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Early detection of SARS-CoV-2 using molecular techniques is paramount to the fight against COVID-19. Due to its high sensitivity and specificity, RT-qPCR is the “gold standard” method for this purpose. However, its technical requirements, processing time and elevated costs hamper its use towards massive and timely molecular testing for COVID-19 in rural and socioeconomically deprived areas of Latin America. The advent and rapid evolution of CRISPR-Cas technology has boosted the development of new pathogen detection methodologies. Recently, DETECTR -a combination of isothermal RT-LAMP amplification and Cas12a-mediated enzymatic detection-has been successfully validated in the Netherlands and the USA as a rapid and low-cost alternative to RT-qPCR for the detection of SARS-CoV-2 from nasopharyngeal swabs. Here, we evaluated the performance of RCSMS, a locally adapted variant of DETECTR, to ascertain the presence of SARS-CoV-2 in saliva samples from 276 patients in two hospitals in Lima, Perú (current status over a total of 350 samples). 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. Our clinical validation shows that RCSMS detects up to 5 viral copies per reaction in 40 min, with sensitivity and specificity of 93.8% and 99.0% in the field, respectively, relative to RT-qPCR. Since CRISPR-Cas biosensors can be easily reprogrammed by using different guide RNA molecules, RCSMS has the potential to be quickly adapted for the detection of new SARS-CoV-2 variants. Notably, estimation of its negative and positive predictive values suggests that RCSMS can be confidently deployed in both high and low prevalence settings. Furthermore, our field study validates the use of lateral flow strips to easily visualize the presence of SARS-CoV-2, which paves the way to deploy RCSMS as a “point of care” test in environments with limited access to state-of-the-art diagnostic laboratories. In sum, RCSMS is a fast, efficient and inexpensive alternative to RT-qPCR for expanding COVID-19 testing capacity in low- and middle-income countries.

Understanding animal cognition has been of interest to scientists for well over a century (e.g., Melrose, 1921). Cognition is broadly defined as the neural and behavioral processes associated with the acquisition, retention, and use of information (Dukas, 2004). Since the discovery of multiple memory systems and of the fundamental role of the hippocampus in relational learning and memory in humans (Penfield and Milner, 1958), one exciting focus of study has been to determine how animals encode, transform, compute and manipulate spatial, temporal, and contextual information from their environment, and how this information is utilized to organize behavioral responses (Cook, 1993). Initial studies used simple visual and acoustic stimuli, such as colored lights and distinct sounds. However, the use of such stimuli hindered the study of animal cognition because it did not allow the subjects to fully engage their full information processing capabilities. To address this issue, researchers started using more complex stimuli, such as objects, photos, and videos. These studies demonstrated a higher level of cognitive processing not previously attributed to animals (Dukas, 2004). As the field of learning and memory advanced, studies started to show remarkable similarities between the cognitive processes of animals and humans. Animals have been found to be even able to learn varied and sophisticated concepts, exhibit mental processes, such as symbol coding and organization, to form spatial, temporal, and numerical abstractions and perceive cause and effect relationships (Wynne, 2001).

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 induce RNA degradation 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-Cas13d is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that both zygotically-expressed and maternally-provided transcripts are efficiently targeted, resulting in an 80% average decrease in transcript level and the 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-Cas13d is an efficient knock-down platform to interrogate gene function 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., This work was supported by Ramon y Cajal program (RyC-2017-23041) and grants PGC2018-097260-B-I00 and MDM-2016-0687 from Spanish Ministerio de Ciencia, Innovación y Universidades and the Springboard program from CABD (M.A.M.-M.) and the Stowers Institute for Medical Research (A.A.B.). M.A.M.-M. was the recipient of the Genome Engineer Innovation 2019 Grant from Synthego. A.A.B. was awarded with Pew Innovation Fund. J.R.M.-M. is supported by BFU2017-86339-P and MDM-2016-0687 grants (Spanish Ministerio de Ciencia, Innovación y Universidades). E.M.-T. and J.A.-N.d.P. are supported by INNOVATE PERÚ grant 168-PNICP-PIAP-2015 and FONDECYT travel grant 043-2019.

Background Prions and amyloid-β (Aβ) 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/β-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 β-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 Aβ oligomers up-regulate the PrP-1/SFK/E-cadherin/β-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 Aβ 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. Electronic supplementary material The online version of this article (doi:10.1186/s13024-016-0076-5) contains supplementary material, which is available to authorized users.

Prions and Amyloid beta (Aβ) 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 Aβ oligomers and prions explains diverse neurotransmission deficits observed in human patients and mouse models of prion and Alzheimer's neurodegeneration. While Aβ, 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.

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.

Reggies (flotillins) are detergent-resistant microdomains involved in the scaffolding of large heteromeric complexes that signal across the plasma membrane. Based on the presence of an evolutionarily widespread motif, reggies/flotillins have been included within the SPFH (stomatin-prohibitin-flotillin-HflC/K) protein superfamily. To better understand the origin and evolution of reggie/flotillin structure and function, we searched databases for reggie/flotillin and SPFH-like proteins in organisms at the base and beyond the animal kingdom, and used the resulting dataset to compare their structural and functional domains. Our analysis shows that the SPFH grouping has little phylogenetic support, probably due to convergent evolution of its members. We also find that reggie/flotillin homologues are highly conserved among metazoans but are absent in plants, fungi and bacteria, where only proteins with "reggie-like" domains can be found. However, despite their low sequence similarities, reggie/flotillin and "reggie-like" domains appear to subserve related functions, suggesting that their basic biological role was acquired independently during evolution.

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.

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.

The early origin of four vertebrate Hox gene clusters during the evolution of gnathostomes was likely caused by two consecutive duplications of the entire genome and the subsequent loss of individual genes. The presumed conserved and important roles of these genes in tetrapods during development led to the general assumption that Hox cluster architecture had remained unchanged since the last common ancestor of all jawed vertebrates. But recent data from teleost fishes reveals that this is not the case. Here, we present an analysis of the evolution of vertebrate Hox genes and clusters, with emphasis on the differences between the Hox A clusters of fish (actinopterygian) and tetrapod (sarcopterygian) lineages. In contrast to the general conservation of genomic architecture and gene sequence observed in sarcopterygians, the evolutionary history of actinopterygian Hox clusters likely includes an additional (third) genome duplication that initially increased the number of clusters from four to eight. We document, for the first time, higher rates of gene loss and gene sequence evolution in the Hox genes of fishes compared to those of land vertebrates. These two observations might suggest that two different molecular evolutionary strategies exist in the two major vertebrate lineages. Preliminary data from the African cichlid fish Oreochromis niloticus compared to those of the pufferfish and zebrafish reveal important differences in Hox cluster architecture among fishes and, together with genetic mapping data from Medaka, indicate that the third genome duplication was not zebrafish-specific, but probably occurred early in the history of fishes. Each descending fish lineage that has been characterized so far, distinctively modified its Hox cluster architecture through independent secondary losses. This variation is related to the large body plan differences observed among fishes, such as the loss of entire sets of appendages and ribs in some lineages.

In tetrapods, the functional (classical) class I and class II B loci of the major histocompatibility complex (Mhc) are tightly linked in a single chromosomal region. In an earlier study, we demonstrated that in the zebrafish, Danio rerio, order Cypriniformes, the two classes are present on different chromosomes. Here, we show that the situation is similar in the stickleback, Gasterosteus aculeatus, order Gasterosteiformes, the common guppy, Poecilia reticulata, order Cyprinodontiformes, and the cichlid fish Oreochromis niloticus, order Perciformes. These data, together with unpublished results from other laboratories suggest that in all Euteleostei, the classical class I and class II B loci are in separate linkage groups, and that in at least some of these taxa, the class II loci are in two different groups. Since Euteleostei are at least as numerous as tetrapods, in approximately one-half of jawed vertebrates, the class I and class II regions are not linked.

Prion diseases encompass a diverse group of lethal neurodegenerative disorders associated with the accumulation of misfolded conformers of the prion protein (PrP) in brain neurons. Modeling these diseases in mice and hamsters has led to major advances in our understanding of prion transmission and pathogenesis. However, laboratory rodents are also expensive, time-consuming, and limiting for systematic studies. Genetically tractable animal models, such as the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the zebrafish Danio rerio, have recently made significant contributions to PrP pathogenesis. Here, we discuss recent applications of these three nonmammalian models to various relevant areas, including PrP processing, trafficking, misfolding, neurotoxicity, as well as unraveling its elusive endogenous function. Now that these alternative models have staked a claim on PrP biology, we anticipate that they will expand their range of applications and contributions in the next few years, including the generation of nonmammalian models of prion transmissibility.

13 páginas, 5 figuras, 1 tabla, 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., Thiswork is supported by a DFGgrant(MA 2525/2-1) andUniversity of Konstanz funds awarded to EMT. ES, TS and AF are funded by EU grants (QLK5-2002-00866 for ES, TS, AF and FOOD-CT-2004-506579 for ES, TS)

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.

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., Author Summary Unlike conventional pathogens, prions are infectious particles devoid of nucleic acids and composed entirely of a misfolded host protein, PrP. It is widely assumed that the neurodegeneration observed in prion disorders may be related to an aberrant function of PrP in the misfolded state. However, the normal physiological function of PrP remains poorly understood, mainly owing to the absence of clear phenotypes in mice lacking PrP. Here, we show that when PrP is depleted in zebrafish embryos, dramatic phenotypes ensue, severely affecting the development of early and late (neural) structures. We examined the mechanisms responsible for some of these defects, and found that fish and mammalian PrPs play conserved roles in cell–cell communication, by directly mediating cell adhesion and by triggering cellular signals that further modulate the function of other adhesion molecules. In the early zebrafish embryo, these activities control not only tissue integrity and cell morphology, but also the complex cellular movements that give rise to germ layers. This study describes—to our knowledge—the first known in vivo function of PrP and its molecular cellular basis, which may provide helpful insights into the role of PrP in the adult brain and its proposed connections to prion-induced neurotoxicity., Knockdown experiments in zebrafish embryos reveal that prion proteins, otherwise known for their involvement in lethal neurodegenerative disease, play important roles in cell-cell adhesion and signaling during embryonic development.

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 vivoas well as axon/neurite extension in hippocampal and N2a neuronsin vitrothrough 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.

The best-known attribute of the prion protein (PrP) is its tendency to misfold into a rogue isoform. Much less understood is how this misfolded isoform causes deadly brain illnesses. Neurodegeneration in prion disease is often seen as a consequence of abnormal PrP function yet, amazingly little is known about the normal, physiological role of PrP. In particular, the absence of obvious phenotypes in PrP knockout mice has prevented scientists from answering this important question. Using knockdown approaches, we previously produced clear PrP loss-of-function phenotypes in zebrafish embryos. Analysis of these phenotypes revealed that PrP can modulate E-cadherin-based cell-cell adhesion, thereby controlling essential morphogenetic cell movements in the early gastrula. Our data also showed that PrP itself can elicit homophilic cell-cell adhesion and trigger intracellular signaling via Src-related kinases. Importantly, these molecular functions of PrP are conserved from fish to mammals. Here we discuss the use of the zebrafish in prion biology and how it may advance our understanding of the roles of PrP in health and disease.

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.

Thy-1 is a developmentally regulated, immunoglobulin superfamily member (IgSF), glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein expressed most strongly in neurons and lymphocytes. Thy-1 is expressed in all vertebrates and has been implicated in a variety of processes, including axon regeneration and transmembrane signaling, but its specific function remains elusive. A Thy-1-like molecule in teleost fish was recently identified, with evidence for its role in lipid-raft based signal transduction linked to optic nerve regeneration. For a better characterization of Thy-1, the evolutionary relationships between novel fish homologues and other vertebrate Thy-1s were analyzed. Although the sequence similarity between fish and mammals is very low, there appeared conservation of gene structure and disrupted but recognizable synteny. In addition, the detailed expression analysis of teleost Thy-1 showed nervous system Thy-1 mainly in sensory systems. Strong Thy-1 expression was detected in the youngest retinal ganglion cells and in some neurons in deeper retinal layers, probably amacrine cells. From the olfactory bulbs, Thy-1-positive cells extended axons into the telencephalon. The vagal lobe stained intensively as well as facial and glossopharyngeal lobes and nerves. Outside the CNS, skin cells, blood vessels, kidney macrophages, swim bladder, spleen, gut-associated nerve fibers and the palatal organ were labeled.

The species flocks of cichlid fishes in the Great East African Lakes are paradigms of adaptive radiation and hence, of great interest to evolutionary biologists. Phylogenetic studies of these fishes have, however, been hampered by the lack of suitable polymorphic markers. The genes of the major histocompatibility complex hold the promise to provide, through their extensive polymorphism, a large number of such markers, but their use has been hampered by the complexity of the genetic system and the lack of definition of the individual loci. In this study we take the first substantial step to alleviate this problem. Using a combination of methods, including the typing of single sperm cells, gyno- or androgenetic individuals, and haploid embryos, as well as sequencing of class II B restriction fragments isolated from gels for Southern blots, we identify the previously characterized homology groups as distinct loci. At least 17 polymorphic class II B loci, all of which are presumably transcribed, have been found among the different species studied. Most of these loci are shared across the various cichlid species and genera. The number of loci per haplotype varies from individual to individual, ranging from 1 to 13. A total of 21 distinct haplotypes differing in the number of loci they carry has thus far been identified. All the polymorphic loci are part of the same cluster in which, however, distances between at least some of the loci (as indicated by recombination frequencies) are relatively large. Both the individual loci and the haplotypes can now be used to study phylogenetic relationships among the members of the species flocks and the mode in which speciation occurs during adaptive radiation.

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.

The prion protein (PrP) has been implicated in many diverse functions, making it difficult to pinpoint its basic physiological role. Our most recent studies in zebrafish, mammalian and invertebrate cells indicate that PrP regulates cell-cell communication, as well cell-matrix interactions at focal adhesions. In addition, we previously have shown that upon antibody-mediated cross-linking, PrP can be induced to cluster in the preformed T-cell cap. Here we review these data and discuss how the spatial link between PrP and the microdomain-forming proteins reggie- 1 (flotillin-2) and reggie-2 (flotillin-1) may contribute to PrP signaling, leading to the local assembly of membrane protein complexes at sites involved in cellular communication, such as cell-cell contacts, focal adhesions, the T-cell cap, and synapses.