1 True S-cones are concentrated in the ventral mouse retina and wired for color detection in 1 the upper visual field 2 3 Francisco M. Nadal-Nicolás 1, *, Vincent P. Kunze 1, †, John M. Ball 1, †, Brian T. Peng 1, †, Akshay 4 Krisnan 1, †, Gaohui Zhou 1, †, Lijin Dong 2 , Wei Li 1, *. 5 6 1 Retinal Neurophysiology Section, National Eye Institute, National Institutes of Health, Bethesda, 7 Maryland, USA. 8 2 Genetic Engineering Facility, National Eye Institute, National Institutes of Health, Bethesda, 9 Maryland, USA. 10 11 12 †Equal contribution 13 *Corresponding authors 14 15 ABSTRACT 16 17 Color, an important visual cue for survival, is encoded by comparing signals from 18 photoreceptors with different spectral sensitivities. The mouse retina expresses a short 19 wavelength-sensitive and a middle/long wavelength-sensitive opsin (S- and M-opsin), forming 20 opposing, overlapping gradients along the dorsal-ventral axis. Here, we analyzed the 21 distribution of all cone types across the entire retina for two commonly used mouse strains. We 22 found, unexpectedly, that “true S-cones” (S-opsin only) are highly concentrated (up to 30% of 23 cones) in ventral retina. Moreover, S-cone bipolar cells (SCBCs) are also skewed towards ventral 24 retina, with wiring patterns matching the distribution of true S-cones. In addition, true S-cones 25 in the ventral retina form clusters, which may augment synaptic input to SCBCs. Such a unique 26 true S-cone and SCBC connecting pattern forms a basis for mouse color vision, likely reflecting 27 evolutionary adaption to enhance color coding for the upper visual field suitable for mice’s 28 habitat and behavior. 29 30 KEYWORDS 31 32 Genuine S-cone, cone distribution, cone cluster, mammalian photoreceptor, S-cone bipolar cells, 33 blue bipolar cells, color vision. 34 2 1. INTRODUCTION 35 Topographic representation of the visual world in the brain originates from the light-sensitive 36 photoreceptors in the retina (Rhim et al., 2017). Although the neuronal architecture of the 37 retina is similar among different vertebrates, the numbers and distributions of photoreceptors 38 vary considerably (Hunt and Peichl, 2014). Such patterns have been evolutionarily selected, 39 adapting to the animal’s unique behavior (diurnal or nocturnal) and lifestyle (prey or predator) 40 for better use of the visual information in the natural environment (Dominy and Lucas, 2001; 41 Gerl and Morris, 2008; Peichl, 2005). Color, an important visual cue for survival, is encoded by 42 comparing signals carried by photoreceptors with different spectral preferences (Baden and 43 Osorio, 2019). While amongst mammals, trichromatic color vision is privileged for some 44 primates (Jacobs et al., 1996; Nathans et al., 1986; Yokoyama and Yokoyama, 1989), most 45 terrestrial mammals are dichromatic (Marshak and Mills, 2014; Puller and Haverkamp, 2011; 46 Jacobs, 1993). The mouse retina expresses two types of cone opsins, S- and M-opsin, with peak 47 sensitivities at 360 nm and 508 nm, respectively (Jacobs et al., 1991; Nikonov et al., 2006). The 48 expression patterns of these two opsins form opposing and overlapping gradients along the 49 dorsal-ventral axis, resulting in a majority of cones expressing both opsins (herein either “mixed 50 cones” or M + S + ) (Applebury et al., 2000; Ng et al., 2001; Wang et al., 2011). Thus, S-opsin 51 enrichment in the ventral retina better detects short-wavelength light from the sky, and M-52 opsin in the dorsal retina perceives the ground (e.g., a grassy field) (Baden et al., 2013; Gouras 53 and Ekesten, 2004; Osorio and Vorobyev, 2005; Szél et al., 1992), while co-expression of both 54 opsins (herein either mixed cones or M + S + ) (Röhlich et al., 1994) broadens the spectral range of 55 individual cones and improves perception under varying conditions of ambient light (Chang et 56 al., 2013). 57 58 This unusual opsin expression pattern poses a challenge for color-coding, particularly so for 59 mixed cones. However, it has been discovered that a small population of cones only expresses 60 S-opsin (“true S-cones”, or S + M - ). These true S-cones are thought to be evenly distributed 61 across the retina (Franke et al., 2019; Haverkamp et al., 2005; Szatko et al., 2019; Wang et al., 62 2011) and to be critical for encoding color, especially in the dorsal retina where they are quasi-63 evenly distributed in a sea of cones expressing only M-opsin (M + S - ), a pattern akin to 64 mammalian retinas in general (Haverkamp et al., 2005; Wang et al., 2011). Nonetheless, 65 subsequent physiological studies revealed that color-opponent retinal ganglion cells (RGCs) are 66 more abundant in the dorsal-ventral transition zone (Chang et al., 2013) and the ventral retina 67 (Joesch and Meister, 2016). Recent large scale two-photon imaging results further 68 demonstrated that color opponent cells were mostly located in the ventral retina (Szatko et al., 69 2019). Intriguingly, a behavior-based mouse study demonstrated that their ability to distinguish 70 color is also restricted to the ventral retina (Denman et al., 2018). These results prompt us to 71 study, at the single-cell level and across the whole retina, the spatial distributions of cone types 72 with different opsin expression configurations and, more importantly, with regard to S-cone 73 bipolar cell connections in order to better understand the anatomical base for the unique color-74 coding scheme of the mouse retina. 75 76 2. RESULTS AND DISCUSSION 77 2.1. True S-cones are highly concentrated in the ventral retina of pigmented mouse. 78 3 In mouse retina, the gradients of S- and M-opsin expression along the dorsal-ventral axis have 79 been well documented (Figure 1A-B) (Applebury et al., 2000; Calderone and Jacobs, 1995; 80 Chang et al., 2013; Haverkamp et al., 2005; Jelcick et al., 2011; Lyubarsky et al., 1999; Ortín-81 Martínez et al., 2014; Szél et al., 1992; Wang et al., 2011), but the distribution of individual cone 82 types with different combinations of opsin expression across the whole retina has not been 83 characterized (but see Baden et al., 2013; Eldred et al., 2020, which we discuss below). We 84 developed a highly reliable algorithm to automatically quantify the different opsins (S and M) 85 and cone types (M + S - , true S, and mixed cones, Figure 2, Figure 2- figure supplement 1) based 86 on high-resolution images of entire flat-mount retinas immunolabeled with S- and M-opsin 87 antibodies (Figure 2- figure supplement 1). As demonstrated in examples of opsin labeling from 88 dorsal, medial, and ventral retinal areas of the pigmented mouse (Figure 1B, left), while M 89 opsin-expressing cones (M + : M + S + + M + S - ) were relatively evenly distributed across three regions, 90 S opsin-expressing cones (S + : M + S + + S + M - ) showed considerable anisotropy, with a high density 91 in the ventral retina and a precipitous drop in the dorsal retina, confirming previous 92 observations (Haverkamp et al., 2005; Jelcick et al., 2011; Ortín-Martínez et al., 2014). 93 Surprisingly, instead of finding an even distribution of true S-cones as previously presumed 94 (Baden et al., 2013; Haverkamp et al., 2005; Wang et al., 2011), we found the ventral region 95 had much more numerous true S-cones (~30% of the local cone population; Figure 1C left, 96 Supplementary file 1A) than did the dorsal region (~1%). This result is evident from density 97 maps of cone types from three examples of pigmented mice, showing highly concentrated true 98 S-cones in the ventral retina (Figure 2A, left column, bottom row). In addition, M + S - -cones were 99 concentrated in the dorsal retina, whereas mixed cones dominated the medial and ventral 100 retina (Figure 1C left and Figure 2A, left column, 4 th and 5 th rows). 101 102 2.2. Despite the vast difference in S-opsin expression pattern, the distribution of true S-cones 103 is strikingly similar between the pigmented and albino mouse. 104 Such a highly skewed distribution of true S-cones conflicts with the general notion that true S-105 cones only account for ~5% of cones and are evenly distributed across the mouse retina (Baden 106 et al., 2013; Franke et al., 2019; Haverkamp et al., 2005; Szatko et al., 2019; Wang et al., 2011); 107 however, it is not unprecedented considering the diverse S-cone patterns seen in mammals 108 (Ahnelt et al., 2000; Ahnelt and Kolb, 2000; Calderone et al., 2003; Hendrickson et al., 2000; 109 Hendrickson and Hicks, 2002; Kryger et al., 1998; Müller and Peichl, 1989; Nadal-Nicolás et al., 110 2018; Ortín-Martínez et al., 2014, 2010; Peichl, 2005; Schiviz et al., 2008; Szél et al., 2000). 111 Therefore, we also examined an albino mouse line to determine whether this observation 112 persists across different mouse strains. Overall, albino retinas had slightly smaller cone 113 populations (Figure 2B, Supplementary file 1B; Ortín-Martínez et al., 2014). Interestingly, while 114 M-opsin expressing cones had similar distributions in both strains, S-opsin expression extended 115 well into the dorsal retina of the albino mouse, exhibiting a greatly reduced gradient of S-opsin 116 expression toward the dorsal retina compared to that seen in pigmented mice (Figure 1B-C, 117 Figure 2A second row; Applebury et al., 2000; Ortín-Martínez et al., 2014). Consequently, most 118 cones in the dorsal retina were mixed cones, and M + S - cones were very sparse (7%, compared 119 to 97% in pigmented mouse, Figure 1C right, Supplementary file 1A, Figure 2A right). However, 120 despite these differences, the percentage and distribution of true S-cones were remarkably 121 conserved between strains. In both strains, true S-cones were extremely sparse in the dorsal 122 4 retina (1%) but highly concentrated in the ventral retina (33% vs 29%, Figure 1C and 123 Supplementary file 1A). Notably, the density maps of true S-cones are nearly identical in both 124 strains (Figure 2A, bottom row). Evaluating the distribution of three main cone populations 125 (mixed, M + S - , and true S-cone) in four retinal quadrants centered upon the optic nerve head 126 reveals different profiles between pigmented and albino strain for mixed and M + S - cones 127 (Figure 2C). For example, in the dorsotemporal (DT) quadrant, we observed an increase of M + S - 128 cones from the center to the periphery (green line) in pigmented mice, compared to a majority 129 of mixed cones (gray line) in albino mice. However, true S-cone profiles (magenta lines) were 130 similar between the two strains in all quadrants, except for a slightly increased density along 131 the edge of the ventronasal (VN) quadrant in pigmented mice. A recent study successfully 132 modeled cone opsin expression and type determination according to graded thyroid hormone 133 signaling in a pigmented mouse strain (C57BL/6) (Eldred et al., 2020). It would be interesting to 134 see whether a different pattern of thyroid hormone and/or receptor distribution could 135 recapitulate a similar true S-cone distribution with a very different form of S-opsin expression. 136 137 2.3. S-cone bipolar cells exhibit a dorsal-ventral gradient with a higher density in the ventral 138 retina. 139 One major concern regarding cone classification based on opsin immunolabeling is that some 140 S + M - cones may instead be mixed cones with low M-opsin expression (Applebury et al., 2000; 141 Baden et al., 2013; Nikonov et al., 2006; Röhlich et al., 1994). Even though a similar cone-type 142 distributions have been observed in mouse retina, it has been assumed that only a fraction of 143 the S + M - cones are ‘true’ S-cones (Baden et al., 2013; Eldred et al., 2020). Out of caution, S + M - 144 cones were only referred to as “anatomical” S-cones due to a lack of confirmation regarding 145 their bipolar connections (Baden et al., 2013). Thus, both true S-cones and S-cone bipolar cells 146 have been generally acknowledged to be evenly distributed across the retina (Haverkamp et al., 147 2005; Wang et al., 2011; Baden et al., 2013; Szatko et al., 2019; Franke et al., 2019; Eldred et al., 148 2020). In order to confirm the distribution of true S-cones, it is critical to uncover the 149 distribution and dendritic contacts of S-cone bipolar cells (type 9, or SCBCs). Previously, SCBCs 150 have only been identified among other bipolar, amacrine and ganglion cells in a Thy1-151 Clomeleon mouse line, rendering the quantification of their distribution across the entire retina 152 impractical (Haverkamp et al., 2005). We generated a Copine9-Venus mouse line, in which 153 SCBCs are specifically marked (Figure 3, Supplementary file 1C), owing to the fact that Cpne9 is 154 an SCBC-enriched gene (Shekhar et al., 2016). In retinal sections, these Venus + bipolar cells have 155 axon terminals narrowly ramified in sub-lamina 5 of IPL (Figure 3A), closely resembling type 9 156 BCs as identified in EM reconstructions (Behrens et al., 2016; Stabio et al., 2018a). In flat-mount 157 view, these bipolar cells are often seen to extend long dendrites to reach true S-cones, 158 bypassing other cone types (Figure 3B-C). The majority of dendritic endings formed enlarged 159 terminals beneath true S-cones pedicles (Figure 3C-c’), but occasional slender “blind” endings 160 were present (arrow in Figure 3C-c”), which have been documented for S-cone bipolar cells in 161 many species (Haverkamp et al., 2005; Herr et al., 2003; Kouyama and Marshak, 1992). 162 Unexpectedly, we found that the distribution of SCBCs was also skewed toward VN retina, 163 albeit with a shallower gradient (Figure 3D-E). To examine the connections between true S-164 cones and SCBCs, we immunolabeled S- and M-opsins in Copine9-Venus mouse retinas. 165 Because M-opsin antibody signals did not label cone structures other than their outer 166 5 segments, we first identified true S-cones at the outer segment level and then traced S-opsin 167 labeling to their pedicles in the outer plexiform layer (OPL), where they connect with SCBCs 168 (Figure 3C, for more details see material and methods). Although convergent as well divergent 169 connections were found between true S-cones and SCBCs in both dorsal and ventral retina (see 170 the source data), we noted different connectivity patterns. While in the dorsal retina, a single 171 true S-cone connected to approximately 4 SCBCs (3.8 ± 0.2, see material and methods), in the 172 ventral retina, a single SCBC contacted approximately 5 true S-cones (4.6 ± 0.4; Figure 3C, 173 Supplementary file 2). These results agree well with the true S-cone to SCBC ratios calculated 174 from cell densities in the DT and VN retina. Specifically, in the dorsal retina, the true S-cone to 175 SCBC ratio was approximately 1:3.6, compared to 5.3:1 in the ventral retina (Supplementary file 176 3). Accordingly, both data sets support the presence of a prevalent divergence of true S-cone to 177 SCBC connections in the dorsal retina, in comparison to a prominent convergence of contacts 178 from true S-cones to SCBCs in VN retina. Critically, the specificity of wiring from true S-cones to 179 SCBCs also confirms the identity of true S-cones as revealed by opsin labeling and further 180 supports the finding that true S-cones are highly concentrated in VN mouse retina. 181 182 2.4. True S-cones in the ventral retina are not evenly distributed but form clusters. 183 As demonstrated above, in the mouse retina, despite a large population of mixed cones, SCBCs 184 precisely connect with true S-cones, preserving this fundamental mammalian color circuitry 185 motif (Behrens et al., 2016; Breuninger et al., 2011; Haverkamp et al., 2005; Mills et al., 2014). 186 However, the increased density of SCBCs in the ventral retina does not match that of true S-187 cones (compare Fig 3D and Figure 2a, last row). Thus, individual SCBCs in the ventral retina may 188 be required to develop more dendrites to maximize the number of contacts made with 189 different S-cone terminals (Supplementary file 2, graphs in Figure 3C). Intriguingly, we 190 discovered in both strains that true S-cones in the ventral retina appeared to cluster together 191 rather than forming an even distribution, as revealed by K-nearest neighbor analysis (Figure 4A-192 B, Supplementary file 2). Ideally, such true S-cone clustering may increase the availability of 193 targets for individual SCBCs in a reduced space. 194 195 To quantify the spatial patterning of true S-cone populations (or their lack thereof), we 196 compared the observed true S-cone distributions within 1-mm diameter VN and DT retinal 197 samples to artificially generated alternative populations (Figure 4C). To this end, we considered 198 two extreme patterning rules: First, one in which the space between true S-cone locations was 199 maximized within the set of actual locations for all cones, creating a relatively uniform (evenly 200 “distributed”) mosaic of true S-cones. At the other extreme, cone identities were permuted 201 randomly (“shuffled”) among observed cone locations (Figure 4C). Repetition of these 202 algorithms generated distributions of patterning metrics for true S-cones (see below) that 203 remain constrained by the observed cone locations and proportions of cone types for each 1-204 mm sample. 205 206 To quantitatively compare the patterning of real true S-cone populations to their artificial 207 counterparts, we first computed two measures of regularity for true S-cones: nearest neighbor 208 and Voronoi diagram regularity indices (NNRI and VDRI, respectively; Reese and Keeley, 2015; 209 Figure 4C-D); larger values of these metrics indicate smaller variability in the spacing between 210 6 cones and thus more regular patterns. Interestingly, far from being regularly distributed, true S-211 cone placement was quite irregular and nearly indistinguishable from shuffled populations 212 (including a slight trend toward regularity measures lower than random, which may indicate a 213 tendency toward clustering, Figure 4D; see Reese, 2008). To further probe the possibility of true 214 S-cone clustering, we computed the ratios of true S-cone neighbors for each cone (denoted 215 here as the S-cone neighbor ratio [SCNR]; see Methods for the calculation of the SCNR search 216 radius for each retinal sample). Intriguingly, SCNRs were significantly larger for true S-cones 217 than for other cone types, which were equal to expected ratios due to random chance—218 especially so in ventral retinas, further indicating a clustering of true S-cones in those areas 219 (Figure 4E). Notably, a more extreme form of clustering of S-cones has been observed in the 220 “wild” mouse (Warwick et al., 2018) and with much lower densities in some felids (Ahnelt et al., 221 2000). Here, such clustering may reflect the mode of true S-cone development in the ventral 222 retina, for example, by “clonal expansion” to achieve unusually high densities (Bruhn and Cepko, 223 1996; Reese et al., 1999). It is tempting to speculate that it may also facilitate the wiring of true 224 S-cones with sparsely distributed SCBCs, which were not observed to cluster in the ventral 225 retina (Figure 3E). Indeed, we observed examples of groups of true S-cones forming clusters 226 whose pedicles in the OPL were tightly congregated in a patch and contacted by a nearby SCBC 227 (Figure 4F). 228 229 2.5. Enriched true S-cones in the ventral retina may provide an anatomical base for mouse 230 color vision. 231 Despite being nocturnal and having a rod-dominated retina (Carter-Dawson and LaVail, 1979; 232 Jeon et al., 1998), mice can detect color (Denman et al., 2018; Jacobs et al., 2004). Although it 233 remains uncertain whether the source of long-wavelength sensitive signals for color opponency 234 arises in rods or M-cones (Baden and Osorio, 2019; Ekesten et al., 2000; Ekesten and Gouras, 235 2005; Joesch and Meister, 2016; Reitner et al., 1991), it is clear that true S-cones provide short-236 wavelength signals for color discrimination. Given the previously-held notion that true S-cones 237 are evenly distributed across the retina (Baden et al., 2013; Franke et al., 2019; Haverkamp et 238 al., 2005; Szatko et al., 2019; Wang et al., 2011), whereas M + S - cones are concentrated in the 239 dorsal retina of pigmented mouse, it is intuitive to speculate that color coding is prevalent in 240 the dorsal retina. However, previous physiological and behavioral studies indicate that, 241 although luminance detection can occur across the mouse retina, color discrimination is 242 restricted to the ventral retina (Breuninger et al., 2011; Denman et al., 2018; Szatko et al., 243 2019). Thus, our discovery of high enrichment of true S-cones in the ventral retina provides a 244 previously missed anatomical feature for mouse color vision that could help to re-interpret 245 these results. From projections mapping true S-cone densities into visual space (Figure 4-figure 246 supplement 1; Sterratt et al., 2013), it is conceivable that high ventral true S-cone density will 247 provide a much higher sensitivity of short-wavelength signals, thus facilitating color detection 248 for the upper visual field. Although the true S-cone signals carried by SCBCs in the dorsal retina 249 might not be significant for color detection, they could certainly participate in other functions, 250 such as non-image forming vision, that are known to involve short-wavelength signals (Altimus 251 et al., 2008; Doyle et al., 2008; Patterson et al., 2020). Interestingly, the overall true S-cone 252 percentage in the mouse retina remains approximately 10% (Figure 2B), and the average true S-253 cone to SCBC ratio across the whole retina is about 1.7:1 (Supplementary file 1B-C), similar to 254 7 what has been reported in other mammals (Ahnelt et al., 2006; Ahnelt and Kolb, 2000; Bumsted 255 et al., 1997; Bumsted and Hendrickson, 1999; Curcio et al., 1991; Hendrickson and Hicks, 2002; 256 Hunt and Peichl, 2014; Kryger et al., 1998; Lukáts et al., 2005; Müller and Peichl, 1989; Ortín-257 Martínez et al., 2010; Peichl et al., 2000; Schiviz et al., 2008; Shinozaki et al., 2010; Szél et al., 258 1988). 259 260 Such a spatial rearrangement of true S-cones and SCBCs likely reflects evolutionary adaption to 261 enhance short-wavelength signaling and color coding for the upper visual field as best suited for 262 the habitat and behavior of mice (Baden et al., 2020). For example, it may facilitate aerial 263 predator detection during daytime (Yilmaz and Meister, 2013). Similarly, skewed S-cone 264 arrangement has been reported for other terrestrial prey mammals (Famiglietti and Sharpe, 265 1995; Juliusson et al., 1994; Röhlich et al., 1994), while zebrafish possess a UV-enriched ventral 266 retina that enhances their predation (Zimmermann et al., 2018). In addition, we observed that 267 the clustering of true S-cones in the ventral retina may allow several neighboring cones of the 268 same type to converge onto the same SCBC (Figure 4F), which could potentially enhance signal-269 to-noise ratios for more accurate detection, as described recently in human fovea (Schmidt et 270 al., 2019). It is also remarkable that despite the very different S-opsin expression patterns in 271 both mouse strains, the true S-cone population and distribution are strikingly similar between 272 pigmented and albino mice, suggesting a common functional significance. 273 274 3. ACKNOWLEDGEMENTS 275 The authors would like to thank the NEI Animal Care team, especially Megan Kopera and Ashley 276 Yedlicka. 277 278 4. COMPETING INTERESTS 279 The authors declare no competing or financial interests. 280 281 5. FUNDING 282 This research was supported by Intramural Research Program of the National Eye Institute, 283 National Institutes of Health to WL. 284 8 6. FIGURES 285 286 287 Figure 1. Cone outer segments across retinal areas. Immunodetection of M and S wavelength-288 sensitive opsins in retinal sections (A) and flat-mount retinas (B) in two mouse strains 289 (pigmented and albino mice, left and right columns respectively). (C) Retinal scheme of S-opsin 290 expression used for image sampling to quantify and classify cones in three different retinal 291 regions. Pie graphs showing the percentage of cones manually classified as M + S - (green), S + M - 292 (true S, magenta) and M + S + (mixed, gray) based on the opsin expression in different retinal 293 areas from four retinas per strain. Black mouse: pigmented mouse strain (C57BL6), white 294 mouse: albino mouse strain (CD1). 295 9 296 Figure 2. Topography and total number of different opsins (M + , S + ) and cone-type populations 297 in the whole mouse retina. (A) Density maps depicting the distributions of different opsins 298 10 expressing cones (M + and S + ) and different cone populations classified anatomically as: All, M + S + 299 (mixed), M + S - , S + M - (true S) cones in pigmented and albino mice (left and right side respectively). 300 Each column shows different cone populations from the same retina and, at the bottom of each 301 map is shown the number of quantified cones. Color scales are shown in the right panel of each 302 row (from 0 [purple] to 17,300 [dark red] for all cone types except to 5,000 cones/mm 2 [dark 303 red] for the true S-cones and M + S - -cone in the albino strain). Retinal orientation depicted by D: 304 dorsal, N: nasal, T: temporal, V: ventral. (B) Histogram showing the mean ± standard deviation 305 of different cone subtypes for eight retinas per strain (Supplementary file 1B). The percentages 306 of each cone subtype are indicated inside of each bar, where 100% indicates the total of the ‘all 307 cones’ group. (C) Opsin expression profile across the different retinal quadrants (retinal scheme, 308 DT: dorsotemporal, DN: dorsonasal, VT: ventrotemporal, VN: ventronasal). Line graphs show 309 the spatial profile of relative opsins expression (mixed [gray], M + S - [green], true S-cones 310 [magenta]), where the sum of these three cone populations at a given distance from the optic 311 nerve (ON) head equals 100%. Black mouse: pigmented mouse strain, white mouse: albino 312 mouse strain. 313 11 314 Figure 2-figure supplement 1. Validation of automatic routine for cone outer segment 315 quantification. (A) Retinal photomontages for M- and S-opsin signal in the same pigmented 316 retina (correspond to second column in Figure 2A). The square depicts an area of interest 317 selected (transition zone of S-opsin expression) to perform the automatic routine validation by 318 comparing manual and automatic quantifications. The images processed by the automatic 319 routine using ImageJ show the selection of positive objects from the corresponding original 320 image. (B) X, Y graph showing the linear correlation (Pearson coefficient, R 2 ) between manual 321 and automatic quantifications. 21,898 M + and 13,705 S + cones were manually annotated while 322 21,689 M + and 13,661 S + cones were automatically identified in 3 random images obtained from 323 5 retinal photomontages. (C) All, mixed, M + S - - and true S-cone populations are extracted from 324 the original M- and S-cone images. All-cones were quantified after overlapping M- and S-signals. 325 mixed (M + S + ) cones were obtained by subtracting the background of the S-opsin image in the 326 M-opsin one. M + S - cones for pigmented mice are obtained after subtracting the S-opsin signal 327 to the M-opsin photomontage. Finally, M + S - cones for albino and true S-cones (S + M - ), in both 328 strains, are manually marked on the retinal photomontage (Adobe Photoshop CC). The B&W 329 images shown the processed image after quantifying automatically. At the bottom of each 330 image is shown the number of quantified cones. Black mouse: pigmented mouse strain. 331 12 332 Figure 3. S-cone Bipolar cells (SCBCs) in Cpne9-Venus mouse retina. (A) Retinal cross section 333 showing the characteristic morphology of SCBCs (Behrens et al., 2016; Breuninger et al., 2011). 334 (B) Detailed view of the selective connectivity between Venus + SCBCs and true S-cone terminals 335 (yellow arrows). Note that SCBCs avoid contacts with cone terminals lacking S-opsin expression 336 13 (M + S - -cone pedicles, identified using cone arrestin), as well as a mixed cone pedicle, marked 337 with an asterisk. In fact, on the contrary, the SCBCs prefer to develop multiple contacts to the 338 same true S-cone pedicle. (C) Images from flat-mount retinas focused on the inner nuclear and 339 outer plexiform layers (INL+OPL) or in the photoreceptor outer segment (OS) layer of the 340 corresponding area. Magnifications showing divergent and convergent connectivity patterns 341 from true S-cone pedicles in dorsal and ventral retinal domains, respectively. In the DT retina, 342 six Venus + SCBCs (cyan circles) contact a single true S-cone pedicle (magenta circle in DT); while 343 one Venus + SCBC contacts at least four true S-cone pedicles in the VN retina (magenta circles in 344 VN), which belong to cones possessing S + M - OSs (yellow circles). Connectivity between true S-345 cones and SCBCs in DT and VN retina was assessed as the average number of true S-cone 346 pedicles contacting a single SCBC per retina (magenta plot) or the average number of SCBCs 347 contacting a single true S-cone pedicle per retina (cyan plot) (p<0.0001, p<0.01, respectively; 348 n=5). (c’) Detailed view of a secondary SCBC bifurcation contacting independently two true S-349 cone pedicles. (c”) Detailed view of a “blind” SCBC process. (D) Density maps depicting the 350 distributions of SCBCs in Cpne9-Venus mice. (d) Venus + SCBCs along the DT-VN axis from a flat-351 mount retina (corresponding to the white frame in D) showing the gradual increase of SCBCs 352 towards the VN retina where true S-cone density peaks (last row in Figure 2A). (E) 353 Demonstration of Venus + SCBC densities color-coded by the k-nearest neighbor algorithm 354 according to the number of other Venus + SCBCs found within an 18 m radius in two circular 355 areas of interest (DT and VN). Although, Venus + SCBCs exhibit a sparse density without forming 356 clusters (circular maps), they were significantly denser in VN retina (p<0.0001; n=8). 357 14 358 Figure 4. Clustering of true S-cones in the ventronasal (VN) retina. (A) Retinal magnifications 359 from flat-mount retinas demonstrating grouping of true S-cones in the VN area, where true S-360 cone density peaks. White dashed lines depict independent groups of true S-cones that are not 361 commingled with mixed cones (M + S + , white outer segments in the merged image). (B) Retinal 362 15 scheme of true S-cones used for selecting two circular areas of interest along the 363 dorsotemporal-ventronasal (DT-VN) axis. Circular maps demonstrate true S-cone clustering in 364 these regions. True S-cone locations are color-coded by the k-nearest neighbor algorithm 365 according to the number of other true S-cones found within an 18 m radius. (C-E) Analytical 366 comparisons of DT and VN populations of true S-cones to their simulated alternatives. C) 367 Example real and simulated true S-cone populations and their quantification. Images depict 368 true S-cone locations (magenta dots) and boundaries of their Voronoi cells (dashed lines) from 369 original and example simulated (“distributed”, “shuffled”) cone populations. Gray dots indicate 370 the locations of other cone types. Observed cone locations were used for all simulated 371 populations; only their cone identities were changed. The annotated features are examples of 372 those measurements used in the calculations presented in D-E. (D) Comparison of sample 373 regularity indices for one albino VN retinal sample to violin plots of those values observed for 374 n=200 simulated cone populations. Note that average regularity indices for true S-cones were 375 lower than that of shuffled populations, whereas those values lay between shuffled and 376 distributed populations when all cones were considered. Plots on the right show values for all 377 actual retinal samples normalized using the mean and standard deviations of their simulated 378 “shuffled” counterparts. The y-axis range corresponding to ± 2.5 standard deviations from the 379 mean (i.e., that containing ~99% of shuffled samples) is highlighted in gray. (E) Comparison of 380 the real average SCNR for the example in C-D to those values for its simulated counterparts. 381 Note that the average SCNR for all cones in this sample was equal to that predicted by random 382 chance (i.e., the ratio of true S-cones to all cones), which in turn was equal to the average for 383 true S-cones for shuffled samples. In contrast, the real true S-cone SCNR was higher. Plot on the 384 right shows true S-cone SCNR values for all samples, normalized as described for D. (F) 385 Convergent connectivity from a true S-cone cluster to a single SCBC in the VN retina. Images of 386 a true S-cone cluster, in a flat-mount retina, focused on the photoreceptor outer segment layer 387 and the inner nuclear-outer plexiform layers (INL+OPL). The upper left panel show the 388 numerical and colored identification of each true S-outer segment in the cluster (note that the 389 number positions indicate the locations where outer segments contact the photoreceptor inner 390 segment). Each true S-cone pedicle belonging to this cluster is outlined and color coded (middle 391 upper panel) and are overlaid upon the SCBC dendritic profile (right upper panel). To identify 392 synaptic contacts between the SCBC and the cone pedicles (maximum intensity projection -393 excluding the SCBC soma- shown in lower left panel), we acquired orthogonal single plane 394 views zooming into putative dendritic tips. An example for the contact with cone #5 is shown in 395 lower middle panel, corresponding to the box area in lower left panel (f). The lower right panel 396 shows dendritic endings of this SBCB (black) contacting the marked cones (#1-6). It also 397 contacts two additional cones outside of the field of view (#7,8). Dashed line depicts the soma 398 of the SCBC. Dendrites from other SCBCs are color coded for differentiation. 399 16 400 Figure 4-figure supplement 1. Reconstruction and mapping of true S-cone densities into visual 401 space. Representative left eye from a 3-month-old pigmented mouse (C57). (A) S-opsin 402 antibody labeling; (B) true s-cone density contour lines separated by quintiles overlaid onto s-403 opsin labeling; (C) quintile heatmap contours of true s-cone density. The top two rows 404 demonstrate the flat-mount retina with marks for edges and relaxing cuts, followed by its 405 reconstruction into uncut retinal space with lines of latitude and longitude that have been 406 projected onto the flat-mount. The bottom two rows show the reconstructed retina inverted 407 into visual space using orthogonal and sinusoidal projections. For these views, eye orientation 408 angles for elevation and azimuth of 22 and 64, respectively, have been used as in (Sterratt et 409 al., 2013). For orthogonal projections, the globe has been rotated forward by 50 to emphasize 410 the relationship of true S-cone densities to the upper pole of the visual field. 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KEY RESOURCES 646 647 Key Resources Table Reagent type (species) or resource Designation Source or reference Identifiers Additional information strain, strain background (Mus musculus, male) C57BL/6J mouse strain Jackson Laboratory Cat#000664, RRID:IMSR_J AX:000664 Pigmented mouse inbred strain strain, strain background (Mus musculus, male) Crl:CD-1(ICR) mouse strain Charles River Cat#022, RRID:IMSR_C RL:022 Albino mouse strain strain, strain background (Mus musculus, male) Copine9- Venus mouse line This paper Material and methods section 8.3.1 antibody anti-OPN1SW (N-20) (Goat polyclonal) Santa Cruz Biotechnology Cat#sc- 14363, RRID:AB_215 8332 IF (1:1200) antibody anti-Opsin Red/Green (Rabbit polyclonal) Millipore/Sigm a Cat#AB5405, RRID:AB_177 456 IF (1:1000) antibody anti-Cone Arrestin (Rabbit polyclonal) Millipore/Sigm a Cat#AB15282 , RRID:AB_116 3387 IF (1:300) antibody anti-GFP (Chicken polyclonal) Millipore/Sigm a Cat#AB16901 , RRID:AB_112 12200 IF (1:100) 24 antibody anti-Rabbit 488 (Donkey polyclonal) Jackson Immunoresear ch Cat#711-547- 003, RRID:AB_234 0620 IF (1:500) antibody anti-Rabbit Cy3 (Donkey polyclonal) Jackson Immunoresear ch Cat#711-165- 152, RRID:AB_230 7443 IF (1:500) antibody anti-Goat 647 (Donkey polyclonal) Jackson Immunoresear ch Cat#705-605- 147, RRID:AB_234 0437 IF (1:500) antibody anti-Goat Cy3 (Donkey polyclonal) Jackson Immunoresear ch Cat#705-166- 147, RRID:AB_234 0413 IF (1:500) antibody anti-Chicken 488 (Donkey polyclonal) Jackson Immunoresear ch Cat#703-545- 155, RRID:AB_234 0375 IF (1:500) sequence-based reagent Copine9_gR NA_L(73/25) This paper 5’GAGACATGA CTGGTCCAA3’ sequence-based reagent Copine9_gR NA_R(62/4.4 0), This paper 5’GCCTCGGAG CGTAGCGTCC 3’ software, algorithm Zen Zeiss Zen lite Black edition 2.3 SP1 software, algorithm FIJI-ImageJ NIH v1.52r https://imagej .nih.gov/ij/ software, algorithm Sigma Plot Systat Software 13.0 25 software, algorithm GraphPad Prism Graph Pad Software 8.3.0 software, algorithm Photoshop Adobe CC 20.0.6 software, algorithm MATLAB MathWorks 2016 software, algorithm R The R Project for Statistical Computing 3.5.3 https://www.r -project.org/ software, algorithm Retina and Visual Space Retistruct Package Sterratt DC et al., PLoS Comput Biol. software, algorithm Zotero Corporation for Digital Scholarship 5.0 https://www.z otero.org/dow nload/ other DAPI ThermoFisher Scientific Cat# D3571, RRID:AB_230 7445 (1ug/ml) 648 8.2. LEAD CONTACT AND MATERIALS AVAILABILITY 649 Further information and requests for resources and reagents should be directed to and will be 650 fulfilled by the Lead Contact, Wei Li (liwei2@nei.nih.gov). 651 652 8.3. METHOD DETAILS 653 654 8.3.1. Animal generation, handling and ethic statement 655 Three months old male pigmented (C57BL/6J, n=5), albino (CD1, n=5) mice were obtained from 656 the National Eye Institute breeding colony. The Venus-Cpne9 mouse line (n=5; based on 657 previous single cell sequencing data (Shekhar et al., 2016)) carries a reporter (Venus) allele 658 under the control of the mouse Cpne9 locus. The reporter allele was created directly in 659 B6.SJL(F1) zygotes using CRISPR-mediated homologous recombination (HR) (Yang et al., 2013). 660 Briefly, a HR targeting template was assembled with PCR fragments of 5’ and 3’ homology arms 661 of 910 bp and 969 bp respectively, flanking exon one, and a Venus expression cassette carrying 662 the bovine growth hormone polyadenylation (bGH-PolyA) signal sequence as the terminator. 663 Homology arms were designed such that integration of the reporter cassette would be at the 664 26 position right after the first codon of the Cpne9 gene in exon one. A pair of guide RNAs (gRNA), 665 with outward orientation (38 bp apart), were synthesized by in vitro transcription as described 666 (Yang et al., 2013) and tested for their efficiency and potential toxicity in a zygote 667 differentiation assay where mouse fertilized eggs were electroporated with SpCas9 protein and 668 gRNA ribonuclear particles. Eggs were cultured in vitro for 4 days in KSOM (Origio Inc, CT) until 669 differentiated to blastocysts. Viability and indel formation were counted respectively. gRNA 670 sequences are (1) Copine9_gRNA_L(73/25), 5’GAGACATGACTGGTCCAA3’; (2) 671 Copine9_gRNA_R(62/4.40), 5’GCCTCGGAGCGTAGCGTCC3’. A mixture of the targeting plasmid 672 (super coiled, 25ng/µl) with two tested gRNAs (25 ng/µl each) and the SpCas9 protein (Life 673 Science technology, 30ng/µl) were microinjected into mouse fertilized eggs and transferred to 674 pseudopregnant female recipients as described elsewhere (Yang et al., 2013). With a total of 15 675 F0 live births from 6 pseudopregnant females, 11 were found to carry the knockin allele by 676 homologous recombination, a HR rate of 73%. F0 founders in B6.SJL F1 (50% C57BL6 genome) 677 were crossed consecutively for 3 generations with C57BL6/J mice to reach near congenic state 678 to C57BL6/J. 679 Mice were housed a 12:12 hours light/dark cycle. All experiments and animal care are 680 conducted in accordance with protocols approved by the Animal Care and Use Committee of 681 the National Institutes of Health and following the Association for Research in Vision and 682 Ophthalmology guidelines for the use of animals in research. 683 684 8.3.2. Tissue collection 685 All animals were sacrificed with an overdose of CO2 and perfused transcardially with saline 686 followed by 4% paraformaldehyde. To preserve retinal orientation, eight retinas per mouse 687 strain/line were dissected as flat whole-mounts by making four radial cuts (the deepest one in 688 the dorsal pole previously marked with a burn signal as described (Nadal-Nicolás et al., 2018; 689 Stabio et al., 2018b). The two remaining retinas were cut in dorso-ventral orientation (14m) 690 after cryoprotection in increasing gradients of sucrose (Sigma-Aldrich SL) and embedding in 691 optimal cutting temperature (OCT; Sakura Finetek). 692 693 8.3.3. Immunohistochemical labeling 694 Immunodetection of flat-mounted retinas or retinal sections was carried out as previously 695 described (Nadal-Nicolás et al., 2018). Importantly, the retinal pigmented epithelium was 696 removed before the immunodetection. First, whole-retinas were permeated (4x10’) in PBS 0.5% 697 Triton X-100 (Tx) and incubated by shaking overnight at room temperature with S-opsin (1:1200) 698 and M-opsin (1:1000) or cone arrestin (1:300) primary antibodies diluted in blocking buffer (2% 699 normal donkey serum). Cpne9-Venus retinas were additionally incubated with an anti-GFP 700 antibody (1:100) to enhance the original Venus signal. Retinas were washed in PBS 0.5% Tx 701 before incubating the appropriate secondary antibodies overnight (1:500). Finally, retinas were 702 thoroughly washed prior to mounting with photoreceptor side up on slides and covered with 703 anti-fading solution. Retinal sections were counterstained with DAPI. 704 705 8.3.4. Image acquisition 706 Retinal whole-mounts were imaged with a 20x objective using a LSM 780 Zeiss confocal 707 microscope equipped with computer-driven motorized stage controlled by Zen Lite software 708 27 (Black edition, Zeiss). M- and S-opsins were imaged together to allow the identification and 709 quantification of different cone types. Magnifications from flat mounts and retinal cross-710 sections (Figure 1) were taken from dorsal, medial and ventral areas using a 63x objective for 711 opsin co-expression analysis. Images from retinal cross-sections were acquired ~1.5mm dorsally 712 or ventrally from the optic disc. 713 714 8.3.5. Sampling and opsin co-expression measurement 715 In four retinas per strain, we acquired images from three 135x135 m samples (63x) per each 716 area of interest (dorsal, medial and ventral). These areas were selected according to the S-opsin 717 gradient in wholemount retinas (see scheme in Figure 1C). Cone outer segments were manually 718 classified as M + S - , true S- (S + M - ) or mixed (M + S + ) cones depending on their opsin expression. 719 Data representation was performed using GraphPad Prism 8.3 software. 720 721 8.3.6. Image processing: manual and automated whole quantification 722 To characterize the distribution of the different cone photoreceptor types in the mouse retina, 723 we developed and validated an automatic routine (ImageJ, NIH) to identify, quantify the total 724 number of outer segments and finally extract the location of each individual cone (Figure 2-725 figure supplement 1A). Briefly, maximum-projection images were background-subtracted and 726 thresholded (background-noise mean value, 9.6±1.2% and 15.2±3.2% for S- and M-opsin 727 respectively, the threshold was applied at 15.7%) to create a binary mask that was then 728 processed using watershed and despeckle filters to isolate individual cones and reduce noise. 729 The “3D Objects Counter” plugin was applied to such images to count cones within fixed 730 parameters (shape and size) and extract their xy coordinates for further analysis. This 731 automation was validated by statistical comparation with manual counting performed by an 732 experienced investigator (Pearson correlation coefficient R 2 = 96-99% for M- or S-opsin 733 respectively, Figure 2-figure supplement 1B). To count cone subtypes, images were pre-734 processed with image processing software (Adobe Photoshop CC) to isolate the desired subtype 735 and then manually marked using Photoshop, or automatically counted using ImageJ as 736 described above. Total cone populations were determined by combining M- and S-opsin 737 channels, while mixed M + S + cones were obtained by masking the M-opsin signal with the S-738 opsin channel. M + S - cones in pigmented mice were obtained by subtracting the S-opsin signal 739 from the M-opsin photomontage. Finally, M + S - cones (in albino samples), true S-cones (both 740 strains) (Figure 2-figure supplement 1C) and Venus + SCBCs (Cpne9-Venus mouse line) were 741 manually marked on the retinal photomontage (Adobe Photoshop CC). 742 743 8.3.7. Topographical distributions. 744 Topographical distributions of cone population densities were calculated from cone locations 745 identified in whole-mount retinas using image processing (see above). From these populations, 746 isodensity maps were created using Sigmaplot 13.0 (Systat Software). These maps are filled 747 contour plots generated by assigning to each area of interest (83.3x83.3 m) a color code 748 according to its cone density, ranging from 0 (purple) to 17,300 cones/mm 2 for all cone types 749 except for true S-cones and M + S - -cone in the albino strain (5,000 cones/mm 2 ), as represented in 750 the last image of each row of Figure 2A, or 1,400 SCBCs/mm 2 (Figure 3D) within a 10-step color-751 scale. These calculations allow as well, the illustration of the number of cones at a given 752 28 position from the ON center. To analyze the relative opsin expression along the retinal surface, 753 we have considered three cone populations (mixed, M + S - - and true S-cones) dividing the retina 754 in four quadrants: dorsotemporal, dorsonasal, ventrotemporal and ventronasal (DT, DN, VT and 755 VN respectively, scheme in Figure 2C). The relative percentage of cone-types are represented in 756 line graphs from four retinas/strain (SigmaPlot 13.0). 757 758 8.3.8. SCBC sampling and ‘true S-cone’ connectivity 759 To characterize the connectivity of Venus + S-cone bipolar cells (Venus + SCBCs) with true S-cone 760 terminals, we acquired images from the same area (260x260 m, 63x) at two focal planes: First, 761 we focused upon the INL+OPL, then the corresponding photoreceptor outer segment (OS) layer, 762 respectively, for two areas of interest (DT and VN). To verify connectivity between Venus + SCBC 763 dendrites and true S-cone pedicles in the OPL, in addition to S-opsin immunodetection, we also 764 labeled retinas using cone arrestin antibodies to discriminate mixed cone pedicles from true S-765 cone pedicles, because true S-cone pedicles contain either low or no cone arrestin (Figure 3B, 766 Haverkamp et al., 2005). In other retinas, SCBC contacts were verified by tracking each cell body 767 from cone pedicles to their respective OS to confirm S + M - opsin labeling (Figure 3C). In five 768 retinas (with S- and M-opsin double immunodetection), we analyzed the connectivity between 769 186 Venus + SCBCs (133 and 53 for DT and VN respectively) and 263 true S-cone pedicles (74 and 770 189, DT and VN respectively). The number of synaptic contacts was assessed by tracking 771 manually each SCBC-branch from the cell body using the Zen lite black visualization package (Z-772 stack with 1m interval). Multiple branch contacts in one true S-cone pedicle from a single 773 SCBC were considered a single contact and counted only once (Figure 3B), while secondary 774 bifurcations were considered as multiple contacts (Figure 3c’). SCBC-blind endings were not 775 counted (Figure 3c”). The average number of contacts per retina was used to calculate the DT 776 and VN means (Supplementary file 2 and graphs in 3C). 777 778 8.3.9. Clustering analysis. K-neighbor maps and variance analysis of Voronoi dispersion. 779 To assess the true S-cones and S-cone bipolar cell (SCBC) clustering, we performed two 780 comparable sets of analyses. First, we extracted two circular areas (1mm diameter) in the DT-781 VN axis at 1mm from the optic disc center (scheme in 4B). A K-nearest neighbor algorithm 782 (Nadal-Nicolás et al., 2014) was used to map the number of neighboring true S-cones within a 783 18 m radius of each true S-cone to a color-code in its retinal position (Figure 4B). Regularity 784 indices were computed for each retinal sample using Voronoi diagrams for cone positions as 785 well as nearest neighbor distances (VDRI and NNRI, respectively (Reese and Keeley, 2015); 786 Figure 4C-E). NNRIs were computed as the ratio of the mean to the standard deviation for the 787 distance from true S-cones to their nearest true S-cone neighbor. true S-cone neighbor ratios 788 (SCNR) were calculated for each retinal sample as the average proportion of true s-cones within 789 a given radius for each cone. This search radius was calculated separately for each sample to 790 correct for sample-to-sample variations in total density: this radius (r) was calculated as r = 3√ 791 (A / (√2 πN)), where A is the circular area of the 1mm diameter retinal sample and N is the total 792 number of cones in that sample. For a highly regular cell mosaic containing N cells filling an area 793 A, this calculation estimates the location of the first minimum in the density recovery profile 794 (Rodieck, 1991), providing the average radius of a circle centered upon a cone that will 795 29 encompass its first tier of cone neighbors (but exclude the second tier) in an evenly distributed 796 mosaic. To minimize edge effects from computations of NNRI, VDRI, SCNR, those values for 797 cones closer to the outer edge of the sample than the SCNR search radius were discarded. To 798 produce simulated cone mosaics for comparison with observed values, cone distributions with 799 evenly “distributed” true S-cones were generated by first using a simple mutual repulsion 800 simulation to maximize the distances between true S-cones, followed by assigning the nearest 801 positions among all cone locations as being “true S”. “Shuffled” populations of true S-cones 802 were generated by permuting cone identities randomly among all cone locations, holding the 803 proportion of true S-cones constant. Voronoi diagrams, neighbor calculations, and mosaic 804 generation and other computations were performed using MATLAB R2016b. 805 806 8.3.10. True S-cone cluster and SCBC synaptic contacts evaluation 807 To characterize the true S-cone cluster connectivity in the VN retina, retinal whole-mounts 808 were imaged with a 63x objective, from the photoreceptor outer segments to the OPL, in a Z-809 stack image with 0.5m interval. To visualize the true S-cone clustering and Venus + SCBC 810 connectivity, we identified numerically, and color coded each true S-outer segment form a 811 cluster. The corresponding true S-pedicles were identified by tracking the cell body from their 812 S + M - OSs. Focusing on the outer plexiform layer (OPL), each individual true S-cone pedicle -that 813 form a cluster- was manually outlined and color coded accordingly. Lastly, the SCBC synaptic 814 terminals, that belong to a single SCBC, were identified by their specific contacts to the 815 respective true S-cone pedicle (Figure 4F). 816 817 8.3.11. Retinal reconstruction and visuotopic projection 818 Retinal images were reconstructed and projected into visual space using R software v.3.5.2 for 819 64-bit Microsoft Windows using Retistruct v.0.6.2 as in Sterratt et al. (2013). Reconstruction 820 parameters from that study were used: namely, a rim angle of 112 (phi0 = 22), and eye 821 orientation angles of 22 (elevation) and 64 (azimuthal). For figure 4-figure supplement 1, true 822 S-cone density contour lines and heatmaps were computed in MATLAB and overlaid onto flat-823 mount retina opsin labeling images using ImageJ prior to processing by Retistruct. 824 825 8.3.12. Statistical analysis 826 Statistical comparisons for the percentage of cones/retinal location, the total cone 827 quantifications (Supplementary file 1) and the DT or VN true S-cones and Venus + SCBCs 828 (Supplementary file 2) were carried out using GraphPad Prism v8.3 for Microsoft Windows. 829 Data are presented as mean ± standard deviation. All data sets passed the D'Agostino-Pearson 830 test for normality, and the comparisons between strains were performed with Student’s t-test. 831 832 For each 1mm retinal sample, VDRI, NNRI, and SCNR values were normalized and compared to 833 the distributions of “shuffled” cone populations. Such comparisons were not performed against 834 “distributed” populations, because in those populations, VDRI and NNRI values were 835 consistently much higher—and SCNR much lower—than in real samples (see Figure 4D-E). The 836 “shuffled” populations for each retinal region produced measurements that were well 837 described by normal distributions (Kolmogorov-Smirnov test, MATLAB). Thus, to allow 838 comparisons across samples, we converted each measurement into a Z-score using the mean 839 30 and standard deviation of those measures from shuffled populations. One-tailed Student’s t-840 tests were performed to compare the normalized measures to the distribution of “randomly 841 shuffled” cone population measures, and significance was determined at the p<0.05 level. 842 843 9. SUPPLEMENTARY MATERIAL 844 845 Supplementary file 1. (A) Cone numbers in different retinal areas along the dorsoventral axis in 846 pigmented and albino mouse. Three images/area (dorsal, medial and ventral) from four 847 retinas/strain. Different cone type quantifications are shown as average ± SD, corresponding to 848 the percentages shown in Fig 1C. The total number of cones analyzed per location and strain 849 are shown in the last column. Total number of cones (B) or S-cone Bipolar cells (SCBCs, C) in 850 eight retinas/mouse strain or line (average ± SD, see also Figure 2B). Significant differences 851 between strains p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****). 852 853 Supplementary file 2. True S-cone terminals and Cpne9-Venus+SCBCs connectivity in 854 dorsotemporal (DT) and ventronasal retina (VN). Quantitative data are shown as mean ± SD 855 from the average of five DT and VN retinal areas (Figure 3C). Significant differences between 856 retinal areas, p<0.01 (**), p<0.0001 (****). 857 858 Supplementary file 3. Numbers of true S-cones (A) and Cpne9-Venus + SCBCs (B) in 859 dorsotemporal (DT) and ventronasal (VN) circular areas (1mm diameter, Figs 3E and 4B). 860 Quantitative data are shown as average ± SD from eight retinas/strain or line. The mean of true 861 S-cones and Venus + SCBCs in these circular areas was used to calculate the DT:VN and true S-862 cone:SCBC (C) ratios. Significant differences between strains p<0.05 (*), p<0.001 (***). True S-863 cones and SCBCs were significant different between DT and VN retina (p<0.0001). 864 Article File