Filippo Del Bene

Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair

Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles. Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.

Optogenetic dissection of a behavioural module in the vertebrate spinal cord.

Locomotion relies on neural networks called central pattern generators (CPGs) that generate periodic motor commands for rhythmic movements. In vertebrates, the excitatory synaptic drive for inducing the spinal CPG can originate from either supraspinal glutamatergic inputs or from within the spinal cord. Here we identify a spinal input to the CPG that drives spontaneous locomotion using a combination of intersectional gene expression and optogenetics in zebrafish larvae. The photo-stimulation of one specific cell type was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. This neuron is the Kolmer–Agduhr cell, which extends cilia into the central cerebrospinal-fluid-containing canal of the spinal cord and has an ipsilateral ascending axon that terminates in a series of consecutive segments. Genetically silencing Kolmer–Agduhr cells reduced the frequency of spontaneous free swimming, indicating that activity of Kolmer–Agduhr cells provides necessary tone for spontaneous forward swimming. Kolmer–Agduhr cells have been known for over 75 years, but their function has been mysterious. Our results reveal that during early development in zebrafish these cells provide a positive drive to the spinal CPG for spontaneous locomotion.

Remote Control of Neuronal Activity with a Light-Gated Glutamate Receptor

The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.

Regulation of neurogenesis by interkinetic nuclear migration through an apical-basal notch gradient.

The different cell types in the central nervous system develop from a common pool of progenitor cells. The nuclei of progenitors move between the apical and basal surfaces of the neuroepithelium in phase with their cell cycle, a process termed interkinetic nuclear migration (INM). In the retina of zebrafish mikre oko (mok) mutants, in which the motor protein Dynactin-1 is disrupted, interkinetic nuclei migrate more rapidly and deeply to the basal side and more slowly to the apical side. We found that Notch signaling is predominantly activated on the apical side in both mutants and wild-type. Mutant progenitors are, thus, less exposed to Notch and exit the cell cycle prematurely. This leads to an overproduction of early-born retinal ganglion cells (RGCs) at the expense of later-born interneurons and glia. Our data indicate that the function of INM is to balance the exposure of progenitor nuclei to neurogenic versus proliferative signals.

Direct interaction of geminin and Six3 in eye development.

Organogenesis in vertebrates requires the tight control of cell proliferation and differentiation. The homeobox-containing transcription factor Six3 plays a pivotal role in the proliferation of retinal precursor cells. In a yeast two-hybrid screen, we identified the DNA replication-inhibitor geminin as a partner of Six3. Geminin inhibits cell-cycle progression by sequestering Cdt1 (refs 4, 5), the key component for the assembly of the pre-replication complex. Here, we show that Six3 efficiently competes with Cdt1 directly to bind to geminin, which reveals how Six3 can promote cell proliferation without transcription. In common with Six3 inactivation, overexpression of the geminin gene (Gem; also known as Gmn) in medaka (Oryzias latipes) induces specific forebrain and eye defects that are rescued by Six3. Conversely, loss of Gem (in common with gain of Six3 (ref. 1)) promotes retinal precursor-cell proliferation and results in expanded optic vesicles, markedly potentiating Six3 gain-of-function phenotypes. Our data indicate that the transcription factor Six3 and the replication-initiation inhibitor geminin act antagonistically to control the balance between proliferation and differentiation during early vertebrate eye development.

Optical control of zebrafish behavior with halorhodopsin

Expression of halorhodopsin (NpHR), a light-driven microbial chloride pump, enables optical control of membrane potential and reversible silencing of targeted neurons. We generated transgenic zebrafish expressing enhanced NpHR under control of the Gal4/UAS system. Electrophysiological recordings showed that eNpHR stimulation effectively suppressed spiking of single neurons in vivo. Applying light through thin optic fibers positioned above the head of a semi-restrained zebrafish larva enabled us to target groups of neurons and to simultaneously test the effect of their silencing on behavior. The photostimulated volume of the zebrafish brain could be marked by subsequent photoconversion of co-expressed Kaede or Dendra. These techniques were used to localize swim command circuitry to a small hindbrain region, just rostral to the commissura infima Halleri. The kinetics of the hindbrain-generated swim command was investigated by combined and separate photo-activation of NpHR and Channelrhodopsin-2 (ChR2), a light-gated cation channel, in the same neurons. Together this “optogenetic toolkit” allows loss-of-function and gain-of-function analyses of neural circuitry at high spatial and temporal resolution in a behaving vertebrate.

Filtering of visual information in the tectum by an identified neural circuit

Small Is Attractive The optic tectum of zebrafish larvae is required for the detection, tracking, and capture of small, highly motile prey. Del Bene et al. (p. 669) applied a combination of optical, genetic, and pharmacological tools to investigate how neural circuits in the optic tectum filter out low-frequency visual information. Most tectal neurons were tuned to respond selectively to small, moving objects in the fishs visual environment and responded very poorly to large stimuli. This spatial filtering mechanism depended on the activity of a small population of GABAergic, inhibitory interneurons at the tectal surface. Inactivation or destruction of these interneurons removed the size selectivity of deeper neurons and the zebrafish lost their ability to catch prey. A neural circuit in zebrafish is preferentially activated by small visual stimuli, facilitating the capture of prey. The optic tectum of zebrafish is involved in behavioral responses that require the detection of small objects. The superficial layers of the tectal neuropil receive input from retinal axons, while its deeper layers convey the processed information to premotor areas. Imaging with a genetically encoded calcium indicator revealed that the deep layers, as well as the dendrites of single tectal neurons, are preferentially activated by small visual stimuli. This spatial filtering relies on GABAergic interneurons (using the neurotransmitter γ-aminobutyric acid) that are located in the superficial input layer and respond only to large visual stimuli. Photo-ablation of these cells with KillerRed, or silencing of their synaptic transmission, eliminates the size tuning of deeper layers and impairs the capture of prey.

A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

Optogenetic localization and genetic perturbation of saccade-generating neurons in zebrafish.

The optokinetic response (OKR) to a visual stimulus moving at constant velocity consists of a series of two alternating components, a slow phase, during which the eyes follow the stimulus, and a quick phase, which resets the eyes to begin a new response cycle. The quick phases of the OKR resemble the saccades observed during free viewing. It is unclear to what extent the premotor circuitry underlying these two types of jerky, conjugate eye movements is conserved among vertebrates. Zebrafish (Danio rerio) larvae, broadly expressing halorhodopsin (NpHR) or channelrhodopsin-2 (ChR2) in most neurons, were used to map the location of neurons involved in this behavior. By blocking activity in localized groups of NpHR-expressing neurons with an optic fiber positioned above the head of the fish and by systematically varying the site of photostimulation, we discovered that activity in a small hindbrain area in rhombomere 5 was necessary for saccades to occur. Unilateral block of activity at this site affected behavior in a direction-specific manner. Inhibition of the right side suppressed rightward saccades of both eyes, while leaving leftward saccades unaffected, and vice versa. Photostimulation of this area in ChR2-transgenic fish was sufficient to trigger saccades that were precisely locked to the light pulses. These extra saccades could be induced both during free viewing and during the OKR, and were distinct in their kinetics from eye movements elicited by stimulating the abducens motor neurons. Zebrafish double indemnity (didy) mutants were identified in a chemical mutagenesis screen based on a defect in sustaining saccades during OKR. Positional cloning, molecular analysis, and electrophysiology revealed that the didy mutation disrupts the voltage-gated sodium channel Scn1lab (Nav1.lb). ChR2 photostimulation of the putative hindbrain saccade generator was able to fully reconstitute saccades in the didy mutant. Our studies demonstrate that an optogenetic approach is useful for targeted loss-of-function and gain-of-function manipulations of neural circuitry underlying eye movements in zebrafish and that the saccade-generating circuit in this species shares many of its properties with that in mammals.

CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish.

The targeted introduction of mutations utilizing sequence specific transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system (RNA-guided nucleases, RGNs) has revolutionized reverse genetic approaches in numerous model organisms. In zebrafish, both systems were successfully applied to generate loss-of-function alleles by targeting open reading frames or deletion and inversion of whole chromosomal regions. In addition to the production of these loss-of-function alleles, genomic engineering by insertion of short sequences utilizing single stranded DNA oligonucleotides as templates for homology based repair was made possible, enabling effective insertion of loxP sites or tags for protein coding genes. Recent studies based on homologous recombination and non-homologous end joining have also broadened the repertoire for genome editing. These approaches allow the targeted insertion of open reading frames or even whole donor vectors. In this review we summarize the use of TALENs and RNA-guided nucleases in the field of zebrafish genetics with a special focus on knock-in approaches.