Targeted chromosomal deletions and inversions in zebrafish.

Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) provide powerful platforms for genome editing in plants and animals. Typically, a single nuclease is sufficient to disrupt the function of protein-coding genes through the introduction of microdeletions or insertions that cause frameshifts within an early coding exon. However, interrogating the function of cis-regulatory modules or noncoding RNAs in many instances requires the excision of this element from the genome. In human cell lines and invertebrates, two nucleases targeting the same chromosome can promote the deletion of intervening genomic segments with modest efficiencies. We have examined the feasibility of using this approach to delete chromosomal segments within the zebrafish genome, which would facilitate the functional study of large noncoding sequences in a vertebrate model of development. Herein, we demonstrate that segmental deletions within the zebrafish genome can be generated at multiple loci and are efficiently transmitted through the germline. Using two nucleases, we have successfully generated deletions of up to 69 kb at rates sufficient for germline transmission (1%-15%) and have excised an entire lincRNA gene and enhancer element. Larger deletions (5.5 Mb) can be generated in somatic cells, but at lower frequency (0.7%). Segmental inversions have also been generated, but the efficiency of these events is lower than the corresponding deletions. The ability to efficiently delete genomic segments in a vertebrate developmental system will facilitate the study of functional noncoding elements on an organismic level.

Global analysis of Drosophila Cys2-His2 zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants.

Cys2-His2 zinc finger proteins (ZFPs) are the largest group of transcription factors in higher metazoans. A complete characterization of these ZFPs and their associated target sequences is pivotal to fully annotate transcriptional regulatory networks in metazoan genomes. As a first step in this process, we have characterized the DNA-binding specificities of 129 zinc finger sets from Drosophila using a bacterial one-hybrid system. This data set contains the DNA-binding specificities for at least one encoded ZFP from 70 unique genes and 23 alternate splice isoforms representing the largest set of characterized ZFPs from any organism described to date. These recognition motifs can be used to predict genomic binding sites for these factors within the fruit fly genome. Subsets of fingers from these ZFPs were characterized to define their orientation and register on their recognition sequences, thereby allowing us to define the recognition diversity within this finger set. We find that the characterized fingers can specify 47 of the 64 possible DNA triplets. To confirm the utility of our finger recognition models, we employed subsets of Drosophila fingers in combination with an existing archive of artificial zinc finger modules to create ZFPs with novel DNA-binding specificity. These hybrids of natural and artificial fingers can be used to create functional zinc finger nucleases for editing vertebrate genomes.

Using defined finger-finger interfaces as units of assembly for constructing zinc-finger nucleases.

Zinc-finger nucleases (ZFNs) have been used for genome engineering in a wide variety of organisms; however, it remains challenging to design effective ZFNs for many genomic sequences using publicly available zinc-finger modules. This limitation is in part because of potential finger-finger incompatibility generated on assembly of modules into zinc-finger arrays (ZFAs). Herein, we describe the validation of a new set of two-finger modules that can be used for building ZFAs via conventional assembly methods or a new strategy-finger stitching-that increases the diversity of genomic sequences targetable by ZFNs. Instead of assembling ZFAs based on units of the zinc-finger structural domain, our finger stitching method uses units that span the finger-finger interface to ensure compatibility of neighbouring recognition helices. We tested this approach by generating and characterizing eight ZFAs, and we found their DNA-binding specificities reflected the specificities of the component modules used in their construction. Four pairs of ZFNs incorporating these ZFAs generated targeted lesions in vivo, demonstrating that stitching yields ZFAs with robust recognition properties.

The Perineuronal Net Protein Brevican Acts in Nucleus Accumbens Parvalbumin-Expressing Interneurons of Adult Mice to Regulate Excitatory Synaptic Inputs and Motivated Behaviors.

BACKGROUND: Experience-dependent functional adaptation of nucleus accumbens (NAc) circuitry underlies the development and expression of reward-motivated behaviors. Parvalbumin-expressing GABAergic (gamma-aminobutyric acidergic) interneurons (PVINs) within the NAc are required for this process. Perineuronal nets (PNNs) are extracellular matrix structures enriched around PVINs that arise during development and have been proposed to mediate brain circuit stability. However, their function in the adult NAc is largely unknown. Here, we studied the developmental emergence and adult regulation of PNNs in the NAc of male and female mice and examined the cellular and behavioral consequences of reducing the PNN component brevican in NAc PVINs. METHODS: We characterized the expression of PNN components in mouse NAc using immunofluorescence and RNA in situ hybridization. We lowered brevican in NAc PVINs of adult mice using an intersectional viral and genetic method and quantified the effects on synaptic inputs to NAc PVINs and reward-motivated learning. RESULTS: PNNs around NAc PVINs were developmentally regulated and appeared during adolescence. In the adult NAc, PVIN PNNs were also dynamically regulated by cocaine. Transcription of the gene that encodes brevican was regulated in a cell type- and isoform-specific manner in the NAc, with the membrane-tethered form of brevican being highly enriched in PVINs. Lowering brevican in NAc PVINs of adult mice decreased their excitatory inputs and enhanced both short-term novel object recognition and cocaine-induced conditioned place preference. CONCLUSIONS: Regulation of brevican in NAc PVINs of adult mice modulates their excitatory synaptic drive and sets experience thresholds for the development of motivated behaviors driven by rewarding stimuli.

Disruption of Eaat2b, a glutamate transporter, results in abnormal motor behaviors in developing zebrafish.

Analysis of zebrafish mutants that have defects in motor behavior can allow entrée into the hindbrain and spinal cord networks that control locomotion. Here, we report that zebrafish techno trousers (tnt) locomotor mutants harbor a mutation in slc1a2b, which encodes Eaat2b, a plasma membrane glutamate transporter. We used tnt mutants to explore the effects of impaired glutamate transporter activity on locomotor network function. Wild-type larvae perform robust swimming behavior in response to touch stimuli at two and four days after fertilization. In contrast, tnt mutant larvae demonstrate aberrant, exaggerated body bends beginning two days after fertilization and they are almost paralyzed four days after fertilization. We show that slc1a2b is expressed in glial cells in a dynamic fashion across development, which may explain the abnormal sequence of motor behaviors demonstrated by tnt mutants. We also show that tnt larvae demonstrate enhanced excitation of neurons, consistent with the predicted effects of excessive glutamate. These findings illustrate the dynamic regulation and importance of glutamate transporters during development. Since glutamate toxicity caused by EAAT2 dysfunction is thought to promote several different neurological disorders in humans, including epilepsy and neurodegenerative diseases, tnt mutants hold promise as a new tool to better understand these pathologies.

Cholinergic neurons constitutively engage the ISR for dopamine modulation and skill learning in mice.

The integrated stress response (ISR) maintains proteostasis by modulating protein synthesis and is important in synaptic plasticity, learning, and memory. We developed a reporter, SPOTlight, for brainwide imaging of ISR state with cellular resolution. Unexpectedly, we found a class of neurons in mouse brain, striatal cholinergic interneurons (CINs), in which the ISR was activated at steady state. Genetic and pharmacological manipulations revealed that ISR signaling was necessary in CINs for normal type 2 dopamine receptor (D2R) modulation. Inhibiting the ISR inverted the sign of D2R modulation of CIN firing and evoked dopamine release and altered skill learning. Thus, a noncanonical, steady-state mode of ISR activation is found in CINs, revealing a neuromodulatory role for the ISR in learning.