Affinity purification of in vivo assembled whirlin-associated protein complexes from the zebrafish retina.

Mutations in WHRN lead to Usher syndrome type 2d or to non-syndromic hearing impairment. The WHRN-encoded gene product whirlin directly interacts with the intracellular regions of the other two Usher syndrome type 2-associated proteins, usherin and ADGRV1. In photoreceptor cells, this protein complex constitutes fibrous links between the periciliary membrane and the connecting cilium. However, the molecular mechanism(s) of retinal degeneration due to compromised formation and function of the USH2-associated protein complex remains elusive. To unravel this pathogenic mechanism, we isolated and characterized whirlin-associated protein complexes from zebrafish photoreceptor cells. We generated transgenic zebrafish that express Strep/FLAG-tagged Whrna, a zebrafish ortholog of human whirlin, under the control of a photoreceptor-specific promoter. Affinity purification of Strep/FLAG-tagged Whrna and associated proteins from adult transgenic zebrafish retinas followed by mass spectrometry identified 19 novel candidate associated proteins. Pull down experiments and dedicated yeast two-hybrid assays confirmed the association of Whrna with 7 of the co-purified proteins. Several of the co-purified proteins are part of the synaptic proteome, which indicates a role for whirlin in the photoreceptor synapse. Future studies will elucidate which of the newly identified protein-protein interactions contribute to the development of the retinal phenotype observed in USH2d patients. SIGNIFICANCE: Since protein-protein interactions identified using targeted in vitro studies do not always recapitulate interactions that are functionally relevant in vivo, we established a transgenic zebrafish line that stably expresses a Strep/FLAG-tagged ortholog of human whirlin (SF-Whrna) in photoreceptor cells. Affinity purification of in vivo-assembled SF-Whrna-associated protein complexes from retinal lysates followed by mass spectrometry, identified 19 novel candidate interaction partners, many of which are enriched in the synaptic proteome. Two human orthologs of the identified candidate interaction partners, FRMPD4 and Kir2.3, were validated as direct interaction partners of human whirlin using a yeast two-hybrid assay. The strong connection of whirlin with postsynaptic density proteins was not identified in previous in vitro protein-protein interaction assays, presumably due to the absence of a biologically relevant context. Isolation and identification of in vivo-assembled whirlin-associated protein complexes from the tissue of interest is therefore a powerful methodology to obtain novel insight into tissue specific protein-protein interactions and has the potential to improve significantly our understanding of the function of whirlin and the molecular pathogenesis underlying Usher syndrome type 2.

Zebrafish models of human eye and inner ear diseases.

Eye and inner ear diseases are the most common sensory impairments that greatly impact quality of life. Zebrafish have been intensively employed to understand the fundamental mechanisms underlying eye and inner ear development. The zebrafish visual and vestibulo-acoustic systems are very similar to these in humans, and although not yet mature, they are functional by 5days post-fertilization (dpf). In this chapter, we show how the zebrafish has significantly contributed to the field of biomedical research and how researchers, by establishing disease models and meticulously characterizing their phenotypes, have taken the first steps toward therapies. We review here models for (1) eye diseases, (2) ear diseases, and (3) syndromes affecting eye and/or ear. The use of new genome editing technologies and high-throughput screening systems should increase considerably the speed at which knowledge from zebrafish disease models is acquired, opening avenues for better diagnostics, treatments, and therapies.

A scientist's guide for submitting data to ZFIN.

The Zebrafish Model Organism Database (ZFIN; zfin.org) serves as the central repository for genetic and genomic data produced using zebrafish (Danio rerio). Data in ZFIN are either manually curated from peer-reviewed publications or submitted directly to ZFIN from various data repositories. Data types currently supported include mutants, transgenic lines, DNA constructs, gene expression, phenotypes, antibodies, morpholinos, TALENs, CRISPRs, disease models, movies, and images. The rapidly changing methods of genomic science have increased the production of data that cannot readily be represented in standard journal publications. These large data sets require web-based presentation. As the central repository for zebrafish research data, it has become increasingly important for ZFIN to provide the zebrafish research community with support for their data sets and guidance on what is required to submit these data to ZFIN. Regardless of their volume, all data that are submitted for inclusion in ZFIN must include a minimum set of information that describes the data. The aim of this chapter is to identify data types that fit into the current ZFIN database and explain how to provide those data in the optimal format for integration. We identify the required and optional data elements, define jargon, and present tools and templates that can help with the acquisition and organization of data as they are being prepared for submission to ZFIN. This information will also appear in the ZFIN wiki, where it will be updated as our services evolve over time.

Modality-specificity of sensory aging in vision and audition: evidence from event-related potentials.

Major accounts of aging implicate changes in processing external stimulus information. Little is known about differential effects of auditory and visual sensory aging, and the mechanisms of sensory aging are still poorly understood. Using event-related potentials (ERPs) elicited by unattended stimuli in younger (M=25.5 yrs) and older (M=71.3 yrs) subjects, this study examined mechanisms of sensory aging under minimized attention conditions. Auditory and visual modalities were examined to address modality-specificity vs. generality of sensory aging. Between-modality differences were robust. The earlier-latency responses (P1, N1) were unaffected in the auditory modality but were diminished in the visual modality. The auditory N2 and early visual N2 were diminished. Two similarities between the modalities were age-related enhancements in the late P2 range and positive behavior-early N2 correlation, the latter suggesting that N2 may reflect long-latency inhibition of irrelevant stimuli. Since there is no evidence for salient differences in neuro-biological aging between the two sensory regions, the observed between-modality differences are best explained by the differential reliance of auditory and visual systems on attention. Visual sensory processing relies on facilitation by visuo-spatial attention, withdrawal of which appears to be more disadvantageous in older populations. In contrast, auditory processing is equipped with powerful inhibitory capacities. However, when the whole auditory modality is unattended, thalamo-cortical gating deficits may not manifest in the elderly. In contrast, ERP indices of longer-latency, stimulus-level inhibitory modulation appear to diminish with age.

Insulin-like growth factor signaling regulates zebrafish embryonic growth and development by promoting cell survival and cell cycle progression.

Although much is known about the global effects of insulin-like growth factor 1 receptor (IGF1R)-mediated signaling on fetal growth and the clinical manifestations resulting from IGF/IGF1R deficiencies, we have an incomplete understanding of the cellular actions of this essential pathway during vertebrate embryogenesis. In this study, we inhibited IGF1R signaling during zebrafish embryogenesis using antisense morpholino oligonucleotides or a dominant-negative IGF1R fusion protein. IGF1R inhibition resulted in reduced embryonic growth, arrested development and increased lethality. IGF1R-deficient embryos had significant defects in the retina, inner ear, motoneurons and heart. No patterning abnormalities, however, were found in the brain or other embryonic tissues. At the cellular level, IGF1R inhibition increased caspase 3 activity and induced neuronal apoptosis. Coinjection of antiapoptotic bcl2-like mRNA attenuated the elevated apoptosis and rescued the retinal and motoneuron defects, but not the developmental arrest. Subsequent cell cycle analysis indicated an increased percentage of cells in G1 and a decreased percentage in S phase in IGF1R-deficient embryos independent of apoptosis. These results provide novel insight into the cellular basis of IGF1R function and show that IGF1R signaling does not function as an anteriorizing signal but regulates embryonic growth and development by promoting cell survival and cell cycle progression.

ERPs differentiate syllable and nonphonetic sound processing in children and adults.

We examined maturation of speech-sound-related indices of auditory event-related brain potentials (ERPs). ERPs were elicited by syllables and nonphonetic correlates in children and adults. Compared with syllables, nonphonetic stimuli elicited larger N1 and P2 in adults and P1 in children. Because the nonphonetics were more perceptually salient, this N1 effect was consistent with known N1 sensitivity to sound onset features. Based on stimulus dependence and independent component structure, children's P1 appeared to contain overlapping P2-like activity. In both subject groups, syllables elicited larger N2/N4 peaks. This might reflect sound content feature processing, more extensive for speech than nonspeech sounds. Therefore, sound detection mechanisms (N1, P2) still develop whereas sound content processing (N2, N4) is largely mature during mid-childhood; in children and adults, speech sounds are processed more extensively than nonspeech sounds 200-400 ms poststimulus.

CYP2K6 from zebrafish (Danio rerio): cloning, mapping, developmental/tissue expression, and aflatoxin B1 activation by baculovirus expressed enzyme.

A full-length zebrafish (Danio rerio) cytochrome P450 (CYP) 2K6 cDNA, was obtained (GenBank accession No. AF283813) through polymerase chain reaction cloning using degenerated primers based on a consensus CYP2 sequence and the heme-binding domain. This first CYP2K family member cloned from zebrafish had 1861 bp which contained 27 bp of 5'-untranslated region (5'-UTR), an open reading frame (ORF) of 1518 bp, and a 300 bp 3'-UTR with a poly A tail. The deduced 506 amino acid sequence of CYP2K6 had 63%, 62% and 59% identity with rainbow trout CYP2K1, CYP2K4 and CYP2K3, respectively; and 45%, 42%, and 42% identity with rabbit CYP2C1, human CYP2C19 and mouse CYP2C39, respectively. CYP2K6 mapped to 107.49cR on LG3 using the LN54 radiation hybrid panel. Its mRNA was detected at 5 days post-fertilization and in the adult liver and ovary among nine tissues examined. The ORF, including the 27 bp of the 5'-UTR, was cloned into pFastBac donor vector and then transferred into the baculovirus genome (bacmid DNA) in DH10Bac competent cells. The recombinant bacmid DNA was used to infect Spodoptera frugiperda insect cells to express the CYP2K6 protein (Bv-2K6). As its ortholog, rainbow trout Bv-2K1 [Yang, Y.H., Miranda, C.L., Henderson, M.C., Wang-Buhler, J.-L., Buhler, D.R., 2000. Heterologous expression of CYP2K1 and identification of the expressed protein (Bv-2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase. Drug Metab. Disp. 28,1279-83.], Bv-2K6 also catalyzed the conversion of aflatoxin B1 (AFB1) to its exo-8,9-epoxide as assessed by the trapping of a glutathione (GSH) adduct in the presence of a specific mouse alpha class glutathione S-transferase. The identity of the AFB1-GSH adduct was verified by liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry-mass spectrometry (MS-MS) analysis. Although rainbow trout Bv-2K1 was capable of oxidizing lauric acid, zebrafish Bv-2K6 protein showed no activity against this substrate.

Dynamic brain sources of visual evoked responses.

It has been long debated whether averaged electrical responses recorded from the scalp result from stimulus-evoked brain events or stimulus-induced changes in ongoing brain dynamics. In a human visual selective attention task, we show that nontarget event-related potentials were mainly generated by partial stimulus-induced phase resetting of multiple electroencephalographic processes. Independent component analysis applied to the single-trial data identified at least eight classes of contributing components, including those producing central and lateral posterior alpha, left and right mu, and frontal midline theta rhythms. Scalp topographies of these components were consistent with their generation in compact cortical domains.

Zebrafish smoothened functions in ventral neural tube specification and axon tract formation.

Sonic hedgehog (Shh) signaling patterns many vertebrate tissues. shh mutations dramatically affect mouse ventral forebrain and floor plate but produce minor defects in zebrafish. Zebrafish have two mammalian Shh orthologs, sonic hedgehog and tiggy-winkle hedgehog, and another gene, echidna hedgehog, that could have overlapping functions. To examine the role of Hedgehog signaling in zebrafish, we have characterized slow muscle omitted (smu) mutants. We show that smu encodes a zebrafish ortholog of Smoothened that transduces Hedgehog signals. Zebrafish smoothened is expressed maternally and zygotically and supports specification of motoneurons, pituitary cells and ventral forebrain. We propose that smoothened is required for induction of lateral floor plate and a subpopulation of hypothalamic cells and for maintenance of medial floor plate and hypothalamic cells.

Analysis and visualization of single-trial event-related potentials.

In this study, a linear decomposition technique, independent component analysis (ICA), is applied to single-trial multichannel EEG data from event-related potential (ERP) experiments. Spatial filters derived by ICA blindly separate the input data into a sum of temporally independent and spatially fixed components arising from distinct or overlapping brain or extra-brain sources. Both the data and their decomposition are displayed using a new visualization tool, the "ERP image," that can clearly characterize single-trial variations in the amplitudes and latencies of evoked responses, particularly when sorted by a relevant behavioral or physiological variable. These tools were used to analyze data from a visual selective attention experiment on 28 control subjects plus 22 neurological patients whose EEG records were heavily contaminated with blink and other eye-movement artifacts. Results show that ICA can separate artifactual, stimulus-locked, response-locked, and non-event-related background EEG activities into separate components, a taxonomy not obtained from conventional signal averaging approaches. This method allows: (1) removal of pervasive artifacts of all types from single-trial EEG records, (2) identification and segregation of stimulus- and response-locked EEG components, (3) examination of differences in single-trial responses, and (4) separation of temporally distinct but spatially overlapping EEG oscillatory activities with distinct relationships to task events. The proposed methods also allow the interaction between ERPs and the ongoing EEG to be investigated directly. We studied the between-subject component stability of ICA decomposition of single-trial EEG epochs by clustering components with similar scalp maps and activation power spectra. Components accounting for blinks, eye movements, temporal muscle activity, event-related potentials, and event-modulated alpha activities were largely replicated across subjects. Applying ICA and ERP image visualization to the analysis of sets of single trials from event-related EEG (or MEG) experiments can increase the information available from ERP (or ERF) data.

Event-related brain response abnormalities in autism: evidence for impaired cerebello-frontal spatial attention networks.

Although under some conditions the attention-related late positive event-related potential (ERP) response (LPC) is apparently normal in autism during visual processing, the LPC elicited by visuospatial processing may be compromised. Results from this study provide evidence for abnormalities in autism in two components of the LPC generated during spatial processing. The early frontal distribution of the LPC which may reflect attention orienting was delayed or missing in autistic subjects during conditions in which attention was to peripheral visual fields. The later parietal distribution of the LPC which may be associated with context updating was smaller in amplitude in autistic subjects regardless of attention location. Both abnormalities suggest disruption of function in spatial attention networks in autism. Evidence that the cerebellar abnormalities in autism may underlie these deficits comes from: (1) similar results in ERP responses and spatial attention deficits in patients with cerebellar lesions; (2) brain-behavior correlations in normally functioning individuals associating the size of the posterior cerebellar vermis and the latency of the frontal LPC; and (3) a previously reported complementary correlation between the size of the posterior vermal lobules and spatial orienting speed. Although the scalp-recorded LPC is thought to be cortically generated, it may be modulated by subcortical neural activity. The cerebellum may serve as a modulating influence by affecting the task-related antecedent attentional process. The electrophysiological abnormalities reported here index spatial attention deficits in autism that may reflect cerebellar influence on both frontal and parietal spatial attention function.

mosaic eyes: a zebrafish gene required in pigmented epithelium for apical localization of retinal cell division and lamination.

For proper function of the retina, the correct proportions of retinal cell types must be generated, they must be organized into cell-specific laminae, and appropriate synaptic connections must be made. To understand the genetic regulation of retinal development, we have analyzed mutations in the mosaic eyes gene that disrupt retinal lamination, the localization of retinal cell divisions to the retinal pigmented epithelial surface and retinal pigmented epithelial development. Although retinal organization is severely disrupted in mosaic eyes mutants, surprisingly, retinal cell differentiation occurs. The positions of dividing cells and neurons in the brain appear normal in mosaic eyes mutants, suggesting that wild-type mosaic eyes function is specifically required for normal retinal development. We demonstrate that mosaic eyes function is required within the retinal pigmented epithelium, rather than in dividing retinal cells. This analysis reveals an interaction between the retinal pigmented epithelium and the retina that is required for retinal patterning. We suggest that wild-type mosaic eyes function is required for the retinal pigmented epithelium to signal properly to the retina.

The Zebrafish Information Network (ZFIN): a resource for genetic, genomic and developmental research.

The Zebrafish Information Network, ZFIN, is a WWW community resource of zebrafish genetic, genomic and developmental research information (http://zfin.org). ZFIN provides an anatomical atlas and dictionary, developmental staging criteria, research methods, pathology information and a link to the ZFIN relational database (http://zfin. org/ZFIN/). The database, built on a relational, object-oriented model, provides integrated information about mutants, genes, genetic markers, mapping panels, publications and contact information for the zebrafish research community. The database is populated with curated published data, user submitted data and large dataset uploads. A broad range of data types including text, images, graphical representations and genetic maps supports the data. ZFIN incorporates links to other genomic resources that provide sequence and ortholog data. Zebrafish nomenclature guidelines and an automated registration mechanism for new names are provided. Extensive usability testing has resulted in an easy to learn and use forms interface with complex searching capabilities.

Four resource centers for fishes: specifies, stocks, and services.

A conference on "Aquaria Fish Models of Human Disease" was held September 20-23, 2000, at Southwest Texas State University, San Marcos, Texas, USA. The meeting was sponsored by the National Cancer Institute (National Institutes of Health), the Roy and Joan Mitte Foundation, and Southwest Texas State University, home of the Xiphophorus Genetic Stock Center. In conjunction with the meeting, the conference organizers asked several participants to describe those components of their research programs that provide services and information to other researchers. This article summarizes their responses.

Removal of eye activity artifacts from visual event-related potentials in normal and clinical subjects.

OBJECTIVES: Electrical potentials produced by blinks and eye movements present serious problems for electroencephalographic (EEG) and event-related potential (ERP) data interpretation and analysis, particularly for analysis of data from some clinical populations. Often, all epochs contaminated by large eye artifacts are rejected as unusable, though this may prove unacceptable when blinks and eye movements occur frequently. METHODS: Frontal channels are often used as reference signals to regress out eye artifacts, but inevitably portions of relevant EEG signals also appearing in EOG channels are thereby eliminated or mixed into other scalp channels. A generally applicable adaptive method for removing artifacts from EEG records based on blind source separation by independent component analysis (ICA) (Neural Computation 7 (1995) 1129; Neural Computation 10(8) (1998) 2103; Neural Computation 11(2) (1999) 606) overcomes these limitations. RESULTS: Results on EEG data collected from 28 normal controls and 22 clinical subjects performing a visual selective attention task show that ICA can be used to effectively detect, separate and remove ocular artifacts from even strongly contaminated EEG recordings. The results compare favorably to those obtained using rejection or regression methods. CONCLUSIONS: The ICA method can preserve ERP contributions from all of the recorded trials and all the recorded data channels, even when none of the single trials are artifact-free.

The olfactory placodes of the zebrafish form by convergence of cellular fields at the edge of the neural plate.

The primary olfactory sensory system is part of the PNS that develops from ectodermal placodes. Several cell types, including sensory neurons and support cells, differentiate within the olfactory placode to form the mature olfactory organ. The olfactory placodes are thought to arise from lateral regions of the anterior neural plate, which separate from the plate through differential cell movements. We determined the origins of the olfactory placodes in zebrafish by labeling cells along the anterior-lateral edge of the neural plate at times preceding the formation of the olfactory placodes and examining the later fates of the labeled cells. Surprisingly, we found that the olfactory placode arises from a field of cells, not from a discrete region of the anterior neural plate. This field extends posteriorly to the anterior limits of cranial neural crest and is bordered medially by telencephalic precursors. Cells giving rise to progeny in both the olfactory organ and telencephalon express the distal-less 3 gene. Furthermore, we found no localized pockets of cell division in the anterior-lateral neural plate cells preceding the appearance of the olfactory placode. We suggest that the olfactory placodes arise by anterior convergence of a field of lateral neural plate cells, rather than by localized separation and proliferation of a discrete group of cells.

Anterior movement of ventral diencephalic precursors separates the primordial eye field in the neural plate and requires cyclops.

A currently favored hypothesis postulates that a single field of cells in the neural plate forms bilateral retinas. To learn how retinal precursors segregate, we followed individual labeled neural plate cells in zebrafish. In the late gastrula, a single field of odd-paired-like-expressing cells contributed to both retinas, bordered posteriorly by diencephalic precursors expressing mariposa. Median mariposa-expressing cells moved anteriorly, separating the eyes, and formed ventral anterior diencephalon, the presumptive hypothalamus. In cyclops mutants, corresponding cells failed to move anteriorly, a ventral diencephalon never formed, and the eyes remained fused. Ablation of the region containing these cells induced cyclopia in wild types. Our results indicate that movement of a median subpopulation of diencephalic precursors separates retinal precursors into left and right eyes. Wild-type cyclops gene function is required for these morphogenetic movements.

Functionally independent components of early event-related potentials in a visual spatial attention task.

Spatial visual attention modulates the first negative-going deflection in the human averaged event-related potential (ERP) in response to visual target and non-target stimuli (the N1 complex). Here we demonstrate a decomposition of N1 into functionally independent subcomponents with functionally distinct relations to task and stimulus conditions. ERPs were collected from 20 subjects in response to visual target and non-target stimuli presented at five attended and non-attended screen locations. Independent component analysis, a new method for blind source separation, was trained simultaneously on 500 ms grand average responses from all 25 stimulus-attention conditions and decomposed the non-target N1 complexes into five spatially fixed, temporally independent and physiologically plausible components. Activity of an early, laterally symmetrical component pair (N1aR and N1aL) was evoked by the left and right visual field stimuli, respectively. Component N1aR peaked ca. 9 ms earlier than N1aL. Central stimuli evoked both components with the same peak latency difference, producing a bilateral scalp distribution. The amplitudes of these components were no reliably augmented by spatial attention. Stimuli in the right visual field evoked activity in a spatio-temporally overlapping bilateral component (N1b) that peaked at ca. 180 ms and was strongly enhanced by attention. Stimuli presented at unattended locations evoked a fourth component (P2a) peaking near 240 ms. A fifth component (P3f) was evoked only by targets presented in either visual field. The distinct response patterns of these components across the array of stimulus and attention conditions suggest that they reflect activity in functionally independent brain systems involved in processing attended and unattended visuospatial events.

Spatial attention deficits in patients with acquired or developmental cerebellar abnormality.

Recent imaging and clinical studies have challenged the concept that the functional role of the cerebellum is exclusively in the motor domain. We present evidence of slowed covert orienting of visuospatial attention in patients with developmental cerebellar abnormality (patients with autism, a disorder in which at least 90% of all postmortem cases reported to date have Purkinje neuron loss), and in patients with cerebellar damage acquired from tumor or stroke. In spatial cuing tasks, normal control subjects across a wide age range were able to orient attention within 100 msec of an attention-directing cue. Patients with cerebellar damage showed little evidence of having oriented attention after 100 msec but did show the effects of attention orienting after 800-1200 msec. These effects were demonstrated in a task in which results were independent of the motor response. In this task, smaller cerebellar vermal lobules VI-VII (from magnetic resonance imaging) were associated with greater attention-orienting deficits. Although eye movements may also be disrupted in patients with cerebellar damage, abnormal gaze shifting cannot explain the timing and nature of the attention-orienting deficits reported here. These data may be consistent with evidence from animal models that suggest damage to the cerebellum disrupts both the spatial encoding of a location for an attentional shift and the subsequent gaze shift. These data are also consistent with a model of cerebellar function in which the cerebellum supports a broad spectrum of brain systems involved in both nonmotor and motor function.