Editor's evaluation: Copy number variation and population-specific immune genes in the model vertebrate zebrafish
Year: 2024
Type: peer-review
Abstract: Full text Figures and data Peer review Side by side Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Appendix 1 Data availability References Article and author information Metrics Abstract Copy number variation in large gene families is well characterized for plant resistance genes, but similar studies are rare in animals. The zebrafish (Danio rerio) has hundreds of NLR immune genes, making this species ideal for studying this phenomenon. By sequencing 93 zebrafish from multiple wild and laboratory populations, we identified a total of 1513 NLRs, many more than the previously known 400. Approximately half of those are present in all wild populations, but only 4% were found in 80% or more of the individual fish. Wild fish have up to two times as many NLRs per individual and up to four times as many NLRs per population than laboratory strains. In contrast to the massive variability of gene copies, nucleotide diversity in zebrafish NLR genes is very low: around half of the copies are monomorphic and the remaining ones have very few polymorphisms, likely a signature of purifying selection. Editor's evaluation This useful study employs a sequence capture approach to characterize the diversity of NLR sequences in wild zebrafish populations. The authors provide solid evidence that wild zebrafish populations harbor several thousand NLR genes in total, with individual fish having a few hundred NLR gene copies. https://doi.org/10.7554/eLife.98058.sa0 Decision letter eLife's review process eLife digest Humans and other animals have immune systems that protect them from bacteria, viruses and other potentially harmful microbes. Members of a family of genes known as the NLR family play various roles in helping to recognize and destroy these microbes. Different species have varying numbers of NLR genes, for example, humans have 22 NLRs, but fish can have hundreds. 400 have been found in the small tropical zebrafish, also known as zebra danios. Zebrafish are commonly used as model animals in research studies because they reproduce quickly and are easy to keep in fish tanks. Much of what we know about fish biology comes from studying strains of those laboratory zebrafish, including the 400 NLRs found in a specific laboratory strain. Many NLRs in zebrafish are extremely similar, suggesting that they have only evolved fairly recently through gene duplication. It remains unclear why laboratory zebrafish have so many almost identical NLRs, or if wild zebrafish also have lots of these genes. To find out more, Schäfer et al. sequenced the DNA of NLRs from almost 100 zebrafish from multiple wild and laboratory populations. The approach identified over 1,500 different NLR genes, most of which, were previously unknown. Computational modelling suggested that each wild population of zebrafish may harbour up to around 2,000 NLR genes, but laboratory strains had much fewer NLRs. The numbers of NLR genes in individual zebrafish varied greatly – only 4% of the genes were present in 80% or more of the fish. Many genes were only found in specific populations or single individuals. Together, these findings suggest that the NLR family has expanded in zebrafish as part of an ongoing evolutionary process that benefits the immune system of the fish. Similar trends have also been observed in the NLR genes of plants, indicating there may be an evolutionary strategy across all living things to continuously diversify large families of genes. Additionally, this work highlights the lack of diversity in the genes of laboratory animals compared with those of their wild relatives, which may impact how results from laboratory studies are used to inform conservation efforts or are interpreted in the context of human health. Introduction The innate immune system of an organism provides the first defense line against pathogens. Immune genes tend to evolve quickly and are often associated with a high degree of genetic variability. Many genes and proteins of the immune system are lineage-specific (limited to specific groups of animals, plants, or other taxa), while others have defense roles in a wide range of species. In particular, proteins containing a large nucleotide-binding domain followed by smaller repeats have an immune function in animals, plants, fungi, and bacteria alike (Ting et al., 2008; Jones and Dangl, 2006; Uehling et al., 2017; Gao et al., 2022). In animals, these repeats are usually leucine-rich repeats (LRRs) and the proteins themselves are classified as NLRs (nucleotide binding domain leucine-rich repeat containing, also known as NOD-like receptors). They have a multitude of functions: some act as pathogen sensors or transcription factors (Almeida-da-Silva et al., 2023), others are components or modulators of inflammasomes, large protein complexes that are assembled within cells as part of the response to biological or chemical danger (Almeida-da-Silva et al., 2023). Plants have their own NLRs that are structurally similar to the ones from animals and also carry out central functions in the immune response (Urbach and Ausubel, 2017; Yue et al., 2012). Their diversity has been extensively characterized in several species, including the thale cress (Arabidopsis thaliana), and vastly different repertoires have been found from different strains or individuals (Van de Weyer et al., 2019b). NLR repertoires can also be referred to as NLRomes, and a species-wide repertoire is called the 'pan-NLRome'. Most knowledge about NLRs in animals comes from studies of humans and rodents, but their NLR repertoires (20–30 genes) are smaller than those of many other species such as the purple sea urchin, the sponge Amphimedon queenslandica, and many fish (Hibino et al., 2006; Yuen et al., 2014; Suurväli et al., 2022). However, even in mice one NLR (Nlrp1) has different copy numbers in different laboratory strains, ranging from 2 to 5 (Lilue et al., 2018). In many fishes, studies have reported NLR repertoires in the range of 10–50 genes (e.g., Rajendran et al., 2012; Li et al., 2016). In others, hundreds of NLRs are present, including in the model species zebrafish (Danio rerio) (Stein et al., 2007; Laing et al., 2008; Tørresen et al., 2018; Adrian-Kalchhauser et al., 2020; Suurväli et al., 2022). The zebrafish reference genome contains nearly 400 NLR genes, two-thirds of which are located on the putative sex chromosome (chromosome 4), in a genomic region associated with extensive haplotypic variation (Howe et al., 2013; Howe et al., 2016; McConnell et al., 2023; Anderson et al., 2012). The majority of fish NLRs represent a fish-specific subtype that was originally labeled NLR-C (Laing et al., 2008), although they can be further divided into at least six groups based on structural similarities and sequence of conserved exons (Howe et al., 2016; Adrian-Kalchhauser et al., 2020). A schematic structure of proteins encoded by zebrafish NLR-C genes is presented in Figure 1A. All of them possess a FISNA domain (fish-specific NACHT-associated domain), which precedes the nucleotide-binding domain NACHT and is encoded by the same large exon near the N-terminus of the protein (Howe et al., 2016). FISNA-NACHT is in some cases preceded by the effector domain PYD, but this is encoded by a separate exon (Howe et al., 2016). Additionally, many NLR-C proteins have a B30.2 domain (also known as PRY/SPRY) at the C-terminal end, separated from FISNA-NACHT by multiple introns and exons containing the LRRs (Figure 1A; Howe et al., 2016). The B30.2 domain functions through protein–protein interaction (Woo et al., 2006) and is also found in a variety of other genes such as the large family of TRIM ubiquitin ligases (van der Aa et al., 2009; Howe et al., 2016; Suurväli et al., 2022) that are often also involved in immunity. Figure 1 Download asset Open asset Structure of zebrafish NLRs and a map showing the origin of wild zebrafish samples. (A) Generalized, schematic representation of the domain architecture of an NLR-C protein. Each box represents a translated exon. The N-terminal repeats, the death-fold domain, as well as the B30.2 domain only occur in subsets of NLR-C genes. The number of N-terminal repeats and leucine-rich repeats can vary. Domains that can be either present or absent in different NLRs are surrounded by square brackets. (B) Sampling sites for wild zebrafish. All sites are located near the Bay of Bengal. Final sequenced sample sizes are indicated in parentheses. The map is based on geographic data collected and published by AQUASTAT from the Food and Agriculture Organization of the United Nations (FAO, 2021). The population DP is marked with an asterisk because its analysis and results are presented only in figure supplements. It is not known why fishes possess so many NLRs, how they evolve, and how much within-species genetic variability they have. The previously observed repeated expansions and contractions of this family suggest it to have a high rate of gene birth and death (Suurväli et al., 2022). Studies have shown that viral and bacterial infections can induce the expression of specific fish NLRs (reviewed in Chuphal et al., 2022). Some of these have PYD or CARD domains and can even form inflammasomes similar to mammalian NLRs (Kuri et al., 2017; Li et al., 2018b). A species-wide inventory of major NLR exons in a model species such as zebrafish would provide valuable insights into the evolution and diversity of this large immune gene family. Results By adapting and modifying a protocol that combines bait-based exon capture with PacBio SMRT technology (Witek et al., 2016), we successfully generated circular consensus sequence (CCS) data for targeted parts of the immune repertoire from 93 zebrafish (of initial 96), representing four wild populations (Figure 1B) and two laboratory strains. With this approach, we aimed to sequence all exons in zebrafish that encode the nucleotide-binding FISNA-NACHT domains and all exons that encode B30.2 domains. Samples of one wild population (DP) suffered from poor sequence coverage and had to be excluded from downstream analyses in order to avoid bias in interpretation. Results involving this population are only shown in figure supplements and not in the main figures. Our protocol used PCR with primers targeting ligated adapters to amplify the below-nanogram amounts of genomic DNA obtained from exon capture. This limited our fragment sizes to the lengths of what the polymerase was able to amplify. Zebrafish NLRs can have their exons spread out across tens of kilobases, so that we cannot know which exons belong to the same gene. However, we were able to use captured sequence surrounding the targeted exons to distinguish among near-identical coding sequences and separate NLR-associated B30.2 domains from B30.2 elsewhere in the genome. The zebrafish pan-NLRome We used an orthology clustering approach on NLR sequences assembled from all populations to create a reference set of NLRs (a pan-NLRome). This resulted in the identification of 1513 unique FISNA-NACHT containing sequences and 567 for NLR-associated B30.2 (NLR-B30.2). Nearly 10% of the sequences (145 FISNA-NACHT and 64 NLR-B30.2) contained pre-mature stop codons that were at least 10 amino acids from the end and led to early truncation of the protein. In total, 101 of the 1513 FISNA-NACHT were preceded by an exon containing the N-terminal effector domain PYD. Nearly all of those (97 out of 101) were found in group 1 NLR-C genes identified by the presence of the characteristic sequence motif GIAGVGKT (Howe et al., 2016). Since the combination of FISNA and NACHT is only present in NLR-C, its count of 1513 can be considered equal to the total number of NLR-C genes in the data. We found each individual zebrafish to have 100–550 NLR genes from the pan-NLRome in at least one copy (Figures 2 and 3), and only 50–75% of these have a high-quality match in the GRCz11 reference genome (Figure 2—figure supplement 2). In general, laboratory zebrafish had less NLRs than wild samples (Figure 2). The number and length of CCS reads and assembled contigs (both prior to orthology clustering) are presented in Figure 2—figure supplement 1. Figure 2 with 3 supplements see all Download asset Open asset Total counts of NLRs found per individual, shown for each population. Black diamonds on the box plots denote means, horizontal lines denote medians. Left side: two laboratory strains; right side: three wild populations. Figure 2—source data 1 Source tables for Figure 2 and its supplements. https://cdn.elifesciences.org/articles/98058/elife-98058-fig2-data1-v2.xlsx Download elife-98058-fig2-data1-v2.xlsx Figure 2—source data 2 Sequences and target locations of RNA baits. https://cdn.elifesciences.org/articles/98058/elife-98058-fig2-data2-v2.xlsx Download elife-98058-fig2-data2-v2.xlsx Figure 3 with 2 supplements see all Download asset Open asset Copy number variation of NLR genes. (A) Sequence data from each individual zebrafish (vertical axis) was aligned to FISNA-NACHT exon sequences of the pan-NLRome (horizontal axis). Grayscale intensity shows, for each NLR, the proportion of NLR-aligning data in each given fish that matches this specific gene. Darker gray indicates a higher likelihood of this NLR being represented in multiple copies in the particular individual. Light gray indicates a single copy, white indicates absence. For clarity, only the 1235 FISNA-NACHT exons for which at least one fish had a minimum of 10 reads mapped to it are shown. (B) Numbers of pan-NLRome sequences (based on FISNACHT diagnosis) found in all three, two, or only one wild population. (C) Relative numbers of fish in which pan-NLRome sequences were found in wild populations. 'Core' pan-NLRome: genes which are found in at least 80% of the sample (from a total of 57 wild fish); 'shell': genes in at least 20%; 'cloud': rare genes found in less than 20% of the sample. (D) Observed and estimated sizes of population-specific pan-NLRomes. Data points (filled circles and squares) show the average number of totally discovered NLR genes (as identified via their FISNA-NACHT domain) when investigating x fish. The dashed line is obtained by non-linear fit of the data to the function given in Equation 2. For all populations, the hypothetical pan-NLRome size – when extrapolating x→∞ – is finite (see Table 1). Figure 3—source data 1 Source tables for Figure 3 and its supplements. https://cdn.elifesciences.org/articles/98058/elife-98058-fig3-data1-v2.xlsx Download elife-98058-fig3-data1-v2.xlsx Whereas FISNA-NACHT is only found in NLRs, B30.2 domains are also found in other gene families. In addition to the 567 NLR-B30.2 domains, we also found 732 B30.2 domains not associated with NLRs. We were able to distinguish between them by utilizing the sequence of a short highly conserved 47 bp exon that appears to precede B30.2 in NLRs, but not in other genes (Figure 2—figure supplement 3). Each individual zebrafish possesses 20–180 NLR-B30.2s n at least one copy (Figure 3—figure supplement 1). Copy number variation in the pan-NLRome Aligning CCS reads to the pan-NLRome revealed a considerable amount of variability in the proportion of reads mapping to them, both between and within populations (Figure 3A). This can be interpreted as the gene being present in different copy numbers. Furthermore, each NLR had its own distinct pattern of copy number variation, although generally the highest copy numbers were observed for the wild populations KG, SN, and CHT (Figure 3A). We also observed some sequencing batch-related differences, but the copy numbers differed even between individuals sequenced in the same batch. Of the 1513 unique FISNA-NACHT and 567 NLR-B30.2 sequences, 880 FISNA-NACHT and 346 NLR-B30.2 (59 and 57%, respectively) were detected in at least one individual from all wild populations (Figure 3B, Figure 3—figure supplement 1). There were also NLR sequences shared between just two wild populations, and some were restricted to a single population (Figure 3B). Moreover, we observed a lot of variability in the distributions of gene copies among fish within populations (Figure 3C). Only around 4% of the genes in the pan-NLRome were found in 80%, or more, of the wild fish. They constitute the core NLRome (Van de Weyer et al., 2019a). Most genes (51%) were found in the so-called shell of the pan-NLRome (20–80% of fish). Almost as many (45%) are found in a few fish (less than 20% of the sample) only. Although 60% of NLR genes occur in all wild populations, only 4% are omnipresent, that is, are in the core pan-NLRome. Thus, there is considerable variation in the NLR repertoires of individuals from the same population. The total number of NLRs identified in a number x of individual fish can be fitted to a harmonic function (Medini et al., 2020). Using this function (see 'Materials and methods'), we estimated the sizes of the NLRomes of the populations (Figure 3D) and found a total of 520 and 570 NLRs in the laboratory strains TU and CGN, respectively (Table 1). For the wild populations, we estimated four times as many: 2283 in KG, ,896 in SN, and 2452 in CHT. Table 1 Values of fitted parameters and saturation limits for FISNA-NACHT and NLR-B30.2 exons, by population. PopulationFISNA-NACHTNLR-B30.2-αβLimitQuantile*αβLimitQuantile*TU178.2741.43356519.54811853.85791.40774164.73164CGN257.2071.62786569.3672378.71561.61283177.24625DP309.141.01231252842930†69.36090.87454∞naKG436.7611.21522288.412060145.7151.14181113.236.41e6SN479.8921.260932152.123907145.5481.101831514.353.75e9CHT416.7121.188932451.811.12e5135.6771.119111218.541.41e8 * Sample size required to capture 90% of the population's pan-NLRome. † DP required sample size refers to only 10% (instead of 90%) of its hypothetical pan-NLRome size. Differences from the reference genome NLRs sequenced in this study were often different from those present in the reference genome GRCz11. Even NLRs sequenced from the strain that the reference genome itself is based on (TU) did not always align well to it. When the exon itself did align, the intronic sequences surrounding it could often be very different from the reference. In only around of NLRs in TU fish aligned to the reference genome GRCz11 with high mapping (Figure 2—figure supplement This number even elsewhere – from of NLRs in which aligned well to the to only around for the wild populations. The majority of NLRs that did not map well had a very poor mapping of 1 (Figure 2—figure supplement Moreover, there were FISNA-NACHT and 10 NLR-associated B30.2 in the pan-NLRome which did not map in the reference genome. on We used the pan-NLRome as a reference for in the data. NLR sequence diversity was with a large of exons not having in of the populations. were present, nucleotide diversity was up to and up to (Figure and In laboratory strains, genetic variability of FISNA-NACHT that of but such pattern was observed for wild populations. B30.2 exons of laboratory strains were also less than B30.2 from wild zebrafish (Figure The proportion of exons was much higher among FISNA-NACHT than among B30.2 (Figure The majority of NLR exons had of less than 1 (Figure indicating an of rare Figure with 1 supplement see all Download asset Open asset variation in NLR nucleotide diversity and of the rate for FISNA-NACHT (A) and NLR-associated B30.2 (B) (C) of exons single nucleotide (D) of Only exons with at least one are shown. The horizontal line a of the and population size. The diamonds on box plots denote means, horizontal lines denote medians. Figure data 1 Source tables for Figure and its Download Discussion We sequenced and assembled the FISNA-NACHT and B30.2 exons of hundreds of NLRs from 93 zebrafish. We were able to capture the diversity of this gene family in three wild populations and two laboratory strains, and coverage NLR data for an wild population the zebrafish from populations other than we found evidence that each genome from a wild individual contains only a of more than identified NLR copies. The number of NLRs found per individual, each with one or more copies, from around Some of the counts were likely to sequencing in specific samples. Since all samples from population DP suffered from their analysis is only shown in figure supplements. targeted sequencing based on between and NLR exons may have been in our This B30.2 exons even more than FISNA-NACHT exons because they are much However, the observed in NLR gene copies per sequenced individual the first few individuals indicates that not many NLRs were The sizes of NLR repertoires between zebrafish individuals in the three wild populations. of NLR counts to Equation 2 suggested that the populations all possess with NLRs in the laboratory strains and around NLRs in the wild populations. The total numbers of NLRs with a B30.2 exon are about in the laboratory strains and between and for the wild populations (Table 1). To the NLRome of wild populations, large samples are based on the we that 90% of the NLRome may up to several hundred thousand fish (Table 1). clustering with the data from DP resulted in 47 FISNA-NACHT exons which did not occur in other population. Our results suggest that the pan-NLRome of the species be vastly than what we have been able to with our limited sample sizes from a limited number of populations. populations – for example, in or the et al., – likely harbor many more NLRs which are not present in the populations we Although a few zebrafish are in addition to the reference for the from the of of those provide a for mapping and NLRs on their of the is the that the majority of NLR genes are located on the to of chromosome which of structural variation et al., 2023). Furthermore, large gene families are to mapping and of copies in a particular genome is not downstream analysis which on and if not sequencing technology and the in studies et al., 2020; and 2020; et al., have to the data in which are from a genome the of a inventory of structural variation of a species up remains to be as shown for the zebrafish NLRs, the availability of a single high-quality reference genome is not to to the diversity of large gene families. of the zebrafish NLRome We have previously a in variation in zebrafish laboratory strains compared to wild populations (Suurväli et al., 2020). we that the copy numbers of the NLRome and their variation are also The most for this is the population which the of laboratory strains. The in copy number variation in the major in a population of was to a as well et al., Additionally, the amount of in a laboratory could to a of genes. For these one has to when from studies on laboratory zebrafish to wild zebrafish. The same also be when results from laboratory to other species, including Studies have shown that even have hundreds of genes with functions that are by copy number variation, even it genes et al., 2023; et al., of the is the which varying numbers of gene copies between species of et al., and has copies in mice (Lilue et al., 2018). However, the number of NLRs in zebrafish with variation et al., and high of what has been found in other animals so A can be found in the NLR genes of the thale cress Our number of NLRs in a zebrafish population is on the same as the NLRs found in the thale cress NLRome (Van de Weyer et al., 2019b). Moreover, copy numbers also greatly between and 2020). A total of were identified in of which were as the core NLRome because they were found in a at least 80% of the (Van de Weyer et al., 2019b). In wild zebrafish, we found a set of 880 NLR genes which were detected in at least one individual from three wild populations, but only NLRs were found in the majority than of wild individuals. Although structural similarities of NLRs in and animals are to be the of evolution et al., it appears that the similarities to their evolutionary However, the number of gene copies as well as the variation in copy numbers within populations and in individual gene repertoires are more in zebrafish than in We that as immune genes, many NLR genes are likely shared between populations because they provide a in the defense against pathogens. The NLRs shared among only some of the wild populations and the population-specific NLRs may represent to Additionally, there could be within the so that different individuals have different NLRs with the same In general, the that hundreds of NLR gene copies are in zebrafish, with a signature of purifying that the evolution of these genes is from Although the expression of fish NLRs is often by pathogen (reviewed in Chuphal et al., the function of most zebrafish NLR-C genes remains It is that some of them in the and of inflammasomes et al., et al., et al., but we only found the N-terminal effector domains or that are involved in this function et al., in a small of NLR-C genes. Although we used the counts of FISNA-NACHT to total numbers of NLRs, we also the B30.2 exons of NLR-C genes. In general, NLR-associated B30.2 exons of copy number variation that are similar to those for For example, about half of the B30.2 sequences are found in all wild populations, similar to the set of 880 FISNA-NACHT exon sequences conserved among populations. the copy number There are at least two which could to the extensive copy number variation among zebrafish it could be to a high degree of haplotypic DNA different of genes and gene copies, similar to the zebrafish et al., haplotypic variation on the of chromosome the containing over two-thirds of all NLRs in zebrafish et al., 2023). would the of the core but not the presence of genes that occur only in a single individual. or the evolution of NLR-C genes could be by et al., and gene (Laing et al., can be by or
Source: eLife
Author: Detlef Weigel
Institution: Max Planck Institute for Biology
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
Award: G154905275
OpenAlex ID: W4399886726