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Data availability
All scRNA-seq data have been deposited at the Gene Expression Omnibus (GSE184933).
Code availability
Codes for processing scRNA-seq files are available at GitHub (https://github.com/dkim195/Ptbp1_Nature_MA).
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Acknowledgements
We thank F. Zhou, A. Fischer, J. Ling, C. Qian and W. Yap for their comments on the manuscript; the staff at the Single Cell & Transcriptomics Core (Johns Hopkins) for sequencing of scRNA-seq libraries. Schematics in Figs. 1 and 2, Extended Data Fig. 3 and Supplementary Figs. 1–6 were created in part using BioRender. This work was supported by the NIH National Eye Institute grants R01EY020560 and U01EY027267 to S.B.; R24EY027283 to S.B.; and the Maryland Stem Cell Research Fund (2019-MSCRFF-5124) to D.W.K. S.B. is supported by a Stein Innovation Award from Research to Prevent Blindness.
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Contributions
T.H., D.W.K. and S.B. conceived and supervised the study. T.H. and D.W.K. generated and analysed immunohistochemistry and scRNA-seq data from the brain, assisted by H.A. M.O. and S.Z. provided reagents. J.K. performed slice electrophysiology of cortical astrocytes. T.H., D.W.K. and S.B. drafted the manuscript. All of the authors contributed to writing the manuscript.
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S.B. is a shareholder in CDI Labs, receives research support from Genentech and is/was a consultant to Third Rock Ventures and Tenpoint Therapeutics.
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Extended data figures and tables
Extended Data Fig. 1 Astrocyte-specific deletion of Ptbp1 does not lead to replenishing lost dopaminergic neurons.
a, Schematic of the experimental timeline for 6-OHDA-induced lesion of dopaminergic neurons, followed by tamoxifen-induced specific deletion of Ptbp1 and lineage tracing of astrocytes. b, Unilateral loss of Th-positive cells in the striatum and midbrain following 6-OHDA lesion. c, Representative immunostaining images of TH and GFP in the striatum and substantia nigra in an intact and lesioned area following 6-OHDA-induced lesion across genotypes. No TH/GFP-double-positive cells were observed in any of the brain regions across genotypes. Note that TH immunostaining signals are only present in non-cell bodies in the striatum (n > = 3 mice/genotype). Scale bars = 50 μm.
Extended Data Fig. 2 RNAScope showing non-astrocytic cells in the lateral ventricular/choroid plexus regions that express GFP and Otx2 mRNA.
a, Low magnification images of GFP mRNA and DAPI in Ptbp1 KO brain sections (n > = 3 mice/genotype). b, Low magnification images of Otx2 mRNA and DAPI in Ptbp1 KO brain section. c,d, High magnification images of GFP and Otx2 mRNA across genotypes. Scale bars = 50 μm.
Extended Data Fig. 3 ScRNA-Seq analysis of doublet-cells from FACS-purified GFP+ cells in mouse brain after astrocyte-specific Ptbp1 deletion.
Violin plots showing expression of neurogenic and cluster-enriched genes in all collected cells in the cortex (a), striatum (b) and substantia nigra (c) across genotypes. Note that Neurog2 and Neurod1 were not detected in the striatum and substantia nigra scRNA-Seq dataset. The drawings of coronal sections were created using BioRender (https://www.biorender.com/).
Supplementary information
Supplementary Information
The legends for Supplementary Figs. 1–6 and Supplementary Tables 1–17, the Supplementary Methods and references.
Supplementary Fig. 1
Ptbp1 immunohistochemistry analysis in the adult mouse brain after astrocyte-specific deletion of Ptbp1. The drawings of coronal sections were created using BioRender (https://www.biorender.com/).
Supplementary Fig. 2
Immunohistochemistry analysis of the adult mouse brain using neuronal markers after astrocyte-specific deletion of Ptbp1. The drawings of coronal sections were created using BioRender (https://www.biorender.com/).
Supplementary Fig. 3
Immunohistochemistry analysis of the adult mouse brain using dopamine markers after astrocyte-specific deletion of Ptbp1. The drawings of coronal sections were created using BioRender (https://www.biorender.com/).
Supplementary Figure 4
scRNA-seq analysis of subsets of astrocytes in the mouse brain after astrocyte-specific Ptbp1 deletion. The drawings of coronal sections were created using BioRender (https://www.biorender.com/).
Supplementary Fig. 5
scRNA-seq analysis of the mouse brain after astrocyte-specific Ptbp1 deletion. The drawings of coronal sections were created using BioRender (https://www.biorender.com/).
Supplementary Fig. 6
scRNA-seq analysis of FACS-purified GFP+ cells in the mouse brain after astrocyte-specific Ptbp1 deletion. The drawings of coronal sections were created using BioRender (https://www.biorender.com/).
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Hoang, T., Kim, D.W., Appel, H. et al. Ptbp1 deletion does not induce astrocyte-to-neuron conversion. Nature 618, E1–E7 (2023). https://doi.org/10.1038/s41586-023-06066-9
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DOI: https://doi.org/10.1038/s41586-023-06066-9
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