß) peptides induce synaptic damage via complex mechanisms that include the pathological alteration of intracellular signaling cascades. The host-encoded cellular prion protein (PrPC) acts as a high-affinity cell surface receptor for both toxic species and it can modulate the endocytic trafficking of the N-methyl D-aspartate (NMDA) receptor and E-cadherin adhesive complexes via Src family kinases (SFKs). Interestingly, SFK-mediated control of endocytosis is a widespread mechanism used to regulate the activity of important transmembrane proteins, including neuroreceptors for major excitatory and inhibitory neurotransmitters. Here we discuss our recent work in zebrafish and accumulating evidence suggesting that subversion of this pleiotropic regulatory mechanism by Aß oligomers and prions explains diverse neurotransmission deficits observed in human patients and mouse models of prion and Alzheimer's neurodegeneration. While Aß, PrPC and SFKs constitute potential therapeutic targets on their own, drug discovery efforts might benefit significantly from aiming at protein-protein interactions that modulate the endocytosis of specific SFK targets.

Activation of zebrafish Src family kinases by the prion protein is an amyloid-ß-sensitive signal that prevents the endocytosis and degradation of E-cadherin/ß-catenin complexes in vivo.

BACKGROUND: Prions and amyloid-ß (Aß) oligomers trigger neurodegeneration by hijacking a poorly understood cellular signal mediated by the prion protein (PrP) at the plasma membrane. In early zebrafish embryos, PrP-1-dependent signals control cell-cell adhesion via a tyrosine phosphorylation-dependent mechanism. RESULTS: Here we report that the Src family kinases (SFKs) Fyn and Yes act downstream of PrP-1 to prevent the endocytosis and degradation of E-cadherin/ß-catenin adhesion complexes in vivo. Accordingly, knockdown of PrP-1 or Fyn/Yes cause similar zebrafish gastrulation phenotypes, whereas Fyn/Yes expression rescues the PrP-1 knockdown phenotype. We also show that zebrafish and mouse PrPs positively regulate the activity of Src kinases and that these have an unexpected positive effect on E-cadherin-mediated cell adhesion. Interestingly, while PrP knockdown impairs ß-catenin adhesive function, PrP overexpression enhances it, thereby antagonizing its nuclear, wnt-related signaling activity and disturbing embryonic dorsoventral specification. The ability of mouse PrP to influence these events in zebrafish embryos requires its neuroprotective, polybasic N-terminus but not its neurotoxicity-associated central region. Remarkably, human Aß oligomers up-regulate the PrP-1/SFK/E-cadherin/ß-catenin pathway in zebrafish embryonic cells, mimicking a PrP gain-of-function scenario. CONCLUSIONS: Our gain- and loss-of-function experiments in zebrafish suggest that PrP and SFKs enhance the cell surface stability of embryonic adherens junctions via the same complex mechanism through which they over-activate neuroreceptors that trigger synaptic damage. The profound impact of this pathway on early zebrafish development makes these embryos an ideal model to study the cellular and molecular events affected by neurotoxic PrP mutations and ligands in vivo. In particular, our finding that human Aß oligomers activate the zebrafish PrP/SFK/E-cadherin pathway opens the possibility of using fish embryos to rapidly screen for novel therapeutic targets and compounds against prion- and Alzheimer's-related neurodegeneration. Altogether, our data illustrate PrP-dependent signals relevant to embryonic development, neuronal physiology and neurological disease.

Restricted expression of reggie genes and proteins during early zebrafish development.

Reggies are plasma membrane-associated proteins and characteristic markers of lipid-raft microdomains. They are highly conserved from flies to humans and have been implicated in axon regeneration and cell process and contact formation, possibly providing functional platforms for cell-signaling in neurons and other cell types. We analyzed reggie mRNA and protein expression patterns during early zebrafish development. All three zebrafish genes, re-1a, -2a, and -2b, span a considerably diverse set of expression patterns, and their proteins are induced maternally, showing ubiquitous expression at early stages. Although re-2a mRNA can be observed in differentiating neurons in the brain, spinal cord, and neurogenic placodes, re-2b is transcribed mainly in head mesoderm, in neural crest derivates, and along somite boundaries. re-1a mRNA is present at high levels in expression domains that overlap with the combined expression pattern of both re-2 genes except at the somites, where it complements the pattern of re-2b. Immunostaining on embryos reveals reggie protein localization at the cell membrane, at cell-cell contacts, and along all early axon tracts. The early phase of reggie expression suggests a basic and ubiquitous function during the first stages of embryogenesis and into the gastrula period. Upon segmentation, a second phase of expression shows distinctly localized expression patterns, indicating tissue-specific roles and an involvement of re-1a/re-2a in neural development.

Complete nucleotide sequence of the mitochondrial genome of a salamander, Mertensiella luschani.

The complete nucleotide sequence (16,650 bp) of the mitochondrial genome of the salamander Mertensiella luschani (Caudata, Amphibia) was determined. This molecule conforms to the consensus vertebrate mitochondrial gene order. However, it is characterized by a long non-coding intervening sequence with two 124-bp repeats between the tRNA(Thr) and tRNA(Pro) genes. The new sequence data were used to reconstruct a phylogeny of jawed vertebrates. Phylogenetic analyses of all mitochondrial protein-coding genes at the amino acid level recovered a robust vertebrate tree in which lungfishes are the closest living relatives of tetrapods, salamanders and frogs are grouped together to the exclusion of caecilians (the Batrachia hypothesis) in a monophyletic amphibian clade, turtles show diapsid affinities and are placed as sister group of crocodiles+birds, and the marsupials are grouped together with monotremes and basal to placental mammals. The deduced phylogeny was used to characterize the molecular evolution of vertebrate mitochondrial proteins. Amino acid frequencies were analyzed across the main lineages of jawed vertebrates, and leucine and cysteine were found to be the most and least abundant amino acids in mitochondrial proteins, respectively. Patterns of amino acid replacements were conserved among vertebrates. Overall, cartilaginous fishes showed the least variation in amino acid frequencies and replacements. Constancy of rates of evolution among the main lineages of jawed vertebrates was rejected.

Evolution of duplicated reggie genes in zebrafish and goldfish.

Invertebrates, tetrapod vertebrates, and fish might be expected to differ in their number of gene copies, possibly due the occurrence of genome duplication events during animal evolution. Reggie (flotillin) genes code for membrane-associated proteins involved in growth signaling in developing and regenerating axons. Until now, there appeared to be only two reggie genes in fruitflies, mammals, and fish. The aim of this research was to search for additional copies of reggie genes in fishes, since a genome duplication might have increased the gene copy number in this group. We report the presence of up to four distinct reggie genes (two reggie-1 and two reggie-2 genes) in the genomes of zebrafish and goldfish. Phylogenetic analyses show that the zebrafish and goldfish sequence pairs are orthologous, and that the additional copies could have arisen through a genome duplication in a common ancestor of bony fish. The presence of novel reggie mRNAs in fish embryos indicates that the newly discovered gene copies are transcribed and possibly expressed in the developing and regenerating nervous system. The intron/exon boundaries of the new fish genes characterized here correspond with those of human genes, both in location and phase. An evolutionary scenario for the evolution of reggie intron-exon structure, where loss of introns appears to be a distinctive trait in invertebrate reggie genes, is presented.

Common themes in PrP signaling: the Src remains the same.

The ability of the cellular prion protein (PrP(C)) to trigger intracellular signals appears central to neurodegeneration pathways, yet the physiological significance of such signals is rather puzzling. For instance, PrP(C) deregulation disrupts phenomena as diverse as synaptic transmission in mammals and cell adhesion in zebrafish. Although unrelated, the key proteins in these events -the NMDA receptor (NMDAR) and E-cadherin, respectively- are similarly modulated by the Src family kinase (SFK) Fyn. These observations highlight the importance of PrP(C)-mediated Fyn activation, a finding reported nearly two decades ago. Given their complex functions and regulation, SFKs may hold the key to intriguing aspects of PrP biology such as its seemingly promiscuous functions and the lack of strong phenotypes in knockout mice. Here we provide a mechanistic perspective on how SFKs might contribute to the uncertain molecular basis of neuronal PrP phenotypes affecting ion channel activity, axon myelination and olfactory function. In particular, we discuss SFK target proteins involved in these processes and the role of tyrosine phosphorylation in the regulation of their activity and cell surface expression.

Essential roles of zebrafish rtn4/Nogo paralogues in embryonic development.

BACKGROUND: As a consequence of gene/genome duplication, the RTN4/Nogo gene has two counterparts in zebrafish: rtn4a and rtn4b. The shared presence of four specific amino acid motifs-M1 to M4-in the N-terminal region of mammalian RTN4, and zebrafish Rtn4b suggests that Rtn4b is the closest homologue of mammalian Nogo-A. RESULTS: To explore their combined roles in zebrafish development, we characterized the expression patterns of rtn4a and rtn4b in a comparative manner and performed morpholino-mediated knockdowns. Although both genes were coexpressed in the neural tube and developing brain at early stages, they progressively acquired distinct expression domains such as the spinal cord (rtn4b) and somites (rtn4a). Downregulation of rtn4a and rtn4b caused severe brain abnormalities, with rtn4b knockdown severely affecting the spinal cord and leading to immobility. In addition, the retinotectal projection was severely affected in both morphants, as the retina and optic tectum appeared smaller and only few retinal axons reached the abnormally reduced tectal neuropil. The neuronal defects were more persistent in rtn4b morphants. Moreover, the latter often lacked pectoral fins and lower jaws and had malformed branchial arches. Notably, these defects led to larval death in rtn4b, but not in rtn4a morphants. CONCLUSIONS: In contrast to mammalian Nogo-A, its zebrafish homologues, rtn4a and particularly rtn4b, are essential for embryonic development and patterning of the nervous system.

Conserved roles of the prion protein domains on subcellular localization and cell-cell adhesion.

Analyses of cultured cells and transgenic mice expressing prion protein (PrP) deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1) the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2) the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP's essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP's ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and zebrafish.