Interpreting human genetic variation with in vivo zebrafish assays

Abstract

Rapid advances and cost erosion in exome and genome analysis of patients with both rare and common genetic disorders has accelerated gene discovery and illuminated fundamental biological mechanisms. The thrill of discovery has been accompanied, however, with the sobering appreciation that human genomes are burdened with a large number of rare and ultra rare variants, thereby posing a significant challenge in dissecting both the effect of such alleles on protein function and the biological relevance of these events to patient pathology. In an effort to develop model systems that are able to generate surrogates of human pathologies, a powerful suite of tools have been developed in zebrafish, taking advantage of the relatively small (compared to invertebrate models) evolutionary distance of that genome to humans, the orthology of several organs and signaling processes, and the suitability of this organism for medium and high throughput phenotypic screening. Here we will review the use of this model organism in dissecting human genetic disorders; we will highlight how diverse strategies have informed disease causality and genetic architecture; and we will discuss relative strengths and limitations of these approaches in the context of medical genome sequencing.

Introduction: Challenges and Opportunities from Human Genetics

Major inflections in genomic advances have always been accompanied by accelerated discovery of lesions associated with human pathologies. The development of the first karyotype led rapidly to the discovery of syndromes of polyploidy [1], while the then nascent technologies of genome mapping, cloning and sequencing yielded early insights into rare disease pathogenesis [2]. As the field progressed, molecular cytogenetics at the sub-Mb and ultimately kb-level resolution revealed the high contribution of copy number variants to both rare and common human genetic disorders [3], while, most recently, whole exome and whole genome sequencing (WES/WGS) have hyper-accelerated disease gene discovery both in historical cohorts and in the real-time clinical setting [4].

Amid the euphoria of discovery and the acutely increased expectation from patients and their physicians that the application of genomics can accelerate diagnosis and focus treatment options, the sobering realization has also emerged that each individual human genome is burdened with a large number of rare and ultra rare alleles. Considering bona fide pathogenic mutations alone in the average human exome, studies have reported a median of 50–150 nonsense mutations, several in homozygosity, while the abundance of unique single nucleotide variants (SNVs) can be in in the low to mid 100s [5]. Importantly, the number of rare and ultra-rare SNVs has continued to increase proportionately to the number of available exomes and genomes [6], indicating that we are unlikely to reach saturation of such alleles soon. These observations have generated a significant interpretive problem for disease gene discovery and for clinical genomics, as population-based arguments alone have been unable to dissect the contribution of the majority of these alleles to clinical phenotypes. Computational algorithms that take into consideration a variety of evolutionary, structural and biophysical properties of proteins have been of some assistance; however, their predictive ability (estimated in the 70 – 80% range [7]) has remained somewhat limited, mandating that definitive assessment of pathogenicity must be carried out through other methods.

Animal studies combine the identification of candidate alleles for human diseases with mutant organisms that recapitulate the human mutation or loss of gene function, and have improved our understanding of the causal link between genetic mutation and phenotypic trait [8]. Numerous animal models have been developed to study both monogenic and complex disease. Each model system has its advantages and limitations, such as genetic and anatomic homology to humans, the size of the genetic toolkit, generation time, and cost. Here we will focus on the application of zebrafish in modeling human genetic disease, an organism that has gained utility by bridging the gap between the high throughput abilities of invertebrates and the orthology of structure of mammals (Tables 1, 2). Although not a panacea, the implementation of zebrafish complementation studies [9] - suppression of the orthologous zebrafish gene and rescue with either a mutant or wild-type human mRNA to determine pathogenicity - in human and medical genomics has facilitated disease gene discovery in both monogenic and complex traits, and has also found application in modeling more intricate (and challenging) genetic lesions that include CNVs and epistatic interactions. We will review the tools available, discuss their possible uses and limitations and place the current vector of development of this model organism in the context of the ever- expanding generation of patient genomic data and the need for their accurate interpretation.

Table 1.

General attributes similarities of laboratory organisms used to model human genetic disease

	C. elegans	D. melanogaster	D. rerio	M. musculus
Percent identity with H. sapiens	43%	61%	70%	80%
Genome size	9.7 x 107 bp	1.3 x 108 bp	1.4 x 109 bp	2.5 x 109 bp
Exome size	28.1 Mb	30.9 Mb	96 Mb	49.6 Mb
Practical attributes
Husbandry demands	$	$	$	$$$
Cost per animal	$	$	$	$$$
Characterized inbred strains	+	+	+	++++
Outbred laboratory strains	+	+	+++	++
Germline/Embryonic cryopreservation	Yes	No	Yes	Yes
Lifespan	2 weeks	0.3 years	2–3 years	1.3–3 years
Generation interval	5.5 days	2 weeks	3 months	6–8 weeks
Number of offspring	300 larva	10–20 eggs	>200 embryos/clutch	10–12 pups/litter
Embryonic development	ex vivo	ex vivo	ex vivo	in utero
Molecular biology tools
Transgenesis *	++++	+++	++++	+++
Gene targeting *	++++	+++	+	++++
Conditional gene targeting	+	++	+	++++
Transient in vivo assays *	+++	++	++++	+
Allelic series from TILLING *	+++	+++	++++	++
Affordability of large scale screens **	++++	++++	+++	+
Cell biology tools
Cell lines and tissue culture	+	++	+	++++
Antibody reagents	+	++	+	++++
In situ probes	+	+++	++++	+++
Disease process
Birth defects	+	++	++++	++++
Adult-onset	++	+	+	++++
Behavioral	++	++	++	++
Aging	+++	++	++	++
Metabolic	++	++	+++	+++

Table adapted from Lieschke and Currie 2007. Nat Rev Genet. 8: 353–367.

^*reverse genetics;

^**forward genetic

Table 2.

Anatomical comparisons between zebrafish and humans

Anatomy	Key similarities	Key differences
Embryology	Cleavage, early patterning, gastrulation, omitogenesis, organogenesis are all represented	Rapid Influence of maternal transcripts Non-placental, involves hatching
Skeletal system	Ossified skeleton comprising cartilage and bone	Lack long bone, cancellous bone, and bone marrow Joints are not weight-bearing
Muscle	Axial and appendicular muscle groups Skeletal, cardiac and smooth muscle cell types, with similar cellular architecture and machinery Fast and slow skeletal muscle fibers	Fast- and slow-twitch muscle topographically separate Tail-driven locomotion depends on alternating contraction of myotomal muscle Appendicular muscle bulk is proportionately small
Nervous system and behavior	Central nervous system anatomy: fore-, mid- and hind-brain, including diencephalon, telencephalon and cerebellum Peripheral nervous system has motor and sensory components; Enteric and autonomic nervous systems Specialized sensory organs, eye, olfactory system and vestibular system, are well conserved Complex behaviors and integrated neural function: memory, conditioned responses and social behaviors (for example, schooling)	Telencephalon has only a rudimentary cortex Fish-specific sensory organs, such as the lateral line Fish behaviors and cognitive function are simplified compared with human behavior Significant difference in population of dopaminergic neurons (telencephalic vs midbrain) Some immediate early genes and neuropeptides not conserved in zebrafish
Hematopoietic and lymphoid/immune systems	Multiple hematopoietic cell types: erythrocytes, myeloid cells (neutrophils, eosinophils, monocytes and macrophages), T- and B-lymphocytes Coagulation cascade for hemostasis; Innate and adaptive humoral and cellular immunity	Erythrocytes are nucleated Possess thrombocytes rather than platelets Kidney interstitium is the hematopoietic site
Cardiovascular system	Multi-chamber heart with an atrium and ventricle Circulation within arteries and veins Separate lymphatic circulation Cardiac differentiation occurs through similar signaling pathways (eg, nkx2.5, bmp2b) Similar electrical properties and conduction patterns (SA node, slow atrial conductance, AV node, fast ventricular conductance)	Has left-right distinctions in cardiac anatomy, but does not have separate left-right circulations, that is, the heart has only two chambers So far no evidence for secondary heart field derivatives Lymph nodes have not been described Embryos are not dependent on functioning CV system for larval development Atria and ventricles express different myosin heavy chains during development (human hearts only later differentiate between atrial and ventricular mhc) Heart has high regenerative capacity, even in adult animals
Respiratory system	Cellular gas exchange; Oxygenation is dependent on circulation and hemoglobin carriage	Respiration occurs in gills, not lungs No pulmonary circulation Endoderm-derived swim bladder (functioning as a variable buoyancy device), which corresponds embryologically but not functionally to the lungs
Gastrointestinal system	Major organs: liver, exocrine and endocrine pancreas, gall bladder Zonal specializations along the length of the absorptive alimentary tract Immune cells in lamina propria	Lack an acidified digestive organ Have an intestinal bulb rather than stomach Intestinal Paneth cell not present
Renal and urinary systems	Glomerular anatomy and function	Filtration occurs in anterior and posterior kidneys Mesonephric rather than metanephric adult kidney No bladder or prostate gland No structure in zebrafish homologous to descending or ascending thin limb of nephron in mammals
Reproductive system	Molecular and embryological biology of germ-cell development Cellular anatomy of germ-cell organs, the testis and ovary	No sex chromosomes; Mechanism of sex determination is uncertain Fertilization is ex vivo (no uterus or the related internal female reproductive organs); Oocytes are surrounded by a chorion, not the zona pellucida, which must be penetrated by sperm Non-lactating; no breast equivalent
Endocrine system	Most endocrine systems represented, including hypothalamic/ hypophyseal axis (glucocorticoids, growth hormone, thyroid hormone, prolactin), parathyroid hormone, insulin and rennin	Differences in anatomical distribution of glands, for example, discrete parathyroid glands do not seem to be present Prolactin has a primary role in osmoregulation
Skin and appendages	Ectodermal derivative; Pigmentation pattern is due to neural-crest-derived pigment cells including melanocytes	Structures unique to fish that are specialized for the aquatic environment (including, elasmoid scales, mucous cells) Lack appendages (hair follicles, sebaceous glands) Additional pigment cell types: xanthophores and iridophores

Table adapted from Lieschke and Currie 2007. Nat Rev Genet. 8: 353–367.

Animal models of human genetic disease

A deep understanding of the genetic architecture of human disease, underlying cellular and molecular mechanisms, and the development of therapeutic paradigms is dependent on model organisms that can robustly capture the pathology under investigation. Mammalian models such as the mouse (Mus musculus), have historically been attractive platforms by virtue of a high level of genomic sequence homology to humans (>80%) [10], highly conserved anatomical and physiological features, and a diverse repertoire of gene targeting strategies to recapitulate human disease phenotypes (for scholarly reviews of this topic see [11, 12]). However, in the context of human genomics, time and cost now present significant drawbacks. By contrast, invertebrate models such as the nematode worm (Caenorhabditis elegans), or the fruit fly (Drosophila melanogaster) offer inexpensive alternatives, and have proven especially powerful for studying orthologous genes of interest through the use of sophisticated gene manipulation strategies (RNAi, transposable insertion elements etc; refs [13, 14] for reviews). Nonetheless, the reduced cost and increased experimental tractability of these models are accompanied with a greater disparity in cellular processes and structures in comparison to humans, in large part due to a decreased percentage of genes shared between species (43% and 61% for worm and fly versus human, respectively [15]). An intermediate model, the zebrafish (Danio rerio), has emerged as strong candidate to achieve the experimental tractability of its invertebrate counterparts, but with the genomic and physiological proximity of a vertebrate for the investigation of human genetic disease.

Zebrafish: an overview

The zebrafish is a tropical teleost that lives in the freshwaters of south-east Asia. In the late 1960s, George Streisinger transitioned this common aquarium species to a model for basic research of embryogenesis and organ development because of its “desirable attributes,” including a relatively short generation time of three to four months, the ability of mating pairs to generate several hundred embryos that develop rapidly and synchronously ex vivo, and the small size of adult fish (3 cm in length), making them easy to care for [16]. Moreover, embryos are transparent, allowing facile microscopic visualization in the first days of development, with major organ formation occurring 24 hours post-fertilization. Zebrafish have a diploid genome, but differ notably from the genomic structure of other vertebrates by the major teleost specific genome duplication that has resulted in sub-functionalization and neo-functionalization of genes [17–19]. Importantly, the biomedical research community now has a publicly available, extensively annotated version of the zebrafish genome at its disposal, of which 70% of genes have an identifiable human ortholog [20]. Additionally, a vast catalog of mutants, transgenic reporters, and gene-specific expression data has been generated from over two decades of dedicated Danio rerio use for “phenotype-driven” forward genetic screens and “gene-driven” reverse genetic approaches. These data are curated in ZFIN (the Zebrafish Model Organism Database), a community-wide resource warehousing genomic information, anatomical atlases, molecular tools and links to zebrafish publications (www.zfin.org and [21]).

Forward genetics: advances in vertebrate developmental biology

Initial forays in zebrafish research predated the precise knowledge of gene content or location within the zebrafish genome, and were not necessarily motivated by targeted questions of human pathology. Rather, most forward screens were conducted to understand vertebrate embryonic development by 1) introducing random mutations throughout the genome; 2) conducting an informative breeding scheme to generate progeny with homozygous recessive mutations; 3) evaluating animals for a measurable phenotypic readout; and 4) identifying the mutation and gene underscoring the phenotype. Used widely across multiple model organisms, the application of this traditionally laborious strategy in zebrafish has been reviewed extensively elsewhere [22].

The first zebrafish screens were reported in the 1980s and involved the application of gamma rays to induce recessive lethal mutations. However, this approach resulted in significant chromosomal breaks that rendered mapping to a single locus difficult [23, 24]. Alkylating agents, primarily N-ethyl-N-nitrosourea (ENU), replaced gamma rays as an effective mutagen and application resulted in discrete genomic mutagenesis in zebrafish germ cells that could be mapped to a single gene (approximately one mutant per genome evaluated) [25, 26]. This discovery led to large-scale efforts by labs in Tübingen and Boston to apply ENU screening to zebrafish. Within two years, their combined efforts led to the characterization of ~4,000 embryonic lethal phenotypes; these include gastrulation [27, 28]; somitogenesis [29]; brain [30–32]; cardiovascular [33]; and craniofacial development mutants [34–36].

Although forward genetic screens in zebrafish contributed significantly to the fundamental understanding of early embryonic development, the impact on such findings to inherited disease in humans has been sporadic. This modest connection can be attributed to four main reasons. First, such screens are unable to capture alleles that confer a dominant negative (antagonizes the wild-type protein function) or gain of function (mutation confers a protein function different from that of wild-type protein) effect. Second, phenotypes must have a measurable phenotypic readout in early embryonic or larval stages, decreasing the possibility of detecting adult-onset or degenerative phenotypes. Third, although such screens were able to uncover discrete gene functions, the odds of generating precisely the same allele by ENU as has been seen in a patient is remote. Finally, this approach is confounded further by the fact that the zebrafish genome underwent a teleost specific duplication [17–19]. Among the genes for which there is an identifiable human ortholog, 47% have a one-to-one orthologous relationship with a human counterpart, while the remainder of zebrafish genes have complicated one-to-many or many-to-one orthology in comparison to the human gene [20]. As a result, duplicated gene function may either be a) retained in both copies making them functionally redundant; b) lost in one of the two copies wherein it becomes a pseudogene; or c) a novel and divergent gene function is acquired by one of the two copies. Therefore, mutations in only one of two functionally redundant orthologs might not display a phenotype. Finally, Nonetheless, ENU mutants have been successful in drawing anatomical correlates for genes implicated in recessive human disorders that cause anatomical birth defects. For instance, the craniofacial mutant crusher^m299 is caused by a nonsense mutation in sec23a [37]; at the same time as this discovery, SEC23A mutations in humans were shown to cause a clinically relevant craniofacial dysmorphology, cranio-lenticulo-sutural dysplasia, bolstering the evidence of causality in both species [38]. Importantly, the recent application of WGS [39], WES [40], and improved mapping strategies [41] to zebrafish ENU mutants has enabled the rapid and cost-effective identification of mutations, justifying the continued use of forward genetics to assist with assigning causality in human genetic disease.

Reverse genetics: from candidate causal gene to physiologically relevant animal model

Forward genetic screening involves the unbiased examination of phenotypes resulting from mutations in the zebrafish genome. However, the randomness of this approach is hampered by the inability to specifically target every coding gene and/or specific mutations implicated in human pathology. To circumvent this problem, the precise targeting of candidate genes and alleles can be achieved through several methods that have been developed over the past ~15 years.

First, transient gene manipulation can be achieved through the injection of either morpholino (MO) antisense oligonucleotides (suppression) or capped in vitro transcribed mRNA (ectopic expression) into zebrafish embryos. MOs are stable molecules that consist of a large, nonribose morpholine backbone with the four DNA bases pairing stably with mRNA at either the translation start site (to disrupt protein synthesis) or at intron-exon boundaries (to disrupt mRNA splicing) [42]. The use of MOs to confer effective gene knockdown was first shown in zebrafish in 2000; Nasevicius and Ekker recapitulated the developmental phenotypes of five different embryonic lethal mutants and also developed models of the human genetic disorders hepatoerythropoietic porphyria and holoprosencephaly through the suppression of urod and shh, respectively [43]. Since this report, MOs have been used broadly to study vertebrate development and disease; co-injection of MO and orthologous mRNA has been employed for the systematic functional testing of alleles identified in humans, offering a powerful approach for analysis of variant pathogenicity and direction of effect [44]. Still, this methodology does have notable drawbacks: 1) MO efficacy is limited to ~3–5 days [43], and similarly, the presence of mRNA is limited to the same embryonic timeframe; 2) with few exceptions [45], injected MOs and mRNAs do not confer spatial or temporal specific activity; and 3) MOs can give rise to spurious phenotypes resulting from off-target effects [46]. Even so, the use of this methodology within the appropriate developmental stage, and with the appropriate experimental controls (a) targeting with a splice-blocking MO to demonstrate incorrectly spliced RNA; b) specific rescue of MO phenotypes with orthologous wild-type mRNA; c) demonstration of a similar phenotype with multiple MOs targeting the same gene; or d) comparison with a mutant when possible, and if appropriate [46]) can allow for the correct interpretation of MO phenotypes to establish relevance to human genetic disease through the recapitulation of loss-of-function or dominant negative effects.

Second, it is possible to readily obtain germ line zebrafish mutants for a gene of interest; this avoids the phenotypic variability associated with MOs, and allows the observation of phenotypes beyond early larval stages. Targeting Induced Local Lesions in Genomes (TILLING) was the first reverse genetic approach to produce germ line mutations in a gene of interest. Similar to forward screens, TILLING involves ENU mutagenesis of adult male zebrafish and generation of F1 families. Sperm from F1 males is then cryopreserved while genomic lesions are screened in target genes, typically in early exons or near exonic regions encoding critical protein domains, through PCR amplicon screening [47]. The completion of the zebrafish genome coupled to next-generation sequencing has increased significantly the throughput of the screening aspect of this strategy. TILLING mutants have been identified for >38% of all known zebrafish protein coding genes (http://www.sanger.ac.uk/Projects/D_rerio/zmp and [48]); this corresponds to ~60% of orthologous genes associated with a human phenotype in the Online Mendelian Inheritance in Man (OMIM; http://www.omim.org) database. The ongoing TILLING efforts hope to generate a comprehensive resource of putative null or hypomorphic models of human genetic disease, however, it is critical to be cognizant of the possibility that ENU may introduce multiple lesions in the genome. Ideally, multiple mutants with different alleles in the same gene should be phenotypically characterized to ensure that the pathology is specific. The same guidelines are true for retrovirus [49] or transposon [50] insertional mutants used in similar reverse genetics approaches.

Both forward ENU screens and TILLING are laborious; alternative approaches have recently expanded the utility of the zebrafish by enabling precise and germ line transmittable gene targeting that does not require excessive downstream screening to identify mutations (see ref [14] in this issue for a detailed review of genome editing endonucleases). First, zinc finger nucleases (ZFN) utilize a zinc finger array to enable target sequence specificity (typically the early exon of a gene), and a FokI endonuclease to guide cleavage and subsequent repair at the target site [51]; this was first utilized to target the gol locus (mutation of which results in absence of pigment), ntl (a regulator of early embryogenesis), and kdr (vascular endothelial growth factor-2 receptor), as visible proof-of-principle phenotypes [52, 53]. Second, transcription activator-like TAL effector nucleases (TALEN) have similarly been optimized to achieve locus-specific genome editing and have been shown to achieve greater specificity of and alteration of target sequences than ZFNs [54]. A third, more recent advancement in zebrafish genome editing technology involves clustered, regularly interspaced, short palindromic repeats (CRISPR), bacterial type II systems that guide RNAs to direct site-specific DNA cleavage by the Cas9 endonuclease [55]. Each of ZFN, TALEN, and CRISPR technologies have expanded the molecular toolkit of the zebrafish (for comparisons, see Table 3), accelerating studies of vertebrate development and improving our understanding of analogous phenotypes to human disease. For example, CRISPR/Cas9 was used to edit the gata5 locus [56], and mutant embryos displayed a cardia bifida phenotype mimicking both the fau^tm236a zebrafish mutant [57], and also humans with congenital heart defects [58–60]. However, there is currently a relative paucity of reports in which human-driven WES/WGS studies have been followed with the generation of such stable mutants; this is largely due to the relative newness of the technology, and/or the amount of time and labor still required to generate and characterize mutants; we anticipate the landscape of the field to change rapidly in the coming months and years.

Table 3.

Comparison of different reverse genetics approaches.

Methodology	Class	Specificity (targeting sequence)	Off target effects	Technology adoption time and costs	Ongoing time and cost	Somatic DNA cutting efficiency	Germline efficacy
Morpholinos	Morpholino oligo	25 nt	Low	Low	Low	n/a	n/a
ZFNs	Protein-DNA	18+ bp (pair)	Low	High	Moderate	Low (~2%)	Low
TALENs	Protein-DNA	30+ bp (pair)	Ultra low	Moderate	Low	Moderate to high (~20– ~50%)	Moderate to high
CRISPR- Cas9	RNA-DNA	12+ bp	Ultra low	Low	Low	Moderate to high (~30%– ~60%)	Moderate to high

Table adapted from Blackburn et al. 2013 Zebrafish. 10: 116–118.

Humanizing the zebrafish to study mutations detected in humans

Taken together, the zebrafish exemplifies a tractable and physiologically relevant tool to model genetic variation in humans. Each of the forward and reverse genetics tools have limitations, and in particular, place significant emphasis towards the study of loss-of-function effects of single genes potentially making them an overly simplistic model to investigate oligogenic or even complex traits. In a growing number of instances, however, it has been possible to balance experimental tractability, specificity, and cross-species phenotypic similarity to establish: 1) physiological relevance of a gene to a human clinical phenotype; 2) allele pathogenicity; and 3) direction of allele effect for a vast array of human genetic disorders with diverse models of inheritance, phenotypes, and ages of onset.

Recessive disease

Disorders that segregate under a recessive mode of inheritance, especially congenital or pediatric-onset disorders with an abnormality in a structure with an anatomical counterpart in the developing zebrafish, have achieved widespread use towards demonstrating physiological relevance. This often represents the extent of functional data presented in instances when the human mutations have an unambiguous loss-of-function effect on the protein (nonsense, frameshift, or splice-site). For instance, causal mutations identified in primary ciliary Dyskinesia (PCD) are almost exclusively null changes, and transient MO-based studies in zebrafish have shown that proteins of a priori unknown function including CCDC39, ARMC4, and ZMYND10 give rise to left-right asymmetry defects phenotypes found in humans [61–63]. In other recessive disorders, such as pontocerebellar hypoplasia (PCH), the zebrafish has assisted in establishing clinical relevance and allele pathogenicity (Figure 1A). Wan et al. identified nonsynonymous changes in EXOSC3, encoding exosome component 3, following WES of four affected siblings; MO-induced suppression resulted in phenotypes that were relevant to the human clinical features of microcephaly and reduced motility in exosc3 morphants. Additionally, in vivo complementation of exosc3 MO phenotypes with either zebrafish or human mRNA harboring the missense mutations found in patients failed to rescue the phenotype, indicating that these were loss-of-function alleles [64]. Even so, transient in vivo complementation assays are not applicable to every gene. Human genes with an open reading frame (ORF) larger than ~6 kb are challenging to transcribe in vitro, likely explaining why large genes such as NBEAL2, encoding neurobeachin-like 2, the novel genetic cause for gray platelet syndrome (ORF of 8.2 kb), was shown to cause a relevant thrombocytopenia phenotype in zebrafish morphants, but the missense mutations identified in patients were not tested [65].

Figure 1. Zebrafish models of human genetic disease.

A. Mutations in pontocerebellar hypoplasia are caused by EXOSC3 and in vivo complementation studies in zebrafish recapitulate the brain phenotypes observed in patients and demonstrate that missense mutations are functional null variants. Left panel, neuroimaging of affected individuals (A1–A4; top row) and control images (A5–A8; bottom row); right panel, whole-mount in situ hybridization in splice blocking MO-injected embryos in lateral view (A9; inset- dorsal view, with rostral to the left) demonstrated diminished expression of dorsal hindbrain progenitor-specific marker atoh1a and cerebellar-specific marker pvalb7 (A10; quantification). Images reproduced from Wan et al 2012. Nat Genet. 44: 704–708.

B. Congenital abnormalities of the kidney and urinary tract are caused by haploinsufficiency of DSTYK. Left, panels B1-B3, with hypoplasia of the left kidney (Panel B1, kidneys outlined by dashed lines), bilateral hydronephrosis (Panel B2, arrows) caused by ureteropelvic junction obstruction detected at birth, and hydronephrosis only of the left kidney (Panel B3, arrow) caused by ureteropelvic junction obstruction. The intravenous pyelogram in Panel B4 shows blunting of fornices on the right side (white arrow); right, MO-induced knockdown of dstyk embryos, live lateral images show absence of the patent pronephric duct opening (arrows). Images reproduced from Sanna-Cherchi et al 2013. NEJM. 369: 621–629.

C. Adult-onset limb-girdle muscular dystrophy is caused by dominant negative mutations in DNAJB6. C1, Transmission electron microscopy showed early disruption of Z-disks (arrows; left) and autophagic pathology (right) in LGMD1D; C2-C10, Lateral views of zebrafish embryos 2 days post fertilization subjected to whole-mount immunofluorescence staining of slow myosin heavy chain display myofiber abnormalities (arrows). Images reproduced from Sarparanta et al 2012. Nat Genet. 44: 450–455.

D. PLS3 overexpression exerts a protective effect on SMN1 deletion to rescue motor neuron defects in spinal muscular atrophy (SMA). D1, pedigrees of SMA-discordant families showing unaffected (gray) and affected (black) SMN1-deleted siblings; D2, Lateral view of zebrafish embryos injected with control MO, smn MO, PLS3 RNA, and smn MO + PLS3 RNA. Motor axons were visualized with znp1 antibody at 36 hours post fertilization and show rescue of smn MO with PLS3 RNA. Images reproduced from Oprea et al 2008. Science. 320: 524–527.

E. SCRIB and PUF60 suppression drive the multisystemic phenotypes of the 8q24.3 copy number variant. E1, photographs five individuals with the 8q23.4 CNV show craniofacial abnormalities and microcephaly; E2, Lateral and dorsal views of control and scrib or puf60a MO-injected embryos at 5 dpf show head size and craniofacial defects observed in affected individuals. Images reproduced from Daubert et al 2013. Am J Hum Genet. 93: 798–811. LOF, loss-of-function; DN, dominant negative.

Dominant disorders

In contrast to recessive disorders, in which the allele effect is typically loss-of-function, traits that segregate under an autosomal dominant inheritance pattern are the result of either a haploinsufficiency, dominant negative, or gain-of-function mechanism. For some dominant pediatric-onset disorders, the genetic evidence of a heterozygous null variant segregating in a large pedigree with fully penetrant disease is sufficient to suggest that the direction of allele effect is haploinsufficiency, and these predictions have been confirmed in zebrafish through MO-induced gene suppression. For example, dilated cardiomyopathy is caused by nonsense, splice-site or missense mutations in the gene encoding heat shock protein co-chaperone BCL2-associated athanogene 3 (BAG3) and gene suppression results in similar cardiac phenotypes in zebrafish embryos [66]. Similarly, congenital abnormalities of the kidney and the urinary tract (CAKUT) associated with a loss-of-function splice site mutation segregating in a dominant pedigree were identified in the dual serine-threonine kinase encoded by DSTYK (Figure 1B); the human phenotypes were recapitulated in dstyk morphant embryos [67]. Transient experiments in zebrafish embryos can also determine allele pathogenicity and capture a dominant negative direction of effect that is isoform-specific. This is exemplified by the transient functional studies of missense mutations in the co-chaperone protein, DNAJB6, associated recently with adult-onset limb girdle muscular dystrophy (Figure 1C) [68]. Co-injection of mutant DNAJB6 mRNA in the presence of equivalent amounts of wild-type transcript resulted in myofiber defects in zebrafish embryos; injection of increasing amounts wild-type mRNA with a fixed concentration of mutant resulted in a phenotypic rescue, indicating the dominant toxicity of the mutant alleles. Given the nature of these mutations (deleterious in heterozygosity), the use of zebrafish for dissecting dominant disorders will likely remain restricted to transient MO- and mRNA-based studies until the development of more sophisticated conditional gene suppression/expression techniques in zebrafish.

De novo variants

Variants that arise de novo as a product of either germline mosaicism or early developmental DNA replication errors are significant contributors to the human mutational burden [69]. Similar to variants that underscore autosomal dominant disorders, de novo changes may give rise to clinical phenotypes produced from falling below a gene dosage threshold, dominant negative effects, or acquisition of a novel function. As such, an unbiased approach towards dissecting the direction of de novo allele effect is critical once physiological relevance has been determined. For instance, transient approaches have been carried out in zebrafish to investigate de novo missense mutations in CACNA1C, encoding the voltage-gated calcium channel Ca_v1.2, in the pathophysiology of Timothy syndrome (TS), a pediatric disorder characterized by cardiac arrhythmias, syndactyly, and craniofacial abnormalities. Ectopic expression of mutant mRNA and suppression of cacna1c in zebrafish embryos not only revealed that the mutation confers a gain-of-function effect, but also demonstrated a novel role for Ca_v1.2 in the non-excitable cells of the developing jaw [70].

CNVs

Frequently arising de novo, copy number variants (CNVs) likewise represent a significant molecular basis for human genetic disease [71]. These variations in genomic structure, can range in size from a few thousand to millions of base pairs, are not identifiable by conventional chromosomal banding, and can encompass from one to hundreds of genes [72]. Although genotype-phenotype correlations among affected individuals with overlapping CNVs can assist in narrowing specific genetic drivers, CNVs have been historically intractable to functional interpretation in animal models, with sparse reports of human CNVs being modeled in the mouse [73]. Zebrafish models have emerged recently as powerful tools to dissect both recurrent and non-recurrent CNVs. First, systematic zebrafish modeling of the 29 genes in the recurrent reciprocal 16p11.2 duplication/deletion CNV— associated with a range of neurocognitive defects— found the main driver of the neuroanatomical phenotypes to be KCTD13, causing mirrored macro- and microcephaly upon suppression or overexpression in zebrafish, respectively [74]. Second, MO-induced suppression of three genes in the 8q24.3 non-recurrent deletion CNV in zebrafish embryos revealed that the planar cell polarity effector SCRIB, and the splicing factor PUF60 could be linked to distinct aspects of the renal, short stature, coloboma, and cardiac phenotypes observed in five individuals with overlapping microdeletions at this locus (Figure 1E) [75].

Second-site modifiers

The demonstration of second-site phenotype modification in primarily recessive human genetic disease has been fuelled by the use of in vivo assays in zebrafish. The ciliopathies, disorders underscored by dysfunction of the primary cilium, have been causally linked with over 50 different loci, can give rise to a constellation of human phenotypes, and have been an ideal system to study epistasis [76]. The recent dissection of the genetic architecture of Bardet-Biedl syndrome (BBS), a ciliopathy hallmarked by retinal degeneration, obesity, postaxial polydactyly, renal abnormalities, and intellectual disability, 1) informed the pathogenic potential of missense BBS alleles contributing to the disorder (null, hypomorphic, or dominant negative); 2) revealed the surprising contribution of dominant negative alleles in oligogenic pedigrees with BBS; and 3) provided sensitivity (98%) and specificity (82%) metrics for the zebrafish in vivo complementation assay to predict allele pathogenicity [77]. Transient zebrafish in vivo complementation assays have similarly been used to identify RPGRIP1L A229T as a modulator of retinal endophenotypes [78], RET as a modifier of Hirschsprung phenotypes in BBS [79], and TTC21B as a frequent contributor to mutational load in ciliopathies [80]. Second-site modification phenomena are not unique to the ciliopathies; for example, overexpression of plastin 3 (PLS3) to mimic the gene expression in unaffected individuals, improved the axon length and growth defects associated with SMN1 deletion in spinal muscular atrophy (SMA). The interaction of these two genes was shown, in part, through modeling of SMA genotype and phenotype correlates in zebrafish embryos (Figure 1D) [81].

Complex traits

Genome wide association studies (GWAS) alone have been hampered by an inability to connect risk association to genes and underlying mechanism. However, the zebrafish has emerged as a tool to dissect genes at or near loci that confer significant risk for the complex trait under investigation. Rare alleles in GWAS hits have been more straightforward to dissect, since the strategy has been similar to that of Mendelian traits. For example, a combination of in vitro analysis of enzyme stability and secretion and vascular integrity in the retina in zebrafish embryos demonstrated a functional role for a rare allele in the gene encoding complement factor I (CFI), thus providing direct evidence for a loss of function role of CFI in AMD [82]. Zebrafish have also been used in the absence of candidate coding changes in GWAS-identified loci. In one example, Liu et al assessed the physiological relevance of candidate genes identified from a GWAS of chronic kidney disease among African Americans populations; these efforts identified KCNQ1 as a functionally relevant candidate due to the glomerular filtration defects observed in kcnq1 morphants [83]. Similarly, functional validation of loci associated with platelet count in cohorts of European ancestry yielded eleven novel genes implicated Danio rerio blood cell formation [84]. Although numerous questions remain regarding the combinatorial effects of GWAS hits, their mechanistic basis for conferring risk, and the physiological relevance of significantly associated sites, both coding and non-coding, the zebrafish offers a tractable tool to begin to dissect existing GWAS data.

Adult Onset Disease

The majority of zebrafish models discussed so far have been used to understand the role of genes and alleles in pediatric and congenital disorders, in large part because transient MO and mRNA analysis is possible only during development. Nonetheless, given the correct tools and appropriate assays, this model organism is also useful for the study of adult onset disorders. AMD was one example described above, the utility of the model being extracted from the ability to model vascular integrity in zebrafish embryos, a phenotype relevant to AMD pathology. There are numerous other examples as well. In particular, phenotypic proxies of human neurodegenerative diseases such as schizophrenia, Huntington, Parkinson, and Alzheimer disease, have been used to examine the role various genes in these diseases (reviewed extensively in [85] and [86]), although in almost all cases these studies involved genetic mutants and/or stable transgenes. In one example, stable transgenic zebrafish expressing human 4-repeat Tau showed Tau accumulation within neuronal cell bodies axons in neurons throughout the adult brain, resembling neurofibrillary tangles [87]. Some defects, such as behavioral phenotypes, are not immediately observable through anatomic or histological methods, making it necessary to employ more sensitive methods of analysis. To this extent, assays for memory and learning impairment [88] and conditioned avoidance [89] allow for quantifiable testing of subtle phenotypes in adult fish.

Conclusion: The road ahead

In the study of human genetics, animal models have provided insight into the genetics and pathophysiology. The efficacy of a model organism always hinges on whether that organism models appropriately the target pathology of humans and whether the experiments necessary to provide burden of proof are tractable and not cost-prohibitive. In the context of human and medical genomics, we anticipate that the entire spectrum of model organisms will continue to be used. Nonetheless, it is clear that models such as zebrafish and possibly other similar organisms, such as Medaka or Xenopus that offer transparency, low cost and the ability to manipulate their genome efficiently will gain a prominent role as the community strives to model thousands of candidate disease-associated genes and alleles.

A key requirement towards the widespread use of the zebrafish to determine pathogenicity of alleles identified by WES/WGS moving forward will be to a) improve throughput of physiologically-relevant models of human disease (through the rapid generation of in vivo complementation assays); and b) automated phenotyping and image analysis. While MOs may still be a tractable option for the evaluation of early developmental phenotypes, the recent ability to generate knock-in mutants [90], conditional zebrafish mutants [91], and multiplexed mutants [92] holds great promise towards accelerating disease modeling throughput. Equally as important, some platforms for high throughput phenotypic screening of zebrafish larvae have been developed recently [93]. For example, one system captures hundreds of three-dimensional morphological features with speed and accuracy, clustering quantitative phenotypic signatures so that multiple phenotypes can be detected and classified simultaneously. Nonetheless, this system is limited to bright field images, rendering marker analysis (by RNA in situ or antibody staining) difficult. Given that embryo phenotyping represents the most significant bottleneck in scaling the use of zebrafish in human genomics, there is an acute need to develop additional transgenic reporter lines to assist with visualization of cellular and anatomical structures of interest [94], as well as imaging and embryo manipulation technologies further.

It is also important to note that zebrafish analysis, like all other genetic and molecular biology tools, has its limitations. MO studies can sometimes generate conflicting data, especially in the context of early developmental phenotypes that are most sensitive to toxic effects. Rescue studies and a minimum of two MOs per gene tested, when possible, are essential to validate findings. Similarly, multiple independent lines from genome editing experiments will need to be studied to ensure that the phenotypes observed are driven by the engineered mutation, not an off-site introduced allele. Moreover, not all human genes and alleles are modelable; some 25–30% of the human transcriptome is not present in zebrafish, while other genes can be difficult to model because of divergent functions or extreme dosage sensitivity (especially transcription factors) rendering them experimentally difficult. Further, for some disorders (e.g. pulmonary fibrosis) there cannot be a credible phenotypic surrogate and distant surrogates might lead to incorrect conclusions. Finally, most zebrafish studies to date have focused on coding variation; modeling non-coding variation is significantly more taxing, yet remain important, not least because regulatory regions are likely to be enriched for alleles that drive GWAS signals [95]. This work is possible, once we recognize that evolutionary constraints might render some data uninterpretable. For example, testing multiple sequences located within a 50 kb block of the regulatory domain of IRX3 (certain variants of which are associated with obesity in humans) resulted in transgenic zebrafish with expression in pancreas [96]; knockdown of irx3 in zebrafish reduced the number of pancreatic beta-cells. However, given current designs, it will be difficult to execute such experiments at the throughout required to address the needs of the human genetics community.

Despite these limitations, modeling human variation in zebrafish embryos has been a significant contributor towards dissecting the causality of genes and alleles in human genetic disorders. Moreover, the development of human disease models will serve as a platform for the discovery of novel therapeutic paradigms by employing high-throughput small molecule screening approaches (reviewed extensively elsewhere [97, 98]). Moving forward, we anticipate that the community, through the combinatorial use of all the tools discussed here, will saturate in the coming years a significant fraction of the morbid human genome. Ultimately, we imagine that such studies will inform the design of improved computational algorithms, probably through the training of thousands of human alleles tested in vivo, which will in turn represent the next inflection point in human and medical genomics.

Highlights.

• Interpretation of rare and novel variants is a current challenge in human genetics

• Zebrafish are a model system to study the pathogenicity of human alleles

• Conserved evolutionary and physiological features make zebrafish an ideal model

• Zebrafish are suitable for moderate to high throughput assays of human disease