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Although single-cell gene expression profiling has been possible for the past two decades, a number of recent technological advances in microfluidic and sequencing technology have recently made the procedure much easier and less expensive. Awatramani and colleagues discuss the use of single-cell gene expression profiling for classifying neuronal cell types. Cellular specialization is particularly prominent in mammalian nervous systems, which are composed of millions to billions of neurons that appear in thousands of different 'flavors' and contribute to a variety of functions. Even in a single brain region, individual neurons differ greatly in their morphology, connectivity and electrophysiological properties. Systematic classification of all mammalian neurons is a key goal towards deconstructing the nervous system into its basic components. With the recent advances in single-cell gene expression profiling technologies, it is now possible to undertake the enormous task of disentangling neuronal heterogeneity. High-throughput single-cell RNA sequencing and multiplexed quantitative RT-PCR have become more accessible, and these technologies enable systematic categorization of individual neurons into groups with similar molecular properties. Here we provide a conceptual and practical guide to classification of neural cell types using single-cell gene expression profiling technologies.

Glaucoma is a common cause of vision loss or blindness and reduction of intraocular pressure (IOP) has been proven beneficial in a large fraction of glaucoma patients. The IOP is maintained by the trabecular meshwork (TM) and the elevation of IOP in open-angle glaucoma is associated with dysfunction and loss of the postmitotic cells residing within this tissue. To determine if IOP control can be maintained by replacing lost TM cells, we transplanted TM-like cells derived from induced pluripotent stem cells into the anterior chamber of a transgenic mouse model of glaucoma. Transplantation led to significantly reduced IOP and improved aqueous humor outflow facility, which was sustained for at least 9 wk. The ability to maintain normal IOP engendered survival of retinal ganglion cells, whose loss is ultimately the cause for reduced vision in glaucoma. In vivo and in vitro analyses demonstrated higher TM cellularity in treated mice compared with littermate controls and indicated that this increase is primarily because of a proliferative response of endogenous TM cells. Thus, our study provides in vivo demonstration that regeneration of the glaucomatous TM is possible and points toward novel approaches in the treatment of this disease.

The E2F transcription factor family determines whether or not a cell will divide by controlling the expression of key cell-cycle regulators. The individual E2Fs can be divided into distinct subgroups that act in direct opposition to one another to promote either cellular proliferation or cell-cycle exit and terminal differentiation. What is the underlying molecular basis of this 'push-me-pull-you' regulation, and what are its biological consequences?

The development of complex tissues requires that mitotic progenitor cells integrate information from the environment. The highly varied outcomes of such integration processes undoubtedly depend at least in part upon variations among the gene expression programs of individual progenitor cells. To date, there has not been a comprehensive examination of these differences among progenitor cells of a particular tissue. Here, we used comprehensive gene expression profiling to define these differences among individual progenitor cells of the vertebrate retina. Retinal progenitor cells (RPCs) have been shown by lineage analysis to be multipotent throughout development and to produce distinct types of daughter cells in a temporal, conserved order. A total of 42 single RPCs were profiled on Affymetrix arrays. In situ hybridizations performed on both retinal sections and dissociated retinal cells were used to validate the results of the microarrays. An extensive amount of heterogeneity in gene expression among RPCs, even among cells isolated from the same developmental time point, was observed. While many classes of genes displayed heterogeneity of gene expression, the expression of transcription factors constituted a significant amount of the observed heterogeneity. In contrast to previous findings, individual RPCs were found to express multiple bHLH transcription factors, suggesting alternative models to those previously developed concerning how these factors may be coordinated. Additionally, the expression of cell cycle related transcripts showed differences among those associated with G2 and M, versus G1 and S phase, suggesting different levels of regulation for these genes. These data provide insights into the types of processes and genes that are fundamental to cell fate choices, proliferation decisions, and, for cells of the central nervous system, the underpinnings of the formation of complex circuitry.

Previous lineage analyses have shown that retinal progenitor cells (RPCs) are multipotent throughout development, and expression-profiling studies have shown a great deal of molecular heterogeneity among RPCs. To determine if the molecular heterogeneity predicts that an RPC will produce particular types of progeny, clonal lineage analysis was used to investigate the progeny of a subset of RPCs, those that express the basic helix–loop–helix transcription factor, Olig2. The embryonic Olig2+ RPCs underwent terminal divisions, producing small clones with primarily two of the five cell types being made by the pool of RPCs at that time. The later, postnatal Olig2+ RPCs also made terminal divisions, which were biased toward production of rod photoreceptors and amacrine cell interneurons. These data indicate that the multipotent progenitor pool is made up of distinctive types of RPCs, which have biases toward producing subsets of retinal neurons in a terminal division, with the types of neurons produced varying over time. This strategy is similar to that of the developing Drosophila melanogaster ventral nerve cord, with the Olig2+ cells behaving as ganglion mother cells.

To better understand the mechanisms that govern the development of retinal neurons, it is critical to gain additional insight into the specific intrinsic factors that control cell fate decisions and neuronal maturation. In the developing mouse retina, Atoh7, a highly conserved transcription factor, is essential for retinal ganglion cell development. Moreover, Atoh7 expression in the developing retina occurs during a critical time period when progenitor cells are in the process of making cell fate decisions. We performed transcriptome profiling of Atoh7+ individual cells isolated from mouse retina. One of the genes that we found significantly correlated with Atoh7 in our transcriptomic data was the E3 ubiquitin ligase, Trim9. The correlation between Trim9 and Atoh7 coupled with the expression of Trim9 in the early mouse retina led us to hypothesize that this gene may play a role in the process of cell fate determination. To address the role of Trim9 in retinal development, we performed a functional analysis of Trim9 in the mouse and did not detect any morphological changes in the retina in the absence of Trim9. Thus, Trim9 alone does not appear to be involved in cell fate determination or early ganglion cell development in the mouse retina. We further hypothesize that the reason for this lack of phenotype may be compensation by one of the many additional TRIM family members we find expressed in the developing retina.

The vertebrate retina uses diverse neuronal cell types arrayed into complex neural circuits to extract, process, and relay information from the visual scene to the higher order processing centers of the brain. Amacrine cells, a class of interneurons, are thought to mediate much of the processing of the visual signal that occurs within the retina. Although amacrine cells display extensive morphological diversity, the molecular nature of this diversity is largely unknown. Furthermore, it is not known how this diversity arises during development. Here, we have combined in vivo genetic labeling, single cell genome-wide expression profiling, and classical birthdating to (i) identify specific molecular types of amacrine cells, (ii) demonstrate the molecular diversity of the amacrine cell class, and (iii) show that amacrine cell diversity arises at least in part through temporal patterning.

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.

The E2F transcription factors play a key role in the regulation of cellular proliferation and terminal differentiation. E2F6 is the most recently identified and the least well understood member of the E2F family. It is only distantly related to the other E2Fs and lacks the sequences responsible for both transactivation and binding to the retinoblastoma protein. Consistent with this finding, E2F6 can behave as a dominant negative inhibitor of the other E2F family members. In this study, we continue to investigate the possible role(s) of E2F6 in vivo. We report the isolation of RYBP, a recently identified member of the mammalian polycomb complex, as an E2F6-interacting protein. Mapping studies indicate that RYBP binds within the known \"repression domain\" of E2F6. Moreover, we demonstrate that endogenous E2F6 and polycomb group proteins, including RYBP, Ring1, MEL-18, mph1, and the oncoprotein Bmi1, associate with one another. These findings suggest that the biological properties of E2F6 are mediated through its ability to recruit the polycomb transcriptional repressor complex.

In mammals, a subset of retinal ganglion cells (RGCs) expresses the photopigment melanopsin, which renders them intrinsically photosensitive (ipRGCs). These ipRGCs mediate various non-image-forming visual functions such as circadian photoentrainment and the pupillary light reflex (PLR). Melanopsin phototransduction begins with activation of a heterotrimeric G protein of unknown identity. Several studies of melanopsin phototransduction have implicated a G-protein of the Gq/11 family, which consists of Gna11, Gna14, Gnaq and Gna15, in melanopsin-evoked depolarization. However, the exact identity of the Gq/11 gene involved in this process has remained elusive. Additionally, whether Gq/11 G-proteins are necessary for melanopsin phototransduction in vivo has not yet been examined. We show here that the majority of ipRGCs express both Gna11 and Gna14, but neither Gnaq nor Gna15. Animals lacking the melanopsin protein have well-characterized deficits in the PLR and circadian behaviors, and we therefore examined these non-imaging forming visual functions in a variety of single and double mutants for Gq/11 family members. All Gq/11 mutant animals exhibited PLR and circadian behaviors indistinguishable from WT. In addition, we show persistence of ipRGC light-evoked responses in Gna11-/-; Gna14-/- retinas using multielectrode array recordings. These results demonstrate that Gq, G11, G14, or G15 alone or in combination are not necessary for melanopsin-based phototransduction, and suggest that ipRGCs may be able to utilize a Gq/11-independent phototransduction cascade in vivo.