Linking transcriptomes with morphological and functional phenotypes in mammalian retinal ganglion cells

Huang, Wenyong; Xu, Qiang; Su, Jing; Tang, Lei; Hao, Zhao-Zhe; Xu, Chuan; Liu, Ruifeng; Shen, Yifeng; Sang, Xuan; Xu, Nana; Tie, Xiaoxiu; Miao, Zhichao; Liu, Xialin; Xu, Ying; Liu, Feng; Liu, Yizhi; Liu, Sheng

doi:10.1016/j.celrep.2022.111322

Cited by 28 publications

(19 citation statements)

References 83 publications

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“…However, the exact role of the A 2A Rs on the development of retinal neurons including different RGC types is still not known. The RGCs are rather heterogeneous and have been classified into over 30 different types, based on their dendritic anatomies, functional characteristics or transcriptomic features (Baden et al, 2016;Bae et al, 2018;Goetz et al, 2022;Huang et al, 2022). Furthermore, retinal A 2A Rs also participate in not only the normal retinal development but also the development under pathological processes in the retina, such as neuroinflammation and inflammation-associated retinal degeneration.…”

Section: Introductionmentioning

confidence: 99%

The adenosine A2A receptor antagonist KW6002 distinctly regulates retinal ganglion cell morphology during postnatal development and neonatal inflammation

Zhang

et al. 2022

Front. Pharmacol.

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Adenosine A2A receptors (A2ARs) appear early in the retina during postnatal development, but the roles of the A2ARs in the morphogenesis of distinct types of retinal ganglion cells (RGCs) during postnatal development and neonatal inflammatory response remain undetermined. As the RGCs are rather heterogeneous in morphology and functions in the retina, here we resorted to the Thy1-YFPH transgenic mice and three-dimensional (3D) neuron reconstruction to investigate how A2ARs regulate the morphogenesis of three morphologically distinct types of RGCs (namely Type I, II, III) during postnatal development and neonatal inflammation. We found that the A2AR antagonist KW6002 did not change the proportion of the three RGC types during retinal development, but exerted a bidirectional effect on dendritic complexity of Type I and III RGCs and cell type-specifically altered their morphologies with decreased dendrite density of Type I, decreased the dendritic field area of Type II and III, increased dendrite density of Type III RGCs. Moreover, under neonatal inflammation condition, KW6002 specifically increased the proportion of Type I RGCs with enhanced the dendrite surface area and volume and the proportion of Type II RGCs with enlarged the soma area and perimeter. Thus, A2ARs exert distinct control of RGC morphologies to cell type-specifically fine-tune the RGC dendrites during normal development but to mainly suppress RGC soma and dendrite volume under neonatal inflammation.

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Section: Introductionmentioning

confidence: 99%

The adenosine A2A receptor antagonist KW6002 distinctly regulates retinal ganglion cell morphology during postnatal development and neonatal inflammation

Zhang

et al. 2022

Front. Pharmacol.

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“…The combined expression of our updated markers was highly type-specific and sufficient to resolve all adult RGC types (Figure S7). Lastly, we curated annotations from two recent studies which used Patch-seq to associate molecular types with functional characteristics (Table 1) 23,24 . Our updated RGC annotations improved type resolution and provide useful information for identifying or gaining genetic access to specific types.…”

Section: Resultsmentioning

confidence: 99%

Section: Rgc Atlas Version 11mentioning

confidence: 99%

Sample multiplexing for retinal single-cell RNA-sequencing

Ma,

Chu,

Polo Prieto

et al. 2024

Preprint

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Single-cell RNA sequencing (scRNA-seq) is a powerful tool for evaluating cell-type specific transcription but can be challenging to apply to rare cell populations. Efficient collection of low abundance cell types can be achieved by pooling biological samples, but this precludes resolution of sample origin together with phenotypic data. This limitation is particularly problematic in experiments in which biological or technical variation is expected to be high (e.g., disease models which vary in phenotypic severity, high throughput genetic perturbation screens, or human patient samples). One solution is sample multiplexing where each sample is tagged with a unique sequence barcode that is resolved bioinformatically. We have established a scRNA-seq sample multiplexing pipeline for mouse retinal ganglion cells (RGCs) using cholesterol-modified-oligos (CMOs). RGCs are a scarce and highly heterogenous cell type (~47 types) that represent less than 1% of all retinal cells. We found that CMOs labeled RGCs efficiently and did not have a significant impact on cell viability, gene expression, or cluster type representation. Through these assignments, we evaluated differential gene expression and identified transcriptional variance between RGCs of individual retinas both as a whole and within specific RGC types. Overall RGC type distributions and transcriptomic correlations between retinas were remarkably similar. Additionally, sample multiplexing enabled the identification of multiplets and resolution of key biological features such as sex-specific gene expression differences. In conclusion, we found that the addition of CMOs is not detrimental to scRNA-seq analysis and enabled the evaluation of differential gene expression among individual biological samples for a rare cell population. As single-cell transcriptomics are becoming a more widely used approach to research development and disease, sample multiplexing represents a useful method to enhance the precision of scRNA-seq analysis.

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“…8 B ; Extended Data Figs. 8-1 to 8-4 ; Bae et al, 2018 ; Goetz et al, 2022 ; Huang et al, 2022 ; Jacobi et al, 2022 ).…”

Section: Resultsunclassified

Differential expression analysis identifies candidate synaptogenic molecules for wiring direction-selective circuits in the retina

Tworig,

Morrie,

Bistrong

et al. 2024

J. Neurosci.

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An organizational feature of neural circuits is the specificity of synaptic connections. A striking example is the direction-selective (DS) circuit of the retina. There are multiple subtypes of DS retinal ganglion cells (DSGCs) that prefer motion along one of 4 preferred directions. This computation is mediated by selective wiring of a single inhibitory interneuron, the starburst amacrine cell (SAC), with each DSGC subtype preferentially receiving input from a subset of SAC processes. We hypothesize that the molecular basis of this wiring is mediated in part by unique expression profiles of DSGC subtypes. To test this, we first performed paired recordings from isolated mouse retina of both sexes to determine that postnatal day 10 (P10) represents the age at which asymmetric synapses form. Second, we performed RNA-sequencing and differential expression analysis on isolated P10 ON-OFF DSGCs tuned for either nasal or ventral motion and identified candidates which may promote direction-specific wiring. We then used a conditional knockout strategy to test the role of one candidate, the secreted synaptic organizer cerebellin-4 (Cbln4), in the development of DS tuning. Using two-photon calcium imaging, we observed a small deficit in directional tuning among ventral-preferring DSGCs lacking Cbln4, though whole-cell voltage clamp recordings did not identify a significant change in inhibitory inputs. This suggests that Cbln4 does not function primarily via a cell-autonomous mechanism to instruct wiring of DS circuits. Nevertheless, our transcriptomic analysis identified unique candidate factors for gaining insights into the molecular mechanisms that instruct wiring specificity in the DS circuit.Significance StatementBy performing mRNA transcriptome analysis on three populations of direction-selective ganglion cells - two preferring horizontal motion and one preferring vertical motion - we identified differentially expressed candidate molecules potentially involved in cell subtype-specific synaptogenesis within this circuit. We tested the role of one differentially expressed candidate, Cbln4, enriched in ventral-preferring DSGCs. Using a targeted knockout approach, the deletion of Cbln4 led to a small reduction in direction-selective tuning while maintaining dendritic morphology and normal strength and asymmetry of inhibitory synaptic transmission. Overall, we have shown that this approach can be used to identify interesting candidate molecules, and future functional studies are required to reveal the mechanisms by which these candidates influence synaptic wiring within specific circuits.

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Linking transcriptomes with morphological and functional phenotypes in mammalian retinal ganglion cells

Cited by 28 publications

References 83 publications

The adenosine A2A receptor antagonist KW6002 distinctly regulates retinal ganglion cell morphology during postnatal development and neonatal inflammation

The adenosine A2A receptor antagonist KW6002 distinctly regulates retinal ganglion cell morphology during postnatal development and neonatal inflammation

Sample multiplexing for retinal single-cell RNA-sequencing

Differential expression analysis identifies candidate synaptogenic molecules for wiring direction-selective circuits in the retina

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