Unified classification of mouse retinal ganglion cells using function, morphology, and gene expression.

Classification and characterization of neuronal types are critical for understanding their function and dysfunction. Neuronal classification schemes typically rely on measurements of electrophysiological, morphological, and molecular features, but aligning such datasets has been challenging. Here, we present a unified classification of mouse retinal ganglion cells (RGCs), the sole retinal output neurons. We use visually evoked responses to classify 1,859 mouse RGCs into 42 types. We also obtain morphological or transcriptomic data from subsets and use these measurements to align the functional classification to publicly available morphological and transcriptomic datasets. We create an online database that allows users to browse or download the data and to classify RGCs from their light responses using a machine learning algorithm. This work provides a resource for studies of RGCs, their upstream circuits in the retina, and their projections in the brain, and establishes a framework for future efforts in neuronal classification and open data distribution.

Molecular signatures of retinal ganglion cells revealed through single cell profiling.

Retinal ganglion cells can be classified into more than 40 distinct subtypes, whether by functional classification or transcriptomics. The examination of these subtypes in relation to their physiology, projection patterns, and circuitry would be greatly facilitated through the identification of specific molecular identifiers for the generation of transgenic mice. Advances in single cell transcriptomic profiling have enabled the identification of molecular signatures for cellular subtypes that are only rarely found. Therefore, we used single cell profiling combined with hierarchical clustering and correlate analyses to identify genes expressed in distinct populations of Parvalbumin-expressing cells and functionally classified RGCs. RGCs were manually isolated based either upon fluorescence or physiological distinction through cell-attached recordings. Microarray hybridization and RNA-Sequencing were employed for the characterization of transcriptomes and in situ hybridization was utilized to further characterize gene candidate expression. Gene candidates were identified based upon cluster correlation, as well as expression specificity within physiologically distinct classes of RGCs. Further, we identified Prph, Ctxn3, and Prkcq as potential candidates for ipRGC classification in the murine retina. The use of these genes, or one of the other newly identified subset markers, for the generation of a transgenic mouse would enable future studies of RGC-subtype specific function, wiring, and projection.

Single cell transcriptome profiling of developing chick retinal cells.

The vertebrate retina is a specialized photosensitive tissue comprised of six neuronal and one glial cell types, each of which develops in prescribed proportions at overlapping timepoints from a common progenitor pool. While each of these cells has a specific function contributing to proper vision in the mature animal, their differential representation in the retina as well as the presence of distinctive cellular subtypes makes identifying the transcriptomic signatures that lead to each retinal cell's fate determination and development challenging. We have analyzed transcriptomes from individual cells isolated from the chick retina throughout retinogenesis. While we focused our efforts on the retinal ganglion cells, our transcriptomes of developing chick cells also contained representation from multiple retinal cell types, including photoreceptors and interneurons at different stages of development. Most interesting was the identification of transcriptomes from individual mixed lineage progenitor cells in the chick as these cells offer a window into the cell fate decision-making process. Taken together, these data sets will enable us to uncover the most critical genes acting in the steps of cell fate determination and early differentiation of various retinal cell types.

Polo-Like Kinase 3 Appears Dispensable for Normal Retinal Development Despite Robust Embryonic Expression.

During retinogenesis seven different cell types are generated in distinct yet overlapping timepoints from a population of retinal progenitor cells. Previously, we performed single cell transcriptome analyses of retinal progenitor cells to identify candidate genes that may play roles in the generation of early-born retinal neurons. Based on its expression pattern in subsets of early retinal cells, polo-like kinase 3 (Plk3) was identified as one such candidate gene. Further characterization of Plk3 expression by in situ hybridization revealed that this gene is expressed as cells exit the cell cycle. We obtained a Plk3 deficient mouse and investigated changes in the retina's morphology and transcriptome through immunohistochemistry, in situ hybridization and gene expression profiling. These experiments have been performed initially on adult mice and subsequently extended throughout retinal development. Although morphological studies revealed no consistent changes in retinogenesis upon Plk3 loss, microarray profiling revealed potential candidate genes altered in Plk3-KO mice. Further studies will be necessary to understand the connection between these changes in gene expression and the loss of a protein kinase such as Plk3.

Expression Profiling of Developing Zebrafish Retinal Cells.

During retinal development, a variety of different types of neurons are produced. Understanding how each of these types of retinal nerve cells is generated is important from a developmental biology perspective. It is equally important if one is interested in how to regenerate cells after an injury or a disease. To gain more insight into how retinal neurons develop in the zebrafish, we performed single-cell mRNA profiling and in situ hybridizations (ISHs) on retinal sections and whole-mount zebrafish. Through the series of ISHs, designed and performed solely by undergraduate students in the laboratory, we were able to retrospectively identify our single-cell mRNA profiles as most likely coming from developing amacrine cells. Further analysis of these profiles will reveal genes that can be mutated using genome editing techniques. Together these studies increase our knowledge of the genes driving development of different cell types in the zebrafish retina.

Transcriptomic analyses of Onecut1 and Onecut2 deficient retinas.

In this article, we further explore the data generated for the research article "Onecut1 and Onecut2 play critical roles in the development of the mouse retina". To better understand the functionality of the Onecut family of transcription factors in retinogenesis, we investigated the retinal transcriptomes of developing and mature mice to identify genes with differential expression. This data article reports the full transcriptomes resulting from these experiments and provides tables detailing the differentially expressed genes between wildtype and Onecut1 or 2 deficient retinas. The raw array data of our transcriptomes as generated using Affymetrix microarrays are available on the NCBI Gene Expression Omnibus (GEO) browser (Reference number GSE57917 and GSE57918GSE57917GSE57918).

Onecut1 and Onecut2 play critical roles in the development of the mouse retina.

The entire repertoire of intrinsic factors that control the cell fate determination process of specific retinal neurons has yet to be fully identified. Single cell transcriptome profiling experiments of retinal progenitor cells revealed considerable gene expression heterogeneity between individual cells, especially among different classes of transcription factors. In this study, we show that two of those factors, Onecut1 and Onecut2, are expressed during mouse retinal development. Using mice that are deficient for each of these transcription factors, we further demonstrate a significant loss (∼70-80%) of horizontal cells in the absence of either of these proteins, while the other retinal cells appear at normal numbers. Microarray profiling experiments performed on knockout retinas revealed defects in horizontal cell genes as early as E14.5. Additional profiling assays showed an upregulation of several stress response genes in the adult Onecut2 knockout, suggesting that the integrity of the retina is compromised in the absence of normal numbers of horizontal cells. Interestingly, melanopsin, the gene coding for the photopigment found in photosensitive ganglion cells, was observed to be upregulated in Onecut1 deficient retinas, pointing to a possible regulatory role for Onecut1. Taken together, our data show that similar to Onecut1, Onecut2 is also necessary for the formation of normal numbers of horizontal cells in the developing retina.

Making of a retinal cell: insights into retinal cell-fate determination.

Understanding the process by which an uncommitted dividing cell produces particular specialized cells within a tissue remains a fundamental question in developmental biology. Many tissues are well suited for cell-fate studies, but perhaps none more so than the developing retina. Traditionally, experiments using the retina have been designed to elucidate the influence that individual environmental signals or transcription factors can have on cell-fate decisions. Despite a substantial amount of information gained through these studies, there is still much that we do not yet understand about how cell fate is controlled on a systems level. In addition, new factors such as noncoding RNAs and regulators of chromatin have been shown to play roles in cell-fate determination and with the advent of "omics" technology more factors will most likely be identified. In this chapter we summarize both the traditional view of retinal cell-fate determination and introduce some new ideas that are providing a challenge to the older way of thinking about the acquisition of cell fates.

Otx2 and Onecut1 promote the fates of cone photoreceptors and horizontal cells and repress rod photoreceptors.

Cone photoreceptors carry out phototransduction in daylight conditions and provide the critical first step in color vision. Despite their importance, little is known about the developmental mechanisms involved in their generation, particularly how they are determined relative to rod photoreceptors, the cells that initiate vision in dim light. Here, we report the identification of a cis-regulatory module (CRM) for the thyroid hormone receptor beta (Thrb) gene, an early cone marker. We found that ThrbCRM1 is active in progenitor cells biased to the production of cones and an interneuronal cell type, the horizontal cell (HC). Molecular analysis of ThrbCRM1 revealed that it is combinatorially regulated by the Otx2 and Onecut1 transcription factors. Onecut1 is sufficient to induce cells with the earliest markers of cones and HCs. Conversely, interference with Onecut1 transcriptional activity leads to precocious rod development, suggesting that Onecut1 is critically important in defining cone versus rod fates.

Single-cell profiling of developing and mature retinal neurons.

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue(1-7). In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions(8). This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells(9). The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself(10). The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development. As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population(11). Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina(2,4,5,12). In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells(8). With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.