SUMMARYWhile the exterior of vertebrate bodies appears bilaterally symmetrical, internal organ positioning and morphology frequently exhibit left-right (L-R) asymmetries. In several vertebrates, including human, mouse, frog and zebrafish, left-right symmetry-breaking during embryonic development is initiated by a ciliated organ called the Node or left-right organizer. Within the Node, a leftward flow of extraembryonic fluid named the Nodal flow mediates the asymmetric expressions of Nodal factors. Although downstream Nodal pathway components leading to the establishment of the embryonic left-right axis are well known, less is known about the development and formation of the embryonic Node itself.Here we reveal a novel role for the Meteorin protein family in the establishment of the left-right axis and in the formation of the Kupffer’s vesicle, the Node equivalent structure in zebrafish. We show that the genetic inactivation of each or all three members of the zebrafish Meteorin family (metrn,metrn-like aandmetrn-like b) leads to defects in properties of the Kupffer’s vesicle, caused by impaired assembly and migration of the Kupffer’s vesicle forming dorsal forerunner cells. In addition, we demonstrate that Meteorins genetically interact with integrins ItgaV and Itgβ1b regulating the dorsal forerunner cell clustering and thatmeteorinsloss-of-function results in disturbed Nodal factor expression and consequently in randomized or symmetric heart looping and jogging.These results identify a new role for the Meteorin protein family in the left-right asymmetry patterning during embryonic vertebrate development.

The zebrafish model has emerged as a reference tool for phenotypic drug screening. An increasing number of molecules have been brought from bench to bedside thanks to zebrafish-based assays over the last decade. The high homology between the zebrafish and the human genomes facilitates the generation of zebrafish lines carrying loss-of-function mutations in disease-relevant genes; nonetheless, even using this alternative model, the establishment of isogenic mutant lines requires a long generation time and an elevated number of animals. In this study, we developed a zebrafish-based high-throughput platform for the generation of F0 knock-out (KO) models and the screening of neuroactive compounds. We show that the simultaneous inactivation of a reporter gene (tyrosinase) and a second gene of interest allows the phenotypic selection of F0 somatic mutants (crispants) carrying the highest rates of mutations in both loci. As a proof of principle, we targeted genes associated with neurodevelopmental disorders and we efficiently generated de facto F0 mutants in seven genes involved in childhood epilepsy. We employed a high-throughput multiparametric behavioral analysis to characterize the response of these KO models to an epileptogenic stimulus, making it possible to employ kinematic parameters to identify seizure-like events. The combination of these co-injection, screening and phenotyping methods allowed us to generate crispants recapitulating epilepsy features and to test the efficacy of compounds already during the first days post fertilization. Since the strategy can be applied to a wide range of indications, this study paves the ground for high-throughput drug discovery and promotes the use of zebrafish in personalized medicine and neurotoxicity assessment.

List of genes up-regulated in LPAR6 knocked down cells.

Description of additional methods and procedures used in the study.

Kinesins are motor proteins involved in microtubule (MT)-mediated intracellular transport. They contribute to key cellular processes, including intracellular trafficking, organelle dynamics and cell division. Pathogenic variants in kinesin-encoding genes underlie several human diseases characterized by an extremely variable clinical phenotype, ranging from isolated neurodevelopmental/neurodegenerative disorders to syndromic phenotypes belonging to a family of conditions collectively termed as 'ciliopathies.' Among kinesins, kinesin-1 is the most abundant MT motor for transport of cargoes towards the plus end of MTs. Three kinesin-1 heavy chain isoforms exist in mammals. Different from KIF5A and KIF5C, which are specifically expressed in neurons and established to cause neurological diseases when mutated, KIF5B is an ubiquitous protein. Three de novo missense KIF5B variants were recently described in four subjects with a syndromic skeletal disorder characterized by kyphomelic dysplasia, hypotonia and DD/ID. Here, we report three dominantly acting KIF5B variants (p.Asn255del, p.Leu498Pro and p.Leu537Pro) resulting in a clinically wide phenotypic spectrum, ranging from dilated cardiomyopathy with adult-onset ophthalmoplegia and progressive skeletal myopathy to a neurodevelopmental condition characterized by severe hypotonia with or without seizures. In vitro and in vivo analyses provide evidence that the identified disease-associated KIF5B variants disrupt lysosomal, autophagosome and mitochondrial organization, and impact cilium biogenesis. All variants, and one of the previously reported missense changes, were shown to affect multiple developmental processes in zebrafish. These findings document pleiotropic consequences of aberrant KIF5B function on development and cell homeostasis, and expand the phenotypic spectrum resulting from altered kinesin-mediated processes.

Expression of LPAR1-5 mRNA levels in peritumoral and HCC cell lines (S1); Cell cycle analysis of LPAR6-knocked-down cells versus controls (S2); Absence of apoptosis in stably LPAR6-knocked-down cells (S3); LPAR6 knockdown impairs HCC migration and actin organization but not cells adhesion (S4); Ectopic expression of LPAR6 in human hepatocarcinoma cell line HLE (S5); LPAR6 knockdown affects the gene expression profile in HCC cells (S6); The PIM3 inhibitor, SGI-1776 decreases tumor growth in vivo and in vitro (S7).

Grouped genes according to their functions (analysis for comparative cellular processes) using the publicly available software ToppCluster multiple gene analyzer.

List of genes down-regulated in LPAR6 knocked down cells.

Legends for Supplementary Tables S1-S3 and Supplementary Figures S1-S7.

Over the last decade, the use of gene editing technologies revolutionized the field of reverse genetics in multiple model organisms, including the zebrafish. In this context, the CRISPR/Cas9 system rapidly became the preferred tool for researchers, mostly because of the experimental accessibility that makes it tailored for a wide range of potential applications, both in basic and preclinical research. Nowadays, a variety of tools are available to the zebrafish community, reaching far beyond the generation of loss of function alleles for the analysis of gene function. Among other, the CRISPR/Cas9 technology can be used to generate conditional knock-out alleles, create tissue-specific reporter lines, introduce specific mutations at desired loci (mimicking human disease-associated mutations), or modulate gene expression through the use of transcriptional activators and repressors binding regulatory target sequences. This book chapter describes all these CRISPR/Cas9-based strategies, evaluates their strengths and limitations and discusses how they have changed modern zebrafish genetics.

A conserved organizational and functional principle of neural networks is the segregation of axon-dendritic synaptic connections into laminae. Here we report that targeting of synaptic laminae by retinal ganglion cell (RGC) arbors in the vertebrate visual system is regulated by a signaling system relying on target-derived Reelin and VLDLR/Dab1a on the projecting neurons. Furthermore, we find that Reelin is distributed as a gradient on the target tissue and stabilized by heparan sulfate proteoglycans (HSPGs) in the extracellular matrix (ECM). Through genetic manipulations, we show that this Reelin gradient is important for laminar targeting and that it is attractive for RGC axons. Finally, we suggest a comprehensive model of synaptic lamina formation in which attractive Reelin counter-balances repulsive Slit1, thereby guiding RGC axons toward single synaptic laminae. We establish a mechanism that may represent a general principle for neural network assembly in vertebrate species and across different brain areas.

The zebrafish (Danio rerio) has emerged in recent years as a powerful vertebrate model to study neuronal circuit development and function, thanks to its relatively small size, rapid external development and translucency. These features allow the easy application of in vivo microscopy analysis and optical perturbation of neuronal function. So far, genetic manipulation in zebrafish has been limited to the generation of constitutive loss-of-function alleles and transgenic models. CRISPR/Cas9 offers unprecedented possibilities for genomic manipulation that can be exploited to study neuronal function. In the past few years, we have successfully used CRISPR/Cas9-based technology in zebrafish to achieve two goals crucial for neuronal circuit analysis by developing two CRISPR/Cas9-based approaches that overcome previous major limitations to the study of gene and neuron functions in zebrafish. The study of gene function via tissue-or cell-specific mutagenesis remains challenging in zebrafish when the study of the function of certain loci might require tight spatiotemporal control of gene inactivation, which is particularly true in studying the function of a particular gene in post mitotic neurons, when the same gene may have had an earlier developmental function. To circumvent this limitation, we developed a simple and versatile protocol to achieve tissue-specific and temporally controlled gene disruption based on Cas9 expression under the control of the Gal4/UAS binary system (Di Donato et al. 2016). This strategy allows us to induce somatic mutations in genetically labeled cell clones or single cells and to follow them in vivo via reporter gene expression. We have also been able to target endogenous genomic loci to specifically label the great variety of neuronal cell types with reporter genes such as the transcriptional activator Gal4 (Auer et al. 2014). As a result, we can specifically target the expression of fluorescent proteins, a genetically encoded calcium indicator or optogenetic actuators in defined neuronal subpopulations. We will present ways that these two methods can be applied to the study of the development of the nervous system in larval zebrafish.

The zebrafish (Danio rerio) has emerged in recent years as a powerful vertebrate model to study neuronal circuit development and function, thanks to its relatively small size, rapid external development and translucency. These features allow the easy application of in vivo microscopy analysis and optical perturbation of neuronal function. So far, genetic manipulation in zebrafish has been limited to the generation of constitutive loss-of-function alleles and transgenic models. CRISPR/Cas9 offers unprecedented possibilities for genomic manipulation that can be exploited to study neuronal function. In the past few years, we have successfully used CRISPR/Cas9-based technology in zebrafish to achieve two goals crucial for neuronal circuit analysis by developing two CRISPR/Cas9-based approaches that overcome previous major limitations to the study of gene and neuron functions in zebrafish. The study of gene function via tissue- or cell-specific mutagenesis remains challenging in zebrafish when the study of the function of certain loci might require tight spatiotemporal control of gene inactivation, which is particularly true in studying the function of a particular gene in post mitotic neurons, when the same gene may have had an earlier developmental function. To circumvent this limitation, we developed a simple and versatile protocol to achieve tissue-specific and temporally controlled gene disruption based on Cas9 expression under the control of the Gal4/UAS binary system (Di Donato et al. 2016). This strategy allows us to induce somatic mutations in genetically labeled cell clones or single cells and to follow them in vivo via reporter gene expression. We have also been able to target endogenous genomic loci to specifically label the great variety of neuronal cell types with reporter genes such as the transcriptional activator Gal4 (Auer et al. 2014). As a result, we can specifically target the expression of fluorescent proteins, a genetically encoded calcium indicator or optogenetic actuators in defined neuronal subpopulations. We will present ways that these two methods can be applied to the study of the development of the nervous system in larval zebrafish.

Axon ensheathment by specialized glial cells is an important process for fast propagation of action potentials. The rapid electrical conduction along myelinated axons is mainly due to its saltatory nature characterized by the accumulation of ion channels at the nodes of Ranvier. However, how these ion channels are transported and anchored along axons is not fully understood. We have identified N-myc downstream-regulated gene 4, ndrg4, as a novel factor that regulates sodium channel clustering in zebrafish. Analysis of chimeric larvae indicates that ndrg4 functions autonomously within neurons for sodium channel clustering at the nodes. Molecular analysis of ndrg4 mutants shows that expression of snap25 and nsf are sharply decreased, revealing a role of ndrg4 in controlling vesicle exocytosis. This uncovers a previously unknown function of ndrg4 in regulating vesicle docking and nodes of Ranvier organization, at least through its ability to finely tune the expression of the t-SNARE/NSF machinery.

CRISPR/Cas9-mediated targeted mutagenesis allows efficient generation of loss-of-function alleles in zebrafish. To date, this technology has been primarily used to generate genetic knockout animals. Nevertheless, the study of the function of certain loci might require tight spatiotemporal control of gene inactivation. Here, we show that tissue-specific gene disruption can be achieved by driving Cas9 expression with the Gal4/UAS system. Furthermore, by combining the Gal4/UAS and Cre/loxP systems, we establish a versatile tool to genetically label mutant cell clones, enabling their phenotypic analysis. Our technique has the potential to be applied to diverse model organisms, enabling tissue-specific loss-of-function and phenotypic characterization of live and fixed tissues.

In the last few years the development of CRISPR/Cas 9-mediated genome editing techniques has allowed the efficient generation of loss-of-function alleles in several model organisms including zebrafish. However, these methods are mainly devoted to target-specific genomic loci leading to the creation of constitutive knock-out models. On the contrary, the analysis of gene function via tissue- or cell-specific mutagenesis remains challenging in zebrafish. To circumvent this limitation, we present here a simple and versatile protocol to achieve tissue-specific gene disruption based on the Cas9 expression under the control of the Gal4/upstream activating sequence binary system. In our method, we couple Cas9 with green fluorescent protein or Cre reporter gene expression. This strategy allows us to induce somatic mutations in genetically labeled cell clones or single cells, and to follow them in vivo via reporter gene expression. Importantly, because none of the tools that we present here are restricted to zebrafish, similar approaches are readily applicable in virtually any organism where transgenesis and DNA injection are feasible.

Abstract The aberrant processes driving hepatocellular carcinoma (HCC) are not fully understood. Lysophosphatidic acid receptors (LPAR) are commonly overexpressed in HCC, but their contributions to malignant development are not well established. In this report, we show that aberrant expression of LPAR6 sustains tumorigenesis and growth of HCC. Overexpression of LPAR6 in HCC specimens associated with poor survival in a cohort of 128 patients with HCC. We took a genetic approach to elucidate how LPAR6 sustains the HCC tumorigenic process, including through an expression profiling analysis to identify genes under the control of LPAR6. RNAi-mediated attenuation of LPAR6 impaired HCC tumorigenicity in tumor xenograft assays. Expression profiling and mechanistic analyses identified Pim-3 as a pathophysiologically relevant LPAR6 target gene. In nonmalignant cells where LPAR6 overexpression was sufficient to drive malignant character, Pim-3 was upregulated at the level of transcription initiation through a STAT3-dependent mechanism. A further analysis of HCC clinical specimens validated the connection between overexpression of LPAR6 and Pim-3, high proliferation rates, and poorer survival outcomes. Together, our findings establish LPAR6 as an important theranostic target in HCC tumorigenesis. Cancer Res; 75(3); 532–43. ©2014 AACR.