The Developmental Progression of Eight Opsin Spectral Signals Recorded from the Zebrafish Retinal Cone Layer Is Altered by the Timing and Cell Type Expression of Thyroxin Receptor β2 (trβ2) Gain-Of-Function Transgenes

Cone distributions in the larval retinas of wild-type and crx:trβ2 transgenics

Suzuki et al. (2013) developed the crx:mYFP-2A-trβ2 gain-of function transgenic as a rescue line to restore red-cones during a morpholino blockade of native trβ2 that abolished their formation. In zebrafish the crx gene promotor becomes activate at the retinal progenitor stage (∼2 dpf; Shen and Raymond, 2004). The gain-of-function crx:trβ2 transgene replaces the missing retinal trβ2, but not in the same cell types or at the same developmental stage. In the experiments of Suzuki et al. (2013), it was nonetheless effective. Red-opsin immunoreactive cones were restored with supernormal density, but there was suppression of green-opsin, blue-opsin, and UV-opsin immunoreactive cones (Suzuki et al., 2013).

In Figure 3A,B, the experiment of Suzuki et al. (2013) is repeated on crx:mYFP-2A-trβ2 larvae without morpholino suppression of native trβ2. In wild-type retinas, immunoreactive mosaics of both UV cones (Fig. 3Ai) and red cones (Fig. 3Aiii) stain with opsin antibodies. Superposition of both mosaics (Fig. 3Aii) show no opsin overlaps, with each cone type expressing a single opsin. The situation is similar for blue cones, which exist in a mosaic pattern separate from red cones (Fig. 3Aiv,v,vi).

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Figure 3.

Opsin distributions and spectral responses in embryonic wild-type and crx:mYFP-2A-trβ2 larval eyes. Ai, UV opsin (SWS1) immunoreactive cones in a WT retina. Aiii, Avi, Red opsin (LWS1, LWS2) immunoreactive cones in WT retinas. Aiv, Blue-opsin (SWS2) immunoreactive cones in a WT retina. Aii, UV and red opsins are expressed in separate cones in a WT retina. Av, Red and blue opsins are expressed in separate cones in a WT retina. Bi, UV-opsin immunoreactive cones in a crx:trβ2 retina. Biii, Bvi, Red-opsin immunoreactive cones in crx:trβ2 retinas. Biv, Blue-opsin immunoreactive cones in a crx:trβ2 retina. There are fewer UV and blue cones in crx:trβ2 retinas than in WT retinas. Bii, One crx:trβ2 cone is immunoreactive for both UV and red opsins (arrowhead). Bv, Arrowhead points to a crx:trβ2 cone immunoreactive for both red and blue opsins. C, Cone signals from a WT larval eye respond to all stimulus wavelengths with largest amplitudes at 490 nm. D, A larval crx:trβ2 retina responds with maximal amplitudes at wavelengths 490, 570, and 650 nm but is less responsive than WT for 330, 410, and 490 nm (arrows), wavelengths that stimulate blue and UV cones. A, B, 5-dpf larvae. C, D, 6-dpf larvae. Perfusion medium contains 20 mm aspartate to isolate photoreceptor signals in the ERG. Five of nine stimulus-protocol wavelengths are illustrated. The stimulus irradiances [units of log(quanta·μm⁻²·s⁻¹)] appear in legends to the right of stacked irradiance-response traces.

Red cones are denser in the retinas of the crx:mYFP-2A-trβ2 larvae (Fig. 3Biii,vi) than in WT (Fig. 3Aiii,vi). Based on Figure 3A,B, the density of red-opsin immunoreactive cones in these 5-dpf crx:trβ2 transgenics (188,000 mm⁻¹) is significantly greater than WT (72,500 mm⁻¹; t₍₁₀₎ = 18.0, p = 6.0 × 10⁻⁹). The density of crx:trβ2 UV opsin immunoreactive cones (7900 mm⁻¹; Fig. 3Bi) is significantly less than WT (42,000 mm⁻¹; Fig. 3Ai; t₍₆₎ = 7.0, p = 0.00043). One of the three crx:trβ2 UV cones (Fig. 3Bi) stains for both UV and red opsins (Fig. 3Bi–iii, arrowhead), indicating co-expression of red and UV opsins. The density of crx:trβ2 blue-opsin immunoreactive cones (13 800 mm⁻¹; Fig. 3Biv) is significantly less than WT (30,500 mm⁻¹; Fig. 3Aiv; t₍₆₎ = 4.7, p = 0.0033). Of four transgenic blue cones illustrated (Fig. 3Biv), one is immunoreactive for red opsin (Fig. 3Biv–vi, arrowhead), evidence for co-expression of red and blue opsins in a single cone. The alterations in densities of cone types resemble findings obtained in the presence of morpholino blockade of native trβ2 (Suzuki et al., 2013) suggesting the crx:trβ2 transgene alters cone developmental patterns regardless of the activity of the native trβ2 gene. The difference is the mixed opsin cones, which were not earlier described.

Cone spectral signals from wild-type and crx:trβ2 retinas in larvae and adults

The spectral pattern of aspartate-isolated cone signals from larval crx:mYFP-2A-trβ2 eyes parallels the altered cone densities. With short and UV wavelength stimulation (410 and 330 nm; Fig. 3C), an isolated WT eye responds with substantial signals for three of the brightest stimulus irradiances (green, dark yellow or red traces) but at these same wavelengths, signals from a crx:trβ2 eye (Fig. 3D) are evident at only the two brightest irradiances (dark yellow and red traces) and are of lesser amplitude than WT. Maximal amplitudes were evoked at 490 nm in the WT eye (Fig. 3C), while amplitudes in the crx:trβ2 eye plateaued at longer wavelengths (490, 570, and 650 nm; Fig. 3D). The loss of amplitudes at 490 nm (Fig. 3D) is not a rod signal loss, as no change in the time to peak of threshold signals is apparent.

Adult spectral differences in crx:mYFP-2A-trβ2 and WT eyecups were even more pronounced than those in larval eyes. In the WT traces, the maximal amplitude occurred at 490 nm (Fig. 4A), whereas the maximal amplitude from a crx:trβ2 eyecup was seen at 650 nm (Fig. 4B). Amplitudes at 410 and 330 nm (Fig. 4B) were greatly reduced as compared with the WT control (Fig. 4A). At these short wavelengths WT signals were more sensitive than crx:trβ2 signals, with the dimmer stimuli (Fig. 4A,B, gray and blue traces) evoking deflections in WT, but only overlapping baselines in crx:trβ2 eyecups. The slower-peaking, mid-spectral threshold signals of rods are not evoked in either WT or crx:trβ2.

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Figure 4.

Adult cone spectral signals in WT and crx:mYFP-2A-trβ2 retinas. A, In WT eyecups, all wavelengths and most irradiances evoke signals, with a maximal response at 490 nm. B, Cone signals from crx:trβ2 eyecups are maximal at 650 nm, where they are relatively greater than WT (up arrow). Response amplitudes to wavelengths that stimulate blue and UV cones (330, 410, and 330 nm) are relatively less than WT (down arrows). In A and B, adults are 8–18 months. The perfusion medium contains 10 mm aspartate to isolate photoreceptor signals. The nested responses are irradiance-response series at each wavelength, with irradiances given in legends to the right of the traces in units of log(quanta·μm⁻²·s⁻¹). C–E, Properties of ERG cone-PIII waveforms in larval and adult, WT and crx:trβ2 retinas. C, Distributions of maximal trough-to-peak amplitudes in larval and adult spectral datasets. D, Cone-signal latency from stimulus onset to the minimum of the ON trough of the cone signal are measured on the mean responses of each 280-stimulus spectral dataset. E, Cone-signal latency to the OFF peak in the mean responses of spectral datasets. OFF latencies are measured from stimulus offset. Asterisks (n.s., not significant) are probabilities that WT and crx:trβ2 distributions differ in larvae or adults (GraphPad Prism convention, statistics given in text).

Wild-type and crx:trβ2 waveforms in larvae and adults

The crx:mYFP-2A-trβ2 transgene subtly changes the amplitudes and kinetics of aspartate-isolated cone signals (cone PIII). In larvae a greater mean amplitude and greater spread of amplitudes were found (Fig. 4C). The mean of maximal peak-to-trough responses in 6-dpf spectral datasets for 10 WT larval eyes was 89.6 ± 8.7 μV (28 datasets, mean and SE), while the mean of maximal amplitudes for 12 crx:trβ2 eyes was 145.0 ± 18.7 μV (29 datasets). The crx:trβ2 amplitudes were somewhat larger (t₍₅₅₎ = 2.65, p = 0.0103; Fig. 4C). The dispersion of larval amplitudes was also significantly greater for crx:trβ2 (F_(4.77,28,27) = 5.6 × 10⁻⁵, p = 0.00011). But on reaching adulthood, crx:trβ2 maximal PIII amplitudes became indistinguishable from WT counterparts (Fig. 4C). In 14 WT eyecups the mean of maximum peak-to-trough amplitudes was 34.4 ± 5.1 μV (20 datasets). In 16 crx:trβ2 eyecups the mean of maximal peak-to-trough amplitudes was 32.4 ± 6.1 μV (21 datasets). The difference was insignificant (t₍₃₈₎ = 0.248, p = 0.806). The variance in maximal amplitudes was similar (F_(1.51,20,19) = 0.813, p = 0.375). Lesser amplitudes occur in adult eyecups than intact larval eyes as photocurrents escape around eyecup edges, whereas the larval eye is an electrically sealed system. Neither larval nor adult crx:trβ2 amplitude distributions (Fig. 4C) show evidence of amplitude loss, a characteristic associated with retinal degenerations.

“PIII ON trough” is the time interval from stimulus onset to the minimum in the vitreal negative phase of the cone PIII signal. In larvae, PIII-ON-trough latencies for the crx:mYFP-2A-trβ2 waveforms were similar to WT, but in adults the latency to the PIII ON trough was shorter (Fig. 4D). Response peak and trough timings were measured on the mean waveform for all 280 responses in a spectral dataset. In this average response, noise is minimized and interferes least with determination of extrema timing. For larval eyes the latency to the cone PIII trough for 29 crx:trβ2 datasets (12 eyes) was 135 ± 6 ms from stimulus onset (mean and SE). The trough time for 28 WT datasets (10 eyes) was 134 ± 5 ms. The WT and transgenic trough times were not significantly different (t₍₅₅₎ = 0.218, p = 0.828) and the trough-time variances were also similar (F_(1.80,28,27) = 0.935, p = 0.133; Fig. 4D). In 16 adult crx:trβ2 eyecups the mean latency to PIII ON troughs was 111 ± 3 ms (21 datasets). In 14 WT eyecups the PIII trough latency was 124 ± 5 ms (20 datasets). The adult crx:trβ2 transgenics were somewhat quicker to peak (t₍₃₉₎ = 2.133, p = 0.039). The variances in onset latencies was similar in crx:trβ2 and WT adults (F_(2.11,19,20) = 0.947, p = 0.106; Fig. 4D). For WT controls, larval and adult trough latencies did not differ significantly (t₍₄₆₎ = 1.45, p = 0.155). For crx:trβ2 transgenics trough times for adults were significantly quicker than for larvae (t₍₄₈₎ = 3.23, p = 0.0022).

“PIII OFF peak” (Fig. 4E) is the time interval from stimulus offset to the maximum of the upward course in the PIII OFF signal. The larval PIII-OFF-peak latencies for crx:mYFP-2A-trβ2 (360 ± 36 ms) did not differ significantly from WT controls (313 ± 17 ms, t₍₅₅₎ = 1.171, p = 0.247), although the variance in crx:trβ2 timing was significantly greater (F_(4.68,28,27) = 0.999, p = 1.3 × 10⁻⁴). In adults the crx:mYFP-2A-trβ2 OFF peak (262 ± 14 ms), like the ON trough, occurred significantly sooner than in WT (329 ± 20 ms, t₍₃₉₎ = 2.728, p = 0.0095). The variances of adult OFF-peak latencies were similar in crx:trβ2 and WT adults (F_(1.99,19,20) = 0.932, p = 0.135; Fig. 4E). For WT controls, larval and adult PIII OFF peak timing was not significantly different (t₍₄₆₎ = 0.600, p = 0.551). For crx:trβ2 transgenics PIII-OFF-peak times for adults were somewhat quicker than for larvae (t₍₄₈₎ = 2.234, p = 0.030). Taken together, the latency distributions (Fig. 4D,E) show little evidence of waveform abnormalities associated with errors in cone phototransduction or retinal disease. The quicker waveforms of crx:trβ2 adults may result from different weighting of contributing cone types.

In crx:trβ2 larvae, red-cone signals increase but UV-cone and blue-cone signals diminish

To determine the larval cone contributions affected by the crx:mYFP-2A-trβ2 transgene, 255 models comprising all combinations of eight cone spectral types, were fit to crx:trβ2 and WT cumulative spectral datasets. For the 6-dpf WT and crx:trβ2 larvae, the cumulative datasets included 1858 response amplitudes (28 datasets) and 1860 amplitudes (29 datasets), respectively. Optimal models differed (WT, #111; crx:trβ2, #202). The residual variance of the WT model (#111) as fit to the crx:trβ2 cumulative dataset differed from the residual variance of the crx:trβ2 dataset fit to its own optimal model 202 (F_{(9.16,1821,1825)} = 1, p = 0), indicating that the WT model is not an equivalently good representation of crx:trβ2 data.

In Figure 5A,B, amplitudes are plotted against irradiance for four of the nine test wavelengths. Continuous curves are calculated from the optimal models for WT and crx:trβ2 datasets. These curves are generated from a global model fit to all wavelength data, not just the illustrated data. The adherence of irradiance-response curves to datapoints for individual wavelengths is an index of the ability of the global models to represent the cumulative spectral data. The distribution of points and curves is more compressed along the irradiance axis for crx:trβ2 larvae (Fig. 5B) than for WT controls (Fig. 5A). The points and curve at 370 nm (magenta) lie to the left of the 490-nm points and curve (green) for WT spectral signals (Fig. 5A) but to the right of the 490-nm curve and points for crx:trβ2 larvae (Fig. 5B), suggesting a loss of sensitivity for 370 nm-sensing cones (UV cones). The log of semi-saturation irradiance calculated for the R1 and R2 cones was 4.56 ± 0.018 for WT larvae and 4.53 ± 0.027 for crx:trβ2 (Fig. 5A,B). These semi-saturation irradiances did not significantly differ (t₍₃₆₈₁₎ = 1.858, p = 0.063).

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Figure 5.

Spectral models of cumulative data from 6-dpf embryonic crx:mYFP-2A-trβ2 and wild-type eyes. A, WT irradiance-response datapoints, SEs, and optimal model (#111) curves as fit to 1858 spectral response amplitudes combined from 28 normalized datasets taken from 10 eyes (Fig. 1A, Eq. 1 algorithm); 370 nm, n = 23–28; 490 nm, n = 27–28; 570 nm; n = 23–28; 650 nm, n = 23–55. B, crx:trβ2 irradiance-response datapoints and optimal model (#202) curves fit to 1860 spectral points combined from 29 datasets taken from 11 eyes. Individual points: 370 nm, n = 22–28; 490 nm, n = 23–28; 570 nm, n = 27–28; 650 nm, n = 24–55. The crx:trβ2 transgene moves curves and points along the irradiance axis as compared with WT. C, In WT, six cone opsin types (gray bars) were detected by the optimal model. In crx:trβ2, four cone opsin signals were detected (magenta bars). Cone saturation amplitudes [Vm_i values (Eq. 1; Fig. 2A) ± SE] are fractions of dataset maximal amplitudes. Asterisks denote significance of differences between WT and crx:trβ2 (one-sample or two-sample t tests; n.s., not significant; GraphPad Prism convention). Vu358 (UV, one-sample test): t₍₁₈₂₂₎ = 17.8, p = 1.2 × 10⁻⁶⁵; Vb415 (B1): t₍₃₆₄₇₎ = 0.958, p = 0.338; Vb440 (B2): t₍₁₈₂₂₎ = 4.27, p = 2.0 × 10⁻⁵; Vg460 (G1): t₍₃₆₄₇₎ = 1.87, p =0.062; Vg500 (G4, one-sample test): t₍₁₈₂₂₎ = 3.33, p = 8.8 × 10⁻⁴; Vr556 (R2): t₍₃₆₄₇₎ = 0.318, p = 0.751; Vr575 (R1): t₍₁₈₂₅₎ = 2.72, p = 0.0067. D, Spectral peaks shift to longer wavelengths for crx:trβ2 (magenta) as compared with WT (black). Spectral curves are the modeled amplitudes that would be evoked by three different irradiances of constant quantal stimulation across the spectrum [3.4, 4.0, and 4.6 log(quanta·μm⁻²·s⁻¹)]. Spectral shapes differ with stimulus brightness. A, B, The log(k_R1R2) values are modeled R1-cone and R2-cone semi-saturation irradiances in log(quanta·μm⁻²·s⁻¹). A–D, 20 mm aspartate medium.

The inferred Vm_i values (Fig. 2, Eq. 1) relate closely to functional strength for the individual cone types comprising the 6-dpf larval cone-PIII signal. In Figure 5C, the modeled Vm_i values are compared for WT and crx:mYFP-2A-trβ2 larvae. Six Vm_i cone signals significantly contributed to WT cone PIII ERGs: Vu358-UV, Vb415-B1, Vb440-B2, Vg460-G1, Vg500-G4, Vr556-R2. Only four cone contributions were identified in crx:trβ2 larvae: Vb415-B1, Vg460-G1, Vr556-R2, Vr575-R1. Missing in the transgenic are UV cone signals (Vu358), B2 cone signals (Vb440), and G4 signals (Vg500). But an extra red-cone signal, R1 (Vr575), not detected in WT, became significant in crx:trβ2 larvae. Unaffected were the R2 (Vr556) cone signal, the B1 signal (Vb415) and the G1 signal (Vg460). Overall, the modeling algorithm shows that UV-cone amplitudes were significantly reduced, red-cone amplitudes significantly increased, and among four blue and green cones signals, two were significantly reduced and two were not significantly affected.

Modeled spectral sensitivities represent the impact not only of Vm_i but also k_i values and include Hill-function response compressions, which flatten cone spectral functions for bright stimulation (Eq.1; Fig. 2A). This is the fullest model representation, but less interpretable in terms of individual cone contributions. The spectral curves of Figure 5D are modeled response amplitudes for three levels of constant quantal stimulation across the spectrum. The strong UV signal in WT larval eyes (Fig. 5A) appears as an ultraviolet spectral peak (∼370 nm) in Figure 5D, regardless of stimulus brightness, despite the larger saturation amplitudes of the R2 (Vr556) cones, which, in WT controls, manifest as a long-wavelength bulge more prominent with greater constant-quantal irradiances, which better stimulate the higher semi-saturation characteristics of R2 physiology. The failure to find UV cone signals in the crx:trβ2 larval transgenics precludes an ultraviolet spectral peak at any quantal-irradiance and allows red-cone signals to create spectral peaks (540–550 nm). The residual blue and green signals of crx:trβ2 cooperate to create a 444-nm peak at the dimmest level of stimulation (Fig. 5D).

With 255 models tested, there is not always a single best-fitting model for each dataset as judged by residual variance. For the larval WT datasets, model 95, also a six-cone model, was indistinguishable from model 111 (F_{(0.99895,1851,1851)} = 0.491, p = 0.982). This model replaces the low amplitude G4 (Vg500) cone amplitude with a similar amplitude G3 (Vg480) signal. The WT five-cone model 103 was also indistinguishable (F_{(0.99719,1851,1852)} = 0.476, p = 0.952). This model omits the low-amplitude G1 (Vg460) cone signal. For the crx:trβ2 datasets, model 201 was indistinguishable from model 202 (F_{(0.999,1856,1856)} = 0.491, p = 0.9822). This model substitutes a low-amplitude UV-cone signal (Vu358) for the low-amplitude B1-cone signal (Vb415).

Adult crx:trβ2 zebrafish are red cone dichromats

Only the red R1 (Vr575) and R2 (Vr556) cone signals made significant contributions to the adult cone-PIII responses of crx:mYFP-2A-trβ2 (Fig. 6C). A five-cone WT model including blue, green, and red cones best represented adult controls. These cone signals are Vb415-B1, Vg460-G1, Vg500-G4, Vr556-R2, Vr575-R1. The cumulative datasets included 14 eyecups from WT adults and 16 from crx:trβ2 adults (WT, 20 datasets, 1400 responses; crx:trβ2, 21 datasets, 1470 responses). When the adult WT model (#234) is fit to the adult crx:trβ2 dataset, the residual variance is larger than the variance for the optimal crx:trβ2 model (#192; F_{(1.923,1443,1445)} = 1, p = 0) indicating different cone combinations are operating. The crx:trβ2 transgene alters the positioning of adult irradiance-response data points and model curves (Fig. 6A,B). The 370- and 490-nm curves and points (magenta and green) lie to the left side of the 570-nm curve and points (yellow) in WT adults but shift to the right side of the 570-nm curve and points in crx:trβ2 adults, a diminution of UV-wavelength and mid-wavelength sensitivity. The calculated log of semi-saturation irradiance for long-wavelength-sensitive cones (R1, R2) was 4.49 ± 0.033 for WT adults and 4.69 ± 0.022 for crx:trβ2 adults (Fig. 6A,B), crx:trβ2 being significantly less sensitive (t₍₂₈₁₈₎ = 5.17, p = 2.5 × 10⁻⁷). The modeled amplitudes of cones with significant signals appear in Figure 6C. In the adult WT model, significant blue-cone and green-cone signals (B1, Vb415 and G1, Vg460) are detected, but these signals are not significant in adult crx:trβ2 transgenics, where there are only red-cone signals (R2, Vr556 and R1, Vr575). In crx:trβ2, R1 cones contribute a significantly larger amplitude signal than R2 cones (180%, t₍₂₈₁₈₎ = 3.92, p = 0.000092), whereas in the WT controls, R1 and R2 signal amplitudes were not significantly different.

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Figure 6.

Adult crx:mYFP-2A-trβ2 transgenics are red-cone dichromats. A, Wild-type cone-PIII irradiance-response curves and datapoints at four stimulus wavelengths. The 370-, 490-, 570-, and 650-nm amplitudes are means (±SE) from the cumulative dataset (n = 20 except at 650 nm where n = 20 or 40). Curves are generated from the optimal model (#234) fit to a cumulative 1400 responses at seven irradiances for each of nine wavelengths as compiled from 20 spectral datasets acquired in 14 WT adult eyecups. B, The crx:trβ2 gain-of-function transgene causes irradiance-response points and model curves to bunch together as compared with WT. The model generating the curves (#192) is fit to 1470 cumulative responses compiled from 21 datasets acquired from 16 crx:trβ2 eyecups. The amplitudes (±SE) are means (370, 490, 570, and 650 nm, n = 21 except for 650 nm, n = 21 or 42). C, Signals from five blue, green, or red cone types were detected in WT adults (gray bars), but signals from only the two red-cone types were detected in crx:trβ2 eyecups (magenta bars). Fit values of cone saturation amplitudes (Vm_i, Eq. 1; Fig. 2A) are plotted on a dataset-normalized scale (±SE). Except for Vr556 (LWS2) cone types differed significantly in amplitudes between WT and crx:trβ2 (asterisks, GraphPad Prism convention, n.s., not significant). Vb415 (B1, one-sample test): t₍₁₃₇₃₎ = 8.77, p = 5.4 × 10⁻¹⁸; Vg460 (G1, one-sample test): t₍₁₃₇₃₎ = 4.36, p = 1.4 × 10⁻⁵; Vg500 (G3, one-sample test): t₍₁₃₇₃₎ = 2.30, p = 0.021; Vr556(R2): t₍₂₈₁₈₎ = 0.782, p = 0.434; Vr575 (R1): t₍₂₈₁₈₎ = 3.91, p = 9.2 × 10⁻⁵. D, Model spectral curves for adult WT (black) and crx:trβ2 (magenta) eyecups. The crx:trβ2 transgene shifts sensitivity peaks to long wavelengths at all stimulus irradiances. Curves are modeled for constant quantal stimuli at 3.4, 4.0, and 4.6 log(quanta·μm⁻²·s⁻¹). A, B, The log(k_R1R2) values are the irradiance semi-saturation values in log(quanta·μm⁻²·s⁻¹) for both R1 and R2 cones. A–D, Eight- to 18-month adults, 10 mm aspartate medium.

The modeled spectral curves for the crx:mYFP-2A-trβ2 adult red-cone dichromat (Fig. 6D) show long-wavelength-peaking functions at 565 nm at all quantal stimulation levels, while WT spectral peaks occur between 430 and 522 nm, depending on stimulus brightness. Except at wavelengths >570 nm, WT control amplitudes are greater than transgenic, due both to the greater sensitivity of WT cones overall, and to the presence of mid-wavelength and short-wavelength (blue and green) cones, absent in the transgenic. As judged by residual variance no other models were indistinguishable from the illustrated ones for either crx:trβ2 or WT adults (F tests for all other models, p < 0.95).

Larval gnat2:trβ2 cone types and spectral signals

The gnat2:mYFP-2A-trβ2;mpv17 transgenic was developed to test whether red cones could be restored to a population of mid and short wavelength cones already differentiated under morpholino blockade of the native trβ2 gene, which prevented red-cone development (Suzuki et al., 2013). The gnat2 locus codes for cone transducin α, a gene product only expressed in differentiated and functional cone cells. It is expressed by four dpf. Suzuki et al. (2013) found the gnat2:trβ2 transgene induced a supra-normal 5-dpf density of red-opsin immunoreactive cones in the absence of native trβ2, but unlike the crx:mYFP-2A-trβ2 rescue, green-opsin (Rh2), blue-opsin (SWS2), and UV-opsin (SWS1) immunoreactive cone densities were normal (Suzuki et al., 2013).

The pattern of embryonic larval and juvenile opsin immunoreactivity in gnat2:trβ2 transgenics is similar to that seen in larvae with morpholino blockade of native trβ2. In the 12-dpf control retina (roy orbison mpv17−/−), both UV and red cones (Fig. 7Ai,iii) stain with opsin antibodies. Superposition of red-cone and UV-cone mosaics (Fig. 7Aii) shows no red-opsin and UV-opsin overlap, with immunoreactivity for each opsin expressed in a separate cone cell. The control larval blue cones also exist in a mosaic pattern separate from red cones (Fig. 7Aiv,v,vi). The gnat2:trβ2 retina shows dense representations of UV-opsin and blue-opsin immunoreactive cones (Fig. 7Bi,iv). Red-opsin immunoreactive cones appear denser in gnat2:trβ2 than in mpv17−/− control retinas (Fig. 7Biii,vi). The gnat2:trβ2 red cone density (95,500 mm⁻¹) was significantly greater than the control strain (51,400 mm⁻¹; t₍₁₄₎ = 6.7, p = 0.000011; Fig. 7Aiii,Avi,Biii,Bvi) . The densities of UV-opsin and blue-opsin immunoreactive cones were similar in gnat2:trβ2 and controls. The gnat2:trβ2 UV-cone density (34,252 mm⁻¹) was not significantly different from mpv17−/− (23,435 mm⁻¹; t₍₆₎ = 1.7, p = 0.143), nor was the gnat2:trβ2 blue-cone density (21,633 mm⁻¹) different from mpv17−/− (27,041 mm⁻¹; t₍₆₎ = 1.6, p = 0.168). In these 12-dpf gnat2:trβ2 juveniles (Fig. 7B), UV-opsin and blue-opsin immunoreactive cones were frequently immunoreactive for red opsins (arrowheads), with 2/3 of cones being mixed opsin types.

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Figure 7.

Juvenile cone distributions and spectral responses in gnat2:mYFP-2A-trβ2;mpv17−/− and mpv17−/− control retinas. Ai, UV opsin (SWS1) opsin immunoreactive cones in control (mpv17−/−) retina. Aiv, Blue-opsin (SWS2) immunoreactive cones in control retina. Aiii, Avi, Red (LWS1, LWS2) opsin immunoreactive cones in control retina. Aii, UV and red opsins are expressed in separate cones in control retina. Av, Blue and red opsins are expressed in separate cones in control retina. Bi, UV opsin immunoreactive cones in gnat2:trβ2 retina. Biv, Blue-opsin immunoreactive cones in gnat2:trβ2 retina. Biii, Bvi, Red-opsin immunoreactive cones in gnat2:trβ2 retina. Bii, Bv, Red opsins are expressed in some blue or UV opsin immunoreactive cones in a gnat2:trβ2 retina (arrowheads, Bi–Bvi). A, B, 12 dpf. C, D, Cone signals in mpv17−/− control and gnat2:trβ2 retinas are robust at all wavelengths, with greatest amplitudes at 490 nm and similar spectral patterns. The perfusion medium contains 20 mm aspartate to isolate cone signals. Stimulus irradiances [log(quanta·μm⁻²·s⁻¹)] appear in the legends to the right of irradiance-response trace stacks; 6-dpf larvae.

Densities of cone types and mixed opsin patterns were similar earlier in development. In 7-dpf larvae, the density of red-opsin immunoreactive cones in gnat2:trβ2 was 70,800 mm⁻¹, significantly greater than the mpv17−/− control (52,300 mm⁻¹; t₍₁₀₎ = 13.0, p = 1.3 × 10⁻⁷). The densities of blue-opsin immunoreactive cones were similar (gnat2:trβ2, 22,000 mm⁻¹; mpv17−/−, 24,900 mm⁻¹, t₍₇₎ = 1.9, p = 0.096), but the density of 7-dpf gnat2:trβ2 UV immunoreactive cones was slightly less (gnat2:trβ2, 36,900 mm⁻¹; mpv17−/−, 46,900 mm⁻¹, t₍₈₎ = 3.4, p = 0.010). Mixed blue-red and UV-red opsin expression was noted in ∼10% of 7-dpf blue-opsin or UV-opsin immunoreactive cones.

Larval and adult cone spectral signals from control and gnat2:trβ2 retinas

Despite greater red-cone density and numerous mixed opsin cones (Fig. 7B), the spectral patterns of larval, aspartate-isolated cone signals (cone-PIII) from in vitro gnat2:mYFP-2A-trβ2;mpv17−/− eyes are similar to the mpv17−/− controls. In the trace recordings of Figure 7C, an mpv17−/− control larval eye produces substantial signals at all wavelengths, with maximal amplitudes evoked by the 490-nm stimulus. A gnat2:trβ2 larval eye gives a similar amplitude pattern for the test wavelengths and irradiances (Fig. 7D), with maximal amplitudes at 490 nm.

In an adult mpv17−/− control eyecup (Fig. 8A), the spectral pattern of cone-PIII ERG waveforms shows a with a broad range of responsiveness across wavelengths, and the 490-nm stimuli yielding an amplitude peak. An adult gnat2:mYFP-2A-trβ2;mpv17−/− eyecup also shows a broadly responsive spectral pattern (Fig. 8B), but greater amplitudes are evoked at long wavelengths (570 and 650 nm; Fig. 8B, arrows), suggesting that the underlying input signals from red, green, blue, and UV cone types have undergone a late developmental transformation under the influence of gnat2:trβ2.

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Figure 8.

Adult cone PIII spectral properties in mpv17−/− and gnat2:mYFP-2A-trβ2;mpv17−/− retinas. A, Cone signals in an mpv17−/− control eyecup are evoked by all stimulus wavelengths and all but the dimmest irradiances (black traces). The largest amplitudes occur at 490 nm. B, Cone signals from a gnat2:trβ2 eyecup are stimulated by all wavelengths and irradiances. Greatest amplitudes occur at 650, 570, and 490 nm, with a larger amplitude spectral pattern at 650 and 570 nm than in the control (up arrows). A, B, Adults are 8–18 months. Eyecups are perfused with medium containing 10 mm aspartate to isolate retinal cone signals. Stimulus irradiances [log(quanta·μm⁻²·s⁻¹)] appear in the legends to the right of irradiance-response waveform stacks. C, Voltage distributions of maximal trough-to-peak amplitudes found in spectral datasets. D, Cone-signal latencies from stimulus onset to the minimum in the ON trough. E, Cone-signal latency from stimulus offset to the OFF peak. C, D, E, The n.s. labels on all distributions indicate that that mpv17−/− controls did not significantly differ from gnat2:trβ2 transgenics in waveform characteristics (t test and p-values given in text). Peak amplitudes (dataset Vmax), trough (PIII ON trough), and peak (PIII OFF peak) latencies are measured on the mean waveforms from each 70-stimulus spectral dataset.

Similar waveforms from control and gnat2:trβ2 larvae and adults

There was little difference in the distribution of amplitudes or waveform kinetics between the gnat2:mYFP-2A-trβ2;mpv17−/− transgenic and the mpv17−/− control strain (Fig. 8C–E). In both larvae and adults, the maximal ERG PIII amplitudes were similar for the gnat2:trβ2 transgenics and controls. The 6-dpf larval mean of dataset-maximum peak-to-trough responses (23 datasets, 15 eyes) for mpv17−/− controls was 110.5 ± 12.1 μV (mean and SE). For gnat2:trβ2 larvae, the mean of dataset-maximum peak-to-trough amplitudes was 130.3 ± 16.5 μV (22 datasets, 17 eyes). Maximal amplitudes of the larval gnat2:trβ2 signals were not significantly different from those of the background strain (t₍₄₃₎ = 0.969, p = 0.338). The variances in maximal dataset amplitudes were similar in transgenic and control larvae (F_(1.79,21,22) = 0.908, p = 0.185).

Adult gnat2:mYFP-2A-trβ2;mpv17−/− maximal cone PIII amplitudes were not distinguishable from controls (Fig. 8C). In 16 mpv17−/− control adult eyecups the mean of dataset-maximum peak-to-trough amplitudes was 22.5 ± 3.1 μV (30 datasets). In 15 gnat2:trβ2 eyecups the mean of dataset-maximum peak-to-trough amplitudes was 28.2 ± 7.2 μV (24 datasets). The difference was not significant (t₍₅₂₎ = 0.780, p = 0.439). The variance in adult transgenic peak amplitudes was greater (F_(4.38,23,29) = 0.9999, p = 0.00025).

Examining kinetics of the larval and adult dataset mean waveforms, the latency to the cone-PIII trough minimum and the variance in its timing differed little between 6-dpf larval gnat2:trβ2 eyes and the mpv17−/− controls (Fig. 8D). The onset time for the larval cone-PIII trough for gnat2:trβ2 eyes (29 datasets, 17 eyes) was 152 ± 5 ms from stimulus onset (mean and SE). The cone-PIII trough latency for larval eyes from the mpv17−/− controls (26 datasets, 16 eyes) was 139 ± 4 ms. The trend, though not significant, for larval gnat2:trβ2 onset kinetics was toward greater delay in the PIII ON trough (t₍₅₃₎ = 1.89, p = 0.065). Larval gnat2:trβ2 eyes trended, though not significantly, toward greater variability in trough latencies (F_(2.09,28,25) = 0.9669, p = 0.067).

For adults the mean time to the PIII ON trough was 122 ± 4 ms in gnat2:trβ2 eyecups (24 datasets, 15 eyes) and 128 ± 4 ms in the mpv17−/− controls (26 datasets, 16 eyes). The differences were not significant (t₍₄₈₎ = 1.11, p = 0.274). The variances in adult trough latencies for gnat2:trβ2 and background strain were similar (F_(1.23,25,23) = 0.689, p = 0.623). Adult trough latencies were significantly quicker than larvae for both mpv17−/− controls and gnat2:trβ2 transgenics (mpv17−/−: t₍₅₀₎ = 2.03, p = 0.047; gnat2:trβ2 t₍₅₁₎ = 4.39, p = 0.000057).

The mean cone-PIII OFF peak timing and variance in larval and adult gnat2:mYFP-2A-trβ2 mpv17−/− did not differ from controls (Fig. 8E). The mean waveforms for the 6-dpf gnat2:trβ2 larval datasets peaked at 321 ± 22 ms after stimulus offset (29 datasets, 15 eyes). For the larval mpv17−/− controls, the OFF peak occurred at 292 ± 18 ms (26 datasets, 16 eyes). These latencies were not significantly different (t₍₅₃₎ = 0.980, p = 0.332). The variances in larval OFF peak timing for transgenic and control were similar (F_(1.83,28,25) = 0.935, p = 0.132). For adult datasets, the OFF peak of the gnat2:trβ2 mean waveforms gave delays of 308 ± 17 ms from stimulus offset (24 datasets, 15 eyes). In the adult mpv17−/− control datasets, the delay was 309 ± 14 ms. The gnat2:trβ2 adult OFF-peak timing did not differ from controls (t₍₄₈₎ = 0.031, p = 0.976). The variances in adult OFF peak timings were not different (F_(1.34,23,25) = 0.763, p = 0.473). Adult OFF-peak latencies did not differ significantly from larvae for either mpv17−/− controls or gnat2:trβ2 transgenics (mpv17−/−: t₍₅₀₎ = 0.75, p = 0.459; gnat2:trβ2: t₍₅₁₎ = 0.43, p = 0.670). Overall, the gnat2:trβ2 transgene has little effect on cone-PIII amplitudes or waveforms. There is no electrical evidence of degenerative disease.

In gnat2:trβ2 larvae the transgene increases R2-cone signals

The larval cone PIII spectral responses of gnat2:mYFP-2A-trβ2;mpv17−/− are similar to the mpv17−/− control strain (Fig. 7C,D). Signal loss in the UV and signal gain at long wavelengths, as seen crx:mYFP-2A-trβ2 larvae (Fig. 3C,D), are not as evident. Modeling suggests subtle changes. The optimal model for gnat2:trβ2 is #79, and optimal control model is #77. Applying the control model 77 to the gnat2:trβ2 dataset gives a greater residual variance than model 79 (F_{(1.21,1506,1511)} = 0.999, p = 0.00021), indicating that cone inputs generating the gnat2:trβ2 cumulative dataset differ from control. In Figure 9A,B, amplitudes are plotted against irradiance for four of the nine test wavelengths and compared with continuous curves calculated from best-fitting models. These are fit to 1945 points (15 eyes, 28 spectral datasets) for mpv17−/− controls and 1540 points (17 eyes, 22 spectral datasets) for gnat2:trβ2. The distribution of points and curves at 570, 490 and 370 nm are more overlapping along the irradiance axis for gnat2:trβ2 larvae than are the same wavelength points and curves for the control strain. Points and curves at 370 and 490 nm (magenta, green) lay to the left of the 570-nm points and curve (yellow) for mpv17−/− controls but coincide with the 570-nm curve for gnat2:trβ2, suggesting less sensitivity from short-wavelength-sensitive and mid-wavelength-sensitive cones. The logs of semi-saturation irradiances for the red (R1, R2) cones were 4.50 ± 0.02 for mpv17−/− larvae and 4.51 ± 0.02 for gnat2:mYFP-2A-trβ2;mpv17−/− (Fig. 9A,B), nearly identical (t₍₃₄₂₂₎ = 0.251, p = 0.802).

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Figure 9.

Embryonic spectral properties of cone signals from gnat2:mYFP-2A-trβ2;mpv17−/− and mpv17−/− control retinas. A, Control irradiance-response points and optimal model curves. The WT model (#77) was fit to 1945 spectral response amplitudes, combined from 28 datasets taken from 15 eyes. The points are subsets taken from the cumulative data. The 370-, 490-, and 570-nm amplitudes are means (±SE), n = 27 or 28; the 650 nm means, n = 28 or 56. B, The gnat2:trβ2 irradiance-response points and model curves at 370, 490, and 570 nm are more bunched together than in the control. The gnat2:trβ2 model (#79) was fit to 1540 spectral points, combined from 22 datasets taken from 17 eyes. The 370-, 490-, and 570-nm amplitudes are means (±SE), n = 22; 650 nm, n = 22 or 44. C, Four cone opsin types contributed significantly to cone-signals isolated from mpv17−/− control eyes (gray bars) but five types proved significant in gnat2:trβ2 eyes (magenta bars). Cone saturation amplitudes (Vm_i, Eq. 1; Fig. 2A) are fractions of maximal dataset amplitudes (±SE). Asterisks represent the significance of amplitude differences between mpv17−/− control and gnat2:trβ2 (GraphPad Prism convention, n.s., not significant). Vu358 (UV): t₍₃₄₂₂₎ = 5.37, p = 8.7 × 10⁻⁸; Vb415 (B1, one-sample test): t₍₁₅₁₁₎ = 2.90, p = 0.0038; Vb440 (B2): t₍₃₄₂₂₎ = 2.62, p = 0.0081; Vg460 (G1): t₍₃₄₂₂₎ = 0.851, p = 0.395; Vr556 (R2): t₍₃₄₂₂₎ = 9.94, p = 5.6 × 10⁻²³. D, Model spectral curves for mpv17−/− controls and gnat2:trβ2 retinas for constant quantal irradiances of 3.4, 4.0, and 4.6 log(quanta·μm⁻²·s⁻¹). There is greater long-wavelength sensitivity, and lesser short-wavelength sensitivity in the embryonic gnat2:trβ2 gain-of-function transgenic, although the gains and deficits are much less severe than in the crx: trβ2 larvae. A, B, The log(k_R1R2) values are the modeled R1-cone or R2-cone semi-saturation irradiances in log(quanta·μm⁻²·s⁻¹). A–D, 6-dpf larvae, 20 mm aspartate medium.

Four cone signals are identified in 6-dpf larval mpv17−/− controls, but five were found in gnat2:mYFP-2A-trβ2 siblings (Fig. 9C). Significantly reduced in gnat2:trβ2 are UV (Vu358) and B2 (Vb440) cone amplitudes. Significantly increased are B1 (Vb415) and R2 (Vr556) cone signals. The G1 cone signal (Vg460) was not significantly affected. The larval mpv17−/− control and gnat2:trβ2 model-generated spectral sensitivities are similar (Fig. 9D), but with increased long-wavelength and decreased short-wavelength sensitivities in gnat2:trβ2. This leads to long-wavelength spectral peaks between 535 and 549 nm for gnat2:trβ2, not seen in the mpv17−/− controls. While there is a definite spectral shift toward long-wavelength sensitivity in gnat2:trβ2 larvae (Fig. 9D), the extent is subtle compared with crx:trβ2 larvae (Fig. 5D).

Of the 255 models fit to the 6-dpf larval gnat2:trβ2 combined dataset none were deemed to fit equally as well as model 79 based on residual variance, that is by an F test with p ≥ 0.95. Two were indistinguishable from the best-fit model 77 for the mpv17−/− controls. Model 103 (F_{(0.9992,1939,1940)} = 0.493, p = 0.986), employed an additional 5^th cone substituting G4 for G1 and adding B1. Model 101 (F_{(0.9972,1940,1940)} = 0.475, p = 0.95) substituted G4 for G1. All three indistinguishable control models agreed on the presence of UV, B2, and R2 cone signals in mpv17−/− controls.

Adult gnat2:trβ2 retinas generate large R1 red-cone signals

To determine the cone-type composition of adult gnat2:trβ2 retinal cone signals we searched for the best models to represent the gnat2:trβ2 cumulative spectral dataset. Trough-to-peak amplitudes for all eyes and datasets (mpv17−/−, 13 eyes, 23 datasets, 1610 responses; gnat2:trβ2, 11 eyes, 20 datasets, 1400 responses) were fit to the 255 combinations of eight cones. Model 219 fit best for mpv17−/− control eyecups, and model 202, for gnat2:trβ2 (Fig. 10A,B). When the adult gnat2:trβ2 cumulative dataset was fit to the control model 202, the residual variance was significantly greater than with the optimal gnat2:trβ2 model, indicating the cones signals contributing to the gnat2:trβ2 gain-of-function dataset differed significantly from the control (F_{(1.942,1370,1374)} = 1, p = 0).

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Figure 10.

Adult spectral signals from gnat2:mYFP-2A-trβ2;mpv17−/− transgenics and mpv17−/− controls. A, Control (mpv17−/−) irradiance-response amplitudes and model curves at four wavelengths. The optimal model (#219) was fit to 1610 responses (all wavelengths) combined from 23 datasets taken from 13 eyecups. The 370-, 490-, and 570-nm amplitudes are means (±SE), n = 23; 650 nm, n = 23 or 46. B, Irradiance-response amplitudes, and model curves, for gnat2:trβ2. The transgene shifts 370- and 490-nm irradiance-response functions from the left of the 570-nm curve (control) to the right of the 570-nm curve (transgenic). The spectral algorithm was fit to 1400 points combined from 20 datasets accumulated from 11 eyecups. The 370-, 490-, and 570-nm amplitudes are means (±SE), n = 20; 650 nm, n = 20 or 40. C, In adult mpv17−/− control eyecups (gray bars), significant signals from six cone types were detected. Four were found in gnat2:trβ2 adults (magenta bars). Cone saturation amplitudes (Vm_i, Eq. 1; Fig. 2A) are fractions of dataset maximum amplitudes (±SE). Asterisks (or n.s., not significant) represent the significance of differences between control and gnat2:trβ2 cone signals (GraphPad Prism convention). Vu358 (UV, one-sample test): t₍₁₅₇₉₎ = 4.81, p = 1.7 × 10⁻⁶; Vb415 (B1): t₍₂₉₈₃₎ = 2.97, p = 0.0030; Vg460 (G1): t₍₂₉₈₃₎ = 2.26, p = 0.024; Vg480 (G3, one-sample test): t₍₁₅₇₉₎ = 6.59, p = 6.1 × 10⁻¹¹; Vr556 (R2): t₍₂₉₈₃₎ = 1.52, p = 0.127; Vr575 (R1): t₍₂₉₈₃₎ = 4.87, p = 1.2 × 10⁻⁶. D, Adult spectral curves for mpv17−/− control and gnat2:trβ2 retinas. The transgene shifts sensitivity peaks to long wavelengths for all constant-quantal irradiances [3.4, 4.0, and 4.6 log(quanta·μm⁻²·s⁻¹)]. A, B, The log(k_R1R2) values are R1 or R2 cone semi-saturation irradiances in log(quanta·μm⁻²·s⁻¹). A–D, Eight- to 18-month adults, 10 mm aspartate medium.

For both points and optimal model curves the gnat2:trβ2 transgene alters adult irradiance-response characteristics (Fig. 10A,B). In the control dataset (mpv17−/−), the 570-nm points and curve (yellow) show less sensitivity than either the 370- or 490-nm points and curves (magenta, green), but show greater sensitivity in gnat2:trβ2. In gnat2:trβ2, brighter stimuli are required to elicit responses in the UV (370 nm) and mid spectrum (490 nm). The fit of log of semi-saturation irradiances for the red cones (R1, R2) were 4.30 ± 0.026 for adult controls and 4.43 ± 0.026 for gnat2:trβ2 adults (Fig. 10A,B), a significantly greater sensitivity (∼35%) than for gnat2:trβ2 adults (t₍₂₉₅₃₎ = 3.44, p = 0.0006).

In the best-fitting model for gnat2:mYFP-2A-trβ2;mpv17−/− adults, the long-wavelength R1 cone (Vr575) contributes the largest amplitude signal with the R2 amplitude (Vr556) being only 22% as large, significantly less than R1 (t₍₂₇₄₈₎ = 5.23, p = 2.6 × 10⁻¹⁷), and the gnat2:trβ2 R1 amplitudes are only marginally significant (t₍₁₃₇₅₎ = 2.02, p = 0.044; Fig. 10C). In the mpv17−/− control, both R1 and R2 red cone amplitudes were highly significant (Vr575-R1, t₍₁₅₈₀₎ = 6.01, p = 4.1 × 10⁻¹⁸; Vr556-R2, t₍₁₅₈₀₎ = 5.52, p = 8.0 × 10⁻¹⁷) and did not significantly differ in amplitude (t₍₃₁₅₈₎ = 0.042, p = 0.966; Fig. 10C). A six-cone model best fit adult control datasets (Vu358-UV, Vb415-B1, Vg460-G1, Vg480-G3, Vr556-R2, Vr575-R1). A four-cone model sufficed for gnat2:trβ2 adults (Vb415-B1, Vg460-G1, Vr556-R2, Vr575-R1). Unlike gnat2:trβ2 larvae (Fig. 9C), UV-cone signals were not significant in gnat2:trβ2 adults (Fig. 10C).

At all levels of constant quantal stimulation, long-wavelength spectral peaks between 560 and 570 nm are modeled for gnat2:mYFP-2A-trβ2;mpv17−/− (Fig. 10D). Models of the mpv17−/− control peak spectrally between 441 and 493 nm depending on stimulus brightness. Except at wavelengths >540 nm, control amplitudes were greater than transgenic, due both to the greater cone sensitivities (lesser half-saturation irradiances), and to the significantly greater amplitudes of numerous mid-wavelength and short-wavelength cones (Vu358-UV, Vb415-B1, Vg460-G1, Vg480-G3).

As judged by residual variance no models were indistinguishable from the illustrated ones for gnat2:mYFP-2A-trβ2;mpv17−/− adults (F tests for other models, p < 0.95). Model 235 was indistinguishable from model 219 (the best model) for the mpv17−/− control adults (F_{(0.9978,1603,1603)} = 0.482, p = 0.96). Models #235 and #219 both employed six cones, but model 235 substituted the G4 (Vg500) for the G3 (Vg480) cone.

The larval dual opsin UV cone functions as a UV cone in trβ2 gain-of-function transgenics

Many cones in gnat2:mYFP-2A-trβ2;mpv17−/− larvae express mixed opsins, overlaying a red opsin on the native expression of a shorter-wavelength opsin (Fig. 7B). A few dual-opsin cones also occur in crx:mYFP-2A-trβ2 larvae (Fig. 3B). The spectral physiology and molecular development of such cones is of interest. The wide spectral separation of red and UV opsins makes the two opsins in dual-opsin UV cones amenable to separate stimulation. One proposal is that zebrafish transgenic dual-opsin cones are analogous to the stable mws-sws “dual physiology” cone configuration seen in rodents (Jacobs et al., 1991; Applebury et al., 2000; Nikonov et al., 2005; Suzuki et al., 2013). Another proposal is that the mixed-opsin cones in gnat2:trβ2 zebrafish are transitional states, later to develop into red cones. The tendency to lose UV-opsin signals in trβ2 gain-of-function transgenics suggests an adverse action on UV opsins, and a comparable model in mouse development is that trβ2 changes differentiated S cones to M cones (Swaroop et al., 2010).

To observe the effects of excess trβ2 on sws1 (UV opsin) gene activity, UV opsin immunoreactivity was examined in double transgenic gnat2:trβ2 larvae to which a fluorescent sws1 opsin reporter gene was added (gnat2:mYFP-2A-trβ2;sws1:nfsBmCherry;mpv17−/−). Confocal micrographs of tangential planes through the cone photoreceptor layers in control and the double transgenic larvae show that at 5 dpf (Fig. 11D,H) red-opsin immunoreactive cones are about twice as dense in gnat2:trβ2 larvae (148,000 ± 12,000 mm⁻¹) as in controls (60,000 ± 14,000 mm⁻¹; t₍₆₎ = 5.52, p = 0.0015) , but at this stage, the density of UV-opsin immunoreactive cones (Fig. 11B,F) is not affected (gnat2:trβ2: 48,000 ± 3400 mm⁻¹; control: 52,000 ± 4100 mm⁻¹; t₍₆₎ = 0.77, p = 0.47).

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Figure 11.

The UV-opsin reporter is inactive in embryonic cones co-expressing UV and red opsins, but these red opsins produce no UV-desensitizing signal. A, UV-opsin (SWS1) immunoreactivity (green) and florescence of a reporter transgene for sws1 (sws1:nfsBmCherry, magenta) colocalize in mpv17−/− control UV cones. B, UV-opsin immunoreactivity in control retina. C, In the control retina UV-opsin (green) and red-opsin (red) immunoreactivity localize in separate cones. D, Red-opsin immunoreactivity in the control retina. E, In gnat2:trβ2 cones, UV-opsin immunoreactivity (green) is always found when there is sws1 reporter gene fluorescence (sws1:nfsBmCherry, magenta), but not all UV-opsin immunoreactive cones show reporter-gene fluorescence (red and green checkered arrowheads). F, UV-opsin immunoreactive cones in the gnat2:trβ2 retina. G, Co-expression of UV-opsin (green) and red-opsin (red) immunoreactivity in gnat2:trβ2 cones. White arrowheads (F, G, H) point to cones expressing both UV opsin (green) and red opsin (red). The dual opsin cones are the UV-opsin immunoreactive cones in E, that lack sws1 reporter fluorescence (red and green checkered arrowheads). H, Red-opsin immunoreactive cones in gnat2:trβ2 retina. I, Saturation amplitudes (V_mi ± SE) of signals from five cone types in control (gray) and gnat2:trβ2 (magenta) eyes. Control: Spectral algorithm fit to 1890 datapoints from 27 datasets recorded in 15 eyes; gnat2:trβ2, 1260 datapoints from 18 datasets recorded in 11 eyes. In each case, the optimal fit was model 79. Asterisks give significance of differences (two sample t tests, n.s., not significant) in cone-signal amplitudes (Vu358: t₍₃₀₉₁₎ = 0.56, p = 0.57; Vb415: t₍₃₀₉₁₎ = 0.25, p = 0.81; Vb440: t₍₃₀₉₁₎ = 1.05, p = 0.29; Vg460: t₍₃₀₉₁₎ = 0.084, p = 0.93; Vr556, t₍₃₀₉₁₎ = 3.39, p = 0.0007). J, Control strain (mpv17−/−) irradiance-response curves at 370 and 650 nm in the presence of infrared (IR; black) or 627-nm (red) backgrounds (bkgd). Points are means ± SE (370-nm stimulus: IR background, n = 27; red background, n = 15; 650-nm stimulus: IR background, n = 54 or 27; red background, n = 30 or 15). K, gnat2:trβ2 irradiance-response curves at 370 and 650 nm in the presence of IR (black) or red backgrounds. Points are means ± SE (370-nm stimulus: IR background, n = 18; red background, n = 12; 650-nm stimulus: IR background, n = 18 or 36; red background, n = 12 or 24). J, K, Hill function curves fit at each background and wavelength are constrained to have equal maximal amplitudes and exponents. Cone-PIII ERG signals isolated with 20 mm aspartate. A–K, Eyes and retinas are from 5-dpf embryos.

For the mpv17−/− control, all UV-opsin immunoreactive cones (Fig. 11A–C, green) express the sws1:nfsBmCherry UV-opsin gene reporter as a magenta inner segment halo surrounding the narrower green immunofluorescence of the UV-cone outer segment (Fig. 11A). UV-opsin and red-opsin immunoreactivities are segregated, being expressed in separate cone cells (Fig. 11C), the native larval opsin expression pattern (Allison et al., 2010). In the larval gnat2:trβ2 double transgenic not all UV-opsin immunoreactive cones show the magenta halo of the sws1:nfsBmCherry reporter gene. In some, the sws1 reporter gene generates no fluorescence (Fig. 11E, red and green arrowheads), suggesting the native sws1 gene locus is inactive, and UV opsin, while still present, is no longer being synthesized. Only legacy SWS1 immunoreactivity remains. In Figure 11G, patterns of gnat2:trβ2 immunoreactivity for UV opsin and red opsin are compared. The arrowheads point to cone cells where both UV and red opsin are co-expressed. A comparison of Figure 11F with E reveals that UV-opsin immunoreactive cones with inactive sws1 reporter genes are the same cones that are double immunoreactive for both UV and red opsins. The expression of red opsin in a UV cone by the gain-of-function gnat2:trβ2 transgene appears incompatible with continued expression of UV-opsin, suggesting that the overabundance trβ2 in a dual UV-red opsin cone either directly or indirectly blocks the sws1 gene. Therefore, co-expression of UV-opsin and red-opsin immunoreactivity cannot continue indefinitely, as in rodents, but would be limited by the catabolism of previously expressed, but not renewed, UV-opsin.

At the same embryonic developmental stage (5 dpf), the functional properties of UV cones with co-expression of red opsin were explored. In this earliest embryonic dataset, the same 5-cone model (#79) was the best fit for both control and gnat2:trβ2 gain-of-function larvae (Fig. 11I), and the residual variance of the gnat2:trβ2 dataset was not significantly greater when modeled with the control amplitude distribution (F_{(1.031,1230,1235)} = 0.705, p = 0.591), although the R2-cone saturation amplitude (Vr556) trended greater (9%) in gnat2:trβ2 (t₍₃₀₉₁₎ = 3.39, p = 0.0007). Saturation amplitudes of shorter-wavelength cone signals (Vu358-UV, Vb415-V1, Vb440-B2, Vg460-G1) in gnat2:trβ2 and control were not significantly different. UV-cone saturation amplitude at 5 dpf appeared unaffected by numerous of its members both lacking sws1 gene activity and co-expressing red opsin.

Red chromatic adaptation should desensitize UV/red mixed opsin cones to UV stimulation. 650 nm red stimuli are not absorbed by UV opsins, but 370-nm UV stimuli are strongly absorbed. We generated irradiance-response functions at 650 and 370 nm in the presence of red or IR backgrounds (Fig. 11J,K). For 370-nm stimuli, the control, irradiance-response curves overlap in both datapoints and Hill-function curve fits regardless of red or IR background illumination (Fig. 11J). Hill function fits give semi-saturation values of 4.53 log(quanta·μm⁻²·s⁻¹) for the IR background and 4.59 for the 627-nm background, not distinguishable (t₍₂₉₀₎ = 1.26, p = 0.210), an expected result as UV-opsin does not absorb either the 627 nm or the IR adapting light. As the 650-nm stimuli (Fig. 11J) are not as efficiently transduced, even by red cones, the 650-nm irradiance-response functions shift to greater irradiances. On the IR background the 650-nm semi-saturation irradiance is 6.38 log(quanta·μm^–2·s⁻¹) but increases to 6.53 log(quanta·μm^–2·s⁻¹) with the 627-nm background, a significant desensitization (t₍₅₈₃₎ = 3.66, p = 0.0003). The absorption of the red background by the red-cone red opsin reduces red-cone sensitivity (Fig. 11J).

What was unexpected was that the 370-nm irradiance functions for the gnat2:trβ2 gain-of-function larvae, with co-expression of red opsin in some UV cones, were unaffected by the red background (Fig. 11K). The red opsin in the mixed-opsin UV cones, which would be activated by the 627-nm background, does not desensitize 370-nm UV signals. For 370-nm stimuli, Hill fits give semi-saturation irradiances of 4.42 log(quanta·μm^–2·s⁻¹) on the IR background and 4.44 log(quanta·μm^–2·s⁻¹) on the 627-nm background, values not significantly different (t₍₂₀₅₎ = 0.300, p = 0.765). But the same red background significantly desensitizes red-opsin signals from gnat2:trβ2 red cones, as seen with 650-nm stimuli (Fig. 11K). The Hill semi-saturation changed from 6.26 log(quanta·μm⁻²·s⁻¹) on the IR background to 6.55 log(quanta·μm²·s⁻¹) on the 627-nm background, a significant sensitivity loss (t₍₄₁₅₎ = 5.01, p = 1.5 × 10⁻¹²), which demonstrates the effectiveness of this background for red opsins (Fig. 11K). The failure of long-wavelength backgrounds to affect UV signals from mixed-opsin cones has also been observed in ERG spectra of rodents (Jacobs et al., 1991). In zebrafish the spectral physiology of UV cones appears, at least early in development at 5 dpf, not to be affected by the introduction of transgenic red opsins into many UV-cone members, or by inactivation of the UV-opsin gene.

Thyroxin receptor β2 gain-of-function transgenes alter cone morphology

Thyroid hormone receptor β2 is required in zebrafish for both the development of red cones and the expression of red opsins (Deveau et al., 2020). Morphologically, adult red cones are the principal members of zebrafish double cones (Engstrom, 1960; Raymond et al., 1993). Double cones failed to develop in trβ2−/− mutants (Deveau et al., 2020). The impact of trβ2 in determining the course of cone morphologic development is seen in transverse optical sections of individual transgenic cones from in vivo 6-dpf larvae (Fig. 12A,C). These are higher magnifications taken from larval retina confocal image stacks such as seen in Figure 1D–F. In the Figure 12 examples, the fluorescent-reporter shapes of trβ2 gain-of-function cones are distinct from the control morphologies of red, blue, and UV cones, the latter marked by the reporter fluorescent proteins expressed in sws2:GFP (blue cones) and sws1:GFP;trβ2:tdTomato (UV and red cones).

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Figure 12.

Thyroxin-receptor-β2 gain-of-function transgenes alter cone morphology. A, Width of cone types identified by transgene markers, is measured at the greatest extent of the inner segment. B, The trβ2 gain-of function cones are significantly wider than red-cones. C, The cone axon length is measured from the base of the inner segment to the apex of the cone pedicle. D, The trβ2 gain-of-function axon lengths are significantly shorter than red-cone axon lengths. B, D, Asterisks indicate significant differences (GraphPad convention, ANOVA and Tukey post hoc p-values given in text). A, C, Images are of 6-dpf in vivo larval fluorescent cones from confocal stacks. Control UV and red cones were imaged in sws1:GFP;trβ2:tdTomato larvae; blue cones, in sws2:GFP larvae; fluorescent trβ2 gain-of-function cones, in crx:mYFP-2A-trβ2 and gnat2:mYFP-2A-trβ2;mpv17−/− larvae. Larvae were anesthetized with MS222 and embedded in agarose after raising to 6 dpf in 300 μm PTU to block melanin formation in the pigment epithelium.

The maximal widths of the five sorts of cone inner segments illustrated differed (ANOVA, F_(4,356) = 36.98, p < 0.00001). The maximal widths for cone inner segments of crx:mYFP-2A-trβ2 labeled cones are significantly wider than those of red cones (post hoc Tukey, p < 0.000001), as are the maximal widths of cone inner segments of gnat2:mYFP-2A-trβ2;mpv17−/− labeled cones (post hoc Tukey, p = 0.00024). Neither trβ2 gain-of-function cone is as wide as the inner segments of UV cones (post hoc Tukey: crx:mYFP-2A-trβ2, p = 0.00005; gnat2:mYFP-2A-trβ2;mpv17−/−, p < 0.000001). The inner segment widths of the crx:mYFP-2A-trβ2 cones were not distinguishable from blue cones (post hoc Tukey: p = 0.126), while gnat2:mYFP-2A-trβ2 widths were marginally narrower than blue cones (post hoc Tukey: p = 0.00764).

The lengths of axons connecting the base of cone inner segments and the apex of cone synaptic pedicles differed among the cone types (ANOVA, F_(4,290) = 9.692, p < 0.00001; Fig. 12D). In the trβ2 gain-of-function cones, the length of the axon is significantly shorter than in red cones (post hoc Tukey: crx:mYFP-2A-trβ2, p = 0 0.00644; gnat2:mYFP-2A-trβ2;mpv17−/−, p = 0.00883; Fig. 12D). The lengths are not distinguishable from blue cone axons (post hoc Tukey: crx:mYFP-2A-trβ2, p = 0.212; gnat2:mYFP-2A-trβ2;mpv17−/−, p = 0.175) or UV cones axons (post hoc Tukey: crx:mYFP-2A-trβ2, p = 0.108; gnat2:mYFP-2A-trβ2;mpv17^-/, p = 0. 134).

On morphometrics, the mYFP-marked trβ2 gain-of-function cones have not attained red-cone shape at 6 dpf, but nonetheless enhance red cone signal amplitudes. Gain-of-function trβ2 morphologically alters larval cones and appears to shift the metrics toward a larval blue-cone pattern.

Impact of excess thyroid hormone receptor β2 on development of zebrafish spectral signals

During zebrafish maturation, patterns of cone opsin mRNA expression change through interactions with a “developmental factor” (Takechi and Kawamura, 2005). Green-cone and red-cone spectral peaks shift from shorter to longer wavelengths by adulthood (Nelson et al., 2019) as different members of gene-duplicated green (Rh2) and red (lws) opsin groups are sequentially expressed. In the present control data, the R2 opsin signal (Vr556) is largest in embryonic (5, 6 dpf) and juvenile (12 dpf) ages but the R1 opsin signal (Vr575) attains equal amplitude status in adults, while, over the same developmental course, the UV cone signal (Vu358) diminishes (Fig. 13A,B, WT and mpv17−/− controls, gray bars). Introduction of gain-of-function trβ2 shifts native developmental patterns of opsin expression. In the adults of both crx:trβ2 and gnat2:trβ2 gain-of-function transgenics, UV-cone (Vu358), blue-cone (Vb415, Vb440), and green-cone (Vg460, Vg480, Vg500) signals are reduced or extinguished by adulthood. In embryos and juveniles, mainly R2 signals (Vr556) increase but in adults, R1 signals (Vr575) increase (Fig. 13A,B). In crx:trβ2 transgenics, as early as 5 dpf, UV-cone (Vu358) and B1-cone (Vb415) signals decrease significantly, to 11% and 20% of WT control, while R2-cone amplitude (Vr556) increases 230% (Fig. 13A). In crx:trβ2, the altered pattern of signal amplitudes from cone types persists throughout development, with UV cone signals becoming undetectable from 6 dpf on through adulthood (Fig. 13A, 6 pdf, 12 dpf, Adult), and there is a failure to detect blue and green cone signals after the 12-dpf juvenile stage (Fig. 13A, Adult). Signals from red cones increase throughout embryonic (5, 6 dpf), juvenile (12 dpf) and adult developmental stages. R2 cone amplitudes (Vr556) increase at both 5 and 12 dpf (12 dpf: t₍₁₅₅₈₎ = 6.29, p = 3.9 × 10⁻¹⁰) and R1 cone signals (Vr575) increase for 6-dpf embryos and for adults (6 dpf: t₍₁₈₂₅₎ = 2.72, p = 0.0067; Adult: t₍₂₈₁₈₎ = 3.92, p = 9.2 × 10⁻⁵; Fig. 13A).

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Figure 13.

Signal development in red, green, blue, and UV cones. A, In crx:mYFP-2A-trβ2 eyes, red-cone signals (Vr575, Vr556) are larger than WT from 5-dpf embryos to adults while blue-cone, green-cone, and UV-cone signals (Vb415, Vb440; Vg460, Vg480, Vg500; Vu360) are smaller, and disappear in adults. B, In 5-dpf embryonic gnat2:mYFP-2A-trβ2;mpv17−/− eyes, the overall signal amplitudes of red (Vr575, Vr556), green (Vg460, Vg480, Vg500), blue (Vb415, Vb440), and UV (Vu358) cones are indistinguishable from mpv17−/− controls (ANOVA in text), but later in development red-cone amplitudes increase while green-cone, blue-cone, and UV-cone signals decrease. A, B, In adults of both transgenics, the largest signal is Vr575, coming from LWS1 cones, while controls show lesser Vr575 amplitudes. “5 dpf” and “6 dpf” are larval or embryonic stages. “12 dpf” is an early juvenile stage, and “Adults” are 8–18 months. In each bar chart, spectral datasets from multiple eyes are combined and fit by the Equation 1 (Fig. 1) algorithm to identify the most likely contributing cone types and their saturation voltages. A cone signal detected only in one of the two strains is evaluated by a one-sample t test and a cone detected in both strains by a two-sample t test. Asterisks denote significance (GraphPad convention, n.s., not significant). A, 5 dpf, 7 WT eyes, 11 crx:trβ2 eyes: Vu358 (UV), t₍₂₄₆₅₎ = 10.4, p = 5.8 × 10⁻²⁵; Vb415 (B1), t₍₂₄₆₅₎ = 5.74, p = 1.1 × 10⁻⁸; Vg460 (G1), t₍₁₆₃₈₎ = 4.54, p = 6.0 × 10⁻⁶; Vg480 (G3), t₍₈₂₇₎ = 10.3, p = 1.6 × 10⁻²³; Vr556 (R2), t₍₂₄₆₅₎ = 9.51, p = 4.3 × 10⁻²¹; Vr575 (R1), t₍₈₂₇₎ = 2.02, p = 0.043. 6 dpf, 10 WT eyes, 11 crx:trβ2 eyes: t tests given in Figure 5. 12 dpf, 3 WT eyes, 6 crx:trβ2 eyes. Vu358 (UV), t₍₂₄₃₎ = 5.12, p = 6.2 × 10⁻⁷; Vb415 (B1), t₍₁₅₅₈₎ = 4.86, p = 1.3 × 10⁻⁶; Vg460 (G1), t₍₁₃₁₅₎ = 2.86, p = 0.00435; Vg480 (G3), t₍₂₄₃₎ = 5.61, p = 5.5 × 10⁻⁸; Vr556 (R2), t₍₁₅₅₈₎ = 6.30, 3.90 × 10⁻¹⁰. Adult, 14 WT eyes, 16 crx:trβ2 eyes: t tests given in Figure 6. B, 5 dpf, 15 mpv17−/− eyes, 11 gnat2:trβ2;mpv17−/− eyes: t tests given in Figure 11. 6 dpf, 15 mpv17−/− eyes; 17 gnat2:trβ2; mpv17−/− eyes: t tests given in Figure 9. 12 dpf, 16 mpv17−/− eyes; 9 gnat2:trβ2;mpv17−/− eyes: Vu358 (UV), t₍₂₅₄₀₎ = 0.59, p = 0.55; Vb415 (B1), t₍₁₇₈₆₎ = 5.31, p = 1.2 × 10⁻⁷; Vg440 (B2), t₍₂₅₄₀₎ = 3.04, p = 0.0023; Vg500 (G4), t₍₁₇₈₆₎ = 5.22, p = 2.0 × 10⁻⁷; Vr556 (R2), t₍₂₅₄₀₎ = 2.65, p = 0.00815; Vr575 (R1), t₍₁₇₈₆₎ = 2.66, p = 0.0079. Adult, 13 mpv17−/− eyes; 11 gnat2:trβ2;mpv17−/− eyes: t tests given in Figure 10. A, B, Fit values of V_mi cone saturation voltages ± SE.

For the gnat2 promoter, the consequences of gain-of-function trβ2 are more gradual in developmental course (Fig. 13B). In the 5dpf gnat2:mYFP-2A-trβ2 embryonic larvae, the overall distribution of signal amplitudes from cone types is not significantly different from those found in the control (mpv17−/−) siblings, but R2 cone signals (Vr556) trend larger (Fig. 13B, 5dpf). At 6dpf, cone signal distribution differs significantly and enhancement of R2 amplitudes increases (6 dpf: 22%, t₍₃₄₂₂₎ = 9.42, p = 5.6 × 10⁻²³). In 12 dpf juveniles, R2 amplitude increases by 50% (t₍₂₅₄₀₎ = 2.65, p = 0.0082), but in adults red-cone enhancement switches from R2 (Vr556) to R1 (Vr575), where a 241% enhancement is seen (t₍₂₉₅₃₎ = 4.86, p = 1.2 × 10⁻⁶), with no significant change seen in R2-cone amplitudes (t₍₂₉₅₃₎ = 1.52, p = 0.127). In gnat2:trβ2, UV-cone signals (Vu358) persist through larval and juvenile stages, with amplitudes either not significantly different (5 dpf: t₍₃₀₉₁₎ = 0.563, p = 0.574; 12 dpf: t₍₂₅₄₀₎ = 0.594, p = 0.553) or less than control (6 dpf: 45%, t₍₃₄₂₂₎ = 5.36, p = 8.7 × 10⁻⁸), but unlike the significant UV signals of control adult (t₍₁₅₇₉₎ = 4.81, p = 1.7 × 10⁻⁶), no UV-signal was detected in gnat2:trβ2 adults (Fig. 13B). B1-cone and G1-cone signals persist in gnat2:trβ2 adults but are significantly reduced in amplitude as compared with controls (B1: 37%, t₍₂₉₅₃₎ = 2.97, p = 0.0030; G1, 50%, t₍₂₉₅₃₎ = 2.23, p = 0.024).