Biology subjects

Shekhar, K.

Publications and source records attributed to Shekhar, K..

24 records · Page 2Linked to original sources

bioRxiv2020

Molecular classification of zebrafish retinal ganglion cells links genes to cell types to behavior

Retinal ganglion cells (RGCs) form an array of feature detectors, which convey visual information to central brain regions. Characterizing RGC diversity is required to understand the logic of the underlying functional segregation. Using single-cell transcriptomics, we systematically classified RGCs in adult and larval zebrafish, thereby identifying marker genes for at least 33 stable and transient cell types. We used this dataset to engineer transgenic driver lines, enabling experimental access to specific RGC types. Strikingly, expression of one or few transcription factors often predicts dendrite morphologies and axonal projections to specific tectal layers and extratectal targets. In vivo calcium imaging revealed that molecularly defined RGCs exhibit highly specific functional tuning. Finally, chemogenetic ablation of eomesa+ RGCs, which comprise melanopsin-expressing types with projections to a small subset of central targets, selectively impaired phototaxis. Together, our study establishes a framework for systematically studying the functional architecture of the visual system.

neuroscience↗

bioRxiv2020

CELL ATLAS OF THE HUMAN FOVEA AND PERIPHERAL RETINA

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.

neuroscience↗

bioRxiv2020

Cell Atlas of Aqueous Humor Outflow Pathways in Eyes of Humans and Four Model Species Provides Insights into Glaucoma Pathogenesis

Increased intraocular pressure (IOP) represents a major risk factor for glaucoma, a prevalent eye disease characterized by death of retinal ganglion cells that carry information from the eye to the brain; 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 (scRNA-seq). 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.

cell biology↗

bioRxiv2019

Single-cell profiles of retinal neurons differing in resilience to injury reveal neuroprotective genes

Neuronal types in the central nervous system differ dramatically in their resilience to injury or insults. Here we studied the selective resilience of mouse retinal ganglion cells (RGCs) following optic nerve crush (ONC), which severs their axons and leads to death of ~80% of RGCs within 2 weeks. To identify expression programs associated with differential resilience, we first used single-cell RNA-seq (scRNA-seq) to generate a comprehensive molecular atlas of 46 RGC types in adult retina. We then tracked their survival after ONC, characterized transcriptomic, physiological, and morphological changes that preceded degeneration, and identified genes selectively expressed by each type. Finally, using loss- and gain-of-function assays in vivo, we showed that manipulating some of these genes improved neuronal survival and axon regeneration following ONC. This study provides a systematic framework for parsing type-specific responses to injury, and demonstrates that differential gene expression can be used to reveal molecular targets for intervention.

neuroscience↗

bioRxiv2018

Molecular Classification and Comparative Taxonomics of Foveal and Peripheral Cells in Primate Retina

High acuity vision in primates, including humans, is mediated by a small central retinal region called the fovea. As more accessible model organisms lack a fovea, its specialized function and dysfunction in ocular diseases remain poorly understood. We used 165,000 single-cell RNA-seq profiles to generate and validate comprehensive cellular taxonomies of macaque fovea and peripheral retina. More than 80% of >65 cell types match between the two regions, but exhibit substantial differences in proportions and gene expression, some of which we relate to functional differences. Comparison of macaque retinal types with those of mice reveals that interneuron types are tightly conserved, but that projection neuron types and programs diverge, despite conserved transcription factor codes. Key macaque types are conserved in humans, allowing mapping of cell-type and region-specific expression of >190 genes associated with 6 human retinal diseases. Our work provides a framework for comparative single-cell analysis across tissue regions and species.

neuroscience↗

bioRxiv2017

T helper cells modulate intestinal stem cell renewal and differentiation

In the small intestine, a cellular niche of diverse accessory cell types supports the rapid generation of mature epithelial cell types through self-renewal, proliferation, and differentiation of intestinal stem cells (ISCs). However, not much is known about interactions between immune cells and ISCs, and it is unclear if and how immune cell dynamics affect eventual ISC fate or the balance between self-renewal and differentiation. Here, we used single-cell RNA-seq (scRNA-Seq) of intestinal epithelial cells (IECs) to identify new mechanisms for ISC-immune cell interactions. Surprisingly, MHC class II (MHCII) is enriched in two distinct subsets of Lgr5+ crypt base columnar ISCs, which are also distinguished by higher proliferation rates. Using co-culture of T cells with intestinal organoids, cytokine stimulations, and in vivo mouse models, we confirm that CD4+ T helper (Th) cells communicate with ISCs and affect their differentiation, in a manner specific to the Th subtypes and their signature cytokines and dependent on MHCII expression by ISCs. Specific inducible knockout of MHCII in intestinal epithelial cells in mice in vivo results in expansion of the ISC pool. Mice lacking T cells have expanded ISC pools, whereas specific depletion of Treg cells in vivo results in substantial reduction of ISC numbers. Our findings show that interactions between Th cells and ISCs mediated via MHCII expressed in intestinal epithelial stem cells help orchestrate tissue-wide responses to external signals.

immunology↗