Development of a high-throughput tailored imaging method in zebrafish to understand and treat neuromuscular diseases

Abstract

The zebrafish (Danio rerio) is a vertebrate species offering multitude of advantages for the study of conserved biological systems in human and has considerably enriched our knowledge in developmental biology and physiology. Being equally important in medical research, the zebrafish has become a critical tool in the fields of diagnosis, gene discovery, disease modeling, and pharmacology-based therapy. Studies on the zebrafish neuromuscular system allowed for deciphering key molecular pathways in this tissue, and established it as a model of choice to study numerous motor neurons, neuromuscular junctions, and muscle diseases. Starting with the similarities of the zebrafish neuromuscular system with the human system, we review disease models associated with the neuromuscular system to focus on current methodologies employed to study them and outline their caveats. In particular, we put in perspective the necessity to develop standardized and high-resolution methodologies that are necessary to deepen our understanding of not only fundamental signaling pathways in a healthy tissue but also the changes leading to disease phenotype outbreaks, and offer templates for high-content screening strategies. While the development of high-throughput methodologies is underway for motility assays, there is no automated approach to quantify the key molecular cues of the neuromuscular junction. Here, we provide a novel high-throughput imaging methodology in the zebrafish that is standardized, highly resolutive, quantitative, and fit for drug screening. By providing a proof of concept for its robustness in identifying novel molecular players and therapeutic drugs in giant axonal neuropathy (GAN) disease, we foresee that this new tool could be useful for both fundamental and biomedical research.

A multilevel screening pipeline in zebrafish identifies therapeutic drugs for GAN

“…To test whether the selected compounds exert their positive effect through the stabilization of the axonal length and NMJ, we designed a cellular‐based method to quantify these parameters in noninjected WT, nontreated, or treated gan morphants at 48 hpf. This was achieved with the development of a novel high‐content pipeline for image acquisition and analysis, using the Opera Phenix™ High Content Screening System confocal and the Harmony software (methodology published in Lescouzères et al , 2022 ). Briefly, pMN axons and neuromuscular synapses of 48 hpf‐old embryos were labeled with synaptotagmin marker (znp1, axons) and alpha‐bungarotoxin (αBTX, postsynaptic AChRs), and embryos were individually placed in 96‐well plates in a lateral position (Fig 6A ).…”

“…Briefly, pMN axons and neuromuscular synapses of 48 hpf‐old embryos were labeled with synaptotagmin marker (znp1, axons) and alpha‐bungarotoxin (αBTX, postsynaptic AChRs), and embryos were individually placed in 96‐well plates in a lateral position (Fig 6A ). To detect the neuromuscular system in the entire animal, we created an automatized protocol to (i) first automatically locate single zebrafish larvae in the well, (ii) create a global image (see details in Materials and Methods section and Lescouzères et al , 2022 ) at low magnification (5×); and (iii) acquire selected regions with segmentation at 20× (see picture in Fig 6A ).The first regionalization filter (1) was developed to enable the automatic detection and quantification of single AChRs clusters (Fig 6B , left panel). The second regionalization filter (2) was generated for the evaluation of the percentage of co‐localization area between these presynaptic NMJ components and axonal region (Fig 6B , right panel).…”

“…Our multilevel pipeline for drug discovery in zebrafish presents the asset of introducing different scales of analysis, from behavior to automated and high‐content imaging‐based methodology, which can be applied to other neuromuscular diseases (Lescouzères et al , 2022 ). Indeed, most screenings in zebrafish have been performed using a unique functional parameter, and very few studies have integrated computational methods of large‐scale data in the discovery of new medical targets (Walker et al , 2012 ; Rennekamp & Peterson, 2015 ).…”

“…Note that the automated imaging protocol used here has been described in detail Lescouzères et al ( 2022 ). Single fluorescent zebrafish larvae were manually cut at the anterior part of the yolk and placed in single wells of a black F‐bottom 96‐well assay plates (μClear, Greiner Bio‐one) in a lateral position to ensure better recognition of the body shape of each larva.…”

“…Then, we conducted an in silico system biology approach to identify the different pharmacological families of Hits and pinpointed recurrence, allowing us to further reduce our candidate to 16 drugs. We subsequently developed a novel imaging‐based filtering method (Lescouzères et al , 2022 ) to identify Hits rescuing the NMJ and axonal deficits in the gan zebrafish. Overall, we established a stepwise methodology to pinpoint two specific pharmacological classes (muscarinic antagonist and α‐adrenergic agonist), whose actions converge in the stabilization of the NMJ units.…”