Zebrafish Genome’s Extra Duplicated Genes Reveal Hidden Rules of Gene Function

The zebrafish has long been prized as a model organism for its genetic similarity to humans, with roughly 70 percent of human protein-coding genes, including most genes implicated in disease, having highly conserved orthologs in the fish. But a new open-access review published in Development Genes and Evolution argues that the species’ greatest scientific value may lie in a feature that researchers often treat as an inconvenience: an extraordinary abundance of duplicated genes. Authors Maliha Tasnim, Preston Wahlquist, and Jonathon T. Hill of the journal’s pages contend that studying both members of zebrafish gene pairs together can expose how protein structure, function, and regulation evolve—insights that are difficult, and in some cases impossible, to obtain from species with single-copy genes.

The source of this duplication is an ancient evolutionary accident. All vertebrates share two rounds of whole genome duplication, known as 1R and 2R, thought to have occurred roughly 500 to 600 million years ago before the radiation of extant vertebrate classes. Teleost fish, the clade that includes zebrafish, underwent a third event, the teleost-specific whole genome duplication, approximately 350 million years ago. This extra round is credited with helping drive the explosive diversification that made ray-finned fishes one of the largest vertebrate groups today. Evidence for the event is substantial: many teleosts, including medaka, pufferfish, and zebrafish, carry seven Hox gene clusters, whereas lobed-finned fishes and most other vertebrates have only four, and genomic analyses identified 3,440 gene pairs—26 percent of the genes analyzed—within double-conserved synteny blocks in the zebrafish genome.

Whole genome duplication is not the whole story. Zebrafish also show a marked propensity for local gene duplication, with one comparative study finding the highest rates of tandem duplicates, defined as copies within 10 kilobases of each other, and intrachromosomal duplicates, copies on the same chromosome but more than 10 kilobases apart, among four teleost species examined. Together, these events have produced duplicates for approximately 5,300 of the 26,206 protein-coding genes identified in zebrafish. The review argues that this reservoir represents what the authors call a 350-million-year natural experiment, one that cannot be reproduced in a laboratory, and that focusing on only one copy of a duplicated pair squanders the information it contains.

When a gene duplicates, one of two initial outcomes follows: loss of one copy or persistence of both. Loss, termed non-functionalization, is the most common result, occurring when random mutations in regulatory or coding regions cause one copy to degenerate into a pseudogene. But even pseudogenes are not necessarily inert. Once dismissed as molecular fossils, they can produce small interfering RNA, competitive endogenous RNA, or antisense transcripts, and pseudogene expression changes have been linked to cancer progression, including kidney cancer, and to disease subtyping with diagnostic and predictive value. Conserved cis-regulatory modules near pseudogenes may also indicate that these elements actively regulate nearby functional genes, with the pseudogene itself a bystander.

When both copies persist, they typically diverge functionally. The most common fate preserving functional paralogs is subfunctionalization, in which the ancestral gene’s expression pattern or domain functions are partitioned between the duplicates. The review highlights the oxr1a and oxr1b pair as an example of spatial division: oxr1a is maternally expressed and ubiquitous early in development, later localizing to the head and, in adults, prominent in the retina and heart, while oxr1b is restricted during the pharyngula period to lateral line ganglia and spinal cord neurons and, in adults, found in the adenohypophysis and neuromasts. Similarly, the pax6a and pax6b paralogs show distinct enhancer elements driving expression in the pancreas versus the brain, knowledge relevant to human PAX6-related conditions such as diabetes mellitus and aniridia.

Subfunctionalization can also act on timing. Morpholino knockdown experiments showed that the nrf2a and nrf2b paralogs, both involved in the response to oxidative stress, act at different developmental stages: nrf2a morphants showed an oxidized glutathione redox potential from 48 hours post-fertilization, while nrf2b knockdown reduced glutathione redox potential at 3 hours post-fertilization, with later stages resembling controls. Protein-level subfunctionalization, once dismissed as rare, also occurs. In developing heart and limb, Tbx5a and Tbx5b are co-expressed, yet the T-box domain of Tbx5b shares only 83 percent sequence identity with its Tbx5a counterpart, well below the typical 95 to 99 percent between paralogous T-box genes in the same subfamily, and mRNA from one paralog could not compensate for loss of the other.

Dosage effects add another layer. In a CRISPR interference study, knocking down znf143a led to a 1.5-fold increase in znf143b mRNA levels, a compensatory mechanism that was not reciprocated when znf143b was knocked down, suggesting tight control of gene dosage is essential for development. The review also points to Dravet syndrome, caused in humans by heterozygous loss-of-function mutations in SCN1A, as a case where zebrafish duplications matter: the scn1lab paralog is the more conserved human ortholog and has been linked to epilepsy phenotypes, but the less-studied scn1laa also influences neuronal excitability, with mutants showing altered brain and forebrain nerve impulse transmission. Studying both paralogs, the authors argue, may reveal compensatory mechanisms relevant to the variability and penetrance of SCN1A-related disorders.

Neofunctionalization, in which one duplicate acquires an entirely new function, is rarer and harder to prove, but confirmed cases exist. The hoxa13a gene exhibits hypermutability, accumulating more mutations than its paralog hoxa13b, and this has been linked to development of the median fin fold, a specialized structure in early teleost development, suggesting a novel rather than ancestral role. Complex fates also occur: hoxb1a and hoxb1b show functional redundancy, each capable of promoting Mauthner neuron differentiation, while also performing unique functions individually. And in a striking example of neofunctionalization, the elnb gene, created during the teleost-specific duplication, became essential for bulbus arteriosus development in zebrafish; rescue experiments with the ancestral elastin gene from the basal actinopterygian Polypterus senegalus failed to restore the knockdown phenotype, implying a genuinely novel function.

The implications extend to human health in several ways. Comparative analysis of duplicate pairs can pinpoint the roles of specific protein domains and enhancers, as with sox9a and sox9b, which have distinct expression patterns and functions in craniofacial and pectoral fin development. Gene cluster comparisons, including the seven or eight Hox clusters of teleosts and the diversified MHC class I genes categorized into U, Z, and L lineages, illuminate development and immunity. Zebrafish also model human diseases caused by duplication itself: HER2 amplification occurs in about 15 to 20 percent of breast cancers, and her2 is duplicated in the zebrafish genome without oncogenic effects, while the historic discovery of DHFR gene duplication in 1978 explained how cancer cells resist methotrexate through increased enzyme production.

The review closes with a practical prescription. A well-designed study of duplicated genes should include identification of each paralog’s expression domain and functional analysis, such as single and double knockouts, to detect individual functions and redundancy, supplemented by rescue experiments, phylogenetic comparison with ancestral genes, and examination of expression across developmental stages. The authors caution that results from manipulating one paralog may not represent the full repertoire of functions for its human ortholog, since expression patterns and protein domain functions may be partitioned between the two copies. Only by comparing paralogs together, they conclude, can researchers accurately model human disease in the zebrafish and fully exploit the unique genetic resource embedded in its genome.

Subject of Research: Duplicated gene evolution and functional divergence in zebrafish

Article Title: Zebrafish: unraveling genetic complexity through duplicated genes

Article References: Tasnim, M., Wahlquist, P., & Hill, J. T. (2024). Zebrafish: unraveling genetic complexity through duplicated genes. Development Genes and Evolution, 234(2), 99-116. https://doi.org/10.1007/s00427-024-00720-6

Image Credits: AI Generated

DOI: 10.1007/s00427-024-00720-6

Keywords: zebrafish, gene duplication, whole genome duplication, subfunctionalization, neofunctionalization, pseudogenes, paralogs, teleost evolution, Dravet syndrome, Hox genes, developmental biology, comparative genomics