Light intensity and opsin sensitivity shape the morphology of cone photoreceptor outer segments

Jingjin Xu; Zihan Chang; Wei Deng; Luwei Qian; Honggang Su; Xun Huang; Yunsi Kang; Haibo Xie; Chengtian Zhao

doi:10.1371/journal.pbio.3003654

. 2026 Feb 18;24(2):e3003654. doi: 10.1371/journal.pbio.3003654

Light intensity and opsin sensitivity shape the morphology of cone photoreceptor outer segments

Jingjin Xu ^1,^2,³, Zihan Chang ^1,³, Wei Deng ^1,³, Luwei Qian ^1,³, Honggang Su ⁴, Xun Huang ^4,⁵, Yunsi Kang ^1,³, Haibo Xie ^1,³, Chengtian Zhao ^1,^2,^3,^*

Editor: Tom Baden⁶

PMCID: PMC12915902 PMID: 41706763

Abstract

Regulation of neural cell morphology remains a fundamental question in neuroscience. Photoreceptor cells, a specialized class of neurons capable of initiating the phototransduction cascade, exhibit distinct structural and morphological characteristics. While the structural and morphological differences between rod and cone photoreceptors have been extensively studied, the variability in the morphology of cone outer segments (OS) remains largely unexplored. Zebrafish possess four distinct cone types, each displaying unique OS morphologies. By modulating opsin expression across cone types, we reveal that the morphology of the cone OS correlates directly with the wavelength sensitivity of the expressed opsins, with cones expressing longer wavelength-sensitive opsins exhibiting elongated OS. This regulatory mechanism is conserved across various vertebrates. Furthermore, we show that alterations in light intensity—induced by ectopic lipid droplet formation in the light path or by changing the environment light intensity—can also modulate OS morphology. Notably, this morphological plasticity is not transient, but rather dependent on long-term neural activity. Based on these findings, we propose a model for the regulation of cone OS length. Our data suggest that both opsin sensitivity and light intensity shape cone OS morphology through long-term neural activity, providing critical insights into neural plasticity in these light-sensitive neurons.

Photoreceptor cells exhibit variability in the morphology of the light-absorbing outer segment, the basis of which is unclear. This study in zebrafish shows that the wavelength sensitivity of the expressed opsin and the intensity of light exposure determine outer segment morphology, with implications for our understanding of neuronal plasticity.

Introduction

Neuronal plasticity, the ability of neuronal cells to alter their structure and shape, plays a fundamental role in brain development, learning, and memory. For example, neurons can remodel their dendrites or axons in response to environmental stimuli or learning experiences. However, the mechanisms regulating these processes remain a central mystery in neurobiology [1–3]. Among specialized sensory neurons, photoreceptors are crucial for vision, converting light into electrical signals that serve as the foundation of visual perception. The OS of photoreceptors is critical for light detection as it contains photopigments that absorb light and initiate the visual process [4,5]. Based on OS morphology, photoreceptors are classified into two types: rods and cones. Rods enable vision in low-light conditions, while cones are responsible for color vision and high visual acuity in bright light. Despite their critical roles, the mechanisms underlying the formation and maintenance of such distinct OS morphology in rods and cones remain poorly understood.

Opsin proteins are the most abundant components of the OS in photoreceptor cells, where they bind to the chromophore retinal to initiate phototransduction. Notably, the OS is a dynamic structure that undergoes continuous and periodic renewal to sustain photoreceptor function [6,7]. Since the OS lacks the machinery for protein synthesis, its components must be synthesized in the cell body and transported to the OS through the connecting cilium—a narrow channel linking the inner and outer segments (OS). The OS can be considered a specialized form of cilium, as it shares several key features with ciliary structures in other cell types [8,9]. The transport of OS components is tightly regulated, and defects in this process are implicated in retinal diseases such as retinitis pigmentosa (RP) [10].

Downstream of opsin activation, rods and cones—despite their morphological differences—share a common phototransduction pathway. This involves the activation of transducin, cGMP phosphodiesterase, and the subsequent closure of cGMP-gated ion channels [4,11]. While rods often employ distinct homologous proteins for phototransduction, cones, irrespective of their subtype, largely utilize the same phototransduction components downstream of opsin activation [12]. The structural and morphological differences between rods and cones have been well-documented across various species [13]. However, the morphological diversity among the OS structures of cone subtypes and the regulatory mechanisms driving these differences remain largely unexplored.

Cone photoreceptors are indispensable for human vision, enabling us to perceive color and adapt to varying light conditions. Humans possess three cone types: S-cones (sensitive to short wavelengths, or blue light), M-cones (sensitive to medium wavelengths, or green light), and L-cones (sensitive to long wavelengths, or red light). In contrast, mice—being nocturnal—have a visual system optimized for low-light conditions and possess only two cone types: S-cones (sensitive to ultraviolet light, UV) and M-cones (green). These cones constitute only about 3% of the total photoreceptors, and their even distribution across the retina makes studying cone function in mice more challenging than studying rods [14]. By comparison, the zebrafish retina is predominantly cone-based, containing approximately 92% cones at the larval stage and about 60% in adults—closely resembling the cone-rich human macula [15–18]. Zebrafish possess four types of cone photoreceptors for color vision, which includes short-wavelength-sensitive (blue) cones, mid-wavelength-sensitive (green) cones, long-wavelength-sensitive (red) cones, and ultraviolet-sensitive (UV) cones [15,19]. These cone types form a highly ordered mosaic in the adult zebrafish retina, with UV and blue cones alternating in rows, and green and red cones alternating in neighboring rows [20,21]. Although zebrafish and human cone subtypes are not directly evolutionarily equivalent—for example, human “green” and “red” cones both express LWS opsins and correspond to zebrafish red cones, whereas human “blue” cones (SWS1) are orthologous to zebrafish UV cones [22,23]—zebrafish nevertheless provide an excellent model for investigating cone photoreceptor morphology and development.

In this study, we used zebrafish as a model organism to explore the mechanisms that govern cone OS morphology. We show that there are significant length differences in the OS among various cone subtypes. By switching the expression of cone opsins between different photoreceptor subtypes, we demonstrate that cone OS morphology is directly regulated by the photosensitivity of the opsin protein. Moreover, changing the light intensity reaching the OS either genetically or environmentally can also modify the morphology of the cone OS. This regulatory mechanism appears to be conserved across multiple species. Our data suggest that cone OS morphology is finely tuned to adapt to light conditions, which underscores the dynamic interplay between structure and function in sensory systems.

Results

Variation in the morphology of cone OS in zebrafish

Similar to humans, the diurnal zebrafish primarily relies on cone photoreceptors for daytime vision. The zebrafish retina contains five types of photoreceptors: one type of rod and four types of cone photoreceptors (Fig 1A). Using wheat germ agglutinin (WGA) staining, we were able to distinguish the OS of the different photoreceptor types, which are organized into distinct layers (Fig 1B). In the apical region near the retinal pigment epithelium (RPE), the rod outer segment (ROS) layer is located at the topmost position, followed by the double- and blue-cone OS layers, with the UV cone OS layer positioned at the most basal location (Fig 1B and 1C).

To further investigate the morphology of the cone OS, we utilized two transgenic zebrafish lines, Tg(sws1:sws1-GFP) and Tg(sws2:HA-tdTomato-CT44), to label the OS of UV and blue cones (Fig 1D and 1E). The OS of double cones (red and green cones) were identified using a combination of WGA and Zpr3 antibody staining. The Zpr3 antibody labels not only rod OS but also the OS of green and red cones [24], whereas WGA selectively labels red cone OS with strong fluorescence (Fig 1F). Measurement of the OS lengths revealed that rods had the longest OS, with an average length of approximately 35 μm (±3.76) (Fig 1C and 1G). Among the cone OS, the UV cone OS was the shortest, measuring approximately 10 μm (±0.49), while the blue cone OS had an average length of 12.5 μm (±1.33). Both red and green cone OSs were significantly longer than those of the blue and UV cones, with red cone OSs being slightly longer than green cone OSs (18.6 μm ± 0.94 versus 16 μm ± 1.13) (Fig 1G).

Additionally, we measured the widths of the different OSs, and found that the width of the cone OS was negatively correlated with its length (Fig 1H). A comparison of the ratio of OS length to width further highlighted significant morphological differences between the OSs of different photoreceptor types (Fig 1I). To further validate these observations, we performed ultrastructural analysis on adult zebrafish, which confirmed the morphological differences (S1 Fig). These results suggest that cone OS morphology in the zebrafish retina exhibits notable variation, both in terms of length and width.

UV opsins are essential for the maintenance of UV cones

To investigate the mechanisms regulating OS length and considering that opsins are the most abundant proteins in the OS, we generated zebrafish mutants lacking cone opsins. We designed sgRNAs targeting the second exon of the UV opsin (opn1sw1, or sws1) gene, resulting in a frameshift mutation that led to the loss of the C-terminal 214 amino acids (Fig 2A). This mutation caused a significant reduction in mutant mRNA expression in 5 days post-fertilization (dpf) larvae, likely due to nonsense-mediated decay (Fig 2B) [25,26]. Immunostaining with WGA revealed that UV cone OSs failed to develop in the mutants at both larval and adult stages (Fig 2C and 2D). In the absence of OSs, UV cones began to degenerate around 7 dpf, as indicated by the loss of GFP expression driven by the sws1 promoter. In adult fish, UV cones were nearly completely absent (S2A–S2E Fig).

Fig 2 — **(A)** Genomic structure of the zebrafish *sws1* gene, with wild-type and *sws1* mutant allele sequences shown below. The premature stop codon due to the frameshift mutation is indicated. **(B)** Whole-mount in situ hybridization showing *sws1* expression in 5 dpf wild-type and *sws1* mutant larvae. The position of primers used for probe synthesis is indicated in panel **(A)**. **(C, D)** Confocal images of photoreceptor OS morphology in 7 dpf **(C)** and adult **(D)** wild-type and *sws1* mutant retinae, visualized with WGA staining. The UV OS layer is absent in mutants (stars). **(E)** Ectopic expression of different cone opsins in the UV cones of adult *sws1* mutants. OS were visualized with WGA staining. Diagrams on the right illustrate the corresponding UV cone morphologies. Enlarged views are shown at the bottom. **(F–H)** Quantification of UV cone OS across different transgenic backgrounds, as indicated. **(I, J)** TEM analysis of UV cone OS morphology in wild-type and *sws1* mutants carrying *Tg(sws1:lws1)* transgene at 20 dpf and adult stages. **(K)** Quantification of the length of UV cone OS. In panels **(C–E)**, DAPI (blue) marks cell nuclei. Scale bars: 200 μm in **(B)**, 10 μm in **(C–E)**, 5 μm in **(I, J**). Data information: In **(F–H)**, each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(wt) = 40, n(+Sws1) = 31, n(+Mws3) = 20, n(+Lws1) = 28. Data were derived from N = 5 zebrafish per group. Statistical significance was determined by one-way ANOVA with Bonferroni’s post hoc test. In (K), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(wt 20 dpf) = 18, n(+Lws1 20dfp) =10, n(wt adult) = 17, n(+Lws1 adult) = 14. Data were derived from N = 3 zebrafish per group. Statistical significance was determined by Kruskal–Wallis with Dunn’s post hoc test. * p < 0.05; **** p < 0.0001; ns, not significant. The data underlying this Figure can be found in S1 Data.

Ectopic expression of cone opsins in UV cones

To examine whether UV opsins can be functionally replaced by other opsin proteins, we first generated a GFP-tagged UV opsin transgene, Tg(sws1:sws1-GFP). Although the GFP fusion protein was successfully targeted to the UV-cone OS, it failed to rescue UV-cone degeneration in the absence of Sws1 (S2F–S2I Fig). In contrast, UV-cone degeneration was fully rescued by the Tg(sws1:sws1) line expressing the full-length, untagged UV opsin (S2J and S2K Fig). These results suggest that the GFP tag may interfere with the structural integrity or proper function of UV opsin required for OS formation and maintenance.

Next, we asked whether ectopic expression of other opsins could rescue UV cone degeneration in the mutants. We generated zebrafish transgenes expressing green (mws3), red (lws1), and rod opsin genes. Strikingly, all these transgenes rescued the loss of UV cone OS in the sws1 mutants (Figs 2E and S3A). The number of UV cones was similar between the transgenes and wild-type siblings (S3B Fig). Remarkably, while the OS length is comparable between control and transgenic lines at larvae stages (S3C Fig), the rescued UV cone OSs exhibited significantly greater elongation in adult zebrafish upon ectopic expression of green or red opsin genes (Fig 2E and 2F). Statistical analysis showed that these rescued OSs resembled those of the green or red cone OSs of the double cones, although they localized to different layers of the retina (Fig 2F–2H versus Fig 1G–1I). Notably, ectopic expression of the rod opsin gene also rescued the cone OS, while the rescued OS resembles the morphology of the UV cone OS (S3A–S3D Fig). We further performed TEM ultrastructural analysis on sws1 mutants carrying the Tg(sws1:lws1) transgene. The rescued “UV-cone” OS was correctly positioned beneath the blue-cone OS; however, its morphology was dramatically altered and closely resembled that of a double cone (Fig 2I–2K). Altogether, these results suggest that ectopic expression of different cone opsins can modify the morphology of UV cone OSs.

Ectopic expression of cone opsins can modify the morphology of rod OSs

To investigate whether ectopic expression of cone opsins can affect the morphology of rod OS, we also generated rhodopsin (rho) mutants and confirmed reduced expression of rhodopsin in the mutant larvae (S4A and S4B Fig). Consistent with previous findings, mutation of the rho gene led to rapid degeneration of rod photoreceptors (S4C and S4D Fig) [27,28]. We then created two transgenic lines, Tg(xops:rhodopsin) and Tg(xops:lws1), which express rhodopsin or lws1 genes under the Xenopus rhodopsin gene promoter [16]. Expression of rhodopsin fully rescued the OS loss phenotype in the rho mutants (S4E–S4G Fig). Remarkably, ectopic expression of the red opsin gene also partially rescued rod OS formation in both larvae and adults (S4E–S4G Fig). Interestingly, these rescued OSs became slender and elongated in adults, resembling red-cone OSs (S4G–S4J Fig). Furthermore, the OSs of red opsin-expressing rod cells were predominantly positioned within the cone-OS layer, as indicated by their reduced distance to the outer plexiform layer (OPL) (S4G and S4K Fig). These results suggest that the cylindrical shape of rod OSs can be modified by simply switching the rod opsin with a cone opsin.

The morphology of cone OS is directly related to the absorption wavelength of corresponding opsins

Given that different opsins absorb light at distinct wavelengths, we hypothesized that the morphology of photoreceptor OSs may be influenced by the wavelength of light absorbed by the opsin. Interestingly, zebrafish UV cones possess the shortest OS, corresponding to the shortest wavelength of light—UV light. In contrast, red cones have the longest OSs, which align with the longest wavelength of light—red light (Fig 3A) [31,32]. This raises the question: would the OS of UV cone cells elongate if they expressed an opsin that absorbs light at a much longer wavelength? To address this, we generated a transgene expressing the long-wave cone opsin (LWS) from the turtle Trachemys scripta elegans [33]. The absorption wavelength of this opsin is significantly longer than that of zebrafish red opsin (620 nm versus 558 nm) (Fig 3A). Remarkably, when turtle LWS was expressed in UV cone cells, these cells maintained their characteristic cone morphology but developed OSs 24 μm (±1.25) in length—substantially longer than the ~20 μm OS observed in zebrafish double-cone cells (Fig 3B and 3C).

Fig 3 — **(A)** Absorption spectra of zebrafish cone opsins and the LWS opsin from *Trachemys scripta elegans* [29,30]. **(B)** Confocal images showing ectopic expression of turtle LWS opsin in the UV cones of *sws1* mutant zebrafish. UV cone OS were visualized with WGA staining. **(C)** Quantification of the length of UV cone OS. **(D)** Schematic diagram illustrating the red-shift in opsin absorption wavelength induced by thyroid hormone (TH) treatment. **(E)** Confocal images showing TH-induced morphological changes in red cone OS, labeled with WGA. **(F)** Quantification of the length of red cone OS after TH treatment. DAPI (blue) marks cell nuclei. Scale bars: 10 μm. Data information: In (C), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(wt) = 17, n(+LWS) = 14. Data for the wt and +LWS transgenic groups were derived from N = 3 and N = 5 zebrafish, respectively. Statistical significance was determined by the Mann–Whitney test. In **(K)**, each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(Ctr) = 23, n(TH) =24. Data were derived from N = 6 zebrafish per group. Statistical significance was determined by the Student t test. **** p < 0.0001. The data underlying this Figure can be found in S1 Data.

Opsins require chromophores (retinal) to absorb light and undergo a conformational change that activates the phototransduction pathway. Notably, many aquatic animals, including fish, amphibians, and some reptiles, possess a Vitamin A1-A2 visual pigment system [34]. For instance, zebrafish photoreceptors predominantly contain Vitamin A1-based pigments, but treatment with thyroid hormone (TH) can induce a conversion to Vitamin A2-based pigments, resulting in the absorption of light at longer wavelengths (red-shifts) (Fig 3D) [35,36]. Strikingly, TH treatments also caused a pronounced elongation of red cone OS due to the red-shifts (Fig 3E and 3F). This further confirms that the length of photoreceptor OSs is linked to the wavelength of light absorbed by the opsin.

Environmental adaptation of cone OS to different light intensities

According to optical theory, the wavelength of light is directly related to its energy, with shorter wavelengths, such as UV light, harboring the highest energy. The observed modification of cone OS morphology in response to changes in light wavelength suggests that light energy may play a critical role in determining OS length. To investigate further, we asked whether altering the intensity of light reaching the OS could similarly influence its length. To test this, we reared zebrafish from one month post-fertilization (mpf) under either standard illumination (356 lx) or reduced illumination (38 lx), both maintained on a standard light-dark cycle (Fig 4A). Remarkably, after one month, zebrafish raised under dim light exhibited a significant elongation of cone OS compared to those reared under standard lighting conditions. This elongation was particularly pronounced in double cones (Fig 4B and 4C).

Fig 4 — **(A)** Schematic diagram of low light intensity treatments of zebrafish from 1 to 2 months post-fertilization. **(B)** Confocal images showing the lengths of cone OS in 2 mpf zebrafish following one month of exposure to normal and dim light. OS were labeled with WGA.(C) Quantitative analysis of cone OS lengths under different light intensities. **(D)** Schematic diagram illustrating ectopic lipid droplet expression in cone photoreceptor cells to modulate light intensity reaching the OS. **(E)** Diagram showing lipid droplet fusion mediated by Cidea and Spdl1 proteins. **(F)** Transgene construct for ectopic lipid droplet expression in UV cones: *Tg(sws1:mScarlet-cidea-P2A-GFP-spdl1).* **(G)** Confocal images of ectopic lipid droplet expression at the inner segments of UV cones in adult zebrafish. UV cone OS were labeled with WGA (magenta), lipid droplets labeled with *Tg(sws1:mScarlet-cidea-P2A-GFP-spdl1)* (magenta and green). **(H)** Quantification of UV cone OS length in adult zebrafish. **(I)** Schematic diagram and live images showing the different living depths of aquaculture-reared (sea box) and wild-caught *Sebastes schlegelii*. **(J)** Confocal images comparing double cone OS morphology between aquaculture-reared and wild-caught *Sebastes schlegelii*. OS were labeled with WGA (magenta), and double cone cell bodies were marked with Zpr1 (green). **(K)** Quantitative analysis of double cone OS length in aquaculture-reared and wild-caught *Sebastes schlegelii*. DAPI (blue) marks cell nuclei. Scale bars: 10 cm in **(I)**;10 μm in **(B, G, J)**. Data information: In **(C)**, each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(Ctr UC) = 28, n(Dim-light UC) = 34, n(Ctr BC) = 24, n(Dim-light BC) = 39, n(Ctr RC) = 26, n(Dim-light RC) = 32. Data were derived from N = 5 zebrafish per group. Statistical significance was determined by one-way ANOVA with Bonferroni’s post hoc test. In (H), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(Ctr) = 27, n(LD+) = 24, n(LD−) = 23. Data for the wt and LD+ transgenic groups were derived from N = 4 and N = 6 zebrafish, respectively. Statistical significance was determined by one-way ANOVA with Bonferroni’s post hoc test. In **(K)**, each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(Sea box DC) = 22, n(Wild DC) = 23. Data were derived from N = 3 *Sebastes schlegelii* per group. Statistical significance was determined by Student t test. **** p < 0.0001. The data underlying this Figure can be found in S1 Data.

In various species, such as birds, cone photoreceptors have evolved a unique optical organelle known as the oil droplet or lipid droplet (LD), located within the inner segment. These oil droplets or LDs play a crucial role in photoreceptor activation by modifying the intensity of light transmitted to the OS [37]. These droplets consist of neutral lipids, primarily triacylglycerol, which act as intermediaries in the light path, thereby regulating the light intensity reaching the OS [37–39]. Consequently, we examined whether inducing the formation of oil droplets in zebrafish cone photoreceptors could modify the morphology of cone OS (Fig 4D). CIDEA (cell death-inducing DFFA-like effector A) is a critical factor in LD fusion and is highly expressed in avian cones [38,40,41]. CIDEA, together with SPDL1-L (spindle apparatus coiled-coil protein 1-L), a long isoform of SPDL1, regulates the formation and positioning of oil droplets in chicken cone photoreceptors [38] (Fig 4E). Differing from the well-studied SPDL1 short isoform (SPDL1-S), SPDL1-L contains a unique C-terminal region, which is essential for LD positioning [38]. Interestingly, the zebrafish Spdl1 locus encodes only the Spdl1-S isoform (S5A Fig). Therefore, we generated a chimeric protein combining zebrafish Spdl1 with the C-terminal sequence of the human SPDL1-L protein (Figs 4F and S5A). We then generated a stable transgenic zebrafish line coexpressing the chimeric construct and zebrafish Cidea under the control of the sws1 promoter, which is expected to induce LD formation specifically in UV cones (Fig 4F). At larval stages (10 dpf), LDs were observed in the inner segment of UV cones, and these LDs persisted in photoreceptors into adulthood (Figs 4G and S5B). Notably, some LDs began to degrade in certain UV cones during development, but a significant number of UV cones retained LDs (Figs 4G and S5B). Next, we compared the OS length of these photoreceptors. While no difference in OS length was observed between control and transgenic larvae at 10 dpf, a marked increase in OS length was noted in adult cones containing LDs (Figs 4H and S5C). Even within the same retina, UV cones containing LDs exhibited significantly longer OSs compared to those without LDs (Fig 4G and 4H). These findings suggest that ectopic LDs can also induce elongation of cone OSs. Although the composition of these LDs likely differs from that of native avian oil droplets, we speculate that they may still influence the light intensity reaching the OS, thereby promoting cone-OS elongation.

Finally, we asked whether such length adaption with light intensity occurs in natural conditions. In the ocean, light intensity decreases dramatically with the increase of depth due to absorption and scattering. The black rockfish (Sebastes schlegelii), a type of teleost found in the Northwest Pacific, can inhabit both deep and surface waters [42–44]. Wild-type black rockfish typically reside at depths ranging from 20 m to over 300 m [43,44]. In contrast, black rockfish is also cultivated as a farming fish in coastal regions, where they are raised in shallow waters (usually less than 10 m in depth). We compared cone OS between wild-type black rockfish, collected at depths of ~50 m, and cultured adult black rockfish raised in sea cages at ~7 m (Fig 4I). Using combined staining with WGA and the double cone-specific antibody Zpr1, we readily identified double cone OS in both wild-type and cultured fish (Fig 4J). Strikingly, the double cone OS in wild-type fish was also significantly longer than in cultured fish (Fig 4J and 4K). Altogether, these findings suggest that variations in light intensity also contribute to the regulation of cone OS length.

Variation in cone OS morphology is conserved across species

We have demonstrated that the morphology of zebrafish cone OS varies significantly among different subtypes, with longer wavelength-sensitive cones having longer OS. Finally, we asked whether this pattern is unique to zebrafish or more broadly conserved. In teleosts, we observed similar length variations in cone OS length, including Pterophyllum scalare, Cyprinus carpio, and Oryzias latipes (S6A–S6F Fig). Remarkably, although cone width was comparable among these species, their length-to-width ratios among different cones differed substantially, potentially reflecting light adaptations to distinct ecological niches. We also examined cone OS morphology in rabbits, which possess two cone types sensitive to blue and green light [45]. Notably, these two cone types exhibited significantly different OS lengths in the rabbit retina (S6G–S6H Fig). Likewise, in the human fovea, S -cones also exhibit shorter OS lengths compared to M/L-cones, as reported in previous studies [46–48]. Together, these observations suggest that cone OS length variation is a conserved feature across a wide range of vertebrate species, from teleosts to mammals.

Discussion

The morphology of photoreceptor OS represents a major structural distinction between rods and cones. Although these differences have been well described, the mechanisms that generate and maintain their distinct OS architectures remain poorly understood. Even less is known about the morphological variations between different cone OS subtypes. One of the primary challenges in addressing this question is the lack of suitable model systems for experimental investigation. As a nocturnal animal, mice possess a limited number of cones, and the co-expression of different cone opsins within the same cones further complicates the analysis of cone OS formation mechanisms [14]. In contrast, the zebrafish retina contains a large number of cones with four distinct subtypes, making it an excellent model for studying cone development.

Here, we show that zebrafish cone OS morphology can be modified simply by altering the photosensitivity of the opsin proteins. Notably, OS elongation does not appear to result from increased opsin accumulation. In sws1 heterozygous mutants, UV cone OSs were comparable in size to those of wild-type adults, despite a substantial reduction in sws1 expression (S7A–S7C Fig). Because specific antibodies against Sws1 are unavailable, we further quantified Sws1 protein levels using data-independent acquisition (DIA) mass spectrometry, which confirmed the decreased protein abundance in heterozygotes (S7D Fig). Moreover, the expression level of ectopic lws1 (which produces a longer OS) was even lower than that of sws1 in the Tg(sws1:sws1) line (which produces a shorter OS) (S7E Fig). Together, these findings indicate that factors beyond total opsin amount contribute to the regulation of cone OS morphology.

We propose several possible mechanisms that may influence OS length. First, subtle structural differences among opsins may affect their folding or interactions within the membrane, thereby altering disc composition or packing within the OS. Second, opsins are trafficked to the OS through a ciliary transport system, and transport efficiency may differ between cone subtypes. Such differences could also influence the recruitment of membrane-trafficking regulators, such as Rab or Arl13b proteins. Finally, the rate of photoreceptor disc shedding by RPE cells may vary across cone types, potentially contributing to differences in OS stability and length.

While all of these factors may contribute, we think the key regulator of OS modification is neural activity. This is supported by the finding that, at larval stages, UV-cone OSs in both the ectopic opsin-expression and LD induction experiments remain similar in length to those of wild-type controls (S8A, S8B, S3C, and S5C Figs). Notably, when Tg(sws1:lws1) fish were reared in complete darkness—conditions under which phototransduction-driven neural activity is minimized—the elongation of the “UV-cone” OS was significantly reduced (S8A and S8B Fig). Furthermore, even in adult fish, dim-light incubation increased cone-OS length (S8C and S8D Fig). These results highlight neural activity as a critical factor governing cone-OS morphological plasticity.

How might neural activity influence cone OS morphology? To transduce visual signals from photoreceptors to downstream bipolar cells, photoreceptors must continuously regulate the release of neurotransmitters such as glutamate at their synaptic terminals. This sustained synaptic output depends on adequate electrical responses generated by the phototransduction cascade. The first step in this cascade is the absorption of photon energy by opsin proteins in the OS (S9A Fig). Consequently, the OS must capture enough light energy to reach the threshold for signal initiation. Photon capture efficiency depends on two main factors: the energy of individual photons and the number of photons captured by photopigments. Short-wavelength photons, which carry higher energy, are therefore likely to activate their corresponding opsins more efficiently. In contrast, switching opsin expression from a short-wavelength to a long-wavelength variant—for example, ectopic expression of Lws1 in UV cones—may reduce activation efficiency during phototransduction. Similarly, a decrease in the number of photons reaching the OS, such as through the presence of additional LDs or under dim-light conditions, would be expected to further diminish opsin activation. To compensate for reduced activation efficiency, animals may adopt an alternative strategy—increasing the amount of opsin available in the light path, thereby enhancing photon capture within the OS. Such a mechanism could drive OS elongation (see detailed discussion in the legend of S9 Fig). Notably, this morphological adaptation likely requires prolonged adjustments and may be mediated by neural activity–dependent mechanisms. Although the molecular and cellular pathways that couple neural activity to OS remodeling remain unclear, feedback signals from downstream bipolar cells could potentially contribute to the regulation of OS length, an idea that merits future investigation.

In summary, our findings demonstrate that the morphology of photoreceptor OS is directly linked to the type of opsin it expresses. The longer the wavelength sensitivity of the opsins, the longer the cone OS length. Such regulation may be achieved via long-term environmental adaptation or neural activity. Our results provide new insights into the development of photoreceptor OS and offer a novel understanding of the factors influencing OS structure.

Methods

Ethics statement

All animal procedures in this study were strictly conducted in accordance with the Guideline for Ethical Review of Animal Welfare (GB/T 35892-2018) of the People’s Republic of China. All animal experiments were approved by the Animal Care Committee of Ocean University of China (Animal protocol number: OUC2012316).

Animals

All zebrafish strains were maintained at 28°C under a 14-hour light/10-hour dark cycle. Embryos were reared at 28.5°C in E3 medium (5 mM NaCl, 0.39 mM CaCl₂, 0.17 mM KCl, 0.67 mM MgSO₄, 0.1% methylene blue) according to standard protocols. To generate zebrafish sws1 and rho mutants, we utilized CRISPR/Cas9 technology. The sgRNA target sequences were 5′-GGGATGGTCCTTGGCTGTTC-3′ and 5′-TGTACACCTCCTTGCACGGC-3′. Cas9 mRNA and sgRNA were synthesized following established protocols [49,50], and were co-injected into zebrafish embryos at the one-cell stage. All information regarding the transgenic zebrafish lines is provided in S1 Table, and the primer sequences are listed in S2 Table. Unless otherwise stated, all analyses on adult zebrafish were conducted using 3- to 6-month-old individuals.

Teleost species, including Pterophyllum scalare, Cyprinus carpio, and medaka (Oryzias latipes), were purchased from a local market in Qingdao, China. Wild-type Sebastes schlegelii were collected by fishing in the East China Sea at a depth of approximately 50 m, while cultured black rockfish were obtained from a local market in Qingdao, China.

Whole-mount in situ hybridization and histological analysis

Whole-mount in situ hybridization experiments were performed following a standard protocol [51]. The primers used to amplify sws1 and rho genes are listed in S2 Table. For histological analysis, zebrafish larvae at 5 dpf were fixed overnight in 4% paraformaldehyde (PFA) at 4°C. After fixation, the larvae were washed in PBST (PBS with 0.1% Tween-20), dehydrated through a graded ethanol series (50%, 75%, 85%, and 95% ethanol, each for 15 min), and embedded in JB-4 embedding medium (Polysciences) as previously described [52]. Cryosections were prepared at a thickness of 6 µm using a Leica cryostat. The sections were examined under a Leica stereomicroscope, and images were captured with a Leica digital camera.

Immunohistochemistry

Larval sections in the manuscript were prepared using the cryosectioning method. For the immunostaining of cryosections, zebrafish larvae were fixed in 4% PFA overnight at 4°C. The fixed larvae were washed twice in PBST for 5 min each, infiltrated with 30% sucrose in PBS overnight at 4°C, embedded in OCT (Leica), frozen, and sectioned at a thickness of 12 µm. The following primary antibodies and dilutions were used: mouse Zpr1 (1:500, Zebrafish International Resource Center), Zpr-3 (1:500, Zebrafish International Resource Center). Although WGA546 (1:100; Invitrogen) is commonly used as a marker for rod OS, we found that it labels both rod and cone OS in zebrafish. Notably, the labeling intensity and pattern varied among cone subtypes, potentially reflecting differences in glycoconjugate composition among photoreceptor classes.

Adult fish sections were prepared using vibratome sectioning. To minimize potential effects from retinomotor movements in teleosts, adult fish were euthanized in ice water during the daytime (between 12:00 and 17:00). Eyes were then enucleated and immediately transferred to L-15 medium (Sigma). Under a stereomicroscope (Motic), the outer cornea and lens were carefully removed using forceps. The remaining retinas were fixed in 4% PFA at 4°C overnight. Retinas were then embedded in 4% low-melting-point agarose, sectioned into 40 µm-thick vibratome slices, and subjected to immunolabeling.

Upon capture, eyes from both wild and cultured black rockfish were immediately enucleated and transferred into L-15 medium (Sigma). The outer cornea and lens were carefully removed using forceps. The remaining retinas were fixed in 4% PFA at 4°C overnight. All subsequent steps, including vibratome sectioning and immunofluorescence staining, were performed using the same protocols as described for zebrafish retinas.

Measurement of photoreceptor OS morphology

Photoreceptor OS were measured only from cone cells with a clearly identifiable OS tip. OS width was measured at the base of the OS, while OS length was measured from the base to the apex. For closely apposed double cones, particular care was taken to accurately define the OS tip to avoid errors caused by signal overlap. To ensure consistency, the OS tip was determined based on the following criteria: (a) The terminal point of WGA lectin labeling, identified as the distal-most location where a continuous and sharply demarcated WGA signal from an individual cone terminated. (b) For cases with significant lateral overlap where tips were indistinguishable in two-dimensional projections, the three-dimensional information from the Z-stack was utilized to trace each OS to its individual terminus.

Transmission electron microscope

Retinae from adult zebrafish were fixed overnight at 4°C in 2.5% glutaraldehyde prepared in 0.1 M PBS. The samples were washed 3 times with PBS for 15 min each, followed by fixation in 1% osmium tetroxide for 2 hours. After fixation, the samples were washed, gradually dehydrated through increasing concentrations of acetone up to 100%, and embedded in Epon812 resin. Ultra-thin sections (70 nm) were cut using a Leica EM UC7 ultramicrotome. The sections were collected and stained with uranyl acetate and lead citrate. Specimens were then examined using a transmission electron microscope (Hitachi HT7700) and imaged with Olympus Soft Imaging Solutions.

Quantitative PCR

Retinal tissues were collected from adult zebrafish. For each biological replicate, two adult zebrafish per group were euthanized by immersion in ice-cold water (<4°C). Four retinas were then dissected, pooled, and immediately processed for RNA extraction using Trizol reagent (Takara). RNA was reverse-transcribed using the HiScript III RT SuperMix for qPCR kit (Vazyme, #R323-01). qPCR was performed on a StepOne Real-Time PCR System (Thermo Fisher Scientific) with EvaGreen Master Mix (ABM). We estimated expression levels using the relative standard curve method, using five serial standard dilutions of cDNA obtained from wild-type adult. Reactions were run in technical triplicate under the following cycling conditions: 95°C for 15 s, then 40 cycles of 95°C for 5 s, 60°C for 15 s, and 72°C for 35 s. Relative gene express levels were determined by the comparative Ct (2^−ΔΔCt) method, with zebrafish β-actin serving as the endogenous control. Primer sequences are listed in S2 Table. Differences between the two groups were assessed using the Mann–Whitney U test, a p-value <0.05 was considered statistically significant.

Data-independent acquisition

To compare the protein expressiong level between wild-type and sws1 heterzygotes groups, we performed Data-independent acquisition mass spectrometry. Briefly, four complete retinas were independently collected and pooled for each samples. Immediately after dissection, retinas were placed in pre-chilled 1.5 mL microcentrifuge tubes, flash-frozen in liquid nitrogen, and stored at −80°C until further processing. Protein extraction, tryptic digestion, and subsequent liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis were performed by Applied Protein Technology (Shanghai, China). The raw DIA data files were processed using DIA-NN software (version 1.8.1). Precursor and protein quantification was performed using the built-in quantification algorithms of DIA-NN. The quantified intensity values for the target protein, UV Opsin (Sws1, encoded by the opn1sw1 gene), were extracted from the report for subsequent statistical analysis between the wild-type and sws1^+/− groups. The raw mass spectrometry data are provided in Supporting Information S1 Data.

TH treatments

The procedures for TH treatments were similar to those previously reported [36]. Briefly, L-thyroxine (Selleck) was dissolved in 0.1 M NaOH to prepare a stock solution at a concentration of 3 × 10⁵μg/L. For treatments of zebrafish, the L-thyroxine stock solution was diluted with fresh rearing system water to final concentrations of 300 μg/L. Zebrafish were maintained under a normal light cycle and feeding schedule, and the system water containing the drug was replaced every 24 hours for a duration of 2–3 weeks.

Lower light exposure treatments

One- or 5-month-old zebrafish were maintained in light-isolated chambers containing 500 mL of system water at 28°C. The control group was exposed to standard-intensity illumination (356 lx) via LED panels (400–830 nm), whereas the low-intensity group was exposed to dim light (38 lx) using calibrated LED light sources. Illumination was provided on a 14 h light/10 h dark cycle, with lights on at 08:30 and off at 22:30. Zebrafish were fed twice per day with freshly hatched Artemia nauplii (brine shrimp). After 30 days of photoperiod conditioning, retinal sections were prepared for immunofluorescence microscopy to assess cone photoreceptor OS morphology.

Dark treatments

Two-week-old wild-type and Tg(sws1:lws1) zebrafish were maintained in light-isolated chambers containing 500 mL of system water at 28°C. The control group was exposed to standard-intensity illumination (356 lx) via LED panels (400–830 nm). Illuminated groups were subjected to a 14 h light/10 h dark cycle, with lights on at 08:30 and off at 22:30. A dark group was kept in constant darkness throughout the experiment. Zebrafish were fed twice daily with freshly hatched Artemia nauplii. After 45 days of photoperiod conditioning, retinal sections were prepared for immunofluorescence microscopy to assess cone photoreceptor OS morphology.

Statistical analysis

Data in all graphs are presented as mean ± standard deviation (SD). Normality was assessed for all quantitative datasets, and the appropriate statistical test was selected accordingly: parametric tests (unpaired Student t test for two groups, one-way ANOVA test followed by Bonferroni’s correction for multiple comparisons.) were used when normality assumptions were met; otherwise, non-parametric alternatives (Mann–Whitney test for two groups, Kruskal–Wallis test followed by Dunn’s correction for multiple comparisons.) were applied. Details on sample sizes (n), test values ,and significance levels are provided in the respective figure legends. A p-value < 0.05 was considered statistically significant. No randomization, blinding, or masking was employed in the animal studies. All experiments were repeated at least three times independently. The underlying data for all statistical analyses can be found in Supporting information S1 Data. Statistical analyses were performed using GraphPad Prism 9 (version 9.5; GraphPad Software, San Diego, CA, USA).

Supporting information

S1 Fig. Ultrastructural analysis of photoreceptor OS in adult zebrafish.

(A1, A2) Transmission electron micrograph (TEM) showing the distribution of cone OS in the adult zebrafish retina. (B–D) Higher magnification TEM images illustrating the ultrastructure of OS in UV cones (B), blue cones (C), and double cones (D). (E–G) Quantitative analyses of OS morphology. Abbreviations: RC, red cone; GC, green cone; BC, blue cone; UC, UV cone. Scale bars: 5 μm in (A); 2 μm in (B–D). Data information: In (E–G), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(UC) = 9, n(BC) = 12, n(GC) = 6, n(RC) = 6. Data were derived from N = 3 zebrafish per group. Statistical significance was determined by one-way ANOVA with Bonferroni’s post hoc test. p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant. The data underlying this Figure can be found in S1 Data.

(TIF)

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S2 Fig. Progressive degeneration of UV cones in sws1 mutants.

(A–D) Confocal images showing the morphology of UV cones in wild-type and sws1 mutant retinas at different developmental stages, as indicated. Enlarged views are shown at the bottom. UV cone cell bodies are labeled with Tg(sws1:GFP) (green). White asterisks indicate regions lacking UV cone cell bodies. In (D), the red rectangle outlines the remaining UV cones in the ciliary marginal zone (CMZ), and the white rectangle highlights a region shown in the enlarged view below. Adult = 4 months. (E) Quantitative analysis of UV cone cell numbers across different developmental stages. (F) Confocal images showing the morphology of UV cones in wild-type (labeled with WGA, red) and Tg(sws1:sws1-GFP) (green). (G) Quantification of UV cone OS length in adult zebrafish. (H) Representative confocal images showing the morphology of UV cones following exogenous expression of Sws1-GFP in the sws1 mutant background. (I) Quantitative analysis of the number of UV cone OS. (J) Confocal micrographs showing that expression of zebrafish Sws1 fully rescues UV cones in sws1 mutants. (K) Quantitative analysis of the number of UV cones in adult zebrafish. DAPI (blue) marks cell nuclei. Scale bars: 25 μm (low-magnification images); 10 μm (high-magnification images). Data information: In panels (E, I, K), each dot represents the number of UV cones from the section of a larvae or adult fish. We only collect one data for each sample. Sample sizes per group (E) are as follows: N(wt 5 dpf) = 4, N(sws1 5 dpf) = 7, N(wt 7 dpf) = 6, N(sws1 7 dpf) = 5, N(wt 21 dpf) = 5, N(sws1 21 dpf) = 4, N(wt 4 month) = 3, N(sws1 4 month) = 3. Statistical significance was determined by Kruskal–Wallis with Dunn’s post hoc test. In panel (I) Sample sizes per group are as follows: N(wt 7 dpf) = 17, N(Sws1-GFP 7 dpf) = 8. Statistical significance was determined by the Mann–Whitney test. In panel (K), sample sizes per group are as follows: N(wt) = 6, N(+Sws1) = 6. Statistical significance was determined by the Mann–Whitney test. Relative # of UV cones per A.U (arbitrary units) is calculated by the number of UV cones per arbituary length of confocal images (116.25 µm). In panel (G), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(wt) = 36, n(Sws1-GFP) = 32. Data were derived from N = 3 zebrafish per group. Statistical significance was determined by the Student t test. **** p < 0.0001; ns, not significant. The data underlying this Figure can be found in S1 Data.

(TIF)

pbio.3003654.s002.tif^{(7.5MB, tif)}

S3 Fig. Rescue of UV cone OS deficiency in sws1 mutants via different opsin proteins.

(A) Confocal images illustrating the morphology of UV cone OS in 5 dpf zebrafish with different transgenic backgrounds, as indicated. (B, C) Quantitative analysis of the number and length of UV cone OS in 5 dpf zebrafish larvae. (D) Confocal images showing the morphology of UV cone OS in adult zebrafish following ectopic expression of rhodopsin. The OS were labeled with WGA. DAPI (blue) marks cell nuclei. Scale bars: 15 μm (low-magnification) and 5 μm (high-magnification) in (A) and (D). Data information: In panel (B), each dot represents the relative number of UV cone OS from the section of a larvae. We only collect one data for each sample. Relative # of UV cone OS per A.U (arbitrary units) is calculated by the number of UV cone OS per arbituary length of confocal images (38.75 µm). Sample sizes per group are as follows: N(wt) = 5, N(+Sws1) = 5, N(+Mws3) = 5, N(+Lws1) = 6, N(+Rho) = 5. In panel (C), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(wt) = 15, n(+Sws1) = 15, n(+Mws3) = 17, n(+Lws1) = 15, n(+Rho) = 15. Data were derived from N = 3–6 independent biological replicates per group. The data underlying this Figure can be found in S1 Data.

(TIF)

pbio.3003654.s003.tif^{(7.4MB, tif)}

S4 Fig. Phenotypic analysis of rho mutants and ectopic cone opsin expression in rod cells.

(A) Genomic structure of the zebrafish rho gene, with wild-type and rho mutant allele sequences shown below. The premature stop codon resulting from the frameshift mutation is indicated. (B) Whole-mount in situ hybridization illustrating rho expression in 5 dpf wild-type and rho mutant larvae. The position of primers used for probe synthesis is indicated in panel (A). (C) Confocal images showing the morphology of rod cell bodies (green) and OS (magenta) in the retinae of 5 dpf wild-type and rho mutant larvae. Rod cell bodies were labeled with the Tg(xops:GFP) transgene, while OS were labeled with the Tg(xops:mCherry-CT44) transgene. (D) Confocal images illustrating photoreceptor OS distribution and morphology in adult wild-type and rho mutant retinas. Rod cell bodies were labeled with Tg(xops:GFP), and OS were visualized with WGA staining. Rod OS are absent in rho mutants (stars). (E) Confocal images illustrating the morphology of rod OS in 7 dpf zebrafish with different transgenic backgrounds, as indicated. Rod OS were visualized with Tg(xops:mCherry-CT44) (red). (F) Quantitative analysis of the number of rod OS per section in 7 dpf zebrafish larvae. (G) Ectopic opsin expression in rod cells of adult rho mutants. Rod OS were visualized with Tg(xops:mCherry-CT44). Compared to the wild-type control and rho mutants, rhodopsin expression rescued rod OS morphology, whereas ectopic red opsin expression can partially rescue rods and induced a cone-like OS morphology. Enlarged views of boxed regions are shown below. (H–J) Quantification of rod OS morphology under different genetic backgrounds. (K) Statistical analysis of the distance from the base of the rod OS to the OPL, as shown in panels (G). DAPI (blue) marks cell nuclei. Scale bars: 200 μm in (B); 20 μm in (C, D, E, G). Data information: In (F), each dot represents the relative number of rod OS from the section of a larvae. Sample sizes per group are as follows: N(wt) = 16, N(rho) = 20, N(+Rho) = 12, N(+Lws1) = 12. Statistical significance was determined by Kruskal–Wallis with Dunn’s post hoc test. In (H–J), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(wt) = 15, n(+Rho) = 14, n(+Lws1) = 12. Data were derived from N = 4 zebrafish per group. Statistical significance was determined by Kruskal–Wallis with Dunn’s post hoc test. In (K), each dot represents one photoreceptor OS. Sample sizes per group are as follows: n(wt) = 14, n(+Rho) = 15, n(+Lws1) = 19. Data were derived from N = 4 zebrafish per group. Statistical significance was determined by Kruskal–Wallis with Dunn’s post hoc test.** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant. The data underlying this Figure can be found in S1 Data.

(TIF)

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S5 Fig. Induction of lipid droplet formation in UV cones.

(A) Schematic representation of the domain structures of SPDL1 in zebrafish (D.Spdl1), chicken (C.SPDL1-L), and human (H.SPDL1-L). The chimeric protein used to induce lipid droplet formation contains the N-terminal of zebrafish Spdl1 (1–547) plus the transmembrane domain of human SPDL1 (558–622). Domain predictions were performed using InterPro. (B) Confocal images showing ectopic lipid droplet expression in UV cones of 10 dpf zebrafish larvae. The split green and magenta channels were shown on the right with arrows indicate ectopic LDs in the cell body of UV cones. (C) Quantification of UV cone OS length in 10 dpf larvae zebrafish. DAPI (blue) marks cell nuclei. Scale bar: 10 μm in (B). Data information: In (C), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(Ctr) = 16, n(LD+) = 16. Data for the wt and LD+ transgenic groups were derived from N = 5 and N = 8 zebrafish, respectively. Statistical significance was determined by the Mann-Whitney test. ns, not significant. The data underlying this Figure can be found in S1 Data.

(TIF)

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S6 Fig. Length variation of cone OS in different species.

(A) Phylogenetic tree depicting the evolutionary relationships among Pterophyllum scalare, Cyprinus carpio, Oryzias latipes, Sebastes schlegelii, and Danio rerio. (B) Confocal images illustrating the morphology of cone OS in different species of teleost fish. The photoreceptor OS were labeled with WGA (red), and double cones were labeled with Zpr1 (green). Nuclei were stained with DAPI (blue). (C–F) Statistical analysis of cone OS lengths in various teleost fish species. (G) Confocal images illustrating the distribution and morphology of photoreceptor OS in adult rabbits. WGA (red) labels rod cell OS, while Zpr3 (green) can label the OS of the two types of cone cells. (H) Statistical analysis of cone cell OS lengths in rabbits. Scale bar: 5 μm in (B);10 μm in (H). Data information: In (C), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(UC) = 30, n(BC) = 26, n(DC) = 51. Data were derived from N = 3 Pterophyllum scalare per group. Statistical significance was determined by one-way ANOVA with Bonferroni’s post hoc test. In (D), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(UC) = 9, n(BC) = 22, n(DC) = 30. Data were derived from N = 3 Cyprinus carpio per group. Statistical significance was determined by one-way ANOVA with Bonferroni’s post hoc test. In (E), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(UC) = 11, n(BC) = 23, n(DC) = 27. Data were derived from N = 3 Oryzias latipes per group. Statistical significance was determined by one-way ANOVA with Bonferroni’s post hoc test. In (F), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(BC) = 33, n(DC) = 23. Data were derived from N = 3 Sebastes schlegelii per group. Statistical significance was determined by the Student t test. In (H), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(BC) = 12, n(GC) = 16. Data were derived from N = 3 rabbits per group. Statistical significance was determined by the Mann–Whitney test. **** p < 0.0001. The data underlying this Figure can be found in S1 Data.

(TIF)

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S7 Fig. Gene expression analysis of ectopic opsins.

(A) Confocal images of UV cone OS in wild-type and sws1^⁺/− heterozygous mutant zebrafish. Nuclei are stained with DAPI (blue). (B) Quantification of UV cone OS length in wild-type and sws1^⁺/− zebrafish. (C) qPCR of relative sws1 mRNA expression levels in wild-type and sws1^⁺/− zebrafish. (D) DIA of relative sws1 protein expression levels in wild-type and sws1^⁺/− zebrafish. (E) Relative mRNA expression of sws1 and lws1 in transgenic rescue lines. Scale bars: 15 μm (low-magnification) and 10 μm (high-magnification) in (A). Data information: In (B), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(wt) = 28, n(sws1^⁺/−) = 28. Data were derived from N = 5 zebrafish per group. Statistical significance was determined by the Student t test. In (C, E), the qPCR for each sample was performed in technical triplicates. ns, not significant. The data underlying this Figure can be found in S1 Data.

(TIF)

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S8 Fig. Elongation of the cone OS is dependent on neural activity.

(A) Confocal images showing the morphology of cone OS in wild-type or sws1 mutants carrying Tg(sws1:lws1) transgene. The schematic diagram of the strategy of the light/dark treatment is shown on top of each figure. Enlarged views of the UV cone OS is shown on the bottom. OS were labeled with WGA. (B) Quantification of UV cone OS lengths under normal light and dark conditions with different genetic background as indicated. (C) Confocal images showing the lengths of cone OS following one month of exposure to normal and dim light starting from 5mpf old adult zebrafish. Schematic diagram of low-light intensity treatments was shown on the top. OS were labeled with WGA. To distinguish the length of blue and double cone OS, different focus planes were shown on the right. Z-stack image shows the maximum intensity projection image. (D) Quantitative analysis of cone OS lengths under different light intensities. DAPI (blue) marks cell nuclei. Scale bars: 10 μm in (A, C). Data information: In panel (B), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(wt 7dpf) = 26, n(+Lws1 7dpf) = 30, n(wt 2mpf) = 40, n(+Lws1 2mpf light) = 39, n(+Lws1 2mpf Dark) = 31. Data were derived from N = 6 zebrafish per group. Statistical significance was determined by one-way ANOVA with Bonferroni’s post hoc test. In (D), each dot represents one photoreceptor OS. Sample sizes per group are as follows: OS n(Ctr UC) = 36, n(Dim-light UC) = 40, n(Ctr BC) = 29, n(Dim-light BC) = 32, n(Ctr RC) = 31, n(Dim-light RC) = 34. Data were derived from N = 5 zebrafish per group. Statistical significance was determined by one-way ANOVA with Bonferroni’s post hoc test. **** p < 0.0001; ns, not significant. The data underlying this Figure can be found in S1 Data.

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S9 Fig. Model for the regulation of cone OS length by Light intensity and opsin sensitivity.

(A) Schematic illustrating the path of light through the layered zebrafish retina. The different arrangement of rod and cone OSs in the photoreceptor layer were shown. (B–E) Schematic diagrams illustrating a model for how cone photoreceptor OS length is regulated. For effective phototransduction, the outer segment must generate sufficient membrane potential through cyclic nucleotide-gated (CNG) channels (right). Closure of these channels may require a minimum number of activated opsin molecules. For example, at least four activated opsins (yellow) may be needed to induce adequate CNG channel closure. Because short-wavelength ultraviolet (UV) light has higher photon energy, it can meet this activation threshold with only a single opsin layer (B). In contrast, long-wavelength red light, which activates red opsins less efficiently, requires additional opsin layers to capture enough photons to reach the threshold (C). Likewise, reduced light intensity—whether caused by lipid droplets or low-light environmental conditions—decreases the likelihood of photon capture, potentially necessitating additional opsin layers along the light path to maintain sufficient activation (D). Finally, in the absence of light, such elongation of the OS may not occur even when long-wavelength red opsins are expressed (E).

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S1 Table. Transgenic zebrafish generated in this work.

(DOCX)

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S2 Table. Primer sequences.

(DOCX)

pbio.3003654.s011.docx^{(18.3KB, docx)}

S1 Data. Raw data used in all figures.

(XLSX)

pbio.3003654.s012.xlsx^{(1.7MB, xlsx)}

Acknowledgments

We would like to thank Dr. Jie Qi for her kind help during the acquirement of the black rockfish. We also thank Dr. Xungang Tan, members of the IEMB and FANG center for their kind help during the preparation of this manuscript. We also gratefully acknowledge the outstanding support from the core facilities of the IEMB and FANG Center at OUC.

Abbreviations

dpf: days post-fertilization
LD: lipid droplet
LWS: long-wave sensitive opsin
mpf: month post-fertilization
OPL: outer plexiform layer
OS: outer segments
PFA: paraformaldehyde
ROS: rod outer segment
RPE: retinal pigment epithelium
TH: thyroid hormone
WGA: wheat germ agglutinin

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

This work was supported by the National Natural Science Foundation of China (https://www.nsfc.gov.cn/) (32125015 to C.Z., W2411026 to C.Z., 32500730 to J.X.) and funds from Laoshan Laboratory (LSKJ202203204 to C.Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Biol. doi: 10.1371/journal.pbio.3003654.r001

Decision Letter 0

Taylor Hart, PhD

1 Jul 2025

Dear Dr Zhao,

Thank you for submitting your manuscript entitled "Light intensity and opsin sensitivity shape the morphology of cone photoreceptor outer segments" for consideration as a Short Report by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I am writing to let you know that we would like to send your submission out for external peer review.

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Taylor Hart, PhD,

Associate Editor

PLOS Biology

thart@plos.org

PLoS Biol. doi: 10.1371/journal.pbio.3003654.r002

Decision Letter 1

Taylor Hart, PhD

14 Aug 2025

Dear Dr Zhao,

Thank you for your patience while your manuscript "Light intensity and opsin sensitivity shape the morphology of cone photoreceptor outer segments" was peer-reviewed at PLOS Biology. It has now been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by several independent reviewers.

In light of the reviews, which you will find at the end of this email, we would like to invite you to revise the work to thoroughly address the reviewers' reports.

As you will see below, the reviewers praise the experimental design and say that the results are interesting. However, R1, R2 and R4 pointed out areas requiring additional support, including through adding control experiments, further quantifications, and addressing issues with the methodological descriptions, statistics, and figures. The reviewers all commented on the limited mechanistic insight, and several of them suggested further experiments that could provide further clarity here. The reviewers also offered suggestions for improving the framing of the study.

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Christian Schnell, Ph.D.

Senior Editor

PLOS Biology

cschnell@plos.org

on behalf of

Taylor

Taylor Hart, PhD,

Associate Editor

PLOS Biology

thart@plos.org

------------------------------------

REVIEWS:

Reviewer #1 (Juan Angueyra signed his report): SUMMARY OF REVIEW:

This paper aims to highlight the role of visual opsins in photoreceptor outer segment (OS) morphology, with the goal of providing insight to outer segment development and plasticity. Through a series of novel experiments in both larval and adult zebrafish, using new mutant and transgenic lines, the authors demonstrate that OS shape is correlated with opsin expression, opsin sensitivity, and light intensity. In addition, the authors carry additional manipulations (hormonal and environmental) and cross-species comparisons known to create differences in opsin expression and find corresponding changes in OS shape.

These results are exciting and interesting.

Finally, the authors propose a model on how photoreceptor OS is regulated by light energy. We believe that some changes in rephrasing this claim, and acknowledging the possibility of other mechanisms would improve the overall impact and readability, especially for non-retina experts. We also suggest the authors refine the figure legends and Methods to ensure the reproducibility.

What are the main claims of the paper and how significant are they for the discipline? Are these claims novel?

Claim 1: Specific opsin expression drives OS morphology.

Novel = Yes. Although the influence of opsin levels on outer segment length is well described, this study includes clever ectopic expression of opsins, which validates a mechanism where specific properties of each opsin control OS morphology

Claim 2: Correlation between OS length and wavelength sensitivity of opsin expressed

Novel = Yes. Cell-specific manipulations added novelty to well-described literature.

Claim 3: Control of OS shape is activity dependent

This mechanism is not well supported by the presented data.

Is this paper outstanding in its discipline?

Yes. This study uses novel and insightful approaches, presents interesting findings, and opens multiple possibilities for follow-up studies. In particular this will lead to experiments to identify mechanisms able to control cell shape, which is relevant to many fields.

Who would find this paper of interest? Why?

This could be of general interest to cell biologists, retinal biologists, and visual neuroscientists. Researchers interested in understanding the mechanisms that govern morphology differences across cell subtypes, particularly in the retina, will find this particularly interesting because it may provide a new bridge between structure and function in neurons.

Are the claims properly placed in the context of the previous literature? Have the authors treated the literature fairly?

Yes, the authors cite relevant literature to their field and everything is discussed in context to previous literature.

Do the data and analyses fully support the claims? If not, what other evidence is required?

Claim 1: Specific opsin expression drives OS morphology.

Supporting evidence: Yes; ectopic, environmental, and hormonal manipulations. In addition, cross-species comparisons. All lines of evidence reach similar conclusions.

Claim 2: Correlation between OS length and wavelength sensitivity of opsin expressed

Supporting evidence: Yes.

Claim 3: Control of OS shape is activity dependent

Supporting evidence: No, the evidence provided does not uniquely support this claim. We recommend the authors to modify the claim as it stands.

Further Evidence Required:

To support this claim, a manipulation that modifies activity is required. For example, mutations in phototransduction proteins that cause blindness could be a first step towards this goal.

Other possible (non-activity dependent) mechanisms need to be excluded. For example, OS morphology could be dictated by sequence differences in opsins, differences in ciliary transport of the misexpressed opsins, or changes rates of disc shedding by the RPE, amongst others.

Would additional work improve the paper? How much better would the paper be if this work were performed and how difficult would it be to do this work?

We recommend revisions of the text, without additional experiments.

If the paper is considered unsuitable for publication in its present form, does the study itself show sufficient potential that the authors should be encouraged to resubmit a revised version?

The authors should resubmit a revised version after thorough revision of the methods section to make the manuscript suitable for publications (see below). In addition, the authors should revise or soften their claim regarding that the mechanism that underlies OS length is activity dependent as the data is suggestive but insufficient to exclude other possible mechanisms.

Are original data deposited in appropriate repositories and accession/version numbers provided for genes, proteins, mutants, diseases, etc.?

N/A

Is the manuscript well organized and written clearly enough to be accessible to non-specialists?

Yes.

Are details of the methodology sufficient to allow the experiments to be reproduced?

No.

Imaging: not enough details provided. We recommend that the authors follow established standards in the field for reporting methods for fluorescent and electron-microscopy images (e.g. https://doi.org/10.1038/s41592-021-01156-w)

qPCR: key steps of protocol, experiments and analysis have been omitted. For example, no primers were provided for the normalization gene, no mention of RNA extraction protocol, of quality checks on cDNA, etc. (e.g. https://www.biorxiv.org/content/10.1101/2024.12.04.626769v1.full.pdf)

Light exposure experiments: no spectral data provided for LEDs used. Given the claims about spectrum and activity-dependent mechanisms, this information is highly relevant.

Statistics: none of the experiments have explicit mention of biological replicates, and relevant details to assess confidence on statistical analysis are omitted, including details (other than p-value) of statistical tests (e.g. https://pmc.ncbi.nlm.nih.gov/articles/PMC6639881/; https://plos.org/resource/how-to-report-statistics/)

Animals: While the results mention specific days post-fertilization, the manuscript lacks clarity regarding which developmental time points were used for comparative analysis with teleost species. Given that there is both larval and adult data presented, explicit mention of age is required for all datasets.

We recommend a thorough revision of the methods section. This should also include revision of figure legends; in particular "Quantitative analysis of …" should be replaced with all the details about statistical tests (n, degrees of freedom, value of t-test, etc.). There is also no description of what each dot in bar plots represents. If each dot represents a single OS, it is required to report how many images were analyzed for each experiment and if the images correspond to different animals or not (which correspond to true biological replicates), to be able to assess if this study has enough statistical power.

PLOS Biology encourages authors to publish detailed protocols and algorithms as supporting information online. Do any particular methods used in the manuscript warrant such treatment?

No.

Other Minor Edits:

Many images are not color-blind friendly. We recommend using magenta instead of red when red-green distinctions are important

Much of the text on the fluorescent images is difficult to discern. For example, the red WGA text can be difficult to read where it overlays the images, and would benefit from improved contrast or alternative labeling approaches.

Unclear if scale bar is consistent, and this is sometimes not mentioned. For example, in Figure 2 the dimensions for the scale bar is not mentioned for F-I even though there is a scale bar for I shown.

Having a key for colors in box plots could be helpful, and could be kept consistent for similar parameters in other plots.

Properly report the replicate size and the sample size.

Figure 2A; does the 10 bp insertion create a premature STOP codon?

Figure 3: Are both WGA and cidea labeled red? Is Cidea expressed in the transgenic cones?

Figure S3A: Why is there no quantification with GFP14? Why 5 dpf for only this experiment?

Figure S3G: Why is there no quantification? Is the rescue full? The example images seem to suggest that there are more UV cones in the rescue than in WT?

Figure S4B: What is relative #? How is this calculated and why is it not absolute?

Page 13: "Similar to humans, zebrafish cones are classified into S-cones (blue), M-cones (green), and L-cones (red) (Perkins & Fadool, 2010). Additionally, zebrafish feature a specialized type of cone that is sensitive to UV light" is misleading since the human S-cone is actually related to the zebrafish UV cone and not the Scone (see Baden et al, 2025: https://doi.org/10.1371/journal.pbio.3003157)

Include the spectrum of LED panel used for light exposure experiment

"Reducing photon energy (e.g., switching to longer wavelengths) or decreasing photon capture (e.g., via additional lipid droplets or in low-light environments) lowers the likelihood of photopigment activation": this is not true. For example, according to the spectral sensitivity curves for L-opsins, switching from blue light to red light (high energy to low energy) increases photopigment activation. Photopigment activation depends on the intensity AND spectrum of the stimulating light AND the spectral sensitivity of the opsin.

Suggestions:

Hebbian plasticity: The classic Hebb's rule is about the connection between neurons, and although famous, not the only example of activity dependence. We find it challenging to understand the direct comparison between Hebbian plasticity between two cells and the changes in cell shape presented in this manuscript.

Restructure the discussion to match flow of the introduction:The authors discuss environmental changes as a factor for altering OS shape. We recommend that the authors move the environmental changes section before discussion about oil droplets. The reviewers believe this structure will benefit the flow of the manuscript. If not, alter the introduction to highlight cellular modifications to improve/alter/enhance particular functions (such as oil droplets in photoreceptors).

Clarify times of experiments and adult vs larval data: The authors provide insightful data from both adult and larval stages of zebrafish, but it becomes unclear which pieces of data are being referred to. For example it is explained that the five photoreceptor subtypes are easily distinguished using wheat germ agglutinin (WGA) staining, but it is unclear at what developmental timepoint this is at. We suggest the authors add age to every figure once and distinguish when different ages are used. This will make the results explicitly clear when the authors are referring to morphology in adult vs. larval/developmental stages.

Suggestion for the discussion: the authors state that OS morphology (mainly length, width, and shape) is directly related to the opsin that is expressed. But does not acknowledge scenarios where opsins are co-expressed in the same cells (mice M/S gradient, cichlid/salmon UV/S transition). Do the authors think that OS morphology changes in hybridized cell identities?

Suggestion for the discussion: The term morphology encompasses quite a bit when looking at photoreceptor cell structure: in addition to the outer segment, did the authors notice any changes in the rest of the cell morphology? (inner segment, cell bodies, ribbon synapses).

Reviewer #2: Photoreceptors have outer segment (OS) regions specialized to absorb photons and create a visual response. The manuscript by Xu et al presents a set of data to support an interesting hypothesis, that cone photoreceptor OS size and shape is directly related to light sensitivity, and is adaptable depending on the conditions. Using zebrafish as a model, the authors characterize the OS dimensions of the four cone subtypes, demonstrating that photoreceptors detecting longer wavelengths have longer and wider OSs. The replacement of UV opsin in the short cones with another cone opsin was sufficient to convert the OS morphology to match the opsin. Surprisingly, rod opsin did not change the UV cone morphology. On the other hand, expression of red opsin in rods lacking rho created rods with OSs shaped similar to red cones and also shifted the OS position to a more inner layer. Expression of a long wavelength turtle opsin in sws1-deficient UV cones led to a tripling in OS length compared to wildtype UV cones. Treatment with thyroid hormone, which promotes the switch from Vitamin A1 to A2-based photopigments (A2 is red-shifted) promoted lengthening of red OSs. Another experiment involved the triggering of lipid droplet formation in the inner segment or growing fish in reduced light conditions, in both cases limiting light passage into the OS. The hypothesis was that the OS would lengthen to accommodate the reduction in light input, which was indeed the finding. Finally, the authors examined a variety of fish species, observing similar relationships between cone height and wavelength. For black rockfish that lives at depth when wild but is cultivated in shallower waters, the cone photoreceptors were longer in the deep-dwelling population. Overall, the authors used multiple experimental approaches to support their hypothesis that cone OSs adapt to their light-detecting functions.

Generally, the paper is well written and organized. Some of the experiments are quite ingenious. The group uses a wide breadth of experiments to support their hypothesis. My comments are mostly about improvements for the experimental analysis.

Major comments:

1) The transgenic line sws1:sws1-GFP is used to mark OS. But the line is massively overexpressing sws1 opsin in these cells. Evidence is needed to show that morphology is unaffected in these cells.

2) The WGA labelling is curious. WGA is typically used as a rod OS marker, so the specific labelling of cone OS is surprising. Can the authors explain or justify the discrepancy? Is there evidence in the literature for its use as a cone OS label, especially for UV cones.

3) What are the data points shown in the graphs throughout the paper? Are they measurements for individual cells, section, eyes, or fish?

4) t-tests are the only statistics described, which is not appropriate for multiple groups compared to one wildtype group, unless with a Bonferroni correction.

5) If the UV cones with swapped opsins remain in the same layer, then TEM could be used to support the conclusion of altered morphology and would be a strong addition, particularly for seeing changes in width of the UV cone OS.

6) The rho-/- retinas with lws1 expression must look vastly different from wt, and even the rho rescue looks disorganized. TEM would again benefit this characterization, and maybe also further immuno characterization of the retinal layering.

7) In Figure 2, Panel G, it looks like the top of the UV cone OSs are cut off, either by the physical or optical section. This is also true for the wt image in Fig 3, panel I and Fig 4B. How is the analysis controlled to ensure that the whole cell is captured? And if these images are correct, then are there changes to OS shape? This is an important point since the length of the OS is a key measurement in the analysis.

Minor comments:

1) There are no page or line numbers, which makes it difficult to provide clear comments.

2) Intro: The OS is not analogous to a dendrite. Receives signals, but very different structure.

3) Intro: The authors state the "OS resemble a modified cilium". The OS is a modified cilium

4) Intro: "By comparison, the retina of the zebrafish is predominantly cone-based, with approximately 92% cones at the larval stage and about 60% in adults, closely resembling the cone-rich fovea found in humans (Fadool, 2003, Masek, Zang et al., 2023). There is a missing reference here.

5) More clarification about ages could be used in the text and on the figures. What is the range of ages described as "adult"?

6) For Figure 2, need lower magnification panels to show position/organization of the cells within the retina. Panel E needs a scale bar.

7) The relationship between width and length of OS doesn't seem to hold for the other species. Are the scales accurate, since the cell size looks massively different between cone subtypes in some of the fish. Please comment on the length-width ratio in the discussion.

8) Discussion is generally underwhelming, and the comparison to Hebbian plasticity is weak and doesn't really add anything to the paper. Neural activity of photoreceptors is not activated by light (the opposite), which should be made clear. And the LTP comparison is nebulous.

Reviewer #3: The manuscript by Xu et al. reports the intriguing finding that the morphology (length and width) of the outer segments (OS), the light-sensitive region of photoreceptors, is regulated by the kind of the opsin expressed in the OS and the intensity and wavelength of incident light. The authors used elegant genetics in the zebrafish model to establish this, and also present data that argue for a conservation of this principle in other fish species as well as mammals. Overall, the experiments are well designed, the data are nicely presented, and the manuscript is written in a very accessible style.

Despite the novelty of their findings, the shortcoming of the paper is that the authors do not present any data or much of an argument on how photoreceptor-specific opsins could be regulating OS morphology at the mechanistic level. Since the OS is essentially an exaggerated elaboration of the connecting cilia membrane, could it be that the opsins, which are OS membrane proteins, regulate membrane biogenesis and organization? For instance, the opsins could act through the effectors of ciliary membrane biogenesis like the Rab proteins and Arl13b? While this manuscript is suitable for PLoS Biology as a short article and I am not expecting the authors to now put together some mechanistic data along these lines in the revision, it would nevertheless be worthwhile to include some speculation on the mechanism in the discussion section.

Reviewer #4 (Michel Cayouette and Michael Housset signed their report): In this manuscript, Xu et al. address how cells adopt different morphologies. To explore this very interesting and poorly studied question, they use primarily the zebrafish photoreceptor cells as a model system, taking advantage of the various morphologies displayed by photoreceptor subtypes and ease of genetic manipulations in zebrafish. Specifically, they examine how opsins of different spectral properties and ambient light intensity influences photoreceptor outer segment (OS) morphology. They report a positive correlation between OS length and the peak absorption wavelength of the expressed opsin, with an inverse correlation between OS base width and absorption peak. This relationship appears conserved across vertebrate species, including different fish species and rabbit. The authors also show that sws1 mutant zebrafish exhibit UV cone degeneration, which is rescued by ectopic expression of sws1 (UV), mws3 (green), or lws1 (red) opsins. Strikingly, the OS morphology of rescued cones varies depending on which opsin subtype is expressed, supporting a role for opsin sensitivity in shaping photoreceptor morphology. They also show that thyroid hormone (TH) treatment, which shifts opsin expression in red cones to a longer wavelength red opsin, further elongates the OS, reinforcing the link between spectral tuning and OS morphology. The authors propose a mode in which OS geometry is an adaptive response to differences in photon energy and opsin activation efficiency, supported by experiments in dim light and comparisons with deep-sea Sebastes schlegelli.

Overall, this is a very interesting paper addressing a key question in neurobiology. The conclusions are largely supported by the results (although see below), the paper is well written, and the figures are clear and generally convincing. This work will be of interest to vision scientists and to the neuroscience community in general. While the study lacks mechanistic insights into how exactly different opsins leads to changes in cell morphology, these results establish a solid base for future investigation in the field and constitute groundbreaking work in this area. There are, however, a few points that should be clarified and some additional experiments that may help better support the conclusions. Specifically, mechanistic clarity is needed regarding the roles of light versus opsin identity, and additional quantitative and optical analyses are needed to strengthen the central claims. These points are detailed below.

Major comments

1) In fish, as well as chicken and xenopus, photoreceptors dynamically elongate or contract in response to light and dark adaptation, but the implication of this phenomenon on the interpretation of the data presented in this paper is not discussed. It is crucial that the authors discuss their findings in the context of retinomotor movements. This background would help distinguish between reversible, transient changes and more stable OS modifications.

2) While the study convincingly links opsin identity to OS morphology, the interpretation that OS length reflects an adaptation to help increase opsin content is not fully substantiated. OS length and width are not an adequate proxy for opsin quantity because the amount of opsin proteins that can accumulate in OS disk membrane depends on volume. A quantification of OS volume and surface area, ideally supported by 3D reconstructions, would provide a more robust test of the authors' hypothesis of a gain in opsin content in the OS.

3) The manuscript would benefit from additional data to strengthen the model. Specifically, it remains unclear whether OS shape is intrinsically determined by the opsin protein itself or requires downstream effects of light activation. These aspects could be experimentally disentangled by raising fish in complete darkness and under monochromatic light of different wavelength. Moreover, the intriguing possibility that opsin identity dictates OS width while photon absorption regulates OS length deserves more extensive exploration. The authors could for example put transgenics Tg (sw1 :Mws3) and Tg (sws1 :Sws1) of the sws1 mutant background in total darkness until analysis. If OS shape is directly influenced by the light-mediated activity, as predicted by the authors' model, there should not be any difference between the two conditions, since there is no light to activate the opsins.

4) The analogy between OS elongation and dendritic arbor refinement is compelling but unclear. The authors should clarify whether dim-light-induced OS elongation occurs during a critical developmental window, as often observed in dendrite refinement, or can be modified post-developmentally by performing experiments in adult animals. If it is not possible to carry out these experiments, this limitation should at least be discussed.

5) In Figure 1:

* Indicate OS location in panel A.

* Improve contrast for the legend in panel F.

* Specify whether OS width is measured at the base or along the full segment.

6) In Figure 2:

* Panel B: Clarify if the region targeted by the probe lies within, upsteam or downstream of the deleted portion of the sws1 gene.

* Correct the legend: "10 µm in (C-E)" → "10 µm in (C-I)."

* The authors discuss protein levels, but only RNA is quantified in Supplementary Figure 5. Western blotting would be important to confirm protein expression. Also, clarify which genes are being quantified in the figure S5 panel B. Please segregate qPCR done for Sws1 gene and qPCR for Lws1 on different graphics.

7) In Figure 3:

* Change Pseudemys scripta elegans for the correct current taxonomy: Trachemys scripta elegans.

* Panel B: Quantification of OS shape (length and width) is missing for the turtle LWS rescue.

* Panel C: Consider replacing the current schematic with one showing that TH induce expression of longer wavelength red opsin Lws1 at the expense of the shorter Lws2 at the genomic locus.

* Panels F-I: The lipid droplet experiment is technically impressive but lacks clear optical implications. The droplet, likely composed of neutral lipids and localized basally in the myoid, is unlikely to absorb or scatter UV light significantly, contrary to the avian lipid droplet found in birds PRs which contains specific lipids and pigments. Without optical modeling or empirical measurement, the impact on photon flow remains speculative. Conclusions should therefore be toned down and limitations of this approach discussed.

8) In Figure 4:

* Quantify OS volume and base width in addition to length (or change all quantification to volume measurements, as suggested in point 2 above).

* Clarify how the OS tip was defined in overlapping WGA-labeled double cones.

* Address potential morphological artifacts due to delayed fixation in wild-caught specimens; consider using a control feature (e.g., nuclear diameter or IS length) for normalization.

9) In Figure 5: The model presented in not clear. The authors are encouraged to provide a refined, more self-explanatory figure. The position of cones within the retinal layers could be integrated to the model as well as potential effects of wavelength-dependent penetration in the tissue. If additional data can be obtained to distinguish between an intrinsic role of light and opsin protein species in OS width and length, as suggested in point 3 above, this should be considered in the model.

Minor comments

* Regarding the failure to rescue sws1 mutants with tagged opsins, please include a schematic of tag placement and fusion constructs. The C-terminal domain of rhodopsin contains essential trafficking signals, particularly the final 15 amino acids. If this is disrupted by the tag, mislocalization of opsin could explain the lack of rescue. This limitation should be discussed or excluded experimentally.

* In Figure S6H, expression of long-wavelength opsins appears to reduce inner segment (IS) length. Please mention impact of opsin on IS length and discuss whether this reflects coordinated regulation of IS and OS dimensions. Also, the limited rescue by Lws1 compared to rhodopsin raises concerns about expression efficiency or structural compatibility. please clarify what happens to non-rescued rods.

* Clearly state "n" values (number of cones vs. number of animals) for each experiment.

* Indicate developmental stage and retinal region for all quantifications.

* In several figures, the authors used a student's t-test for comparison between multiple conditions, which is not appropriate statistics. ANOVA with post hoc corrections must instead be used.

PLoS Biol. 2026 Feb 18;24(2):e3003654. doi: 10.1371/journal.pbio.3003654.r003

Author response to Decision Letter 2

16 Dec 2025

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PLoS Biol. doi: 10.1371/journal.pbio.3003654.r004

Decision Letter 2

Taylor Hart, PhD

23 Jan 2026

Dear Dr Zhao,

Thank you for your patience while we considered your revised manuscript "Light intensity and opsin sensitivity shape the morphology of cone photoreceptor outer segments" for publication as a Short Report at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor, and three of the original reviewers.

Based on the reviews, we are likely to accept this manuscript for publication, provided you satisfactorily address the remaining points raised by the reviewers. Please also make sure to address the following data and other policy-related requests.

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Taylor Hart, PhD,

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PLOS Biology

Reviewer remarks:

Reviewer #1 [Juan Angueyra]: The authors addressed our comments satisfactorily, including reassessing parallels with Hebbian plasticity, improving description of methods and structural rearrangements to improve readability. The concerns of other reviewers have also been sufficiently addressed. We believe these edits have improved the paper and strengthened their models to explain the mechanisms that may dictate the structure of photoreceptor outer segments in relation to the environment. We only have two minor suggestions for revisions of the specific interpretations that won't require any additional experiments, and that we leave to the authors discretion.

First, the following paragraph is still inaccurate and/or confusing:

"Light energy acquisition depends on two main factors: the energy of individual photons and the number of photons captured by photopigments. Reducing photon energy (e.g., using longer wavelengths) or limiting photon capture (e.g., through additional lipid droplets or under dim-light conditions) lowers the probability of opsin activation".

As we previously stated, just using longer wavelengths does not guarantee less opsin activation, which this sentence seems to imply. If the authors intended "to emphasize that, for a given opsin at its optimal wavelength, higher-energy photons generally have a higher probability of causing isomerization than lower-energy photons at their respective optimal wavelengths", this should be incorporated in the manuscript, although we still find even this explanation confusing. Since the "number of photons captured by photopigments" depends on photon energy/wavelength AND spectral sensitivity of the photopigment, this phrasing hides an important logical step. I would argue that it's clearer to say that photon capture depends on two main factors: the total number of photons and the relation between the spectral composition of these photons and the spectral sensitivity of the photopigment. Lowering opsin activation requires decreasing the number of photons that reach the opsins or decreasing the spectral overlap between the stimulus and the opsin.

Second, it might be worth elaborating or clarifying a bit more on what the authors consider as the most likely biological implementation of how "neural activity" leads to changes in OS length. As briefly mentioned, transducin activation and glutamate release in photoreceptors are inversely related, so that "when fish are raised in darkness", there is indeed minimal transducin activation but glutamate release by photoreceptors is at its highest, which could be considered maximal neural activity? Do the authors assume that this could be processed through circuits in the ON pathway, which flips the polarity of signals? Is that more likely than a cell-autonomous mechanism depending directly on the number of activated transducins in individual cones? Disclosing glutamate release in the final model figure may make this explicit for readers.

Reviewer #2: The revised paper is strong and acceptable for publication, with a couple of suggested minor changes.

1. The scale bar in Fig4 labelled as "10 cm in (D)" should be "10 cm in (I)".

2. The WGA staining in the manuscript is clearly labelling cones. However, this is unusual. I'm unclear if that's a result of a different WGA preparation, more concentrated solution, or imaging parameters. It should be mentioned in the manuscript that WGA is typically used a rod OS marker, but that the researchers found it could be adapted for cone labelling. Otherwise, it will confuse the reader.

3. Also, it appears in many of the panels that WGA fails to label the top portion of the UV cone OS. If so, this should be mentioned.

4. The panels and labels in Fig S6 are very small. The figure could be reorganized to enlarge panels.

Reviewer #4 [Michel Cayouette and Michael Housset]: The authors have done a wonderful job addressing our comments both with text changes and new experiments. We have no further comments. Congratulations on a very interesting study.

PLoS Biol. 2026 Feb 18;24(2):e3003654. doi: 10.1371/journal.pbio.3003654.r005

Author response to Decision Letter 3

28 Jan 2026

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PLoS Biol. doi: 10.1371/journal.pbio.3003654.r006

Decision Letter 3

Taylor Hart, PhD

30 Jan 2026

Dear Dr Zhao,

Thank you for the submission of your revised Short Report "Light intensity and opsin sensitivity shape the morphology of cone photoreceptor outer segments" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Tom Baden, I am pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

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Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study.

Sincerely,

Taylor

Taylor Hart, PhD,

Associate Editor

PLOS Biology

thart@plos.org

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Ultrastructural analysis of photoreceptor OS in adult zebrafish.

(TIF)

pbio.3003654.s001.tif^{(7.6MB, tif)}

S2 Fig. Progressive degeneration of UV cones in sws1 mutants.

(TIF)

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S3 Fig. Rescue of UV cone OS deficiency in sws1 mutants via different opsin proteins.

(TIF)

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S4 Fig. Phenotypic analysis of rho mutants and ectopic cone opsin expression in rod cells.

(TIF)

pbio.3003654.s004.tif^{(7.4MB, tif)}

S5 Fig. Induction of lipid droplet formation in UV cones.

(TIF)

pbio.3003654.s005.tif^{(4.6MB, tif)}

S6 Fig. Length variation of cone OS in different species.

(TIF)

pbio.3003654.s006.tif^{(4.9MB, tif)}

S7 Fig. Gene expression analysis of ectopic opsins.

(TIF)

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S8 Fig. Elongation of the cone OS is dependent on neural activity.

(TIF)

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S9 Fig. Model for the regulation of cone OS length by Light intensity and opsin sensitivity.

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S1 Table. Transgenic zebrafish generated in this work.

(DOCX)

pbio.3003654.s010.docx^{(15.3KB, docx)}

S2 Table. Primer sequences.

(DOCX)

pbio.3003654.s011.docx^{(18.3KB, docx)}

S1 Data. Raw data used in all figures.

(XLSX)

pbio.3003654.s012.xlsx^{(1.7MB, xlsx)}

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Data Availability Statement

All relevant data are within the paper and its Supporting information files.

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PERMALINK

Light intensity and opsin sensitivity shape the morphology of cone photoreceptor outer segments

Jingjin Xu

Zihan Chang

Wei Deng

Luwei Qian

Honggang Su

Xun Huang

Yunsi Kang

Haibo Xie

Chengtian Zhao

Roles

Abstract

Introduction

Results

Variation in the morphology of cone OS in zebrafish

Fig 1. Morphological diversity of cone OS in the zebrafish retina.

UV opsins are essential for the maintenance of UV cones

Fig 2. Phenotypic analysis of sws1 mutants and ectopic cone opsin expression in UV cones.

Ectopic expression of cone opsins in UV cones

Ectopic expression of cone opsins can modify the morphology of rod OSs

The morphology of cone OS is directly related to the absorption wavelength of corresponding opsins

Fig 3. Modification of cone OS morphology by the absorption wavelength of corresponding opsins.

Environmental adaptation of cone OS to different light intensities

Fig 4. Modification of cone OS morphology by light intensity.

Variation in cone OS morphology is conserved across species

Discussion

Methods

Ethics statement

Animals

Whole-mount in situ hybridization and histological analysis

Immunohistochemistry

Measurement of photoreceptor OS morphology

Transmission electron microscope

Quantitative PCR

Data-independent acquisition

TH treatments

Lower light exposure treatments

Dark treatments

Statistical analysis

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Taylor Hart, PhD

Roles

Decision Letter 1

Taylor Hart, PhD

Roles

Author response to Decision Letter 2

Decision Letter 2

Taylor Hart, PhD

Roles

Author response to Decision Letter 3

Decision Letter 3

Taylor Hart, PhD

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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