The significance of non-mammalian model organisms has been recognized for quite some time ^[1][6]. The Nobel Prize in Physiology or Medicine has been awarded to researchers for their discoveries in apoptosis (2022) and RNA interference using worms (2006).

C. elegans is an unsegmented pseudocoelomate, lacking circulatory and respiratory systems, whose body segments are serially repeated one after the other ^[7]. C. elegans was used primarily for neuronal development research in 1963 and has since seen extensive use as a model organism for research into neural and molecular mechanisms of learning, memory, mating behavior, chemotaxis, thermotaxis, and mechano-transduction ^[8]. In addition, the C. elegans model has provided insights into finer mechanistic details of human health, including inter-cellular signaling pathways (e.g., Notch signaling), intra-cellular pathways (e.g., autophagy), molecular machines (e.g., spliceosome), and multi-cellular processes (e.g., basement membrane biology) ^[9].

In 2019, the first C. elegans multicellular organism underwent whole-genome sequencing and is the only organism to have completed its connectome (the “wiring diagram” of neurons) ^[10][11][12]. There are 20,512 protein-coding genes ^[13], and 83% of the worm proteome is found to have human homologous genes ^[14]. Only 11% or less contain roundworm-specific genes. These findings provide the basis by which C. elegans has served as a suitable model organism for human gene functional research ^{[15][16][17][18]}. In the case of rare disease modeling, C. elegans provides an ideal system to study human diseases.

Drosophila melanogaster is a species of fly in the Drosophila family (taxonomic order Diptera). For the past century, the Drosophila melanogaster has been used as a model organism to understand the fundamentals of genetics, developmental biology, immunity, and neuroscience ^[19][20]. Five Nobel Prizes have been awarded to fruit fly scientists for their work with the animal in 2017. Drosophila melanogaster has emerged as an important model system for dissecting and understanding the molecular mechanisms underlying rare human diseases, due to its rapid life cycle, relatively simple genetics (only four pairs of chromosomes), and large number of offspring per generation. This rise is owed in part to the first genome-wide survey of ~1000 genes registered in the Online Mendelian Inheritance in Man (OMIM), which found that 75% of disease-causing genes in humans are conserved in Drosophila ^[21]. Of the approximately 4000 human disease-related genes currently displayed in OMIM, ~85% have homologues in Drosophila. Considering that ~65% of protein-coding genes are conserved between humans and flies, the data suggest that genes conserved between these species are more likely to implicate genetic disease in humans ^[22][23]. In addition to being used as a tool for dissecting rare disease mechanisms and exploring potential therapeutic avenues, flies have emerged as a key tool in explaining variants of uncertain significance found in patients ^[24][25].

The zebrafish (Danio rerio) is a small freshwater fish, 3-to-4 cm long, with a lifespan of about 2 years. Zebrafish have become increasingly important in scientific research since the 1960s, due to several distinct advantages over other vertebrate models.

Because of the simplicity of their natural habitat, lab maintenance of a zebrafish colony is much easier than simulating the conditions necessary for mammals. Therefore, zebrafish can be grown in a cost-effective manner. Their short generation time of 3 months helps accelerate experimental progress ^[26], and ex utero development facilitates the observation and rapid experimental manipulation of embryos. In addition, zebrafish have large clutch sizes, ranging from 200 to 300 embryos per adult mating pair, which ensures a robust stock of animals for research work. Due to these features, combined with the relatively small size of the embryo/larva/adult, zebrafish are well-suited for high-throughput screening of potential neuroactive compounds.

The zebrafish possesses many characteristics that make it an invaluable model to study human diseases ^[27]; however, one of its unique advantages is the unparalleled optical clarity of the embryo, which allows for the visualization of individual genes (fluorescently stained or labeled) throughout development, using non-invasive imaging techniques ^{[28][29][30][31]} (Figure 1A). This transparency of the embryo also facilitates genetic manipulation, such that gene function can readily be studied by the injection of synthetic mRNA or plasmid DNA into early stage zebrafish embryos generating transgenic zebrafish lines or altering gene function through genome editing techniques, such as the inclusion of zinc finger nucleases (ZFNs), transcription activation-like effector nucleases (TALENs), and the clustered regularly interspaced repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system ^[32][33][34]. Furthermore, the zebrafish genome has been sequenced, and 71% of human genes and 82% of human disease-related genes have orthologs in zebrafish ^[35]. Both ZFNs and TALENs require the creation of customized protein compositions for each target site, and these systems are not suitable for large-scale applications. However, the CRISPR/Cas9 system relies on target-site recognition by a custom guide RNA (gRNA) molecule and requires only one oligonucleotide to be designed for each target site. The success of CRISPR-mediated transgenic transformation in zebrafish can largely be attributed to frame-shift-generating null alleles via non-homologous extremity joint (NHEJ)-mediated CRISPR-induced DNA break repair ^[35].

Interestingly, similarities between the zebrafish and human nervous system (anatomy and physiological signaling) have been reported ^[33]. The zebrafish brain is composed of the forebrain (telencephalon), midbrain (optic tectum), and hindbrain (cerebellum) (Figure 1B); and many cells, including astrocytes, oligodendrocytes, microglia, cerebellar Purkinje cells, myelin, and motor neurons, are also similar to human cells. Further studies of spinal nerve patterning, neural differentiation, and vertebrate network development in adult zebrafish revealed similarities to higher vertebrates ^[33]. Due to these characteristics, zebrafish are widely used to validate candidate disease genes and to elucidate the molecular mechanisms and pathophysiology of neurological disease. The continued increase in the use of zebrafish in biomedical research publications reflects their expanding popularity ^[33] (Table 2).