Behavioral Assays Dissecting NMDA Receptor Function in Zebrafish.

Zebrafish are a powerful system to study brain development and to dissect the activity of complex circuits. One advantage is that they display complex behaviors, including prey capture, learning, responses to photic and acoustic stimuli, and social interaction (Dreosti et al., Front Neural Circuits 9:39, 2015; Bruckner et al., PLoS Biol 20:e3001838, 2022; Zoodsma et al., Mol Autism 13:38, 2022) that can be probed to assess brain function. Many of these behaviors are easily assayed at early larval stages, offering a noninvasive and high-throughput readout of nervous system function. Additionally, larval zebrafish readily uptake small molecules dissolved in water making them ideal for behavioral-based drug screens. Together, larval zebrafish and their behavioral repertoire offer a means to rapidly dissect brain circuitry and can serve as a template for high-throughput small molecule screens.NMDA receptor subunits are highly conserved in zebrafish compared to mammals (Zoodsma et al., Mol Autism 13:38, 2022; Cox et al., Dev Dyn 234:756-766, 2005; Zoodsma et al., J Neurosci 40:3631-3645, 2020). High amino acid and domain structure homology between humans and zebrafish underlie conserved functional similarities. Here we describe a set of behavioral assays that are useful to study the NMDA receptor activity in brain function.

Developmental loss of NMDA receptors results in supernumerary forebrain neurons through delayed maturation of transit-amplifying neuroblasts.

Developmental neurogenesis is a tightly regulated spatiotemporal process with its dysregulation implicated in neurodevelopmental disorders. NMDA receptors are glutamate-gated ion channels that are widely expressed in the early nervous system, yet their contribution to neurogenesis is poorly understood. Notably, a variety of mutations in genes encoding NMDA receptor subunits are associated with neurodevelopmental disorders. To rigorously define the role of NMDA receptors in developmental neurogenesis, we used a mutant zebrafish line (grin1-/-) that lacks all NMDA receptors yet survives to 10 days post-fertilization, offering the opportunity to study post-embryonic neurodevelopment in the absence of NMDA receptors. Focusing on the forebrain, we find that these fish have a progressive supernumerary neuron phenotype confined to the telencephalon at the end of embryonic neurogenesis, but which extends to all forebrain regions during postembryonic neurogenesis. This enhanced neuron population does not arise directly from increased numbers or mitotic activity of radial glia cells, the principal neural stem cells. Rather, it stems from a lack of timely maturation of transit-amplifying neuroblasts into post-mitotic neurons, as indicated by a decrease in expression of the ontogenetically-expressed chloride transporter, KCC2. Pharmacological blockade with MK-801 recapitulates the grin1-/- supernumerary neuron phenotype, indicating a requirement for ionotropic signaling. Thus, NMDA receptors are required for suppression of indirect, transit amplifying cell-driven neurogenesis by promoting maturational termination of mitosis. Loss of suppression results in neuronal overpopulation that can fundamentally change brain circuitry and may be a key factor in pathogenesis of neurodevelopmental disorders caused by NMDA receptor dysfunction.

Perfluorooctane Sulfonate (PFOS) Negatively Impacts Prey Capture Capabilities in Larval Zebrafish.

Per- and polyfluoroalkyl substances (PFAS) are widely used in many industrial and domestic applications, which has resulted in unintentional human exposures and bioaccumulation in blood and other organs. Perfluorooctane sulfonate (PFOS) is among the most prevalent PFAS in the environment and has been postulated to affect brain functions in exposed organisms. However, the impacts of PFOS in early neural development have not been well described. We used zebrafish larvae to assess the effects of PFOS on two fundamental complex behaviors, prey capture and learning. Zebrafish exposed to PFOS concentrations ranging from 2 to 20 µM for differing 48-h periods were viable through early larval stages. In addition, PFOS uptake was unaffected by the presence of a chorion. We employed two different experimental paradigms; first we assessed the impacts of increasing organismal PFOS bioaccumulation on prey capture and learning, and second, we probed stage-specific sensitivity to PFOS by exposing zebrafish at different developmental stages (0-2 vs. 3-5 days post fertilization). Following both assays we measured the amount of PFOS present in each larva and found that PFOS levels varied in larvae from different groups within each experimental paradigm. Significant negative correlations were observed between larval PFOS accumulation and percentage of captured prey, whereas nonsignificant negative correlations were observed between PFOS accumulation and experienced-induced prey capture learning. These findings suggest that PFOS accumulation negatively affects larval zebrafish's ability to perform complicated multisensory behaviors and highlights the potential risks of PFOS exposure to animals in the wild, with implications for human health. Environ Toxicol Chem 2024;43:847-855. © 2023 SETAC.

Loss of NMDA receptor function during development results in decreased KCC2 expression and increased neurons in the zebrafish forebrain.

Developmental neurogenesis is a tightly regulated spatiotemporal process with its dysregulation implicated in neurodevelopmental disorders. NMDA receptors are glutamate-gated ion channels that are widely expressed in the early nervous system, yet their contribution to neurogenesis is poorly understood. Notably, a variety of mutations in genes encoding NMDA receptor subunits are associated with neurodevelopmental disorders. To rigorously define the role of NMDA receptors in developmental neurogenesis, we used a mutant zebrafish line ( grin1 -/- ) that lacks all NMDA receptors yet survives to 10 days post-fertilization, offering the opportunity to study post-embryonic neurodevelopment in the absence of NMDA receptors. Focusing on the forebrain, we find that these fish have a progressive supernumerary neuron phenotype confined to the telencephalon at the end of embryonic neurogenesis, but which extends to all forebrain regions during postembryonic neurogenesis. This enhanced neuron population does not arise directly from increased numbers or mitotic activity of radial glia cells, the principal neural stem cells. Rather, it stems from a lack of timely maturation of transit-amplifying neuroblasts into post-mitotic neurons, as indicated by a decrease in expression of the ontogenetically-expressed chloride transporter, KCC2. Pharmacological blockade with MK-801 recapitulates the grin1 -/- supernumerary neuron phenotype, indicating a requirement for ionotropic signaling. Thus, NMDA receptors are required for suppression of indirect, transit amplifying cell-driven neurogenesis by promoting maturational termination of mitosis. Loss of suppression results in neuronal overpopulation that can fundamentally change brain circuitry and may be a key factor in pathogenesis of neurodevelopmental disorders caused by NMDA receptor dysfunction.

Disruption of grin2B, an ASD-associated gene, produces social deficits in zebrafish.

BACKGROUND: Autism spectrum disorder (ASD), like many neurodevelopmental disorders, has complex and varied etiologies. Advances in genome sequencing have identified multiple candidate genes associated with ASD, including dozens of missense and nonsense mutations in the NMDAR subunit GluN2B, encoded by GRIN2B. NMDARs are glutamate-gated ion channels with key synaptic functions in excitatory neurotransmission. How alterations in these proteins impact neurodevelopment is poorly understood, in part because knockouts of GluN2B in rodents are lethal. METHODS: Here, we use CRISPR-Cas9 to generate zebrafish lacking GluN2B (grin2B-/-). Using these fish, we run an array of behavioral tests and perform whole-brain larval imaging to assay developmental roles and functions of GluN2B. RESULTS: We demonstrate that zebrafish GluN2B displays similar structural and functional properties to human GluN2B. Zebrafish lacking GluN2B (grin2B-/-) surprisingly survive into adulthood. Given the prevalence of social deficits in ASD, we assayed social preference in the grin2B-/- fish. Wild-type fish develop a strong social preference by 3 weeks post fertilization. In contrast, grin2B-/- fish at this age exhibit significantly reduced social preference. Notably, the lack of GluN2B does not result in a broad disruption of neurodevelopment, as grin2B-/- larvae do not show alterations in spontaneous or photic-evoked movements, are capable of prey capture, and exhibit learning. Whole-brain imaging of grin2B-/- larvae revealed reduction of an inhibitory neuron marker in the subpallium, a region linked to ASD in humans, but showed that overall brain size and E/I balance in grin2B-/- is comparable to wild type. LIMITATIONS: Zebrafish lacking GluN2B, while useful in studying developmental roles of GluN2B, are unlikely to model nuanced functional alterations of human missense mutations that are not complete loss of function. Additionally, detailed mammalian homologies for larval zebrafish brain subdivisions at the age of whole-brain imaging are not fully resolved. CONCLUSIONS: We demonstrate that zebrafish completely lacking the GluN2B subunit of the NMDAR, unlike rodent models, are viable into adulthood. Notably, they exhibit a highly specific deficit in social behavior. As such, this zebrafish model affords a unique opportunity to study the roles of GluN2B in ASD etiologies and establish a disease-relevant in vivo model for future studies.

Comprehensive assessment of chemical residues in surface and wastewater using passive sampling, chemical, biological, and fish behavioral assays.

Effluents from ten full-scale municipal wastewater treatment plants (WWTPs) that discharge into the Hudson River, surface waters, and wild-caught fish samples were analyzed using liquid chromatography with tandem mass spectrometry (LC/MS/MS) to examine the influence of wastewater discharge on the concentrations of contaminants of emerging concern (CECs) and their ecological impacts on fish. Analysis was based on targeted detection of 41 pharmaceuticals, and non-targeted analysis (suspect screening) of CECs. Biological effects of treated WWTP effluents were assessed using a larval zebrafish (Danio rerio) swimming behavior assay. Concentrations of residues in surface waters were determined in grab samples and polar organic chemical integrative samplers (POCIS). In addition, vitellogenin peptides, used as biomarkers of endocrine disruption, were quantified using LC/MS/MS in the wild-caught fish plasma samples. Overall, 94 chemical residues were identified, including 63 pharmaceuticals, 10 industrial chemicals, and 21 pesticides. Eight targeted pharmaceuticals were detected in 100% of effluent samples with median detections of: bupropion (194 ng/L), carbamazepine (91 ng/L), ciprofloxacin (190 ng/L), citalopram (172 ng/L), desvenlafaxine (667 ng/L), iopamidol (3790 ng/L), primidone (86 ng/L), and venlafaxine (231 ng/L). Over 30 chemical residues were detected in wild-caught fish tissues. Notably, zebrafish larvae exposed to chemical extracts of effluents from 9 of 10 WWTPs, in at least one season, were significantly hyperactive. Vitellogenin expression in male or immature fish occurred 2.8 times more frequently in fish collected from the Hudson River as compared to a reference site receiving no direct effluent input. Due to the low concentrations of pharmaceuticals detected in effluents, it is likely that chemicals other than pharmaceuticals measured are responsible for the behavioral changes observed. The combined use of POCIS and non-target analysis demonstrated significant increase in the chemical coverage for CEC detection, providing a better insight on the impacts of WWTP effluents and agricultural practices on surface water quality.

Comparative transcriptomics implicate mitochondrial and neurodevelopmental impairments in larval zebrafish (Danio rerio) exposed to two selective serotonin reuptake inhibitors (SSRIs).

Pharmaceuticals and personal care products are emerging contaminants that are increasingly detected in the environment worldwide. Certain classes of pharmaceuticals, such as selective serotonin reuptake inhibitors (SSRIs), are a major environmental concern due to their widespread use and the fact that these compounds are designed to have biological effects at low doses. A complication in predicting toxic effects of SSRIs in nontarget organisms is that their mechanism of action is not fully understood. To better understand the potential toxic effects of SSRIs, we employed an ultra-low input RNA-sequencing method to identify potential pathways that are affected by early exposure to two SSRIs (fluoxetine and paroxetine). We exposed wildtype zebrafish (Danio rerio) embryos to 100 µg/L of either fluoxetine or paroxetine for 6 days before extracting and sequencing mRNA from individual larval brains. Differential gene expression analysis identified 1550 genes that were significantly affected by SSRI exposure with a core set of 138 genes altered by both SSRIs. Weighted gene co-expression network analysis identified 7 modules of genes whose expression patterns were significantly correlated with SSRI exposure. Functional enrichment analysis of differentially expressed genes as well as network module genes repeatedly identified various terms associated with mitochondrial and neuronal structures, mitochondrial respiration, and neurodevelopmental processes. The enrichment of these terms indicates that toxic effects of SSRI exposure are likely caused by mitochondrial dysfunction and subsequent neurodevelopmental effects. To our knowledge, this is the first effort to study the tissue-specific transcriptomic effects of SSRIs in developing zebrafish, providing specific, high resolution molecular data regarding the sublethal effects of SSRI exposure.

Lrrk2 modulation of Wnt signaling during zebrafish development.

Mutations in leucine-rich repeat kinase 2 (lrrk2) are the most common genetic cause of Parkinson's disease. Difficulty in elucidating the pathogenic mechanisms resulting from disease-associated Lrrk2 variants stems from the complexity of Lrrk2 function and activities. Lrrk2 contains multiple protein-protein interacting domains, a GTPase domain, and a kinase domain. Lrrk2 is implicated in many cellular processes including vesicular trafficking, autophagy, cytoskeleton dynamics, and Wnt signaling. Here, we generated a zebrafish lrrk2 allelic series to study the requirements for Lrrk2 during development and to dissect the importance of its various domains. The alleles are predicted to encode proteins that either lack all functional domains (lrrk2sbu304 ), the GTPase, and kinase domains (lrrk2sbu71 ) or the kinase domain (lrrk2sbu96 ). All three lrrk2 mutants are viable, morphologically normal, and display wild-type-like locomotion. Because Lrrk2 modulates Wnt signaling in some contexts, we assessed Wnt signaling in all three mutant lines. Analysis of Wnt signaling by studying the expression of target genes using whole mount RNA in situ hybridization and a transgenic Wnt reporter revealed wild-type domains of Wnt activity in each of the mutants. However, we found that Wnt pathway activation is attenuated in lrrk2sbu304/sbu304 , which lacks both scaffolding and catalytic domains, but not in the other alleles during late embryogenesis. This supports a model in which Lrrk2 scaffolding functions are key to a context-dependent role in promoting canonical Wnt signaling.

A Model to Study NMDA Receptors in Early Nervous System Development.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play critical roles in neuronal development and nervous system function. Here, we developed a model to study NMDARs in early development in zebrafish, by generating CRISPR-mediated lesions in the NMDAR genes, grin1a and grin1b, which encode the obligatory GluN1 subunits. While receptors containing grin1a or grin1b show high Ca2+ permeability, like their mammalian counterpart, grin1a is expressed earlier and more broadly in development than grin1b Both grin1a-/- and grin1b-/- zebrafish are viable. Unlike in rodents, where the grin1 knockout is embryonic lethal, grin1 double-mutant fish (grin1a-/-; grin1b-/-), which lack all NMDAR-mediated synaptic transmission, survive until ∼10 d dpf (days post fertilization), providing a unique opportunity to explore NMDAR function during development and in generating behaviors. Many behavioral defects in the grin1 double-mutant larvae, including abnormal evoked responses to light and acoustic stimuli, prey-capture deficits, and a failure to habituate to acoustic stimuli, are replicated by short-term treatment with the NMDAR antagonist MK-801, suggesting that they arise from acute effects of compromised NMDAR-mediated transmission. Other defects, however, such as periods of hyperactivity and alterations in place preference, are not phenocopied by MK-801, suggesting a developmental origin. Together, we have developed a unique model to study NMDARs in the developing vertebrate nervous system.SIGNIFICANCE STATEMENT Rapid communication between cells in the nervous system depends on ion channels that are directly activated by chemical neurotransmitters. One such ligand-gated ion channel, the NMDAR, impacts nearly all forms of nervous system function. It has been challenging, however, to study the prolonged absence of NMDARs in vertebrates, and hence their role in nervous system development, due to experimental limitations. Here, we demonstrate that zebrafish lacking all NMDAR transmission are viable through early development and are capable of a wide range of stereotypic behaviors. As such, this zebrafish model provides a unique opportunity to study the role of NMDAR in the development of the early vertebrate nervous system.

CoRest1 regulates neurogenesis in a stage-dependent manner.

BACKGROUND: Developmental processes, including neuronal differentiation, require precise regulation of transcription. The RE-1 silencing transcription factor (Rest), is often called a "master neuronal regulator" due to its large number of neural-specific targets. Rest recruits CoRest (Rcor) and Sin3 corepressor complexes to gene regulatory sequences. CoRest not only associates with Rest, but with other transcription regulators. In this study, we generated zebrafish rcor1 mutants using transcription activator-like effector nucleases (TALENS), to study its requisite role in repression of Rest target genes as well as Rest-independent Rcor1 developmental functions. RESULTS: While rcor1 mutants have a slight decrease in fitness, most survived and produced viable offspring. We examined expression levels of RE1-containing genes in maternal zygotic rcor1 (MZrcor1) mutants and found that Rcor1 is generally not required for the repression of Rest target genes at early stages. However, MZrcor1 mutants undergo more rapid neurogenesis compared to controls. We found that at gastrula stages, Rcor1 acts as a repressor of her gene family, but at later stages, her6 decreased in the MZrcor1 mutant. CONCLUSIONS: Based on these findings, the central role of CoRest1 in neurogenesis is likely due to a Rest-independent role rather than as a Rest corepressor.

Varying the exposure period and duration of neuroactive pharmaceuticals and their metabolites modulates effects on the visual motor response in zebrafish (Danio rerio) larvae.

Pharmaceuticals and personal care products are emerging contaminants that are increasingly detected in surface waters around the world. Despite the rise in environmental detections, measured concentrations are still typically low, raising the importance of environmental risk assessments that focus on ecologically relevant sublethal endpoints, such as altered behavior. Neuroactive pharmaceuticals, like mental health medications, pain killers, etc., may be particularly potent in this regard as they are specifically designed to cause behavioral changes without causing physiologic impairment in mammalian systems. We screened 15 different popular neuroactive pharmaceuticals, ranging from antidepressants (including 3 major antidepressant metabolites), anxiety medications, and pain killers, under three different exposure scenarios (repeated, late acute and early transient exposure) to look for behavioral effects in larval zebrafish using the visual motor response (VMR). Drugs were screened at 0, 1, 10, and 100 µg/L in the repeated exposure scenario, and at 0 and 100 µg/L in the late acute and early transient exposure scenarios. Eight of the 15 compounds tested, specifically the antidepressants amitriptyline, fluoxetine, nor-fluoxetine, paroxetine, sertraline, nor-sertraline, venlafaxine, and the antipsychotic drug haloperidol decreased swimming activity by 25% to 40% under repeated exposure conditions. Five of the compounds (amitriptyline, fluoxetine, nor-fluoxetine, paroxetine, and sertraline) also significantly decreased activity by 17% to 31% in the late acute exposure paradigm. Three compounds (fluoxetine, paroxetine and venlafaxine) significantly altered swimming activity with early transient exposure, however creating a hyperactive response and increasing activity from 24% to 28%, while haloperidol significantly decreased activity by 31%. This paper is, to our knowledge, the first to screen so many neuroactive pharmaceuticals, including major metabolites, in parallel under multiple exposure conditions. We show that antidepressants most consistently alter VMR swimming activity. Additionally, we show that major antidepressant metabolites can potentially alter behavior as much as their parent compounds. Furthermore, we show that the magnitude and direction of behavioral effect is dependent on the exposure duration and period, indicating that a more diverse experimental approach might be needed to more accurately assess the risk these compounds pose to the environment.

Zebrafish sin3b mutants are viable but have size, skeletal, and locomotor defects.

BACKGROUND: The transcriptional co-repressor Sin3 is highly conserved from yeast to vertebrates and has multiple roles controlling cell fate, cell cycle progression, and senescence programming. Sin3 proteins recruit histone deacetylases and other chromatin modifying factors to specific loci through interactions with transcription factors including Myc, Rest, p53 and E2F. Most vertebrates have two Sin3 family members (sin3a and sin3b), but zebrafish have a second sin3a paralogue. In mice, sin3a and sin3b are essential for embryonic development. Sin3b knockout mice show defects in growth as well as bone and blood differentiation. RESULTS: To study the requirement for Sin3b during development, we disrupted zebrafish sin3b using CRISPR-Cas9, and studied the effects on early development and locomotor behavior. CONCLUSIONS: Surprisingly, Sin3b is not essential in zebrafish. sin3b mutants show a decrease in fitness, small size, changes to locomotor behavior, and delayed bone development. We did not detect a role for Sin3b in cell proliferation. Our analysis of the sin3b mutant revealed a more nuanced requirement for zebrafish Sin3b than would be predicted from analysis of mutants in other species. Developmental Dynamics 246:946-955, 2017. © 2017 Wiley Periodicals, Inc.

Maternal Rest/Nrsf Regulates Zebrafish Behavior through snap25a/b.

UNLABELLED: During embryonic development, regulation of gene expression is key to creating the many subtypes of cells that an organism needs throughout its lifetime. Recent work has shown that maternal genetics and environmental factors have lifelong consequences on diverse processes ranging from immune function to stress responses. The RE1-silencing transcription factor (Rest) is a transcriptional repressor that interacts with chromatin-modifying complexes to repress transcription of neural-specific genes during early development. Here we show that in zebrafish, maternally supplied rest regulates expression of target genes during larval development and has lifelong impacts on behavior. Larvae deprived of maternal rest are hyperactive and show atypical spatial preferences. Adult male fish deprived of maternal rest present with atypical spatial preferences in a novel environment assay. Transcriptome sequencing revealed 158 genes that are repressed by maternal rest in blastula stage embryos. Furthermore, we found that maternal rest is required for target gene repression until at least 6 dpf. Importantly, disruption of the RE1 sites in either snap25a or snap25b resulted in behaviors that recapitulate the hyperactivity phenotype caused by absence of maternal rest Both maternal rest mutants and snap25a RE1 site mutants have altered primary motor neuron architecture that may account for the enhanced locomotor activity. These results demonstrate that maternal rest represses snap25a/b to modulate larval behavior and that early Rest activity has lifelong behavioral impacts. SIGNIFICANCE STATEMENT: Maternal factors deposited in the oocyte have well-established roles during embryonic development. We show that, in zebrafish, maternal rest (RE1-silencing transcription factor) regulates expression of target genes during larval development and has lifelong impacts on behavior. The Rest transcriptional repressor interacts with chromatin-modifying complexes to limit transcription of neural genes. We identify several synaptic genes that are repressed by maternal Rest and demonstrate that snap25a/b are key targets of maternal rest that modulate larval locomotor activity. These results reveal that zygotic rest is unable to compensate for deficits in maternally supplied rest and uncovers novel temporal requirements for Rest activity, which has implications for the broad roles of Rest-mediated repression during neural development and in disease states.

Rest mutant zebrafish swim erratically and display atypical spatial preferences.

The Rest/Nrsf transcriptional repressor modulates expression of a large set of neural specific genes. Many of these target genes have well characterized roles in nervous system processes including development, plasticity and synaptogenesis. However, the impact of Rest-mediated transcriptional regulation on behavior has been understudied due in part to the embryonic lethality of the mouse knockout. To investigate the requirement for Rest in behavior, we employed the zebrafish rest mutant to explore a range of behaviors in adults and larva. Adult rest mutants of both sexes showed abnormal behaviors in a novel environment including increased vertical swimming, erratic swimming patterns and a proclivity for the tank walls. Adult males also had diminished reproductive success. At 6 days post fertilization (dpf), rest mutant larva were hypoactive, but displayed normal evoked responses to light and sound stimuli. Overall, these results provide evidence that rest dysfunction produces atypical swimming patterns and preferences in adults, and reduced locomotor activity in larvae. This study provides the first behavioral analysis of rest mutants and reveals specific behaviors that are modulated by Rest.

The histone chaperone Spt6 coordinates histone H3K27 demethylation and myogenesis.

Histone chaperones affect chromatin structure and gene expression through interaction with histones and RNA polymerase II (PolII). Here, we report that the histone chaperone Spt6 counteracts H3K27me3, an epigenetic mark deposited by the Polycomb Repressive Complex 2 (PRC2) and associated with transcriptional repression. By regulating proper engagement and function of the H3K27 demethylase KDM6A (UTX), Spt6 effectively promotes H3K27 demethylation, muscle gene expression, and cell differentiation. ChIP-Seq experiments reveal an extensive genome-wide overlap of Spt6, PolII, and KDM6A at transcribed regions that are devoid of H3K27me3. Mammalian cells and zebrafish embryos with reduced Spt6 display increased H3K27me3 and diminished expression of the master regulator MyoD, resulting in myogenic differentiation defects. As a confirmation for an antagonistic relationship between Spt6 and H3K27me3, inhibition of PRC2 permits MyoD re-expression in myogenic cells with reduced Spt6. Our data indicate that, through cooperation with PolII and KDM6A, Spt6 orchestrates removal of H3K27me3, thus controlling developmental gene expression and cell differentiation.

Zebrafish churchill regulates developmental gene expression and cell migration.

BACKGROUND: Regulation of developmental signaling pathways is essential for embryogenesis. The small putative zinc finger protein, Churchill (ChCh) has been implicated in modulation of both TGF-ß and FGF signaling. RESULTS: We used zinc finger nuclease (ZFN) mediated gene targeting to disrupt the zebrafish chch locus and generate the first chch mutations. Three induced lesions produce frameshift mutations that truncate the protein in the third of five ß-strands that comprise the protein. Surprisingly, zygotic and maternal zygotic chch mutants are viable. Mutants have elevated expression of mesodermal markers, but progress normally through early development. chch mutants are sensitive to exogenous Nodal. However, neither misregulation of FGF targets nor sensitivity to exogenous FGF was detected. Finally, chch mutant cells were found to undergo inappropriate migration in cell transplant assays. CONCLUSIONS: Together, these results suggest that chch is not essential for survival, but functions to modulate early mesendodermal gene expression and limit cell migration.

Silencer-delimited transgenesis: NRSE/RE1 sequences promote neural-specific transgene expression in a NRSF/REST-dependent manner.

BACKGROUND: We have investigated a simple strategy for enhancing transgene expression specificity by leveraging genetic silencer elements. The approach serves to restrict transgene expression to a tissue of interest - the nervous system in the example provided here - thereby promoting specific/exclusive targeting of discrete cellular subtypes. Recent innovations are bringing us closer to understanding how the brain is organized, how neural circuits function, and how neurons can be regenerated. Fluorescent proteins enable mapping of the 'connectome', optogenetic tools allow excitable cells to be short-circuited or hyperactivated, and targeted ablation of neuronal subtypes facilitates investigations of circuit function and neuronal regeneration. Optimally, such toolsets need to be expressed solely within the cell types of interest as off-site expression makes establishing causal relationships difficult. To address this, we have exploited a gene 'silencing' system that promotes neuronal specificity by repressing expression in non-neural tissues. This methodology solves non-specific background issues that plague large-scale enhancer trap efforts and may provide a means of leveraging promoters/enhancers that otherwise express too broadly to be of value for in vivo manipulations. RESULTS: We show that a conserved neuron-restrictive silencer element (NRSE) can function to restrict transgene expression to the nervous system. The neuron-restrictive silencing factor/repressor element 1 silencing transcription factor (NRSF/REST) transcriptional repressor binds NRSE/repressor element 1 (RE1) sites and silences gene expression in non-neuronal cells. Inserting NRSE sites into transgenes strongly biased expression to neural tissues. NRSE sequences were effective in restricting expression of bipartite Gal4-based 'driver' transgenes within the context of an enhancer trap and when associated with a defined promoter and enhancer. However, NRSE sequences did not serve to restrict expression of an upstream activating sequence (UAS)-based reporter/effector transgene when associated solely with the UAS element. Morpholino knockdown assays showed that NRSF/REST expression is required for NRSE-based transgene silencing. CONCLUSIONS: Our findings demonstrate that the addition of NRSE sequences to transgenes can provide useful new tools for functional studies of the nervous system. However, the general approach may be more broadly applicable; tissue-specific silencer elements are operable in tissues other than the nervous system, suggesting this approach can be similarly applied to other paradigms. Thus, creating synthetic associations between endogenous regulatory elements and tissue-specific silencers may facilitate targeting of cellular subtypes for which defined promoters/enhancers are lacking.

Zebrafish rest regulates developmental gene expression but not neurogenesis.

The transcriptional repressor Rest (Nrsf) recruits chromatin-modifying complexes to RE1 'silencer elements', which are associated with hundreds of neural genes. However, the requirement for Rest-mediated transcriptional regulation of embryonic development and cell fate is poorly understood. Conflicting views of the role of Rest in controlling cell fate have emerged from recent studies. To address these controversies, we examined the developmental requirement for Rest in zebrafish using zinc-finger nuclease-mediated gene targeting. We discovered that germ layer specification progresses normally in rest mutants despite derepression of target genes during embryogenesis. This analysis provides the first evidence that maternal rest is essential for repression of target genes during blastula stages. Surprisingly, neurogenesis proceeds largely normally in rest mutants, although abnormalities are observed within the nervous system, including defects in oligodendrocyte precursor cell development and a partial loss of facial branchiomotor neuron migration. Mutants progress normally through embryogenesis but many die as larvae (after 12 days). However, some homozygotes reach adulthood and are viable. We utilized an RE1/NRSE transgenic reporter system to dynamically monitor Rest activity. This analysis revealed that Rest is required to repress gene expression in mesodermal derivatives including muscle and notochord, as well as within the nervous system. Finally, we demonstrated that Rest is required for long-term repression of target genes in non-neural tissues in adult zebrafish. Our results point to a broad role for Rest in fine-tuning neural gene expression, rather than as a widespread regulator of neurogenesis or cell fate.

The transcriptional repressor REST/NRSF modulates hedgehog signaling.

The spatial and temporal control of gene expression is key to generation of specific cellular fates during development. Studies of the transcriptional repressor REST/NRSF (RE1 Silencing Transcription Factor or Neural Restrictive Silencing Factor) have provided important insight into the role that epigenetic modifications play in differential gene expression. However, the precise function of REST during embryonic development is not well understood. We have discovered a novel interaction between zebrafish Rest and the Hedgehog (Hh) signaling pathway. We observed that Rest knockdown enhances or represses Hh signaling in a context-dependant manner. In wild-type embryos and embryos with elevated Hh signaling, Rest knockdown augments transcription of Hh target genes. Conversely, in contexts where Hh signaling is diminished, Rest knockdown has the opposite effect and Hh target gene expression is further attenuated. Epistatic analysis revealed that Rest interacts with the Hh pathway at a step downstream of Smo. Furthermore, we present evidence implicating the bifunctional, Hh signaling component Gli2a as key to the Rest modulation of the Hh response. The role of Rest as a regulator of Hh signaling has broad implications for many developmental contexts where REST and Hh signaling act.

Churchill and Sip1a repress fibroblast growth factor signaling during zebrafish somitogenesis.

Cell-type specific regulation of a small number of growth factor signal transduction pathways generates diverse developmental outcomes. The zinc finger protein Churchill (ChCh) is a key effector of fibroblast growth factor (FGF) signaling during gastrulation. ChCh is largely thought to act by inducing expression of the multifunctional Sip1 (Smad Interacting Protein 1). We investigated the function of ChCh and Sip1a during zebrafish somitogenesis. Knockdown of ChCh or Sip1a results in misshapen somites that are short and narrow. As in wild-type embryos, cycling gene expression occurs in the developing somites in ChCh and Sip1a compromised embryos, but expression of her1 and her7 is maintained in formed somites. In addition, tail bud fgf8 expression is expanded anteriorly in these embryos. Finally, we found that blocking FGF8 restores somite morphology in ChCh and Sip1a compromised embryos. These results demonstrate a novel role for ChCh and Sip1a in repression of FGF activity.