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IJMSInternational Journal of Molecular Sciences
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6 March 2026

21 Pages

miRNA 183 Knockout Alters Cone Subtype Distribution, Transcriptional Activity and ERG Signals in the Tetrachromatic Zebrafish Visual System

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1
Ophthalmic Molecular Genetics Section, Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA
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National Institute of Neurological Disorders and Stroke, National Institutes of Health, Rockville, MD 20892, USA
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Department of Biology and Center for Neuroscience and Behavior, American University, Washington, DC 20016, USA
4
Visual Function Core, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA
Int. J. Mol. Sci.2026, 27(5), 2433;https://doi.org/10.3390/ijms27052433
This article belongs to the Special Issue RNA in Biology and Medicine (2nd Edition)
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Abstract

miRNA 183 is part of the miRNA-183/96/182 cluster, which is known to play a decisive role in fine-tuning the activity of gene expression in sensory systems, particularly in the retina. Although miR-183 is essential for retinal gene expression in mammals, the contributions of miR-183 to mRNA expression and photoreceptor development and function in other classes of animals have not been fully elucidated. Danio rerio have a diverse photoreceptor system, with cone photoreceptors sensitive to red, green, blue and ultraviolet (UV) light. We generated knockout zebrafish by deleting the whole seed sequence of miR-183. RNAscope results show no expression of mature miR-183 and decreased expression of miR-182 in both dorsal and ventral KO retinas. The number of UV and blue photoreceptors decreased, and the photoreceptors showed shortening or loss of their outer segments. In the absence of miR-183, the transcription levels of phototransduction genes were altered differentially at 3 and 12 months of age. Finally, photoreceptor-only electroretinogram (PIII) signals showed attenuated amplitudes of red and green-sensitive photoreceptor subtypes while the b-wave amplitudes reflecting second order retinal neuron activity, were decreased in response to the UV-, blue-, and red-stimulating wavelengths. These results reveal a novel microRNA regulatory network in teleost fish and indicate that miR-183 plays a facilitative role in retinal development and function, especially for short-wavelength-sensitive photoreceptor subtypes.

1. Introduction

MicroRNAs (miRNAs) are small RNA molecules that play a critical role in controlling gene expression by binding to target messenger RNA (mRNA) to inhibit its translation and/or initiate mRNA decay [1]. In some cases, regulation of mRNA expression by miRNAs has been shown to include modulation of alternative splicing or even transcriptional regulation [2]. The approximately 22 nucleotide (nt) miRNA guide the Argonaute complex (AGO) to a specific target mRNA area via complementary base pairing to recruit a silencing complex (miRISC) [3]. The miRISC contains a 182 kDa glycine–tryptophan repeat-containing protein (GW182, TNRC6A) and a miRNA-guided AGO protein [4].
An increasing number of identified miRNAs are involved in retinal development and the maintenance of retinal morphology and phototransduction. Among retinal miRNAs, the highly conserved miR-183/96/182 cluster (miR-183C) is required for the maturation of sensory organs, especially the photoreceptor cells [5]. Disruption of miRNA-183-96-182 expression results in irregularities of the thickness of the photoreceptor and inner nuclear layers in mice [6]. Cone-specific conditional knockout of Drosha/DGCR8, which cleaves pri-miRNAs to form pre-miRNAs, in mice causes disruption of cone outer segments with reduced B-wave amplitude, which can be rescued by replacing miRNA-182 and miRNA-183 [7]. miR-183C KO mouse models show multisensory defects, including incomplete retinal development with extinguished ERG responses and retinal degeneration [8,9,10]. Recently, miR-183C was reported to be expressed in both sensory nerves (CSNs) and corneal resident myeloid cells (CRMCs), regulating tear production and corneal sensitivity by modulating corneal inflammatory/immune responses [11]. Collectively, these findings verify the critical regulatory role of miR-183C in the mammalian visual system.
However, the role of miR-183 in regulating retinal development in general, and especially in non-mammalian retina photoreceptor development and function, has not been fully delineated. The zebrafish is a good candidate for exploring photoreceptor cells in non-mammalian animals [12]. Expression of miR-183 in the eye field is low at 1 dpf but increases approximately 9-fold by 3 dpf and 12-fold by 5 dpf [13]. Compared with mammals, which have lost many functions during evolution, zebrafish retain a variety of photoreceptor cells that are sensitive to various wavelengths of light. Unlike the mammalian retina, in which rods predominate, about 60% of photoreceptors in adult zebrafish retina are cones, arranged in a precise mosaic pattern [14]. In addition, cones account for 92% of the photoreceptors in larval zebrafish retinas. Previous studies have revealed that all members of the miR-183 cluster are expressed in photoreceptors, and that their expression levels increase rapidly within 20 days after birth in the mouse, which is also a critical time for the development of photoreceptor cells. This suggests that the miR-183 cluster is important for photoreceptor development [15]. However, Fogarty et al. previously showed that deletion of the mir-183 cluster and its components did not cause major structural retinal abnormalities in zebrafish [13]. Here, as an initial step in a detailed examination of the roles of individual members of miR183C and their combinations on retinal architecture and visual function, we generate a zebrafish model deleting the entire seed sequence of miR-183 and explore its effects. No mature miR-183 was detected in the miR-183 KO. Short-wavelength-sensitive photoreceptors, especially blue opsin and UV opsin cones, show reduced cell numbers and abnormal collapsed morphology, being shorter in height and wider in diameter with shorter outer segments. Photoreceptor-only ERG responses, obtained by blocking signals from the inner retinal neurons with L-aspartic acid show reduced ERG amplitudes in response to different stimulating wavelengths, suggesting specific photoreceptor deficits. These morphological and functional changes are accompanied by the disruption of transcriptional profiles in various photoreceptor subtypes. Overall, our results reveal a novel impact of miR-183 in teleost fish that is specific to certain cone photoreceptor subtypes and their higher order neuronal connections.

2. Results

2.1. miR-183 Seed Sequence Deletion in Zebrafish

miR-183, -96, and -182 are grouped together as a miR-183 cluster as they share similar seed sequences and overlapping targets. In mammals, the miR-183 mature sequence and its seed sequence are highly conserved (Figure 1A). Although the mature miR-183 is relatively less conserved in non-mammals, showing some divergence in lancelets (Branchiostoma) and sea urchins (Strongylocentrotus purpuratus), its seed sequence is highly conserved even in these distant species (Figure 1B), suggesting that it serves a fundamental role in the regulation of gene expression in these diverse systems. Transcription activator-like effector nuclease (TALEN) technology was utilized to generate a KO zebrafish model, termed miR-183−/− (Figure 1C,D). RNAscope targeting of the mature sequence of miR-183 validated the absence of this microRNA expression in both the dorsal and ventral side of the KO retina (Figure 2 and Figure S1), demonstrating that mature miR-183 was completely depleted. This is also the case for the AB strain miR-183 KO described in detail in Fogarty et al. [13] (Figure S2).
Figure 1. Generation of the miR-183−/− zebrafish model. (A) Conservation of mature and seed sequences of miR-183 in mammals. (B) Conservation of mature and seed sequences of miR-183 in non-mammals. The mature sequence of miR-183 is slightly different in non-mammals, but the seed sequence is highly conserved. (C) Transcription activator-like effector nuclease (TALEN) technology was used to generate the miR-183−/− zebrafish model. The left and right recognition sequences of TALEN were designed to flank and delete the seed sequence. The NLS sequence helps guide TALEN into the cell nucleus and target the genome, improving editing efficiency. (D) Genotyping of miR-183 knockout zebrafish. A total of 11 bp, including the seed sequence, was deleted. NLS: nuclear localization domain. RVD: repeat-variable di-residue.
Figure 2. miR-183 cluster expression in WT and KO TL strain zebrafish retinas. (A,B) RNAscope results with probes specifically binding to miR-183, 96, and 182. Neither the dorsal (A) nor ventral (B) side of the mutant retina show binding with the miR-183 probe. The miR-96 probe binds the retina normally while miR-182 shows decreased binding in the miR-183 KO retina. Magenta indicates probe binding to the retina. The darker the color, the higher the miRNA expression level. Blue indicates nuclei and brown is intrinsic pigment of the RPE. (C,D) Statistical results of RNAscope signal intensity of the above images, the values are the comparison of each miRNA with WT. The results show that miR-183 expression is completely knocked out in this zebrafish model and that miR-182 expression is partially reduced. WTTL: wild-type zebrafish of a TL background, N = 6. miR-183−/−: miR183 11 bp deletion including whole seed sequence, N = 10. Bars in the graph represent s.e.m. ** p < 0.01, *** p < 0.001. Student’s t-test.

2.2. MiR-183 KO Decreases the Number of Short-Wavelength-Sensitive Photoreceptors in the Zebrafish Retina

Similar to previously reported results [13], miR-183−/− zebrafish retinas have a normal retinal structure, although they do show mild degenerative changes in the photoreceptors and RPE (Figure S3). At 1 month of age there were no significant changes in the length or number of blue or red/green cones (Figure S4A–C). However, at 10 months of age, in the absence of mature miR-183, the number of short-wavelength-sensitive cone photoreceptors, expressing UV (Figure 3A,C) and blue opsin (Figure 3B,C), were significantly decreased (p < 0.005), whereas the number of rods and green/red cones were minimally impacted (Figure S4D–G). In addition to showing fewer blue and UV cones, the mir-183 KO retinas show decreased staining of the outer segments in these cone subtypes, and the length of UV cone outer segments is decreased, while a suggestive but not statistically significantly decrease is seen in blue cone outer segment length (Figure 3D,E). In contrast, no changes were obvious in the number or cell morphology of rods and green/red cones (Figure S4D–G). TUNEL stains of WTTL and miR183−/− zebrafish from 1–12 months of age show no apoptosis (Figure S5). Taken together, these results confirm the importance of miR-183 for photoreceptor development and demonstrate that short-wavelength photoreceptors are a major regulatory target of miR-183 in zebrafish retina.
Figure 3. Morphology and numbers of short-wavelength-sensitive photoreceptor subtypes in the absence of miR-183 at 10 months old WTTL and miR-183−/− fish. (A) Immunostaining with anti-dre-UV-opsin antibody of retinal flat mounts (left) and cross sections (right). Nuclei and UV cones are depicted in light grey (DAPI) and light blue respectively. The KO retina shows fewer UV cones and decreased length and staining of the cone outer segments. (B) Fluorescent image of retinal cross sections with anti-dre-blue-opsin. The KO retina shows fewer blue cones and decreased length and staining of cone outer segments. (C) Statistical results of UV and blue cone numbers. The value indicates the number of cones per 100 μm in an 8 μm thick section. (D) Cone outer segment lengths for UV and Blue cones. (E) Normalized fluorescence intensity for UV and Blue opsins. WT: N = 5. miR-183−/−: N = 6. Bars in the graph represent s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001. Student’s t-test.

2.3. miR-183 Shows a Differential Effect on Zebrafish Cone Opsin mRNA Levels That Is Independent of Cone Subtype Numbers

Quantitative RT-PCR results of mRNAs expressed in specific retinal cell types show alterations in the expression levels of a subset of photoreceptor genes (Figure 4), although these are not directly related to the changes in the cell subtypes. gfap (expressed in glia, including Müller cells) is increased in expression from 3 dpf to 3 M, but not significantly after that. Blue (sws2) and UV short-wavelength opsin (sws1) mRNA levels are largely unchanged, although sws2 shows apparent decreases at 14 dpf and 12 M. Rhodopsin (rods), is significantly increased only at 3 M and gnat1 (rods) is increased at 6 and 12 M, while grk1a (rods and cones) is also increased at 6 and 12 M. Expression changes in green cones are varied, with rh2-1 increased at 3–12 M, rh2-2 and rh2-3 decreased at 12 M, while rh2-4 is increased at 3 and 12 M. Finally, lws1 and lws2 (red cones) are increased at 6 M, while arr3a (red/green cones) is increased at 3 M and arr3b (blue and UV cones) is increased at 14dpf. It should be noted that the values shown are relative to those in WT zebrafish. Thus, not only does miR-183 have a facilitative effect on the differentiation or survival of cone subtypes but also affects gene expression in photoreceptor cells independently of the cell number. This is perhaps best shown by UV cones having the largest decrease in number, while there is no change in expression of the UV opsin gene. Conversely, while there is no change in the number of red/green cones the levels of the rh2-1 pigment are greatly increased.
Figure 4. Expression levels of photoreceptor-related genes in WT and miR-183−/− retinas. Expression levels are shown at 3dpf, 14dpf, 3M, 6M, and 12M of age. Relative levels are not shown for ages at which neither WT nor miR-183−/− retinas showed values significantly differing from 0. WT: N = 6. miR-183−/−: N = 10. Bars in the graph represent s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001 by Student’s t-test.
In addition, potential targets of miR-183 were examined by qRT-PCR, although, as discussed in the introduction, the mRNA levels of these targets might not reflect protein levels (Figure S6). Interestingly, gm6b mRNA levels were increased at all developmental stages while ncam1a and celf2 mRNAs are increased after 6 months of age, csmd1a mRNA is increased at 6 months of age and vsx2 mRNA levels are decreased after 3 months of age.

2.4. Finding the Cone Combinations Best Representing Spectral Datasets

Eight cone opsins are expressed in zebrafish retina [16]. The algorithm to identify the cone opsin signals generating the ERG spectral shape is based on the axiom that the tiny radial photocurrents of individual cones sum linearly in the extracellular space to produce a net extracellular current which, by traveling through extracellular resistivity, generates ERG photovoltages. We assume that individual cone photocurrents relate to irradiance through Hill functions of exponent 1.0 and semi-saturation irradiance σ [17], and that σ varies with wavelength in inverse proportion to each opsin absorbance. This scheme is represented in Equation (1).
V = ∑ i = 1 8 V m i × I I + k i A w l m a x i ,   w l
V is the net summed photovoltage in the cone ERG (cone PIII) which depends on I, the stimulus irradiance in quanta and wl, the wavelength. The Vmi values are non-linear fit values, the maximal or saturation voltages for each of the i cone types. The semi-saturation irradiance for the ith cone is ki, as evaluated at the ith cone absorbance maximum. The ki values differ among cone types relative to each other. The relative ki values, expressed as log(ki) relative to UV-cone sensitivity, are literature values [18] listed in Figure 5B. The UV-cone value for log(k) is fit by the algorithm; the others are given relative to UV. A(wlmaxi, wl) is absorbance as a function of wavelength (wl, Figure 5B) for the ith cone, whose wavelength maximum is wlmaxi. The maximum wavelengths are also literature values [18]. The Dartnall nomogram [19] is used as the absorbance function, A(wlmaxi, wl). This approximation has the convenience of making opsin absorbance across wavelengths a function of a single variable, wlmaxi. The Dartnall nomogram posits that opsin absorbance shape is constant when plotted on a reciprocal wavelength axis. The template nomogram functions derive from suction electrode recordings of cones in Danio aequipinnatus [20], a relative of the zebrafish (Danio rerio). These templates are represented as 8th order polynomials. We use a single template polynomial [21] for all red, green, and blue cones listed in Figure 5B, but a separate polynomial for UV cones [20]. The resulting absorbance shapes appear in Figure 5A. Altogether there are nine parameters fit by the algorithm: eight Vmi values, and a single ki value (kUV). The spectral algorithm (Equation (1)) sums signals from 8 cone types (Figure 5A,B). These include a UV cone, two blue cones (B1, B2), three green cones (G1, G3, G4) and two red cones (R1, R2). The gene equivalencies of this nomenclature and the functionally measured wavelength peaks are given in Figure 5B. The spectral algorithm chooses among 28-1 or 255 unique cone combinations that might best represent a cumulative ERG spectral dataset. Each cone combination is called a model. The best model is chosen based on four constraints: (1) The model iteration must converge; (2) all model Vmi values must be significantly greater than zero (t-test, p ≤ 0.05); (3) all Vmi values must be less than 2.0, so as not to greatly exceed the largest amplitudes in the cumulative datasets; and (4) the value of r2 for the fit must be larger than that of any other model, as determined by the F-test for residual variance. If equivalent models are found (F-test, p ≥ 0.95), they will be noted. A similar modeling algorithm has been used to determine the cone combinations impinging on larval ganglion cell impulse discharges [22].
Figure 5. Algorithm for determining the signal strengths of the different cone types contributing to ERG spectral data. Aspartate-isolated cone signals from zebrafish eyes are a summation of signals from 8 cone types, each distinguished by different maximal amplitudes (Vmi), semi-saturation irradiances (ki), and opsin peak absorbances (wlmaxi). There are 255 unique combinations of 8 cones, each combination is a candidate to best model cone spectral responses in the ERG. (A) Spectral shapes of opsin absorbances [A(wlmaxi, wl)] are generated from 8th-order template polynomials [20,21] using Dartnall nomogram translations along the wavelength axis to represent opsins of different peak wavelengths [19] (Dartnall, 1953). The dashed line is the sum of the individual spectra. (B) Parameters for each of the i cone types are numbered in order from short to long wavelengths. SWS, short-wavelength-sensitive opsins; RH2, rhodopsin-like green-cone opsins; LWS, long-wavelength-sensitive opsins.

2.5. Knockout of miR-183 Leads to Attenuated ERG Responses

The ERG b-wave mainly represents the electrical signals of the massed downstream retinal ON bipolar cells, including cone-to-bipolar cell synapse changes in b-waves [23]. We also analyzed regular white-light b-wave ERG responses and cone PIII responses at 10 mpf. Cone PIII responses represent the isolated cone-only ERG responses obtained by blocking signals from inner retinal neurons using L-aspartic acid to block photoreceptor synapses. The time-course of the b-wave of miR-183 KO fish is altered for white-light stimuli, which causes a significant delay in miR-183 b-wave peak times (Figure 6A), while the overall b-wave amplitude over varying intensities is not altered (Figure 6B). Spectrally, the miR-183 KO shows loss of both long- and short-wavelength wave sensitivity in response to 330–410 and 570–650 nm stimulating wavelengths, but little difference in the mid spectrum (Figure 6C). The model was used to infer the manner in which cones’ signals operate in concert to produce the eye’s b-wave spectrum. The optimal model for the WTTL background strain uses signals from only three of the eight zebrafish cones: the Vr556 LWS2 cone (R2), the Vg460 RH2-1 cone (G1), and the Vb415 SWS2 cone (B1). The miR-183 KO is best represented by a different model, one which contains the Vr556 LWS2 cone and the Vg460 RH2-1 cone, but not the Vb415 SWS2 blue-cone signal. logk is an additional model parameter. This is the number of quanta to half-saturate cones. miR-183 KO fish have a 0.17 log unit or about 75% larger logk value, meaning that the miR-183 KO is overall 75% less sensitive to the constant quantal stimulus (Figure 6C).
Figure 6. Cone PIII and b-wave responses in WT and miR-183−/− 10-month-old zebrafish. (A–C) The mean white light b-wave responses for miR-183 mutant (red traces) and WTTL (black traces) background strain eyes. (A) b-wave peak time, the median peak time of WTTL for all intensities was 160 ms compared to 205 ms for MiR183 (U = 1518, p < 0.05). (B) B-wave amplitude measured a-wave trough to b-wave peak. (C) Mean b-wave traces with light intensity (given in log units white light attenuation). miR-183, mean of 5 eyes; WTTL control, mean of 20 eyes. (D–F) Isolated photoreceptor-only ERG responses (cone PIII) obtained by blocking signals from inner retinal neurons using L-aspartic acid. (D) Cone PIII peak time, (E) cone PIII amplitude, (F) mean cone PIII traces at different light intensities (given in log units white light attenuation). miR-183 (red traces), mean of 16 eyes; WTTL control (black traces), mean of 12 eyes. (G) Adult b-wave spectral sensitivity. Asterisks are Mann–Whiney U tests comparing the intensity response datasets at each measured wavelength and at all wavelengths, miR-183, 1050 datapoints; WTTL, 1820 datapoints. (H) Adult cone PIII spectral sensitivity with Mann–Whitney U tests at each wavelength and at all wavelengths, MiR-183,1820 datapoints; WTTL 1260 datapoints. Fish are 10 mpf. Error bars in graphs are s.e.m. * p < 0.05, ** p < 0.01, **** p < 0.0001. In (A,B,D,E). t-tests are used at individual irradiances and Mann–Whitney U tests for the overall intensity functions.
Interestingly, cone PIII responses of the miR-183 KO appear slightly lower in amplitude than WTTL, but are not statistically distinguishable, either by individual t-tests at each stimulus brightness, or in the overall intensity response datasets using Mann–Whitney U tests (Figure 6D,E, Table 1 and Table 2). Hence, cone phototransduction does not appear to be responsible for the change in white-light b-wave responses of the miR-183 KO. Furthermore, the cone PIII spectrum does not show a significant difference at wavelengths shorter than 490 nm, although there is a lot of scatter in the data (Figure 6F). The decreases in sensitivity become significant at stimulating wavelengths longer than 490 nm, which corresponds well with the loss in long-wavelength b-wave sensitivity. In contrast to the model for the b-wave, the modeled curve fits for PIII use seven of eight cone types represented for both WTTL and the miR-183 KO. Thus, in the miR-183 KO zebrafish retina there are blue cone signals in the cone layer, but these are apparently not transmitted efficiently to the bipolar cells (Figure 6F).
Table 1. Intensity response statistics at each wavelength and comparison of modeled cone signals in the absence of miR-183. The top panel of the table shows the results of the cone PIII analysis for the miR-183−/− and WTTL wild type mouse retinas. The bottom panel shows similar data for the b-wave response. The Mann–Whitney U test was used to analyze significant changes in intensity response characteristics at each tested wavelength on both b-wave and cone PIII spectral responses. * p < 0.05, ** p < 0.01, **** p < 0.0001, Mann–Whitney U test.
Table 2. Modeling was used to infer the amplitude of signals from different cone opsins. For the cone layer of both the miR183 mutant and the WTTL background, the modeling finds significant signal amplitudes for seven of the eight zebrafish cone opsins. The b-wave, which represents ON bipolar cells, selects only a few of these cone signals seen in the photoreceptor layer. For the WTTL background, significant signals are seen for three of the eight spectral types: The Vr556, LWS2 red cones, the Vg460, Rh2-1 green cones, and the Vb415, SWS2 blue cones. The miR183 mutant b-wave retains the same red and green cone signals but drops the blue cone signal, even though the blue cone signal is available in the sensory layer. The Mann–Whitney U test was used to analyze significant changes in intensity response characteristics at each tested wavelength on both b-wave and cone PIII spectral responses. ** p < 0.01, *** p < 0.001, **** p < 0.0001, Mann–Whitney U test.
Totally, there are significant changes in intensity response characteristics at each tested wavelength on both b-wave and cone PIII spectral responses. For cone PIII, in response to all wavelengths, there is a small but significant 11% reduction in the median amplitude (Figure 6F). This reduction appears concentrated at the longer test wavelengths, 530, 570, and 650. On these purely statistical measures the overall b-wave amplitude is much more significantly reduced, for all wavelengths, by 44%. The amplitude reduction is significant across both long and short wavelengths but not middle wavelengths (Table 1). Interestingly, the Vg460 green cone b-wave signal is significantly larger in the miR-183 KO than in WTTL control, even though the corresponding cone PIII is the same or a bit smaller (Table 2). In summary, the miR-183 KO ERG appears to affect primarily the b-wave with a smaller effect on the cone sensory layer in zebrafish. In more detail, the short-wavelength-sensitive cones seem to be more regulated by miR-183.

3. Discussion

In this study, we found a novel impact of miR-183 on specific wavelength-sensitive photoreceptor subtypes in teleost fish. The miR-183 cluster and its individual members have been well studied in mammals in recent decades. In the mouse, knockout of the entire cluster or miR-183 and miR-96 in combination leads to retinal degeneration associated with dysfunction of multiple additional organs [8,9,10]. Conversely, absence of individual genes, such as 183 or 182 alone, results in a relatively intact retinal structure, with only a slight decrease in ERG amplitudes. Initial results in the zebrafish have shown minimal effects on the retina but auditory hair cell degeneration and early death [13]. Here we show that knockout of miR-183 alone results in complex alterations in the cone populations, vision-related gene expression, and spectral sensitivity in the zebrafish retina as well as decreasing levels of miR-182.
The polycistronic microRNAs miR-183, -96, and -182 are grouped together as a family because they are clustered in the genome, share similar seed sequences and all prove to be extremely important for retinal development and homeostasis. However, the role of each gene in non-mammals, especially the seed sequence, is not yet fully understood. In the zebrafish the spatial expression pattern of each miRNA in the retina is similar (Figure 2 and Figure S1) and expression of the miRNA-183 cluster is consistent with that reported for expression of the miR-183 cluster and the β-geo cassette reported by Lumayag et al. in the mouse retina [9,15]. In miRNA binding, the seed sequence (nucleotides g2–g7 or 8 at the miRNA 5′ end,) is the minimal element needed for specificity and targeting efficacy, with over 80% of miRNA pairing occurring through seed sequence binding [24,25]. Previous studies have shown that knocking out the miR-183 cluster, or a combination of miR-183 and miR-96, causes retinal degeneration and point mutations in the seed region of miR-96, resulting in non-syndromic hearing loss in both human and mice [26,27].
Because the seed sequences of miRNAs are the primary determinant for targeting, one might suspect that the miR-183 cluster forms a redundant system, which is largely consistent with the above results. Further, all three members of the miR-183 cluster have a similar distribution within the retinal layers (Figure 2). However, members of this family are differentially expressed in time in the zebrafish retina [13], and have shown different effects in the auditory system [13,25,26]. The sequence of miR-183 differs from those of miR-182 and miR-96 at the 5′ base of the seed sequence and more in the 3′ sequence, with miR-183 and miR-182 being more closely related, and miR-96, particularly in zebrafish, being further removed (Figures S2 and S7), suggesting the three miRNAs might have different actions in the retina. In order to explore this possibility, we created a zebrafish model in which 11 bp within the miR-183 gene was deleted, removing the entire seed sequence, and compared it to a pre-existing model [13].
As might be expected from the preceding discussion, the effects of knocking out miR-183 are complex and appear to act at multiple levels of the retina. As with our previous study knocking out the entire miR-183 complex, the overall structure of the retina shows little change and there are minimal signs of retinal degeneration (Figure S3) and TUNEL stains of WTTL and miR183−/− zebrafish from 1–12 months of age show no apoptosis (Figure S5). However, knocking out miR-183 has a number of impacts on development of the zebrafish retina. Perhaps the least expected of these is the 40–50% decrease in miR-182 levels in miR-183 KO fish retinas. This observation was confirmed in both the dorsal and ventral retina and in both TL and AB fish strains. Upon careful inspection, it is also seen to some extent in the results of Fogerty et al., although it was not commented upon [13]. The mechanism through which this occurs is unclear. The lack of change in miR-96 levels suggests that it might not be at the transcriptional level. However, pri-miRNAs undergo extensive processing by DROSHA/DGCR8 to pre-miRNAs that are further processed by DICER to the miRNA duplex and finally AGO to form the mature miRNA, providing many potential regulatory points [1]. Finally, while miRNAs generally have a long half-life, they can be differentially degraded by target-directed miRNA degradation [1]. Regardless of the mechanism, it seems likely that the reduction in miR-182 levels might contribute to the phenotype observed in the miR-183 KO fish, and might in part explain the similar retinal degeneration caused by the miR-183 complex and combined miR-183/miR-96 KO mouse models [8,9,10].
Phenotypic changes in the miR-183 KO zebrafish occurred at three levels: photoreceptor number and types, mRNA levels of photoreceptor-associated genes, and ERG timing and amplitude for the b- and PIII waves. Each of these is distinct and their intercorrelation is not straightforward. Perhaps the most basic alteration is the decrease in UV and blue cones in the miR-183 KO retinas, while there are no apparent changes in red/green cones or rod photoreceptor density or outer segment length. Taken together with the lack of signs of apoptosis, this suggests that the affected photoreceptors might be failing to specify, failing to properly differentiate, or undergoing fate switching to an unexamined subtype. This is in contrast to the miR-183 c KO mouse model, in which M opsin-containing cones were decreased in the superior retina [9]. The latter is consistent with results suggesting, that in rats and gerbils, M-cones differentiate from S-cones [28] and that in mice S- and M-cones form an inferior-to-superior gradient with cones in the transitional area expressing both opsins [29]. Consistently, all cones in TRβ2-deficient mice express S-opsin, while none express M-opsin [30]. Similarly, mice lacking Rorb are deficient in rods but overproduce S-cones lacking outer segments, suggesting that Rorb might act upstream of Nrl and then Nr2e2 in rod differentiation [31,32]. Photoreceptor dysfunction with an increase in S cones relative to L and M cones has also been shown in human patients with TRβ mutations [33,34], and these findings have been recapitulated in human retinal organoids [35].
In contrast to mammals, zebrafish have four types of cones [16], generated from specific precursors through symmetrical terminal divisions in which trβ2 drives L-cone development [36]. Six7 has been shown to have two roles in zebrafish photoreceptor development, extending mitosis of photoreceptor progenitors to increase rods and inducing cell death of green cone precursors [37]. Development of photoreceptor progenitors depends on a complex interaction of gdf6a, which reduces blue and red cones, thrβ, which reduces red cones but increases UV and blue cones, and tbxb2, which reduces UV cones in gdf6a mutants [38]. Mutant trβ2 fish show absence of red cones as well as a decrease in red and an increase in UV cone activity on ERG and optokinetic responses [39]. It seems likely that miR-183 acts by modulating expression of one or more of these pathways.
Alterations in mRNA expression of photoreceptor-related genes in miRNA183−/− fish follow a somewhat different pattern from the changes in cone types (Figure 4). Additionally, as miRNAs, which have a variety of mechanisms of action, exert much of their effect at a translational level, the observed changes in mRNA levels presumably represent downstream effects of miRNA 183 activity [1]. Finally, as the retina is extremely complex at a cellular level, the analysis was mostly restricted to genes specific to or enriched in specific photoreceptor types. At 3 months of age there are no decreases in short-wavelength opsin mRNAs as might be expected, but rather increases in rhodopsin, gfap, and rh2-1 and rh2-4, two of the medium wavelength opsins found in green cones. The increase in gfap suggests an increase in macroglial activity including Müller cells and astroglia, which provide structural support for the retina as well as vascularization and metabolic support for retinal ganglion cells [40]. By 12 months of age the KO zebrafish retinas show large increases in rh2-1 and arr3a as well as a continuing moderate increase in rh2-4 expression. Levels of grk1a and gnat1 are also somewhat increased, perhaps reflecting rod activity, although rho mRNA is only suggestively increased. Conversely, the decrease in sws2 mRNA is consistent with the decrease in blue cones, although sws1 is not significantly changed. Finally, rh2-2 and rh2-3 are both decreased, perhaps reflecting shifts within the green cone population. The large increase in arr3a is also consistent with the similarly large increase in rh2-1, with which it pairs, resulting in an overall increase in cone opsins. It is interesting that the changes in photoreceptor-related mRNAs between 3 and 12 months of age suggest the continuing activity of miRNA 183 beyond the initial period of retinal development, consistent with the findings of Busskamp et al. in adult mice lacking miRNAs 182 and 183 [7].
Similarly, ERG changes in the miR 183 KO fish are not well correlated with the cone number or gene expression in a straightforward way. The b-wave peak time is slightly increased, and these results are consistent with those of Lumyag et al. in the miR-183 cluster KO mouse [9]. However, unlike that mouse model the miR-183 KO zebrafish did not show any decrease in the white-light b-wave amplitude, which probably relates to the presence of retinal degeneration in the mouse but not the zebrafish model. In contrast, the b-wave amplitude across the wavelength spectrum is altered in the miR-183 KO fish, showing decreased amplitude in the 330–420 nm range, and somewhat surprisingly also in the 570–650 nm range, which reflects stimulation of the red cone opsins lws2 and lws1, which show no change in mRNA levels. However, the PIII amplitudes, which directly reflect stimulation of the cone responses, also show a decreased amplitude across this region, as well as a suggestive but not statistically significant decrease in the 410–570 nm range, suggesting that the b-wave changes might result from decreases in red cone activity. Surprisingly, considering the decreased number of UV and blue cone numbers, the converse holds for responses to stimulating wavelengths in the 330–410 nm range. Here the PIII amplitude is not significantly different in KO and control fish, while the b-wave amplitude is significantly decreased, suggesting that transmission from the UV and blue photoreceptors to second order retinal neurons, and especially ON bipolar cells might be decreased [39,41].
While miRNAs can affect gene expression at multiple points, it seems most likely that miR-183 primarily exerts its effects by blocking translation or splicing of target mRNAs [1]. From the miRbase (https://www.mirbase.org/, accessed on 1 October 2025) [42] and miRDB (https://mirdb.org/index.html, accessed on 1 October 2025) [43] there are 565 predicted targets for has-miR-183-5p (Table S2), of which 62 are expressed in the retina and, of these, 46 are also targets of miR-182. Among these, myocyte enhancer factor 2C (MEF2C) [44,45,46], visual system homeobox2 (VSX2) [47,48], profilin 2 (PFN2), CUB and Sushi multiple domains 1 (CSMD1) [49] have known roles that might affect retinal development and function. In particular, Mef2c has been shown to influence expression of a number of photoreceptor genes, including Arr3 and Oph1mw, and overexpression of Mef2c in retinal explants promotes cone photoreceptor generation at the expense of bipolar and amacrine cells and delays photoreceptor maturation [44,46]. Similarly, the homeodomain transcription factor Vsx2 maintains multipotency in retinal progenitors by repressing Vsx1 and Ath5, which subsequently affect retinal cell lineages [47]. These target genes provide strong candidate mechanistic pathways for miR-183 effects on retinal and especially photoreceptor development.
In conclusion, miR-183 supports the differentiation of short-wavelength-sensitive photoreceptors, including blue and UV opsin cones. This is accompanied by changes in the expression of genes specific to various photoreceptors. Finally, ERG responses demonstrate changes in the transmission of visual signals to inner retinal cells and photoreceptor-only ERG responses show reduced ERG amplitudes of several specific wavelength-sensitive photoreceptor subtypes. Overall, our results reveal a novel impact of miR-183 in zebrafish, especially regarding the specification of specific wavelength-sensitive photoreceptor subtypes and their higher order neuronal connections.

4. Materials and Methods

4.1. Generation of the miR-183 Knockout Zebrafish Model

Transcription activator-like effector nuclease (TALEN) technology was utilized to generate the miR-183−/− zebrafish model. TALEN requires two arms for binding to the 5′ and 3′ ends of the target to achieve the final DNA cleavage effect. We designed the TALEN binding domain (left arm target sequence: 5′-TGACAGACTCCTGTTCT-3′, right arm target sequence: 5′-TTTCACAGTGAATTCTA-3′) and synthesized the plasmids according to procedures prescribed previously [50]. RNA for zebrafish egg microinjection was synthesized using T7 mMESSAGE mMACHINE kit (ThermoFisher, Waltham, MA, USA). The freshly synthesized TALEN RNA was dissolved in injection buffer (0.03% phenol red, 20 mM HEPES, pH 7.4) and injected into zebrafish egg embryos at the 1–2 cell stage. For genotyping, zebrafish tail genomic DNA was extracted and assayed using polymerase chain reaction (PCR) primer sets (Supplementary Table S1) specific for the miR-183−/− allele. AB strain zebrafish with members of the miR-183 cluster knocked out were constructed as previously described [13].

4.2. Zebrafish Maintenance and Breeding

Zebrafish were maintained in an automated fish housing system following standard protocols approved by the NEI ACUC, proposal NEI-659, last approved on 14 November 2024. Embryos were maintained with fish embryo medium in an incubator (50125590, Thermal Fisher Scientific, Inc., Waltham, MA) at 28.5 °C.

4.3. Preparation and Perfusion of Isolated Eyes for ERG Recording

Adult eyecups were prepared from eyes removed from fish decapitated with a fresh, single-edged razor. Corneas and lenses were removed from the isolated eyes that were then mounted upright on a 5–10 mm square of black nitrocellulose paper, and the preparation was placed in a recording chamber. A perfusion applicator (28-guage microfilm syringe needle, MF28G67; World Precision Instruments, Sarasota, FL, USA) was placed directly above the retina, oxygenating the vitreal surface either with MEM for b-wave recording, or MEM containing 10 mM L-aspartate for the recording of isolated cone signals (cone PIII). The perfusion rate was set at 0.3 mL/min using a syringe pump (New Era 500L; Braintree Scientific, Braintree, MA, USA). Tungsten microelectrodes (Frederick Haer, Bowdoin, ME, USA, 75 mm, #30031) were placed in the eyecup to record ERG signals. ERG signals from the microelectrode were amplified by 10,000 (World Precision Instruments, Sarasota, FL, USA, DAM80, 0.1 Hz-1k Hz bandpass), and digitized (2000 Hz) with an Axon instruments 1440A (Molecular Devices, San Jose, CA, USA) using Clampex 10 software.

4.4. White-Light ERG Stimulus Protocol

A staircase of seven white-light intensities (150 W OFR Xenon arc source, Science-Lighting, Seattle, WA, USA) was presented using UV-compliant neutral density filters (0.5 log unit increments covering 3 log units, Andover Corporation, Salem, NH, USA, each intensity step repeated 4 times). The intensity staircase was repeated 10 times (280 stimuli total, the ‘white light’ dataset). The optical source was divided into two channels gated by Uniblitz shutters (Vincent Associates, Rochester, NY, USA). The first (300 ms flashes) provided the ERG stimuli. The second provided infrared background illumination for observation. The stimulus channel contained three software-driven Sutter filter wheels and a UV–visible-compliant liquid light guide (Sutter Instruments, Novato, CA, USA) coupled to the epifluorescence port of a BX51WI upright microscope (Olympus-Life Science Solutions, Tokyo, Japan) and a 4× UPlanSApo/0.16 objective. Two of the stimulus-channel filter wheels adjusted intensity, the other selected either wavelengths or white light. The interval between stimuli varied between 2.5 and 6 s, with the longer intervals separating the brighter stimuli. White-light intensity response series are means acquired from white-light protocols.

4.5. Spectral ERG Stimulus Protocol

Eyecups were stimulated with nine wavelengths ranging from 330–650 nm (20 nm half-width interference filters, 40 nm increments, Chroma Technology, Bellows Falls, VT, USA). Seven intensities were presented at each wavelength (as above). A calibrated photodiode (Newport Corporation, Greensboro, NC, USA) was used to determine stimulus irradiance in quantal units. This was placed in the plane of the retina. Spectral model calculations are based on absolute, wavelength-specific photodiode calibrations of quanta delivered. The spectral protocol was a fixed software-driven sequence of 280, 300 ms, monochromatic light flashes of different irradiances and wavelengths. Among these stimuli were 64 unique irradiance–wavelength combinations and 6 replicates delivered 10 min apart as an amplitude stability check during the 20 min protocol. The 280 stimuli created a set of 70, 4X-averaged, electroretinogram (ERG) responses, the ‘spectral dataset’. The maximal irradiances in log(quanta·µm−2·s−1) in the stimulus protocol were 7.4 (650 nm), 6.4 (610 nm), 6.5 (570 nm), 6.5 (530 nm), 6.7 (490 nm), 6.5 (450 nm), 6.1 (410 nm), 6.1 (370 nm), and 5.9 (330 nm). The spectral protocol does not record zebrafish rod signals as the retinas are not dark adapted, saturating stimuli are used, and the inter-stimulus intervals are short [51]. There are four rods surrounding each UV cone in adult zebrafish [52]. Slow-peaking (~400 ms) rod b-waves are only recorded after extensive dark adaptation in adults by avoiding suprathreshold stimuli and with interstimulus intervals of 15 s or greater [51].

4.6. Analysis of ERG Signals

Datasets were imported into Origin analysis software and processed using Origin LabTalk scripts (Origin, version 8.5; Originlab Corporation, Northhampton MA, USA). The 4 replicate waveforms at each of the 70 wavelength and irradiance combinations were averaged and boxcar filtered (17 ms, one 60 Hz line-frequency cycle). Trough-to-peak b-wave amplitudes were calculated during 300 ms following the stimulus onset and cone PIII (aspartate-treated) trough-to-peak amplitudes were extracted during 850 ms following the stimulus onset, an interval including both the initial hyperpolarizing trough and post-stimulus repolarizing peak. Each amplitude was associated in Origin with the wavelengths and irradiances of stimulation, providing 70 wavelength, irradiance, and amplitude data points for each spectral dataset. Datasets with unstable response amplitudes over the collection period were excluded. For controls and KOs, datasets from multiple eyes were each normalized to the maximal eye response and combined to form a cumulative dataset, which was fit to a 255-member set of spectral models. The amplitude normalization weighted the individual datasets making up the cumulative dataset equally. Combining datasets from many eyes separated the trends in genetic alterations of spectral properties from the variations among individual eyes. Non-linear model fits to spectral datasets used the Levenberg–Marquardt iteration algorithm provided by Origin (www.OriginLab.com, Northampton, MA, USA). The analysis finds the best spectral model and extracts the signal amplitudes of contributing cone types.

4.7. miRNA Expression Level and Localization Measurement by miRNAscope Assay

The miRNAscope Assay (miRNAscope™ HD Reagent Kit-RED, Cat. No. 324500. Advanced Cell Diagnostics, Newark, CA, USA) was used to visualize specific miRNA expression following the manufacturer’s directions. Each probe’s information was as follows: dr-mir-183-S1 (1206971-S1) targeting 3-21 of mature mir-183; dr-mir-96 (1206981-S1) targeting 2-23 of mature mir-96; and dr-mir-182 (1206991-S1) targeting 2-22 of mature mir-182. Briefly, the RNAscope procedure includes two steps. Step 1, sample preparation, includes preparation and pretreatment of the eye cup. Eye cups were fixed with freshly prepared 4% paraformaldehyde (PFA) in 1X PBS and then immersed in 10% sucrose in 1X PBS at 4 °C overnight, followed by sequential immersion in 20% and 30% sucrose, freezing in OCT and storage at −80 °C. The slides were air dried for 120 min, washed with PBS, and baked for 1 h at 60 °C. They were then post fixed by chilling in 10% NBF for 15 min and the tissue was dehydrated by immersion sequentially in 50%, 70%, and 100% EtOH for 5 min. Samples were post-fixed with fresh 10% NBF overnight at RT. RNAscope hydrogen peroxide was applied to tissues for 10 min at RT and the samples were then washed with distilled water. The tissues were then incubated with Protease III at 40 °C followed by washing with distilled water. Step2 includes the miRNAscope assay. Samples were incubated with miR-183, -96, 182 and control probes for 15 min at 40 °C in a water bath. The probes were then hybridized by incubating the probes with samples in a HybEZ™ Oven (Advanced Cell Diagnostics, Newark, CA, USA) for 2 h at 40 °C, followed by washing the slides with 1X wash buffer. The samples were then incubated with Amp 1 in an oven for 30 min at 40° and washed with the wash buffer. The samples were then incubated with Amp 1 in an oven for 30 min at 40 °C and then washed with 1X wash buffer. The samples were then sequentially incubated with Amp 2, Amp 3, Amp 4, Amp 5, and Amp 6 in an oven at 40 °C for 15–30 min. The red working solution was prepared by mixing a 1:60 ratio of Fast Red B with Fast Red A. The samples were incubated with the red working solution for 10 min at room temperature, and the slides were then washed with wash buffer. The miR-183, -96, and 182 probe signals were evaluated using the RNAscope scoring guidelines.

4.8. Zebrafish Histology and Immunostaining of Cryo-Sections

Fixed tissues of 4% paraformaldehyde were equilibrated in 30% sucrose and embedded in OCT (Neg-50, Thermal Fisher Scientific, Inc., Waltham, MA). Frozen sections of 8 μm thick were performed on a cryostat (CM3050, Leica Microsystems, Wetzler, Germany). They were then blocked with 5% goat serum and incubated with anti-Zpr-3 (1:300, Zebrafish International Resource Center, Eugene, OR, USA), or anti opsins (anti-UV, 1:1000; anti-blue 1:250; anti-green, 1:500; anti-red, 1:500, ZIRC, Eugene, OR, USA). After the sections were rinsed with PBS, slides were incubated with Alexa594- or Alexa488-conjugated IgG (1:300, ThermoFisher, Waltham, MA, USA) and with DAPI (Invitrogen) for nuclear staining. Samples were imaged using a Zeiss (Oberkochen, Germany) LSM 700 confocal microscope. Sections without primary antibody exposure were used as negative controls. Numbers of photoreceptor cells were measured using ImageJ software (version 1.54p, https://imagej.net/ij/ accessed on 30 April 2025, used on the NIH platform). An unpaired Student’s t test statistic t was used for comparisons, and p < 0.05 was considered statistically significant.

4.9. RNA Isolation and qRT-PCR

Total RNA was extracted from the zebrafish retina. The retinas were dissected, and RNA was extracted with TRIzol reagent (15596026, ThermoFisher, Waltham, MA, USA). RNA concentration and quality were measured using a NanoDrop ND-2000 spectrophotometer (Thermal Fisher Scientific, Waltham, MA, USA). qRT-PCR was performed using the ViiA 7 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) to detect photoreceptor-related gene expression. M-MLV reverse transcriptase (28025021, Thermal Fisher Scientific, Inc.) was used to reverse transcribe 1 μg total RNA to cDNA according to the manufacturer’s protocol. An amount of 50 ng cDNA was mixed with PowerTrack™ SYBR Green Master Mix (A46012, Thermal Fisher Scientific, Waltham, MA, USA) and photoreceptor-related gene primers (Supplementary Table S1) at a final concentration of 1 μM to produce a 15 μL reaction mixture. Standard cycling steps were performed as previously described. Briefly, first: initial denaturation, 94 °C, 2 min. Second: denaturation, 94 °C, 15 sec; annealing, extension, and read fluorescence, 60 °C, 1 min (40 cycles). Third: 4 °C. The 2−ΔΔCt method was used to analyze the relative changes in photoreceptor-related genes expression.

4.10. TUNEL Assay

The Click-iT™ Plus TUNEL Assay (Cat. No. 324500, Thermal Fisher Scientific, Waltham, MA, USA) was used to visualize cell death or apoptosis following the manufacturer’s directions.

4.11. Statistical Analyses

For zebrafish white-light b-wave and Cone PIII responses, t-tests were used at individual irradiances and Mann–Whitney U tests for the overall intensity functions. To determine the most appropriate spectral models, F-tests on residual variances for different spectral models were employed. The difference between WTTL and miR-183 knockout zebrafish are compared using Mann–Whiney U tests comparing the intensity response datasets at each measured wavelength. Student’s t-test was performed to analyze the immunostaining and real-time quantitative reverse transcription PCR statistical significance. Error bars of all of the results represent the SEM. Significance levels are indicated by * for p < 0.05; ** for p < 0.01; and *** for p < 0.001.

5. Conclusions

Overall, these results suggest that miR-183 acts at multiple levels, either directly or by affecting activity of a pathway, to promote differentiation of retinal cone populations and to influence the expression of genes active in retinal photoreceptors and probably the transmission of signals from the photoreceptors to second-order retinal neurons. While the pattern of effects is complex, this is not surprising considering the multiple levels on which microRNAs act and the partially overlapping targets of the miR-183 complex. This analysis is complicated by the partial dependence of miR-182 expression on miR-183 and the difficulty of delineating direct actions of miR-183 from downstream influences and even compensatory changes, all of which are likely occurring in the miR-183 KO fish retinas. In spite of these caveats, these results demonstrate the importance of miR-183 for the development and maintenance of non-mammal short-wavelength-sensitive photoreceptors in the zebrafish tetrachromatic visual system, providing new insights into the miR-183 target regulation in the retina.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms27052433/s1.

Author Contributions

Conceptualization, R.C., X.J., L.D., B.D.P. and J.F.H.; formal analysis, R.F.N. and R.C.; investigation, R.C., R.F.N., X.J. and J.F.H.; methodology, R.C., R.F.N., G.Z. and V.P.C.; supervision, J.F.H.; writing—original draft, R.C.; writing—review and editing, R.C., G.Z., B.D.P. and J.F.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Eye Institute, ZIA EY000272-33.

Institutional Review Board Statement

All work included here was as last approved on 14 November 2024 by the Animal Care and Use Committee (ACUC) of the National Eye Institute in NIH ASP NEI-659 and adhered to the Association for Research in Vision and Ophthalmology (ARVO) statement on animal use in ophthalmic and vision research.

Data Availability Statement

All relevant data are included in the manuscript and Supplementary Materials.

Acknowledgments

This research was supported [in part] by the Intramural Research Program of the National Institutes of Health (NIH) and the National Eye Institute. The contributions of the NIH authors were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered works of the United States Government. However, the findings and conclusions presented in this paper are those of the authors and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. We would like to acknowledge the National Eye Institute Genetic Engineering Core Facility and the Biological Imaging Core Facility.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
miRNAMicroRNA
AGOArgonaute complex
KOKnockout
UVUltraviolet
ERGElectroretinogram
PIIIPhotoreceptor-only electroretinogram
NtNucleotide
miRISCSilencing complex
CSNSensory nerves
CRMCsCorneal resident myeloid cells
TALENTranscription activator-like effector nucleases
WTTLWild-type zebrafish of a TL background

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