Can we model autism using zebrafish?

Autism spectrum disorder (ASD) is one of the most common, heritable neuropsychiatric disorders in the world, affecting almost 1% of the population. The core symptoms used to diagnose ASD are decreased social interaction and increased repetitive behaviors. Despite the large number of affected individuals, the precise mechanisms that cause this disorder remain unclear. The identification of genes and environmental factors associated with ASD allows the study of the underlying mechanisms in animal models. Although ASD presents as a human disorder, based on recent advances in understanding their brain anatomy, physiology, behavior, and evolutionary conservation of neuronal cell types, I propose that zebrafish may provide novel insights into the etiology.

Unraveling Presenilin 2 Functions in a Knockout Zebrafish Line to Shed Light into Alzheimer's Disease Pathogenesis.

Mutations in presenilin 2 (PS2) have been causally linked to the development of inherited Alzheimer's disease (AD). Besides its role as part of the γ-secretase complex, mammalian PS2 is also involved, as an individual protein, in a growing number of cell processes, which result altered in AD. To gain more insight into PS2 (dys)functions, we have generated a presenilin2 (psen2) knockout zebrafish line. We found that the absence of the protein does not markedly influence Notch signaling at early developmental stages, suggesting a Psen2 dispensable role in the γ-secretase-mediated Notch processing. Instead, loss of Psen2 induces an exaggerated locomotor response to stimulation in fish larvae, a reduced number of ER-mitochondria contacts in zebrafish neurons, and an increased basal autophagy. Moreover, the protein is involved in mitochondrial axonal transport, since its acute downregulation reduces in vivo organelle flux in zebrafish sensory neurons. Importantly, the expression of a human AD-linked mutant of the protein increases this vital process. Overall, our results confirm zebrafish as a good model organism for investigating PS2 functions in vivo, representing an alternative tool for the characterization of new AD-linked defective cell pathways and the testing of possible correcting drugs.

The microbiota promotes social behavior by modulating microglial remodeling of forebrain neurons.

Host-associated microbiotas guide the trajectory of developmental programs, and altered microbiota composition is linked to neurodevelopmental conditions such as autism spectrum disorder. Recent work suggests that microbiotas modulate behavioral phenotypes associated with these disorders. We discovered that the zebrafish microbiota is required for normal social behavior and reveal a molecular pathway linking the microbiota, microglial remodeling of neural circuits, and social behavior in this experimentally tractable model vertebrate. Examining neuronal correlates of behavior, we found that the microbiota restrains neurite complexity and targeting of forebrain neurons required for normal social behavior and is necessary for localization of forebrain microglia, brain-resident phagocytes that remodel neuronal arbors. The microbiota also influences microglial molecular functions, including promoting expression of the complement signaling pathway and the synaptic remodeling factor c1q. Several distinct bacterial taxa are individually sufficient for normal microglial and neuronal phenotypes, suggesting that host neuroimmune development is sensitive to a feature common among many bacteria. Our results demonstrate that the microbiota influences zebrafish social behavior by stimulating microglial remodeling of forebrain circuits during early neurodevelopment and suggest pathways for new interventions in multiple neurodevelopmental disorders.

A conserved transcriptional fingerprint of multi-neurotransmitter neurons necessary for social behavior.

BACKGROUND: An essential determinant of a neuron's functionality is its neurotransmitter phenotype. We previously identified a defined subpopulation of cholinergic neurons required for social orienting behavior in zebrafish. RESULTS: We transcriptionally profiled these neurons and discovered that they are capable of synthesizing both acetylcholine and GABA. We also established a constellation of transcription factors and neurotransmitter markers that can be used as a "transcriptomic fingerprint" to recognize a homologous neuronal population in another vertebrate. CONCLUSION: Our results suggest that this transcriptomic fingerprint and the cholinergic-GABAergic neuronal subtype that it defines are evolutionarily conserved.

Egr1 Is Necessary for Forebrain Dopaminergic Signaling during Social Behavior.

Finding the link between behaviors and their regulatory molecular pathways is a major obstacle in treating neuropsychiatric disorders. The immediate early gene (IEG) EGR1 is implicated in the etiology of neuropsychiatric disorders, and is linked to gene pathways associated with social behavior. Despite extensive knowledge of EGR1 gene regulation at the molecular level, it remains unclear how EGR1 deficits might affect the social component of these disorders. Here, we examined the social behavior of zebrafish with a mutation in the homologous gene egr1 Mutant fish exhibited reduced social approach and orienting, whereas other sensorimotor behaviors were unaffected. On a molecular level, expression of the dopaminergic biosynthetic enzyme, tyrosine hydroxylase (TH), was strongly decreased in TH-positive neurons of the anterior parvocellular preoptic nucleus. These neurons are connected with basal forebrain (BF) neurons associated with social behavior. Chemogenetic ablation of around 30% of TH-positive neurons in this preoptic region reduced social attraction to a similar extent as the egr1 mutation. These results demonstrate the requirement of Egr1 and dopamine signaling during social interactions, and identify novel circuitry underlying this behavior.

Late onset of Synaptotagmin 2a expression at synapses relevant to social behavior.

As they form, synapses go through various stages of maturation and refinement. These steps are linked to significant changes in synaptic function, potentially resulting in emergence and maturation of behavioral outputs. Synaptotagmins are calcium-sensing proteins of the synaptic vesicle exocytosis machinery, and changes in Synaptotagmin proteins at synapses have significant effects on vesicle release and synaptic function. Here, we examined the distribution of the synaptic vesicle protein Synaptotagmin 2a (Syt2a) during development of the zebrafish nervous system. Syt2a is widely distributed throughout the midbrain and hindbrain early during larval development but very weakly expressed in the forebrain. Later in development, Syt2a expression levels in the forebrain increase, particularly in regions associated with social behavior, and most intriguingly, around the time social behavior becomes apparent. We provide evidence that Syt2a localizes to synapses onto neurons implicated in social behavior in the ventral forebrain and show that Syt2a is colocalized with tyrosine hydroxylase, a biosynthetic enzyme in the dopamine pathway. Our results suggest a developmentally important role for Syt2a in maturing synapses in the forebrain, coinciding with the emergence of social behavior.

Correction: yippee like 3 (ypel3) is a novel gene required for myelinating and perineurial glia development.

[This corrects the article DOI: 10.1371/journal.pgen.1008841.].

yippee like 3 (ypel3) is a novel gene required for myelinating and perineurial glia development.

Hypomyelination, a neurological condition characterized by decreased production of myelin sheets by glial cells, often has no known etiology. Elucidating the genetic causes of hypomyelination provides a better understanding of myelination, as well as means to diagnose, council, and treat patients. Here, we present evidence that YIPPEE LIKE 3 (YPEL3), a gene whose developmental role was previously unknown, is required for central and peripheral glial cell development. We identified a child with a constellation of clinical features including cerebral hypomyelination, abnormal peripheral nerve conduction, hypotonia, areflexia, and hypertrophic peripheral nerves. Exome and genome sequencing revealed a de novo mutation that creates a frameshift in the open reading frame of YPEL3, leading to an early stop codon. We used zebrafish as a model system to validate that YPEL3 mutations are causative of neuropathy. We found that ypel3 is expressed in the zebrafish central and peripheral nervous system. Using CRISPR/Cas9 technology, we created zebrafish mutants carrying a genomic lesion similar to that of the patient. Our analysis revealed that Ypel3 is required for development of oligodendrocyte precursor cells, timely exit of the perineurial glial precursors from the central nervous system (CNS), formation of the perineurium, and Schwann cell maturation. Consistent with these observations, zebrafish ypel3 mutants have metabolomic signatures characteristic of oligodendrocyte and Schwann cell differentiation defects, show decreased levels of Myelin basic protein in the central and peripheral nervous system, and develop defasciculated peripheral nerves. Locomotion defects were observed in adult zebrafish ypel3 mutants. These studies demonstrate that Ypel3 is a novel gene required for perineurial cell development and glial myelination.

Rapid Progressive Social Development of Zebrafish.

Zebrafish (Danio rerio) are highly social animals that engage in a diverse variety of nonreproductive social behaviors that emerge as early as 14 days postfertilization (dpf). However, we observe considerable behavioral variability at this stage, and comparisons across studies are potentially complicated both by chronological gaps in measurements and inconsistencies in developmental staging. To address these issues, we adapted our assay for social orienting and cueing in the adult zebrafish and used it to probe behavior in a critical window of larval development. In addition, we performed measurements of body length and tested a cohort of larvae with impaired growth to understand if this morphological feature is predictive of individual sociality. We report that zebrafish exhibit increasingly complex social behaviors between 10 and 16 dpf, including place preference, orienting, and social cueing. Furthermore, social behavior is related to standard length on an individual basis beginning at 14 dpf, such that developmentally stunted 14 dpf zebrafish raised on dry feed do not exhibit social behaviors, suggesting some morphological features are more predictive than chronological age. This highly variable and early stage in development provides an opportunity to further understand how genetic and environmental factors affect the assembly of neural circuits underlying complex behaviors.

Grxcr1 Promotes Hair Bundle Development by Destabilizing the Physical Interaction between Harmonin and Sans Usher Syndrome Proteins.

Morphogenesis and mechanoelectrical transduction of the hair cell mechanoreceptor depend on the correct assembly of Usher syndrome (USH) proteins into highly organized macromolecular complexes. Defects in these proteins lead to deafness and vestibular areflexia in USH patients. Mutations in a non-USH protein, glutaredoxin domain-containing cysteine-rich 1 (GRXCR1), cause non-syndromic sensorineural deafness. To understand the deglutathionylating enzyme function of GRXCR1 in deafness, we generated two grxcr1 zebrafish mutant alleles. We found that hair bundles are thinner in homozygous grxcr1 mutants, similar to the USH1 mutants ush1c (Harmonin) and ush1ga (Sans). In vitro assays showed that glutathionylation promotes the interaction between Ush1c and Ush1ga and that Grxcr1 regulates mechanoreceptor development by preventing physical interaction between these proteins without affecting the assembly of another USH1 protein complex, the Ush1c-Cadherin23-Myosin7aa tripartite complex. By elucidating the molecular mechanism through which Grxcr1 functions, we also identify a mechanism that dynamically regulates the formation of Usher protein complexes.

4.1Ba is necessary for glutamatergic synapse formation in the sensorimotor circuit of developing zebrafish.

During the process of synapse formation, thousands of proteins assemble at prospective sites of cell-cell communication. Although many of these proteins have been identified, the roles they play in generating functional connections during development remain unknown. 4.1 scaffolding proteins have been implicated in synapse formation and maturation in vitro, but in vivo studies for some family members have suggested these proteins are not important for this role. We examined the role of family member 4.1B because it has been implicated in glutamatergic synaptogenesis, but has not been described in vivo. We identified two 4.1B genes in zebrafish, 4.1Ba and 4.1Bb, by sequence comparisons and synteny analysis. In situ hybridization shows these genes are differentially expressed, with 4.1Ba expressed primarily in the nervous system and 4.1Bb expressed in the nervous system and muscle, but not the spinal cord. We focused our studies on 4.1Ba in the spinal cord. 4.1Ba knockdown reduced the number of glutamatergic synapses at caudal primary motor neurons and caused an increase in the duration of touch-evoked coiling. These results suggest 4.1Ba is important for the formation of functional glutamatergic synapses in the developing zebrafish spinal cord.

Forebrain Control of Behaviorally Driven Social Orienting in Zebrafish.

Deficits in social engagement are diagnostic of multiple neurodevelopmental disorders, including autism and schizophrenia [1]. Genetically tractable animal models like zebrafish (Danio rerio) could provide valuable insight into developmental factors underlying these social impairments, but this approach is predicated on the ability to accurately and reliably quantify subtle behavioral changes. Similarly, characterizing local molecular and morphological phenotypes requires knowledge of the neuroanatomical correlates of social behavior. We leveraged behavioral and genetic tools in zebrafish to both refine our understanding of social behavior and identify brain regions important for driving it. We characterized visual social interactions between pairs of adult zebrafish and discovered that they perform a stereotyped orienting behavior that reflects social attention [2]. Furthermore, in pairs of fish, the orienting behavior of one individual is the primary factor driving the same behavior in the other individual. We used manual and genetic lesions to investigate the forebrain contribution to this behavior and identified a population of neurons in the ventral telencephalon whose ablation suppresses social interactions, while sparing other locomotor and visual behaviors. These neurons are cholinergic and express the gene encoding the transcription factor Lhx8a, which is required for development of cholinergic neurons in the mouse forebrain [3]. The neuronal population identified in zebrafish lies in a region homologous to mammalian forebrain regions implicated in social behavior such as the lateral septum [4]. Our data suggest that an evolutionarily conserved population of neurons controls social orienting in zebrafish.

Correction: A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate Expression Constructs with Diverse Research Applications.

[This corrects the article DOI: 10.1371/journal.pone.0159277.].

Redundant Postsynaptic Functions of SynCAMs 1-3 during Synapse Formation.

Investigating the roles of synaptogenic adhesion molecules during synapse formation has proven challenging, often due to compensatory functions between additional family members. The synaptic cell adhesion molecules 1-3 (SynCAM1-3) are expressed both pre- and postsynaptically, share highly homologous domains and are synaptogenic when ectopically presented to neurons; yet their endogenous functions during synaptogenesis are unclear. Here we report that SynCAM1-3 are functionally redundant and collectively necessary for synapse formation in cultured hippocampal neurons. Only triple knockdown (KD) of SynCAM1-3 using highly efficient, chained artificial microRNAs (amiRNAs) reduced synapse density and increased synapse area. Electrophysiological recordings of quantal release events supported an increase in synapse size caused by SynCAM1-3 depletion. Furthermore, a combinatorial, mosaic lentiviral approach comparing wild type (WT) and SynCAM1-3 KD neurons in the same culture demonstrate that SynCAM1-3 set synapse number and size through postsynaptic mechanisms. The results demonstrate that the redundancy between SynCAM1-3 has concealed their synaptogenic function at the postsynaptic terminal.

A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate Expression Constructs with Diverse Research Applications.

Recombination-based cloning is a quick and efficient way to generate expression vectors. Recent advancements have provided powerful recombinant DNA methods for molecular manipulations. Here, we describe a novel collection of three-fragment MultiSite Gateway cloning system-compatible vectors providing expanded molecular tools for vertebrate research. The components of this toolkit encompass a broad range of uses such as fluorescent imaging, dual gene expression, RNA interference, tandem affinity purification, chemically-inducible dimerization and lentiviral production. We demonstrate examples highlighting the utility of this toolkit for producing multi-component vertebrate expression vectors with diverse primary research applications. The vectors presented here are compatible with other Gateway toolkits and collections, facilitating the rapid generation of a broad range of innovative DNA constructs for biological research.

Improved knockdown from artificial microRNAs in an enhanced miR-155 backbone: a designer's guide to potent multi-target RNAi.

Artificial microRNA (amiRNA) sequences embedded in natural microRNA (miRNA) backbones have proven to be useful tools for RNA interference (RNAi). amiRNAs have reduced off-target and toxic effects compared to other RNAi-based methods such as short-hairpin RNAs (shRNA). amiRNAs are often less effective for knockdown, however, compared to their shRNA counterparts. We screened a large empirically-designed amiRNA set in the synthetic inhibitory BIC/miR-155 RNA (SIBR) scaffold and show common structural and sequence-specific features associated with effective amiRNAs. We then introduced exogenous motifs into the basal stem region which increase amiRNA biogenesis and knockdown potency. We call this modified backbone the enhanced SIBR (eSIBR) scaffold. Using chained amiRNAs for multi-gene knockdown, we show that concatenation of miRNAs targeting different genes is itself sufficient for increased knockdown efficacy. Further, we show that eSIBR outperforms wild-type SIBR (wtSIBR) when amiRNAs are chained. Finally, we use a lentiviral expression system in cultured neurons, where we again find that eSIBR amiRNAs are more potent for multi-target knockdown of endogenous genes. eSIBR will be a valuable tool for RNAi approaches, especially for studies where knockdown of multiple targets is desired.

Transcriptomes of post-mitotic neurons identify the usage of alternative pathways during adult and embryonic neuronal differentiation.

BACKGROUND: Understanding the mechanisms by which neurons are generated and specified, and how they integrate into functional circuits is key to being able to treat disorders of the nervous system and acute brain trauma. Much of what we know about neuronal differentiation has been studied in developing embryos, but differentiation steps may be very different during adult neurogenesis. For this reason, we compared the transcriptomes of newly differentiated neurons in zebrafish embryos and adults. RESULTS: Using a 4tU RNA labeling method, we isolated and sequenced mRNA specifically from cells of one day old embryos and adults expressing the transgene HA-uprt-mcherry under control of the neuronal marker elavl3. By categorizing transcript products into different protein classes, we identified similarities and differences of gene usage between adult and embryonic neuronal differentiation. We found that neurons in the adult brain and in the nervous system of one day old embryos commonly use transcription factors - some of them identical - during the differentiation process. When we directly compared adult differentiating neurons to embryonic differentiating neurons, however, we found that during adult neuronal differentiation, the expression of neuropeptides and neurotransmitter pathway genes is more common, whereas classical developmental signaling through secreted molecules like Hedgehog or Wnt are less enriched, as compared to embryonic stages. CONCLUSIONS: We conclude that both adult and embryonic differentiating neurons show enriched use of transcription factors compared to surrounding cells. However, adult and embryonic developing neurons use alternative pathways to differentiate. Our study provides evidence that adult neuronal differentiation is distinct from the better characterized embryonic neuronal differentiation process. This important insight and the lists of enriched genes we have identified will now help pave the way to a better understanding of the mechanisms of embryonic and adult neuronal differentiation and how to manipulate these processes.

Synapse assembly and neurodevelopmental disorders.

In this review we examine the current understanding of how genetic deficits associated with neurodevelopmental disorders may impact synapse assembly. We then go on to discuss how the critical periods for these genetic deficits will shape the nature of future clinical interventions.

Temporally and spatially restricted gene expression profiling.

Identifying gene function in specific cells is critical for understanding the processes that make cells unique. Several different methods are available to isolate actively transcribed RNA or actively translated RNA in specific cells at a chosen time point. Cell-specific mRNA isolation can be accomplished by the expression of transgenes in cells of interest, either directly from a specific promoter or using a modular system such as Gal4/UAS or Cre/lox. All of the methods described in this review, namely thiol-labeling of RNA (TU-tagging or RABT), TRAP (translating ribosome affinity purification) and INTACT (isolation of nuclei tagged in specific cell types), allow next generation sequencing, permitting the identification of enriched gene transcripts within the specific cell-type. We describe here the general concept of each method, include examples, evaluate possible problems related to each technique, and suggest the types of questions for which each method is best suited.

Complexes of Usher proteins preassemble at the endoplasmic reticulum and are required for trafficking and ER homeostasis.

Usher syndrome (USH), the leading cause of hereditary combined hearing and vision loss, is characterized by sensorineural deafness and progressive retinal degeneration. Mutations in several different genes produce USH, but the proximal cause of sensory cell death remains mysterious. We adapted a proximity ligation assay to analyze associations among three of the USH proteins, Cdh23, Harmonin and Myo7aa, and the microtubule-based transporter Ift88 in zebrafish inner ear mechanosensory hair cells. We found that the proteins are in close enough proximity to form complexes and that these complexes preassemble at the endoplasmic reticulum (ER). Defects in any one of the three USH proteins disrupt formation and trafficking of the complex and result in diminished levels of the other proteins, generalized trafficking defects and ER stress that triggers apoptosis. ER stress, thus, contributes to sensory hair cell loss and provides a new target to explore for protective therapies for USH.