(A) Sketch of the zebrafish retina. RGCs, the innermost retinal neurons, transmit visual information to the rest of the brain through the optic nerve. Unique patterns of dendritic stratification in the inner plexiform layer (IPL), which is divided into two halves that subserve ON and OFF light responses, enables distinct RGC types (colors) to receive presynaptic input from specific interneuron types, rendering individual RGC types sensitive to distinct visual features. (B) Left: RGC projectome (Robles et al., 2014). RGC types are defined by stereotyped combinations of dendritic stratification patterns in the retina and axonal projections to retinorecipient nuclei, named arborization fields (AFs 1–9) and tectum. Right: within the tectum, RGC axons project to nine or ten laminae, including SO (stratum opticum), SFGS (stratum fibrosum et griseum superficiale) 1–6, SGC (stratum griseum centrale), and the boundary between SAC/SPV (stratum album centrale/stratum periventriculare). Each AF or tectal lamina is innervated by a unique set of RGC morphotypes, depicted by colors. (C) Tg(isl2b:tagRFP) labels RGCs. Left: section of an adult eye immunostained for RFP, synaptotagmin (syt2) as a neuropil stain, and DAPI counterstain of somata. Middle: magnified retinal section highlighting RFP-labeled RGCs. Right: overlay of all markers in the retinal section. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars, 500 μm (left) and 50 μm (middle and right). (D) Left: confocal plane covering the RFP-immunostained adult Tg(isl2b:tagRFP) tectum with DAPI counterstain. Right: magnified area. A, anterior; M, medial. For layer abbreviations, see (A). Scale bar, 100 μm. (E) t-distributed stochastic neighbor embedding (tSNE) visualization of 32 transcriptional clusters (colors) of 32,679 adult zebrafish RGCs (points). Clusters are numbered in the order of decreasing relative frequency. (F) Relative frequency (y axis) of adult RGC clusters (x axis), ordered from highest to lowest. Clusters are colored as in (E). (G) Dot plot showing the expression patterns of three RGC markers (rows) across adult clusters (columns). The area of each circle depicts the percentage of cells expressing the gene, and the color depicts the Z-scored expression in cells with non-zero transcripts. Clusters are ordered based on their global transcriptional relatedness depicted using a dendrogram (top), computed using hierarchical clustering. (H) Dot plot showing expression patterns of markers (rows) that are selectively enriched in adult RGC clusters (columns). Column ordering and expression depiction were as in (G).

(A and B) Dot plots highlighting examples of variably expressed TFs in adult RGC clusters, subdivided into broad (A) and restricted (B) categories. Representation is as in Figure 1G. Full list is provided in Table S2. (C) Dot plot highlighting key cell-surface and secreted molecules selectively expressed in adult RGC clusters. (D) Dot plot highlighting neuropeptides selectively expressed in adult RGC clusters.

(A) Global transcriptional relatedness (dendrogram, left) of larval RGC clusters (rows) identifies two groups, corresponding to mature and immature RGCs (gray cluster labels and shaded horizontal bar). The dot plot highlights expression of pan-RGC markers rbpms2b, isl2b, and robo2, as well as the top differentially expressed genes (n = 8) between immature and mature RGC clusters. (B) Expression patterns of markers (columns) that are selectively enriched in larval RGC clusters (rows), ordered as in (A). (C) tSNE visualization of 23 mature RGC clusters (colors) comprising 7,298 cells (points). The median silhouette score was computed for each graph. The score ranges from −1 to 1, with higher values indicating tighter cluster boundaries. (D) Transcriptional correspondence between adult and mature larval RGC clusters. Circles and colors indicate the proportional representation of adult cluster identities (rows) in mature larval clusters (column) based on a supervised classification analysis using the xgboost algorithm. Each row is normalized to sum to 100%. Blue circles highlight six instances of a 1:1 corresponding pair of adult and larval clusters, which are separately analyzed in (E)–(G). (E) Dot plot showing shared patterns of gene expression between the six 1:1 cluster pairings selected from the classification model (blue circles in D). Colored bars (left) indicate matching cluster pairs. (F) Heatmap showing differentially expressed genes (rows) between adult and larval stages identified from the six 1:1 matching clusters shown in (D). Columns correspond to individual RGCs grouped by age. Values are row-wise Z-scored gene-expression values. (G) tSNE visualization of 6 immature RGC clusters (colors) comprising 4,108 cells (points). The median silhouette score was computed as in (C). (H) Transcriptional correspondence between mature (rows) and immature (columns) larval RGC clusters using an xgboost classifier trained on immature larval RGCs. Representation is as in (D). (I) Model of RGC type diversification. Larval clusters (LCs) and adult clusters (ACs) are arranged by their transcriptional correspondences shown in (D) and (H). RGC precursors give rise to immature (early intermediate) RGC clusters and mature (late intermediate) larval clusters, which further diversify into mature adult clusters.

(A) Dot plot showing expression patterns of mafaa, tbr1b, and eomesa (rows) in larval clusters (columns) ordered as in Figure 3A. Cluster numbers (top) correspond to immature (gray) and mature (black) RGC clusters. (B) Marker intersection refines genetic access to TF⁺ RGC types. In a TF:QF2 driver line, TF⁺ cells activate expression of GFP through a QUAS:switchNTR reporter. Combination with the pan-RGC Tg(ath5:Cre) line results in TF⁺ RGCs switching to RFP expression, while TF⁺ non-RGCs continue to express GFP. (C) Visualization of RGC types, shown here for mafaa⁺ RGCs (arrows, red labeling), by immunostaining in a triple-transgenic Tg(TF:QF2, QUAS:switchNTR, ath5:Cre) larva. Scale bar, 100 μm. (D–O) Anatomical characterization of RGC types labeled by mafaa (D–G), tbr1b (H–K), and eomesa (L–O) using quadruple-transgenic Tg(TF:QF2, QUAS:switchNTR, ath5:Cre, isl2b:GFP) larvae. In each case Tg(isl2b:GFP) serves as a label for landmarks of RGC projections. Confocal visualizations showing a single plane (left) and RGC soma distribution (maximum z projection, right) in en face views of the immunostained retina (D, H, and L), in vivo images of axonal projections in the tectum (E, I, and M), fluorescence profile across retinotectal laminae measured from the pan-RGC reporter isl2b and marker-specific RGC axons (F, J, and N) as well as a schematic representation of the soma distribution in the retina and the projection pattern indicating TF⁺ RGCs in red against all RGCs in blue (G, K, and O). Asterisks (∗) in (E, I, and M) denote layers innervated by TF⁺ RGCs. D, dorsal; T, temporal; A, anterior; M, medial. SZ, strike zone enrichment. VR, ventral retina enrichment. Scale bar in (D) for (D), (H), and (L), 50 μm. Scale bar in (E) for (E), (I), and (M), 50 μm.

(A) Dot plot showing selective co-expression of tbx3a in larval tbr1b⁺ cluster 1. (B) Immunostained retina of a Tg(tbx3a:QF2, QUAS:switchNTR, ath5:Cre, isl2b:GFP) larva shows diffuse dendrites of tbx3a⁺ RGCs in the IPL. GCL, ganglion cell layer; IPL, inner plexiform layer. Scale bar, 10 μm. (C) Confocal plane of a live Tg(tbx3a:QF2, QUAS:switchNTR, ath5:Cre, isl2b:GFP) larva shows tbx3a⁺ RGC axons terminating in a deep SFGS layer. A, anterior; M, medial. Scale bar, 50 μm. (D) Schematic representation of the soma distribution and projection patterns of the RGC type labeled by tbx3a (red) against all RGCs (blue). (E) Dot plot showing specific expression of tbx20 in larval eomesa⁺ cluster 4. (F) Immunostained retinal section of a quadruple-transgenic Tg(eomesa:QF2, QUAS:GFP, tbx20:Gal4, UAS:NTR-mCherry) larva showing GFP-labeled eomesa⁺ RGCs (left), one of which also expresses tbx20⁺ based on RFP-staining (right, star indicates the co-labeled cell). Scale bar, 20 μm. (G) Confocal plane of a live Tg(tbx20:Gal4, UAS:Dendra) larval retina with a BODIPY neuropil counterstain shows tbx20⁺ RGCs exhibiting monostratified dendrites in the ON sublayer of the IPL. Scale bar, 5 μm. (H) Confocal image of GFP-immunostained eomesa+ RGC axons and RFP-immunostained eomesa⁺tbx20⁺ RGC axons, innervating AF4 and AF9. D, dorsal; P, posterior. Scale bar, 20 μm. (I) 3D side view of the optic tract imaged in a live Tg(tbx20:Gal4, UAS:Dendra, isl2b:tagRFP) larva, showing that tbx20⁺ RGC axons innervate AF4 and terminate in AF9. D, dorsal; P, posterior. Scale bar, 50 μm. (J) Schematic of soma distribution and axon projections of the RGC type labeled by tbx20 (red) against all eomesa⁺ RGCs (blue). (K) Single mafaa⁺ RGC in the periphery of the immunostained retina with bistratified dendrite (arrowheads) in the IPL. This stratification pattern matches the B2 morphology (Robles et al., 2014). (L) Dense cluster of immunostained mafaa⁺ RGCs in the SZ with dendritic arborizations that extend throughout the width of the IPL (indicated by bracket). Some mafaa⁺ RGCs appear to have D1 morphology (Robles et al., 2014).

(A) Functional imaging of RGC types. Neuronal activity was recorded using two-photon calcium imaging from immobilized larvae expressing GCaMP6s in RGC axon terminals during presentation of a battery of visual stimuli displayed on a projection screen. (B) 3D projection of the optic tract indicating the imaging planes in both ventral and dorsal subdivisions of AF9. D, dorsal; P, posterior. Scale bar, 50 μm. (C) Single imaging plane in AF9. L, lateral; P, posterior. Scale bar, 20 μm. (D) Hierarchical relationship of RGC subpopulations used for functional imaging: isl2b labels all RGCs, eomesa marks a subclass in dorsal AF9, wherein tbx20 is expressed by a single type among eomesa⁺ RGCs. (E) Diversity of isl2b⁺ RGC responses to visual stimuli in AF9-projecting axons. Neural activity recordings derived from single pixels were clustered using affinity propagation to reduce noise, resulting in 345 clusters represented by exemplars (STAR methods). Hierarchical clustering divided exemplar activity into eight major response groups (dendrogram, top). Heatmap (bottom) depicts calculated score of exemplars (columns) to each component of visual stimulus (rows). Sustained (s) and transient (t) activity was observed in responses to changing luminance levels. (F) Activity traces of eight classified response groups shown in (E) aligned with the visual stimulus sequence. Shown are the normalized averaged activity traces (colored lines) and all representing exemplars that fall into the group (gray lines) over time. Response groups encompass different numbers of exemplars depending on their abundance. (G–I) Relative frequencies of the eight response groups in isl2b⁺ RGCs (G), eomesa⁺ RGCs (H), and tbx20⁺ RGCs (I). Error bars represent SEM.

(A) Dot plots showing type-specific expression of melanopsin in larval RGCs. Left: of five melanopsin homologs (columns), only opn4xa and opn4b have discernible expression in specific larval RGC clusters (rows). Right: opn4xa⁺ and opn4b⁺ clusters include eomesa⁺ RGC types, but not mafaa⁺ or tbr1b⁺ types. An opn⁺eomesa^- RGC type is marked by the co-expression of onecut1 and shisa9b. Larval clusters are ordered as in Figure 3A. Expression patterns are conserved in adult RGC types (Figure S7D). (B) Phototaxis assay. Larvae are placed in a light-dark choice arena, and their positions are tracked over time. A phototaxis index (PI) quantifies attraction toward the light source. (C–E) Representative traces and PI values of an MTZ-treated control larva (C), ath5⁺ RGC-ablated blind larva (D), and eomesa+ RGC-ablated larva (E). (F) PI values for NTR^- Tg(ath5:QF2) and Tg(eomesa:QF2) control siblings as well as ath5⁺ RGC ablated blind fish, and eomesa⁺ RGC-ablated larvae. Shown is a bar plot with a superimposed dot plot, where each dot represents one fish. Error bars represent SEM. ∗p < 0.05, ∗∗∗p < 0.001 (Dunn post hoc test). (G) Quantification of optomotor response in MTZ-treated controls and eomesa⁺ RGC-ablated larvae, plotted as in (F). (H) Escape probability of MTZ-treated controls and eomesa⁺ RGC-ablated larvae to a looming disc. Each dot represents the mean value at a given stimulus expansion rate. Error bars represent SEM. (I) Quantification of locomotor activity of MTZ-treated controls and eomesa⁺ RGC-ablated larvae, plotted as in (F).

Percentage of mitochondrial genes in adult clusters.

Removing cells with a mitochondrial percentage greater than 10% does not significantly impact cluster discovery.

Percentage of mitochondrial genes in larval clusters.