It has been known for over a century that the basic organization of the retina is conserved across vertebrates. It has been equally clear that retinal cells can be classified into numerous types, but only recently have methods been devised to explore this diversity in unbiased, scalable, and comprehensive ways. Advances in high-throughput single-cell RNA sequencing (scRNA-seq) have played a pivotal role in this effort. In this article, we outline the experimental and computational components of scRNA-seq and review studies that have used them to generate retinal atlases of cell types in several vertebrate species. These atlases have enabled studies of retinal development, responses of retinal cells to injury, expression patterns of genes implicated in retinal disease, and the evolution of cell types. Recently, the inquiry has expanded to include the entire eye and visual centers in the brain. These studies have enhanced our understanding of retinal function and dysfunction and provided tools and insights for exploring neural diversity throughout the brain.

The development and connectivity of retinal ganglion cells (RGCs), the retina's sole output neurons, are patterned by activity-independent transcriptional programs and activity-dependent remodeling. To inventory the molecular correlates of these influences, we applied high-throughput single-cell RNA sequencing (scRNA-seq) to mouse RGCs at six embryonic and postnatal ages. We identified temporally regulated modules of genes that correlate with, and likely regulate, multiple phases of RGC development, ranging from differentiation and axon guidance to synaptic recognition and refinement. Some of these genes are expressed broadly while others, including key transcription factors and recognition molecules, are selectively expressed by one or a few of the 45 transcriptomically distinct types defined previously in adult mice. Next, we used these results as a foundation to analyze the transcriptomes of RGCs in mice lacking visual experience due to dark rearing from birth or to mutations that ablate either bipolar or photoreceptor cells. 98.5% of visually deprived (VD) RGCs could be unequivocally assigned to a single RGC type based on their transcriptional profiles, demonstrating that visual activity is dispensable for acquisition and maintenance of RGC type identity. However, visual deprivation significantly reduced the transcriptomic distinctions among RGC types, implying that activity is required for complete RGC maturation or maintenance. Consistent with this notion, transcriptomic alternations in VD RGCs significantly overlapped with gene modules found in developing RGCs. Our results provide a resource for mechanistic analyses of RGC differentiation and maturation, and for investigating the role of activity in these processes.

The development of axonal arbors is a critical step in the establishment of precise neural circuits, but relatively little is known about the mechanisms of axonal elaboration in the neocortex. We used in vivo two-photon time-lapse microscopy to image axons in the neocortex of green fluorescent protein-transgenic mice over the first 3 wk of postnatal development. This period spans the elaboration of thalamocortical (TC) and Cajal-Retzius (CR) axons and cortical synaptogenesis. Layer 1 collaterals of TC and CR axons were imaged repeatedly over time scales ranging from minutes up to days, and their growth and pruning were analyzed. The structure and dynamics of TC and CR axons differed profoundly. Branches of TC axons terminated in small, bulbous growth cones, while CR axon branch tips had large growth cones with numerous long filopodia. TC axons grew rapidly in straight paths, with frequent interstitial branch additions, while CR axons grew more slowly along tortuous paths. For both types of axon, new branches appeared at interstitial sites along the axon shaft and did not involve growth cone splitting. Pruning occurred via retraction of small axon branches (tens of microns, at both CR and TC axons) or degeneration of large portions of the arbor (hundreds of microns, for TC axons only). The balance between growth and retraction favored overall growth, but only by a slight margin. Given the identical layer 1 territory upon which CR and TC axons grow, the differences in their structure and dynamics likely reflect distinct intrinsic growth programs for axons of long projection neurons versus local interneurons.

Background

Neurons assemble into a functional network through a sequence of developmental processes including neuronal polarization and synapse formation. In Caenorhabditis elegans, the serine/threonine SAD-1 kinase is essential for proper neuronal polarity and synaptic organization. To determine if SAD-1 activity regulates the establishment or maintenance of these neuronal structures, we examined its temporal requirements using a chemical-genetic method that allows for selective and reversible inactivation of its kinase activity in vivo.

Results

We generated a PP1 analog-sensitive variant of SAD-1. Through temporal inhibition of SAD-1 kinase activity we show that its activity is required for the establishment of both neuronal polarity and synaptic organization. However, while SAD-1 activity is needed strictly when neurons are polarizing, the temporal requirement for SAD-1 is less stringent in synaptic organization, which can also be re-established during maintenance.

Conclusion

This study reports the first temporal analysis of a neural kinase activity using the chemical-genetic system. It reveals that neuronal polarity and synaptic organization have distinct temporal requirements for SAD-1.

The genesis of broad neuronal classes from multipotential neural progenitor cells has been extensively studied, but less is known about the diversification of a single neuronal class into multiple types. We used single-cell RNA-seq to study how newly born (postmitotic) mouse retinal ganglion cell (RGC) precursors diversify into ~45 discrete types. Computational analysis provides evidence that RGC transcriptomic type identity is not specified at mitotic exit, but acquired by gradual, asynchronous restriction of postmitotic multipotential precursors. Some types are not identifiable until a week after they are generated. Immature RGCs may be specified to project ipsilaterally or contralaterally to the rest of the brain before their type identity emerges. Optimal transport inference identifies groups of RGC precursors with largely nonoverlapping fates, distinguished by selectively expressed transcription factors that could act as fate determinants. Our study provides a framework for investigating the molecular diversification of discrete types within a neuronal class.

Transgenic mouse lines are routinely employed to label and manipulate distinct cell types. The transgene generally comprises cell-type specific regulatory elements linked to a cDNA encoding a reporter or other protein. However, off-target expression seemingly unrelated to the regulatory elements in the transgene is often observed, it is sometimes suspected to reflect influences related to the site of transgene integration in the genome. To test this hypothesis, we used a proximity ligation-based method, Targeted Locus Amplification (TLA), to map the insertion sites of three well-characterized transgenes that appeared to exhibit insertion site-dependent expression in retina. The nearest endogenous genes to transgenes HB9-GFP, Mito-P, and TYW3 are Cdh6, Fat4 and Khdrbs2, respectively. For two lines, we demonstrate that expression reflects that of the closest endogenous gene (Fat4 and Cdh6), even though the distance between transgene and endogenous gene is 550 and 680 kb, respectively. In all three lines, the transgenes decrease expression of the neighboring endogenous genes. In each case, the affected endogenous gene was expressed in at least some of the cell types that the transgenic line has been used to mark and study. These results provide insights into the effects of transgenes and endogenous genes on each other's expression, demonstrate that mapping insertion site is valuable for interpreting results obtained with transgenic lines, and indicate that TLA is a reliable method for integration site discovery.

Distinct neuronal types connect in complex ways to generate functional neural circuits. The molecular diversity required to specify this connectivity could be supplied by multigene families of synaptic recognition molecules, but most studies to date have assessed just one or a few members at a time. Here, we analyze roles of cadherins (Cdhs) in formation of retinal circuits comprising eight neuronal types that inform the brain about motion in four directions. We show that at least 15 classical Cdhs are expressed by neurons in these circuits and at least 6 (Cdh6-10 and 18) act individually or in combinations to promote specific connectivity among the cells. They act in part by directing the processes of output neurons and excitatory interneurons to a cellular scaffold formed by inhibitory interneurons. Because Cdhs are expressed combinatorially by many central neurons, similar interactions could be involved in patterning circuits throughout the brain.

Increased intraocular pressure (IOP) represents a major risk factor for glaucoma, a prevalent eye disease characterized by death of retinal ganglion cells; lowering IOP is the only proven treatment strategy to delay disease progression. The main determinant of IOP is the equilibrium between production and drainage of aqueous humor, with compromised drainage generally viewed as the primary contributor to dangerous IOP elevations. Drainage occurs through two pathways in the anterior segment of the eye called conventional and uveoscleral. To gain insights into the cell types that comprise these pathways, we used high-throughput single-cell RNA sequencing (scRNAseq). From ∼24,000 single-cell transcriptomes, we identified 19 cell types with molecular markers for each and used histological methods to localize each type. We then performed similar analyses on four organisms used for experimental studies of IOP dynamics and glaucoma: cynomolgus macaque (Macaca fascicularis), rhesus macaque (Macaca mulatta), pig (Sus scrofa), and mouse (Mus musculus). Many human cell types had counterparts in these models, but differences in cell types and gene expression were evident. Finally, we identified the cell types that express genes implicated in glaucoma in all five species. Together, our results provide foundations for investigating the pathogenesis of glaucoma and for using model systems to assess mechanisms and potential interventions.

Most irreversible blindness results from retinal disease. To advance our understanding of the etiology of blinding diseases, we used single-cell RNA-sequencing (scRNA-seq) to analyze the transcriptomes of ~85,000 cells from the fovea and peripheral retina of seven adult human donors. Utilizing computational methods, we identified 58 cell types within 6 classes: photoreceptor, horizontal, bipolar, amacrine, retinal ganglion and non-neuronal cells. Nearly all types are shared between the two retinal regions, but there are notable differences in gene expression and proportions between foveal and peripheral cohorts of shared types. We then used the human retinal atlas to map expression of 636 genes implicated as causes of or risk factors for blinding diseases. Many are expressed in striking cell class-, type-, or region-specific patterns. Finally, we compared gene expression signatures of cell types between human and the cynomolgus macaque monkey, Macaca fascicularis. We show that over 90% of human types correspond transcriptomically to those previously identified in macaque, and that expression of disease-related genes is largely conserved between the two species. These results validate the use of the macaque for modeling blinding disease, and provide a foundation for investigating molecular mechanisms underlying visual processing.

Visual information is delivered to the brain by >40 types of retinal ganglion cells (RGCs). Diversity in this representation arises within the inner plexiform layer (IPL), where dendrites of each RGC type are restricted to specific sublaminae, limiting the interneuronal types that can innervate them. How such dendritic restriction arises is unclear. We show that the transcription factor Tbr1 is expressed by four mouse RGC types with dendrites in the outer IPL and is required for their laminar specification. Loss of Tbr1 results in elaboration of dendrites within the inner IPL, while misexpression in other cells retargets their neurites to the outer IPL. Two transmembrane molecules, Sorcs3 and Cdh8, act as effectors of the Tbr1-controlled lamination program. However, they are expressed in just one Tbr1⁺ RGC type, supporting a model in which a single transcription factor implements similar laminar choices in distinct cell types by recruiting partially non-overlapping effectors.