Vision-related convergent gene losses reveal SERPINE3’s unknown role in the eye
Henrike Indrischek Juliane Hammer Anja Machate Nikolai Hecker Bogdan Kirilenko Juliana Roscito Stefan Hans Caren Norden Michael Brand Michael Hiller
Specific evolutionary gene loss signatures indicated an eye-related function for the uncharacterized SERPINE3 gene, which was experimentally confirmed by zebrafish gene knockouts, showing how comparative genomics can provide insights into the function of uncharacterized genes.
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eLife
Disorders affecting eyes range from subtle vision impairment to blindness and are among the most prevalent diseases in the human population (Sheffield and Stone, 2011). For example, an estimated 76 million people worldwide suffer from glaucoma (Allison et al., 2020), a disease involving optic nerve damage, and about 1.8 million people are blind due to age-related macular degeneration, a degenerative disease of the central retina affecting retinal pigment epithelium (RPE) and photoreceptor cells (Mitchell et al., 2018; GBD 2019 Blindness and Vision Impairment Collaborators and Vision Loss Expert Group of the Global Burden of Disease Study, 2021).

Extensive research in the past decades identified many protein-coding genes with crucial roles in development and maintenance of different tissues and cell types in the eye as well as numerous genes that are associated with genetic eye disorders (Sheffield and Stone, 2011; Moore et al., 2018). For example, the RetNet database (Daiger et al., 2022) lists 271 genes associated with heritable retinal diseases. Even though the eye probably represents one of the best studied organs, our knowledge of the genes underlying eye diseases and disorders is still incomplete. For example, linkage analysis in patients with cataract, microcornea, microphthalmia, and iris coloboma identified new genomic loci linked to the diseases; however, the disease-causing genes have remained elusive (Wang et al., 2007; Sabir et al., 2010; Abouzeid et al., 2009). Similarly, the Cat-Map database (Shiels et al., 2010) lists several additional cataract-associated loci where the underlying disease-causing gene has not been identified. Furthermore, there are still thousands of genes that have not been experimentally investigated in detail, leaving many genes where potential eye-related functions remain to be discovered. Indeed, systematic knockouts of 4,364 genes in mouse detected ocular phenotypes for 347 genes, with 75% of them not been known as eye-related before (Moore et al., 2018). This indicates that vision-related genes as well as genes associated with genetic eye disorders remain to be identified and characterized.

Interestingly, many genes that are linked to human eye diseases are inactivated (lost) in non-human mammals that naturally exhibit poor vision (Emerling et al., 2017; Sharma and Hiller, 2020). For example, subterranean mammals, such as the blind mole rat, naked mole rat, star-nosed mole, and cape golden mole, exhibit gene-inactivating mutations in genes implicated in cataract, retinitis pigmentosa, color or night blindness, or macular degeneration in human (e.g. ABCA4, BEST1, CRYBA1, EYS, GJA8, GNAT2, PDE6C, ROM1, and SLC24A1) (Sharma and Hiller, 2020; Kim et al., 2011; Fang et al., 2014; Emerling and Springer, 2014; Prudent et al., 2016; Partha et al., 2017; Emerling, 2018). Symptoms that characterize these human eye diseases resemble traits found in these subterranean mammals, such as highly degenerated retinae and lenses and sometimes blindness. Similarly, losses of the short wave sensitive opsin (OPN1SW), which is linked to color blindness in humans, occurred in several mammalian lineages such as cetaceans, bats, sloths, and armadillos that are consequently inferred to have monochromatic vision (Emerling et al., 2017; Meredith et al., 2013; Zhao et al., 2009; Emerling and Springer, 2015; Sadier et al., 2018; Springer et al., 2016). In addition to losses of vision-related genes, subterranean mammals also exhibit widespread sequence and transcription factor binding site divergence in eye-related regulatory elements (Roscito et al., 2018; Langer et al., 2018; Langer and Hiller, 2019). Such mutations can cause mis-expression of target genes in ocular tissues such as the lens (Roscito et al., 2021). Loss and divergence of vision-related genes and regulatory elements in these mammals is likely caused by the lack of natural selection on maintaining functional eyes in a dark environment. Taken together, previous studies established a clear association between naturally occurring poor vision phenotypes and regressive evolution of the genetic machinery required for functional eyes (Emerling et al., 2017; Partha et al., 2017; Roscito et al., 2018; Langer et al., 2018).

Here, we performed a genome-wide screen for genes preferentially lost in independent mammalian lineages with a low visual acuity with the goal of revealing currently uncharacterized genes, where vision-related gene loss patterns would predict vision-related functions. In addition to identifying losses of known vision-related genes, our screen revealed previously unknown losses of several functionally uncharacterized genes, among them SERPINE3, which is lost in 18 mammalian lineages that often do not use vision as the primary sense. We show that SERPINE3 is specifically expressed in eyes of zebrafish and mouse. Furthermore, by knockout of serpine3 in zebrafish, we show that the gene is required for maintenance of a proper retinal lamination and overall eye shape, which confirms the predicted vision-related function. Collectively, our results confirm that SERPINE3 has functions in vertebrate eyes and our discovery-driven study demonstrates how specific evolutionary divergence patterns can reveal novel insights into gene function (Stephan et al., 2022).

Our study combines comparative genomics to predict genes having vision-related functions and experiments in zebrafish to confirm this prediction for a top-ranked candidate, the uncharacterized SERPINE3 gene. By conducting a genome-wide screen for genes that are preferentially lost in mammals with low visual acuity values, we uncovered both known vision-related genes as well as several genes that have no known eye-related function. One of the top-ranked candidates is SERPINE3, which we found to be independently lost at least 18 times in mammalian evolution, preferentially in species that do not use vision as the primary sense. For mouse and zebrafish, we show that the highest SERPINE3 expression is in the eye, which is corroborated by available expression data of other vertebrates. By generating the zebrafish serpine3cbg17 and serpine3cbg18 knockout (KO) lines, we show that inactivation of this gene results in abnormal eye shape and retinal lamination, revealing an eye-related function for serpine3. This is further supported by SERPINE3-linked polymorphisms that are associated with eye-related traits in human.

In zebrafish, serpine3 is expressed in a fraction of Müller glia (MG) cells. MG are the major retinal macroglia and perform numerous functions. By removing waste products and secreting (neuro)trophic substances and signaling molecules, they maintain the blood-retinal barrier and regulate vascularization (Reichenbach and Bringmann, 2013). Most importantly, MGs are essential for the long-term viability of photoreceptors and other neuronal cell types (Bringmann et al., 2009). Our serpine3 KO fish exhibit a disorganization of RPE cells, a phenotype that resembles those previously observed in experiments that perturb MG function. For example, selective ablation of MG in adult mice results in eye defects including aggregation of RPE cells and displacement of pigment granules in the ganglion cell layer (Shen et al., 2012). Interestingly, we also observed that serpine3 KO affects eye shape, which cannot be readily explained by a direct MG-mediated effect. However, MG span the entire retina, connecting the extracellular space of retinal neurons, the vitreous and the capillaries at the apical retina (Reichenbach and Bringmann, 2013; Klimczak et al., 2009). It is therefore possible that a secreted protein, as predicted for serpine3 based on its conserved signal peptide, may also affect other eye subtissues. Finally, in zebrafish, MG are able to regenerate retinal neurons upon injury. However, serpine3 does not seem to be involved in this process as during stress-induced regeneration, it is upregulated in resting MG that do not proliferate (Hoang et al., 2020).

In mammals, the SERPINE3 gene loss pattern and expression profile in several species also support an eye-related function; however, the cell type expression pattern may differ between mammals and zebrafish. While our experiments in zebrafish show serpine3 expression in MG, which is in agreement with Hoang et al., 2020, in mouse and human, SERPINE3 seems to be expressed in RPE (Hoang et al., 2020; Hadziahmetovic et al., 2012; Cowan et al., 2020) indicating that cell type specificity may differ between mammals and fish. As SERPINE3 is likely a secreted extracellular protein, it is possible that it has a similar function in mammals and zebrafish, despite secretion by different cell types. Whether this is the case or whether SERPINE3 function and protein expression pattern in the mammalian eye differs remains to be explored in future studies. At the molecular level, this question may be addressed by investigating whether the molecular targets of SERPINE3 are conserved among vertebrates.

Of particular interest is elucidating the functional role of SERPINE3 in human. Several pieces of evidence indicate a potential role of this gene in anti-inflammatory processes and retinal survival. SERPINE3 is upregulated in human patients with age-related macular degeneration (Newman et al., 2012), a progressive eye disease that is linked to chronic inflammation and wound healing. Consistent with a role in retinal survival, mouse Serpine3 is upregulated after experimental overexpression of neurotrophin-4, a neuroprotective factor that promotes retinal survival (Machalińska et al., 2013). Furthermore, SERPINE3 is a hallmark gene of differentiated, healthy human RPE cells (Radeke et al., 2015; Boles et al., 2020; Sripathi et al., 2021) that also have neurotrophic functions in the retina. It is thus conceivable that perturbation of proper SERPINE3 expression or function may influence age-related diseases or human eye phenotypes, as indicated by human polymorphisms that are linked to SERPINE3.

In addition to SERPINE3, our screen also revealed other candidate genes for which unknown eye-related functions are plausible. The LACTBL1 gene encodes a putative serine proteinase (Liobikas et al., 2006) and has an expression pattern similar to retina phototransduction genes (Uhlen et al., 2010). Furthermore, 20 kb upstream of LACTBL1 is a linked SNP (rs10158878) that is associated with refractive error in human (GWAS catalog, Hysi et al., 2020). Another uncharacterized candidate gene with a strong visual acuity associated loss pattern is SAPCD1. A missense mutation (rs6905572) within SAPCD1 is associated with macular degeneration (dbSNP, Chen et al., 2010). The LRIT2 gene is linked to SNPs (rs12217769 and rs745480) that are associated with macular thickness and refractive error in human. Whereas this gene was not functionally characterized at the time our screen was conducted, a recent study showed that lrit2 knockdown in zebrafish led to a reduction in eye size (Chiang et al., 2020). Finally, a high-throughput screen in mouse found that Pkd1l2 KO leads to abnormalities in lens morphology (Dickinson et al., 2016), but no follow-up experiments exist. A potential role of this gene in human disease is also indicated by an intronic SNP (rs873693), which is associated with age onset of myopia (GWAS catalog, Hysi et al., 2020; Tedja et al., 2018). Thus, in addition to SERPINE3, for which we performed an initial functional characterization in zebrafish, our screen uncovered other promising, uncharacterized candidates that may have an eye-related function. Overall, this highlights the potential of comparative genomics to shed light on the functional roles of less characterized genes and to help to further identify human disease-causing genes (Meadows and Lindblad-Toh, 2017).