Cholinergic waves have a modest influence on the transcriptome of retinal ganglion cells
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Abstract
In the early stages of development, correlated activity known as retinal waves causes periodic depolarizations of retinal ganglion cells (RGCs). The β2KO mouse, which lacks the β2 subunit of the nicotinic acetylcholine receptor, serves as a model for understanding the role of these cholinergic waves. β2KO mice have disruptions in several developmental processes of the visual system, including reduced retinotopic and eye-specific refinement of RGC axonal projections to their primary brain targets and an impact on the retinal circuits underlying direction selectivity. However, the effects of this mutation on gene expression in individual functional RGC types remain unclear. Here, we performed single-cell RNA sequencing on RGCs isolated at the end of the first postnatal week from wild-type and β2KO mice. We found that in β2KO mice, the molecular programs governing RGC differentiation were not impacted and the magnitude of transcriptional changes was modest compared to those observed during two days of normal postnatal maturation. This contrasts with the substantial transcriptomic changes seen in downstream visual system areas under wave disruption in recent studies. However, we identified ∼238 genes whose expression was altered in a type-specific manner. We confirmed this result via in situ hybridization and whole-cell recording by focusing on one of the downregulated genes in aRGCs, Kcnk9 , which encodes the two-pore domain leak potassium channel TASK3. Our study reveals a limited transcriptomic impact of cholinergic signaling in the retina and instead of affecting all RGCs uniformly, these waves show subtle modulation of molecular programs in a type-specific manner.
SIGNIFICANCE STATEMENT
Spontaneous retinal waves are critical for the development of the mammalian visual system. However, their role in transcriptional regulation in the retina across the diverse retinal ganglion cell (RGC) types that underpin the detection and transmission of visual features is unclear. Using single-cell RNA sequencing, we analyzed RGC transcriptome from wild-type mice and mice with disrupted retinal waves. We identified several genes that show RGC-type-specific regulation in their expression, including multiple neuropeptides and ion channels. However, wave-dependent changes in the transcriptome were more subtle than developmental changes, indicating that spontaneous activity-dependent molecular changes in retinal ganglion cells are not primarily manifested at the transcriptomic level.
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Surprisingly, intersectional analysis revealed that none of these DEGs were wave regulated (Fig. 4C), suggesting that spontaneous activity-mediated DSGC wiring does not occur by modulating cell-intrinsic genetic programs. While this result may also reflect the limited resolution of single-cell sequencing, which captures only about 15% of the cell’s transcriptome (79), it is also possible that retinal waves exerts their influence through post-transcriptional or translational processes in DSGCs (80).
This to me is of the major thought-provoking findings of this paper and suggests that our understanding of how early activity shapes circuit development is worth expanding. I agree that one interpretation of the seemingly wave-independent DEGs could be that there may be post-transcriptional modifications or local protein synthesis at …
Surprisingly, intersectional analysis revealed that none of these DEGs were wave regulated (Fig. 4C), suggesting that spontaneous activity-mediated DSGC wiring does not occur by modulating cell-intrinsic genetic programs. While this result may also reflect the limited resolution of single-cell sequencing, which captures only about 15% of the cell’s transcriptome (79), it is also possible that retinal waves exerts their influence through post-transcriptional or translational processes in DSGCs (80).
This to me is of the major thought-provoking findings of this paper and suggests that our understanding of how early activity shapes circuit development is worth expanding. I agree that one interpretation of the seemingly wave-independent DEGs could be that there may be post-transcriptional modifications or local protein synthesis at synapses. But we also are assuming that major functional changes need to be reflected in transcriptional signatures, and it might be worth exploring the possibility that other mechanisms are at play, such as micro-circuit properties (e.g. synaptic organization), ion channel localization, or something else.
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