Towards modelling mental disorders in zebrafish. Neurexins severely modulate anxiety, affiliative social behaviour and aggression
Q. Nguyen, F. Guo, J. Das, O. Hatzimanolis, B. Mowry, Alexandre S. Cristino & J. Giacomotto
Molecular Psychiatry (2026) Cite this article
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Abstract
Neurexin (nrxn) genes encode synaptic cell-adhesion molecules that have been repetitively associated with neurodevelopmental and mental disorders. While the zebrafish animal model offers tremendous advantages for dissecting neural development/function, no complete loss-of-function (LOF) nrxn zebrafish models are currently available. In this study, we generated the first collection of zebrafish knockout lines for each nrxn gene, with mutations ranging from transmembrane domain- to full genomic locus-deletions. Surprisingly, all homozygous lines developed normally, presenting no gross neurodevelopmental or obvious early behavioural abnormalities. However, this absence of early phenotypes translated into profound, paralog-specific behavioural alterations emerging during later developmental stages. All neurexin knockouts affected mating behaviour, complicating the generation and maintenance of homozygous lines. Except for this shared behavioural alteration, nrxn1, but not nrxn2 or nrxn3, led to marked changes in affiliative social behaviour and aggression. In contrast, nrxn2 mutants exhibited severe anxiety-like behaviours, including bottom-dwelling and repetitive freezing/seizure events. Strikingly, nrxn1 full-locus deletion mutants showed opposing behaviour, spending most of their time near the surface. The two also displayed opposite responses to open/closed field transitions; confinement alleviated nrxn2 anxiety but enhanced nrxn1 surface-dwelling. Meanwhile, nrxn3 mutants behaved normally in all our initial tests. In summary, our study introduces a complete set of zebrafish mutants covering the whole nrxn gene family, presenting striking adult behavioural alterations despite the absence of noticeable early defects; echoing the delayed onset of human psychiatric disorders such as schizophrenia. This work confirms the value of zebrafish to study mental disorders and unlock a novel platform to unravel the pathogenic contribution of neurexins and associated subtle neurodevelopmental changes/timing that drive the emergence of mental illnesses.
Introduction
Neurexins, NRXN1, NRXN2 and NRXN3 in humans, code for transmembrane cell-adhesion molecules essential for synaptic formation, maintenance and signalling [1, 2]. These genes have repeatedly been associated with diverse mental and neurodevelopmental disorders such as Schizophrenia (SCZ), Autism (ASD), bipolar disorder (BD) and even recently with Parkinson (PD) [1,2,3,4,5,6]. A vast number of rodent models have been developed over the last two decades, which have significantly contributed to gaining tremendous insights into the biology of this complex gene family [2, 4]. Despite this tremendous effort, their pathogenic role and association with the aforementioned diseases remain unclear. Expanding the tools available to broaden our ability to interrogate these genes is still essential. Surprisingly, while the zebrafish seems to be a model of choice to tackle the function of these genes, especially in investigating their role in the developing brain, there is currently a lack of complete LOF models for research.
Indeed, complementing their rodent counterparts, the zebrafish offers a unique set of tools for studying neurodevelopment and greater versatility for drug discovery [7]. They also exhibit extra-utero development and a very convenient optical transparency, enabling direct visualisation and manipulation of their brain even during the very early developing stages. In addition, the emergence of optogenetics and calcium imaging techniques with this organism offers unique ways to strengthen the ongoing effort aiming at understanding the normal and pathogenic role of this gene family in the developing and mature brain [8, 9]. Despite these tremendous advantages, no complete LOF zebrafish neurexin models are currently available for research. This small animal presents highly conserved neurexin orthologs, which have been ancestrally duplicated -neurexin 1 (nrxn1a and nrxn1b), neurexin 2 (nrxn2a and nrxn2b) and neurexin 3 (nrxn3a and nrxn3b) [10], and although two zebrafish studies have investigated individual neurexin genes, these models target limited genomic regions using traditional small indels and therefore do not eliminate the full repertoire of neurexin isoforms [11, 12].
Indeed, these genes are transcribed into hundreds of isoforms due to multiple promoters and complex, still poorly understood, alternative splicing [13]. This complexity makes the generation of robust LOF mutations challenging. Traditional small indels -generating premature stop-codons and truncated proteins- are at high risk of being genetically compensated by de-novo and/or cryptic splicing. Let alone genetic compensation, it is not straightforward to target all isoforms at once due to the multiple promoters leading to a variety of α-long and β-short isoforms. Exemplifying these issues, the only available studies in zebrafish generated mutants affecting only the long α-isoforms, leaving the β-isoforms intact [11, 12]. Koh et al. [11] also reported that their indel triggered the use of a cryptic splice site, resulting in the removal of most of the exon carrying the mutation -confirming the presence of complex alternative splicing that complicates the study of these genes’ functions-[11]. Although aberrant splicing and compensatory mechanisms may contribute to disease pathogenesis and warrant closer investigation [14], we aimed here at first generating a set of robust zebrafish knockout lines properly missing the associated gene/protein function, and to subsequently assess their effect on zebrafish development and behaviour in order to establish a solid foundation for future mechanistic and complex genetic investigations.
Here, we are using an innovative CRISPR/Cas9 strategy to generate a complete set of zebrafish mutants covering the whole neurexin gene family and conduct a first systematic phenotyping comparison of each homozygous mutant line from embryonic to adult stage. We show that loss of these genes does not markedly affect early development or basic motor function, but results in striking and paralog-specific behavioural alterations in adulthood. Given that most mental health disorders (such as schizophrenia) emerge without detectable developmental or anatomical/structural abnormalities, while being accepted to be neurodevelopmental in origin, the presented data suggest that the zebrafish could be a valuable model to uncover pathogenic mechanisms in play prior to the symptoms. Our study also validates the zebrafish as a powerful tool to uncover the complex role of neurexins in brain development and function.
