Cone Photoreceptors Research Articles

Restoration of cone-circuit functionality in the regenerating adult zebrafish retina

Unlike humans, teleosts like zebrafish exhibit robust retinal regeneration after injury from endogenous stem cells. However, it is unclear if regenerating cone photoreceptors regain physiological function and integrate correctly into post-synaptic circuits. We used two-photon calcium imaging of living adult retina to examine photoreceptor responses before and after light-induced lesions. To assess functional recovery of cones and downstream outer retinal circuits, we exploited color opponency; UV cones exhibit intrinsic Off-response to blue light, but On-response to green light, which depends on feedback signals from outer retinal circuits. Accordingly, we assessed the presence and quality of Off- vs. On-responses and found that regenerated UV cones regain both Off-responses to short-wavelength and On-responses to long-wavelength light within 3months after lesion. Therefore, physiological circuit functionality is restored in regenerated cone photoreceptors, suggesting that inducing endogenous regeneration is a promising strategy for human retinal repair.

Leukemia Inhibitory Factor Protects against Degeneration of Cone Photoreceptors Caused by RPE65 Deficiency.

Retinal pigment epithelium (RPE) 65 is a key enzyme in the visual cycle involved in the regeneration of 11-cis-retinal. Mutations in the human RPE65 gene cause Leber's congenital amaurosis (LCA), a severe form of an inherited retinal disorder. Animal models carrying Rpe65 mutations develop early-onset retinal degeneration. In particular, the cones degenerate faster than the rods. To date, gene therapy has been used successfully to treat RPE65-associated retinal disorders. However, gene therapy does not completely prevent progressive retinal degeneration in patients, possibly due to the vulnerability of cones in these patients. In the present study, we tested whether leukemia inhibitory factor (LIF), a trophic factor, protects cones in rd12 mice harboring a nonsense mutation in Rpe65. LIF was administered to rd12 mice by intravitreal microinjection. Apoptosis of retinal cells was analyzed by TUNEL assay. The degeneration of cone cells was evaluated by immunostaining of retinal sections and retinal flat-mounts. Signaling proteins regulated by LIF in the retinal and cultured cells were determined by immunoblotting. Intravitreal administration of LIF activated the STAT3 signaling pathway, thereby inhibiting photoreceptor apoptosis and preserving cones in rd12 mice. Niclosamide (NCL), an inhibitor of STAT3 signaling, effectively blocked STAT3 signaling and autophagy in cultured 661W cells treated with LIF. Co-administration of LIF with NCL to rd12 mice abolished the protective effect of LIF, suggesting that STAT3 signaling and autophagy mediate the protection. LIF is a potent factor that protects cones in rd12 mice. This finding implies that LIF can be used in combination with gene therapy to achieve better therapeutic outcomes for patients with RPE65-associated LCA.

Structural and functional characterization of the nucleotide-binding domains of ABCA4 and their role in Stargardt disease

ABCA4 is an ATP-binding cassette (ABC) transporter that prevents the buildup of toxic retinoid compounds by facilitating the transport of N-retinylidene-phosphatidylethanolamine across membranes of rod and cone photoreceptor cells. Over 1500 missense mutations in ABCA4, many in the nucleotide binding domains (NBDs), have been genetically linked to Stargardt disease (STGD1). Here, we show by Cryo-electron microscopy that ABCA4 is converted from an open outward conformation to a closed conformation upon the binding of AMP-PNP. Structural information and biochemical studies were used to further define the role of the NBDs in the functional properties of ABCA4 and the mechanisms by which mutations lead to the loss in activity. We show that ATPase activity in both NBDs is required for the functional activity of ABCA4. Mutations in Walker A asparagine residues cause a severe reduction in substrate-activated ATPase activity due to the loss in polar interactions with residues within the D-loops of the opposing NBD. The structural basis for how disease mutations in other NBD residues including the R1108C, R2077W, R2107H and L2027F affect the structure and function of ABCA4 is described. Collectively, our studies provide insight into the structure and function of ABCA4 and mechanisms underlying STGD1.

Exploring autism via the retina: Comparative insights in children with autism spectrum disorder and typical development.

Autism spectrum disorder (ASD) is a widely recognized neurodevelopmental disorder, yet the identification of reliable imaging biomarkers for its early diagnosis remains a challenge. Considering the specific manifestations of ASD in the eyes and the interconnectivity between the brain and the eyes, this study investigates ASD through the lens of retinal analysis. We specifically examined differences in the macular region of the retina using optical coherence tomography (OCT)/optical coherence tomography angiography (OCTA) images between children diagnosed with ASD and those with typical development (TD). Our findings present potential novel characteristics of ASD: the thickness of the ellipsoid zone (EZ) with cone photoreceptors was significantly increased in ASD; the large-caliber arteriovenous of the inner retina was significantly reduced in ASD; these changes in the EZ and arteriovenous were more significant in the left eye than in the right eye. These observations of photoreceptor alterations, vascular function changes, and lateralization phenomena in ASD warrant further investigation, and we hope that this work can advance interdisciplinary understanding of ASD.

Retinoic Acid-Dependent Loss of Synaptic Output from Bipolar Cells Impairs Visual Information Processing in Inherited Retinal Degeneration.

In retinitis pigmentosa (RP), rod and cone photoreceptors degenerate, depriving downstream neurons of light-sensitive input, leading to vision impairment or blindness. Although downstream neurons survive, some undergo morphological and physiological remodeling. Bipolar cells (BCs) link photoreceptors, which sense light, to retinal ganglion cells (RGCs), which send information to the brain. While photoreceptor loss disrupts input synapses to BCs, whether BC output synapses remodel has remained unknown. Here we report that synaptic output from BCs plummets in RP mouse models of both sexes owing to loss of voltage-gated Ca2+ channels. Remodeling reduces the reliability of synaptic output to repeated optogenetic stimuli, causing RGC firing to fail at high-stimulus frequencies. Fortunately, functional remodeling of BCs can be reversed by inhibiting the retinoic acid receptor (RAR). RAR inhibitors targeted to BCs present a new therapeutic opportunity for mitigating detrimental effects of remodeling on signals initiated either by surviving photoreceptors or by vision-restoring tools.

Exonic Short Interspersed Nuclear Element Insertion in FAM161A Is Associated with Autosomal Recessive Progressive Retinal Atrophy in the English Shepherd.

Progressive retinal atrophies (PRAs) are a genetically heterogeneous group of inherited eye diseases that affect over 100 breeds of dog. The initial clinical sign is visual impairment in scotopic conditions, as a consequence of rod photoreceptor cell degeneration. Photopic vision degeneration then follows, due to progression of the disease to the cone photoreceptors, and ultimately results in complete blindness. Two full-sibling English Shepherds were diagnosed with PRA at approximately 5 years old and tested clear of all published PRA genetic variants. This study sought to identify the novel PRA-associated variant segregating in the breed. We utilised a combined approach of whole genome sequencing of the probands and homozygosity mapping of four cases and 22 controls and identified a short interspersed nuclear element within an alternatively spliced exon in FAM161A. The XP_005626197.1 c.17929_ins210 variant was homozygous in six PRA cases and heterozygous or absent in control dogs, consistent with a recessive mode of inheritance. The insertion is predicted to extend exon 4 by 39 aberrant amino acids followed by an early termination stop codon. PRA is intractable to treatment, so the development of a genetic screening test, based on the associated variant, is significant, because it provides dog breeders/owners with a means of reducing the frequency of the disease variant within this breed as well as minimising the risk of breeding puppies that will develop this blinding disease.

Cone photoreceptor differentiation regulated by thyroid hormone transporter MCT8 in the retinal pigment epithelium

The key role of a thyroid hormone receptor in determining the maturation and diversity of cone photoreceptors reflects a profound influence of endocrine signaling on the cells that mediate color vision. However, the route by which hormone reaches cones remains enigmatic as cones reside in the retinal photoreceptor layer, shielded by the blood-retina barrier. Using genetic approaches, we report that cone differentiation is regulated by a membrane transporter for thyroid hormone, MCT8 (SLC16A2), in the retinal pigment epithelium (RPE), which forms the outer blood-retina barrier. Mct8-deficient mice display hypothyroid-like cone gene expression and compromised electroretinogram responses. Mammalian color vision is typically facilitated by cone types that detect medium-long (M) and short (S) wavelengths of light but Mct8-deficient mice have a partial shift of M to S cone identity, resembling the phenotype of thyroid hormone receptor deficiency. RPE-specific ablation of Mct8 results in similar shifts in cone identity and hypothyroid-like gene expression whereas reexpression of MCT8 in the RPE in Mct8-deficient mice partly restores M cone identity, consistent with paracrine-like control of thyroid hormone signaling by the RPE. Our findings suggest that in addition to transport of essential solutes and homeostatic support for photoreceptors, the RPE regulates the thyroid hormone signal that promotes cone-mediated vision.

A combination treatment based on drug repurposing demonstrates mutation-agnostic efficacy in pre-clinical retinopathy models

Inherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6βrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration.

Long-term exposure to prometryn damages the visual system and changes color preference of female zebrafish (Danio rerio)

Color vision, initiated from the cone photoreceptors, is essential for fish to obtain environmental information. Although the visual impairment of triazine herbicide prometryn has been reported, data on the effect of herbicide such as prometryn on natural color sensitivity of fish is scarce. Here, zebrafish were exposed to prometryn (0, 1, 10, and 100 μg/L) from 2 h post-fertilization to 160 days post-fertilization, to explore the effect and underlying mechanism of prometryn on color perception. The results indicated that 10 and 100 μg/L prometryn shortened the height of red-green cone cells, and down-regulated expression of genes involved in light transduction pathways (arr3a, pde6h) and visual cycle (lrata, rpe65a); meanwhile, 1 μg/L prometryn increased all-trans-retinoic acid levels in zebrafish eyes, and up-regulated the expression of genes involved in retinoid metabolism (rdh10b, aldh1a2, cyp26a1), finally leading to weakened red and green color perception of female zebrafish. This study first clarified how herbicide such as prometryn affected color vision of a freshwater fish after a long-term exposure from both morphological and functional disruption, and its hazard on color vision mediated-ecologically relevant tasks should not be ignored.

Modeling Human Macular Cone Photoreceptor Spatial Distribution.

The purpose of this study was to investigate the spatial distribution of human cone photoreceptors and examine cone density differences between the retinal meridians and quadrants. Using adaptive optics scanning laser ophthalmoscopy, the maculae were imaged in 17 eyes of 11 subjects with normal chorioretinal health aged 54 to 72 years. We measured cone density at 325 points within the central 10degrees radius of the retina. Cone density spatial distributions along the primary retinal meridians and in four macular quadrants (superior-nasal, superior-temporal, inferior-temporal, and inferior-nasal) were analytically modeled using the polynomial function to assess the meridional and quadrantal difference. The mean and 95% confidence interval for the prediction of cone density along the primary retinal meridians was modeled with a 7-degree one-variable polynomial (R2 = 0.9761, root mean squared error [RMSE] = 0.0585). In the 4 retinal quadrants, cone density distribution was described by a 2-variable polynomial with X degree 3 and Y degree 4 (R² = 0.9834, RMSE = 0.0377). The models suggest no statistically significant difference between medians and between quadrants. However, cone density difference at corresponding spatial locations in different areas can be up to 25.6%. The superior-nasal region has more areas with high cone density, followed by quadrants of inferior-nasal, inferior-temporal, and superior-temporal. Analytical modeling provides comprehensive knowledge of cone distribution across the entire macula. Although modeling analysis suggests no statistically significant difference between medians and between quadrants, the remarkable cone density discrepancies in certain regions should be accounted for in applications requiring sensitive detection of cone variation.

RDH12 allows cone photoreceptors to regenerate opsin visual pigments from a chromophore precursor to escape competition with rods

Capture of a photon by an opsin visual pigment isomerizes its 11-cis-retinaldehyde (11cRAL) chromophore to all-trans-retinaldehyde (atRAL), which subsequently dissociates. To restore light sensitivity, the unliganded apo-opsin combines with another 11cRAL to make a new visual pigment. Two enzyme pathways supply chromophore to photoreceptors. The canonical visual cycle in retinal pigment epithelial cells supplies 11cRAL at low rates. The photic visual cycle in Müller cells supplies cones with 11-cis-retinol (11cROL) chromophore precursor at high rates. Although rods can only use 11cRAL to regenerate rhodopsin, cones can use 11cRAL or 11cROL to regenerate cone visual pigments. We performed a screen in zebrafish retinas and identified ZCRDH as a candidate for the enzyme that converts 11cROL to 11cRAL in cone inner segments. Retinoid analysis of eyes from Zcrdh-mutant zebrafish showed reduced 11cRAL and increased 11cROL levels, suggesting impaired conversion of 11cROL to 11cRAL. By microspectrophotometry, isolated Zcrdh-mutant cones lost the capacity to regenerate visual pigments from 11cROL. ZCRDH therefore possesses all predicted properties of the cone 11cROL dehydrogenase. The human protein most similar to ZCRDH is RDH12. By immunocytochemistry, ZCRDH was abundantly present in cone inner segments, similar to the reported distribution of RDH12. Finally, RDH12 was the only mammalian candidate protein to exhibit 11cROL-oxidase catalytic activity. These observations suggest that RDH12 in mammals is the functional ortholog of ZCRDH, which allows cones, but not rods, to regenerate visual pigments from 11cROL provided by Müller cells. This capacity permits cones to escape competition from rods for visual chromophore in daylight-exposed retinas.

Transcriptomic Analysis Reveals That Excessive Thyroid Hormone Signaling Impairs Phototransduction and Mitochondrial Bioenergetics and Induces Cellular Stress in Mouse Cone Photoreceptors.

Thyroid hormone (TH) plays an essential role in cell proliferation, differentiation, and metabolism. Experimental and clinical studies have shown a potential association between TH signaling and retinal degeneration. The suppression of TH signaling protects cone photoreceptors in mouse models of retinal degeneration, whereas excessive TH signaling induces cone degeneration, manifested as reduced light response and a loss of cones. This work investigates the genes/transcriptomic alterations that might be involved in TH-induced cone degeneration in mice using single-cell RNA sequencing (scRNAseq) analysis. One-month-old C57BL/6 mice received triiodothyronine (T3, 20 µg/mL in drinking water) for 4 weeks as a model of hyperthyroidism/excessive TH signaling. At the end of the experiments, retinal cells were dissociated, and cell viability was analyzed before being subjected to scRNAseq. The resulting data were analyzed using the Seurat package and visualized using the Loupe browser. Among 155,866 single cells, we identified 14 cell clusters, representing various retinal cell types, with rod and cone clusters comprising 76% and 4.1% of the total cell population, respectively. Cone cluster transcriptomes demonstrated the most alterations after the T3 treatment, with 450 differentially expressed genes (DEGs), accounting for 38.5% of the total DEGs. Statistically significant changes in the expression of genes in the cone cluster revealed that phototransduction and oxidative phosphorylation were impaired after the T3 treatment, along with mitochondrial dysfunction. A pathway analysis also showed the activation of the sensory neuronal/photoreceptor stress pathways after the T3 treatment. Specifically, the eukaryotic initiation factor-2 signaling pathway and the cAMP response element-binding protein signaling pathway were upregulated. Thus, excessive TH signaling substantially affects cones at the transcriptomic level. The findings from this work provide an insight into how excessive TH signaling induces cone degeneration.

FMRI and gene therapy in adults with CNGB3 mutation

Achromatopsia is an inherited retinal disease that affects 1 in 30,000–50,000 individuals and is characterised by an absence of functioning cone photoreceptors from birth. This results in severely reduced visual acuity, no colour vision, marked sensitivity to light and involuntary oscillations of the eyes (nystagmus). In most cases, a single gene mutation prevents normal development of cone photoreceptors, with mutations in the CNGB3 or CNGA3 gene being responsible for ∼80 % of all patients with achromatopsia. There are a growing number of studies investigating recovery of cone function after targeted gene therapy. These studies have provided some promise for patients with the CNGA3 mutation, but thus far have found limited or no recovery for patients with the CNGB3 mutation. Here, we developed colour-calibrated visual stimuli designed to isolate cone photoreceptor responses. We combined these with adapted fMRI techniques and pRF mapping to identify if cortical responses to cone-driven signals could be detected in 9 adult patients with the CNGB3 mutation after receiving gene therapy. We did not detect any change in brain activity after gene therapy when the 9 patients were analysed as a group. However, on an individual basis, one patient self-reported a change in colour perception, corroborated by improved performance on a psychophysical task designed to selectively identify cone function. This suggests a level of cone sensitivity that was lacking pre-treatment, further supported by a subtle but reliable change in cortical activity within their primary visual cortex.

The First Steps of the Visual Cycle in Human Rod and Cone Photoreceptors.

Light detection destroys the visual pigment. Its regeneration, necessary for the recovery of light sensitivity, is accomplished through the visual cycle. Release of all-trans retinal by the light-activated visual pigment and its reduction to all-trans retinol comprise the first steps of the visual cycle. In this study, we determined the kinetics of all-trans retinol formation in human rod and cone photoreceptors. Single living rod and cone photoreceptors were isolated from the retinas of human cadaver eyes (ages 21 to 90 years). Formation of all-trans retinol was measured by imaging its outer segment fluorescence (excitation, 360nm; emission, >420nm). The extent of conversion of released all-trans retinal to all-trans retinol was determined by measuring the fluorescence excited by 340 and 380nm. Measurements were repeated with photoreceptors isolated from Macaca fascicularis retinas. Experiments were carried out at 37°C. We found that ∼80% to 90% of all-trans retinal released by the light-activated pigment is converted to all-trans retinol, with a rate constant of 0.24 to 0.55 min-1 in human rods and ∼1.8 min-1 in human cones. In M. fascicularis rods and cones, the rate constants were 0.38 ± 0.08 min-1 and 4.0 ± 1.1 min-1, respectively. These kinetics are several times faster than those measured in other vertebrates. Interphotoreceptor retinoid-binding protein facilitated the removal of all-trans retinol from human rods. The first steps of the visual cycle in human photoreceptors are several times faster than in other vertebrates and in line with the rapid recovery of light sensitivity exhibited by the human visual system.

Male germ cell-associated kinase is required for axoneme formation during ciliogenesis in zebrafish photoreceptors.

Vertebrate photoreceptors are highly specialized retinal neurons that have cilium-derived membrane organelles called outer segments, which function as platforms for phototransduction. Male germ cell-associated kinase (MAK) is a cilium-associated serine/threonine kinase, and its genetic mutation causes photoreceptor degeneration in mice and retinitis pigmentosa in humans. However, the role of MAK in photoreceptors is not fully understood. Here, we report that zebrafish mak mutants show rapid photoreceptor degeneration during embryonic development. In mak mutants, both cone and rod photoreceptors completely lacked outer segments and underwent apoptosis. Interestingly, zebrafish mak mutants failed to generate axonemes during photoreceptor ciliogenesis, whereas basal bodies were specified. These data suggest that Mak contributes to axoneme development in zebrafish, in contrast to mouse Mak mutants, which have elongated photoreceptor axonemes. Furthermore, the kinase activity of Mak was found to be critical in ciliary axoneme development and photoreceptor survival. Thus, Mak is required for ciliogenesis and outer segment formation in zebrafish photoreceptors to ensure intracellular protein transport and photoreceptor survival.

HIGH-RESOLUTION IMAGING OF THE OUTER RETINA IN TYPE 2 ACUTE MACULAR NEURORETINOPATHY.

The purpose of this study was to investigate the outer retinal changes in a patient with type 2 acute macular neuroretinopathy (AMN). A 35-year-old White woman complaining of a unilateral blind spot was imaged using various retinal imaging modalities including clinical optical coherence tomography (OCT), OCT-angiography, fundus fluorescein angiography, and adaptive optics (AO). Fundus examination revealed multiple paracentral reddish brown petaloid lesions in the symptomatic left eye, while the other eye was unremarkable. Clinical OCT showed areas of hyperreflectance at the outer plexiform layer/outer nuclear layer complex with a disrupted inner/outer segment junction, which are characteristic features of type 2 AMN. AO imaging further revealed either shortening or absence of cone outer segments within the AMN lesions attributing to the darker features observed in the en face images from fundus photography and scanning laser ophthalmoscopy. The AO findings indicate that the petaloid lesions in type 2 AMN are caused by a combination of the shortening and absence of the outer segment in individual cone photoreceptors.

Glutamate transporters are involved in direct inhibitory synaptic transmission in the vertebrate retina.

In the central nervous system of vertebrates, glutamate serves as the primary excitatory neurotransmitter. However, in the retina, glutamate released from photoreceptors causes hyperpolarization in post-synaptic ON-bipolar cells through a glutamate-gated chloride current, which seems paradoxical. Our research reveals that this current is modulated by two excitatory glutamate transporters, EAAT5b and EAAT7. In the zebrafish retina, these transporters are located at the dendritic tips of ON-bipolar cells and interact with all four types of cone photoreceptors. The absence of these transporters leads to a decrease in ON-bipolar cell responses, with eaat5b mutants being less severely affected than eaat5b/eaat7 double mutants, which also exhibit altered response kinetics. Biophysical investigations establish that EAAT7 is an active glutamate transporter with a predominant anion conductance. Our study is the first to demonstrate the direct involvement of post-synaptic glutamate transporters in inhibitory direct synaptic transmission at a central nervous system synapse.

Insights into the evolution of photoreceptor oil droplets in frogs and toads.

Photoreceptor oil droplets (ODs) are spherical organelles placed most commonly within the inner segment of the cone photoreceptors. Comprising neutral lipids, ODs can be either non-pigmented or pigmented and have been considered optically functional in various studies. Among living amphibians, ODs were only reported to occur in frogs and toads (Anura), while they are absent in salamanders and caecilians. Nonetheless, the limited understanding of their taxonomic distribution in anurans impedes a comprehensive assessment of their evolution and relationship with visual ecology. We studied the retinae of 134 anuran species, extending the knowledge of the distribution of ODs to 46 of the 58 currently recognized families, and providing a new perspective on this group that complements the available information from other vertebrates. The occurrence of ODs in anurans shows a strong phylogenetic signal, and our findings revealed that ODs evolved at least six times during the evolutionary history of the group, independently from other vertebrates. Although no evident correlation was found between OD occurrence, adult habits and diel activity, it is inferred that each independent origin involves distinct scenarios in the evolution of ODs concerning photic habits. Furthermore, our results revealed significant differences in the size of the ODs between nocturnal and arrhythmic anurans relative to the length of the cones' outer segment.

High temporal frequency light response in mouse retina requires FAT3 signaling in bipolar cells.

Vision is initiated by the reception of light by photoreceptors and subsequent processing via downstream retinal neurons. Proper cellular organization depends on the multi-functional tissue polarity protein FAT3, which is required for amacrine cell connectivity and retinal lamination. Here we investigated the retinal function of Fat3 mutant mice and found decreases in physiological and perceptual responses to high frequency flashes. These defects did not correlate with abnormal amacrine cell wiring, pointing instead to a role in bipolar cell subtypes that also express FAT3. The role of FAT3 in the response to high temporal frequency flashes depends upon its ability to transduce an intracellular signal. Mechanistically, FAT3 binds to the synaptic protein PTPσ, intracellularly, and is required to localize GRIK1 to OFF-cone bipolar cell synapses with cone photoreceptors. These findings expand the repertoire of FAT3's functions and reveal its importance in bipolar cells for high frequency light response.

Limited Parallelism in Genetic Adaptation to Brackish Water Bodies in European Sprat and Atlantic Herring.

The European sprat is a small plankton-feeding clupeid present in the northeastern Atlantic Ocean, in the Mediterranean Sea, and in the brackish Baltic Sea and Black Sea. This species is the target of a major fishery and, therefore, an accurate characterization of its genetic population structure is crucial to delineate proper stock assessments that aid ensuring the fishery's sustainability. Here, we present (i) a draft genome assembly, (ii) pooled whole genome sequencing of 19 population samples covering most of the species' distribution range, and (iii) the design and test of a single nucleotide polymorphism (SNP)-chip resource and use this to validate the population structure inferred from pooled sequencing. These approaches revealed, using the populations sampled here, three major groups of European sprat: Oceanic, Coastal, and Brackish with limited differentiation within groups even over wide geographical stretches. Genetic structure is largely driven by six large putative inversions that differentiate Oceanic and Brackish sprats, while Coastal populations display intermediate frequencies of haplotypes at each locus. Interestingly, populations from the Baltic and the Black Seas share similar frequencies of haplotypes at these putative inversions despite their distant geographic location. The closely related clupeids European sprat and Atlantic herring both show genetic adaptation to the brackish Baltic Sea, providing an opportunity to explore the extent of genetic parallelism. This analysis revealed limited parallelism because out of 125 independent loci detected in the Atlantic herring, three showed sharp signals of selection that overlapped between the two species and contained single genes such as PRLRA, which encodes the receptor for prolactin, a freshwater-adapting hormone in euryhaline species, and THRB, a receptor for thyroid hormones, important both for metabolic regulation and the development of red cone photoreceptors.