Ptbp1 is not required for retinal neurogenesis and cell fate specification

Haley Appel
Rogger P Carmen-Orozco
Clayton P Santiago
Thanh Hoang
Seth Blackshaw author has email address

Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, United States
Department of Ophthalmology and Visual Sciences, University of Michigan School of Medicine, Ann Arbor, United States
Department of Cell and Developmental Biology, University of Michigan School of Medicine, Ann Arbor, United States
Michigan Neuroscience Institute, University of Michigan School of Medicine, Ann Arbor, United States
Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, United States
Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, United States
Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, United States
Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine, Baltimore, United States

https://doi.org/10.7554/eLife.108331.1

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Figures and data

Ptbp1 is successfully deleted in Chx10-Cre;Ptbp1^lox/lox mice in developing and mature retina.
(A) A schematic diagram of the generation of specific deletion of Ptbp1 in early RPCs using Chx10-Cre. (B) Representative immunostaining for Ptbp1 expression in Chx10-Cre;Ptbp1^+/+ (Ptbp1-Ctrl) and Chx10-Cre;Ptbp1^lox/lox(Ptbp1-KO) mice at E14.5, (C) P1, (D) and P30. (E) Quantification of Ptbp1-positive cells in Ptbp1-Ctrl and Ptbp1-KO retinas (n=8/genotype). Significance was determined via unpaired t test: **** p<0.0001. Each data point was calculated from an individual retina. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar= 100 μm.

BulkRNA-seq analysis reveals accelerated differentiation and altered RNA splicing in Chx10-Cre;Ptbp1^lox/lox mice at E16.5.
(A) Volcano plot showing 2075 differentially expressed genes between heterozygous Chx10-Cre;Ptbp1^lox/+ and homozygous Chx10-Cre;Ptbp1^lox/lox mutants as measured by bulk RNA-seq. (B) Heatmap of select differentially expressed genes in wildtype Chx10-Cre;Ptbp1^+/+, heterozygous Chx10-Cre;Ptbp1^lox/+, and homozygous Chx10-Cre;Ptbp1^lox/lox samples. (C) Gene set enrichment analysis showing activated and repressed gene ontology terms in the homozygous Ptbp1 mutant samples. (D) Barplot showing 864 splicing changes in homozygous Ptbp1 mutant samples by splicing class including retained-introns (54 events), alternative 5’ splice site (32 events), alternative 3’ splice site (67 events), mutually-exclusive exons (128 events) and skipped-exons (583 events). (E) Track plots showing examples of alternative splicing of exons (arrows) in the Ptbp1 mutant samples. (F) Bar plot showing that 7% of neuron-enriched and 35% of rod-specific splicing events overlap with splicing changes observed in Ptbp1 mutants.

Ptbp1 deletion does not alter retinal progenitor proliferation.
(A) A schematic diagram of the experimental timeline for labeling dividing cells using EdU. (B) Representative immunostaining for Ptbp1 expression and EdU labeling in Ptbp1-Ctrl and Ptbp1-KO mice at P0. (C) Quantification of EdU-positive cells in Ptbp1-Ctrl and Ptbp1-KO retinas (n=2/genotype). Significance was determined via unpaired t test: ns=p>0.05. Each data point was calculated from an individual retina. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar=100 μm.

Ptbp1 deletion does not alter retinal neurogenesis.
(A) Representative immunostaining for Ptbp1, (B) Sox9, (C) HuC/D+NeuN, Otx2, (D) Rbpms, and (E) Cone-arrestin in Ptbp1-Ctrl and Ptbp1-KO mice at P30. (F) Quantification of Sox9, HuC/D/NeuN, Otx2, Rbpms, and Cone-arrestin-positive cells in Ptbp1-Ctrl and Ptbp1-KO retinas (n≥4/genotype). Significance was determined via multiple unpaired t tests: ns=p>0.05. Each data point was calculated from an individual retina. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar= 100 μm.

ScRNA-seq analysis reveals modest transcriptional changes in Mϋller glial gene expression in Chx10-Cre;Ptbp1^lox/lox mice.
(A) Uniform manifold approximation and projection (UMAP) plots showing cell types captured in the Chx10-Cre;Ptbp1^+/+ (Ptbp1-Ctrl) and Chx10-Cre;Ptbp1^lox/lox (Ptbp1-KO) merged dataset. (B) UMAP plots showing cell types separated by genotype with the percentage of Mϋller glia in each group. (C) Barplot showing cell proportions for each cell type in both genotypes. (D) Volcano plot showing differentially expressed genes in Mϋller glia between Ptbp1-Ctrl and Ptbp1-KO samples. (E) Venn diagram comparison of upregulated and downregulated differentially expressed genes for Mϋller glia between this dataset and previously published data analyzing Mϋller glia-specific loss of function of Ptbp1 (Hoang et al. 2022). (F) Violin plot showing no significant changes in the expression of canonical Mϋller glia markers in Ptbp1-KO sample compared to Ptbp1-Ctrl.

Cellular expression patterns of Ptbp1 and Ptbp2 during mouse and human retinal development.
(A) 3D UMAP plot showing cell types from combined timepoints (E14-Adult) of mouse retinal development utilizing publicly available data from (Clark et al. 2019). (B) Gene plots for Ptbp1 and Ptbp2 expression during mouse retinal development. (C) Heatmaps of Ptbp1 and Ptbp2 expression patterns in RPCs at varying time points during mouse retinal development. (D) Heatmaps of Ptbp1 and Ptbp2 expression patterns in different mouse retinal cell types. (E) 3D UMAP plot showing cell types from combined timepoints (Hgw9-Adult) of human retinal development utilizing publicly available data from (Lu et al. 2020). (F) Gene plots for Ptbp1 and Ptbp2 expression during human retinal development. (G) Heatmaps of Ptbp1 and Ptbp2 expression patterns in RPCs at varying time points during human retinal development. (H) Heatmaps of Ptbp1 and Ptbp2 expression patterns in different human retinal cell types.

Immunostaining does not detect changes in cell composition in E14 or P0 Ptbp1-deficient retina.
(A) Representative immunostaining for, HuC/D+NeuN and (B) Otx2 in Ptbp1-Ctrl and Ptbp1-KO mice at E14. (C) Representative immunostaining for Opn1sw and (D) Otx2 in Ptbp1-Ctrl and Ptbp1-KO mice at P0. (E) Quantification of Otx2-positive cells in Ptbp1-Ctrl and Ptbp1-KO retinas at P0 (n≥4/genotype). Significance was determined using an unpaired t test: ns=p>0.05. Each data point was calculated from an individual retina. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar= 100 μm.

BulkRNA-seq analysis reveals altered gene expression and splicing patterns in E16 Chx10-Cre;Ptbp1^lox/loxmice retina.
(A) Bar charts showing Ptbp1 and (B) Ptbp2 expression in various cell types and tissues using the ASCOT browser (Ling et al. 2020). Highlighted samples are rods photoreceptors at P2, heart tissue showing low Ptbp1 expression and cochlear hair cells showing high Ptbp1 expression. (C) Heatmap of select differentially expressed genes between individual replicates samples for wildtype (WT), heterozygous Chx10-Cre;Ptbp1^lox/+ (Het1, Het2, Het3), and homozygous Chx10-Cre;Ptbp1^lox/lox (KO1, KO2, KO3) Ptbp1 mutant retinal samples analyzed in this study, along with publicly available bulk RNA-seq datasets including embryonic day 16 retina, postnatal day 28 retina, Thy1-positive neurons, astrocytes, microglia, hair cells and heart tissue. (D) Pearson correlation plot for identified differentially expressed genes between Het and KO samples, plotted across all samples including publically available datasets used. (E) Heatmap showing PSI values and (F) pearson correlation plot for splicing events identified as differentially spliced between Het and KO samples, plotted across all samples including publicly available datasets.

ScRNA-seq analysis identifies differentially expressed genes in major adult retinal cell types following developmental loss of function of Ptbp1.
(A) Dot Plot of cell type-specific markers used to identify clusters for each major retinal cell type. (B) Volcano plot showing differentially expressed genes for bipolar cells (BC), (C) amacrine cells (AC), (D) rod photoreceptors and (E) cone photoreceptors between Ptbp1-Ctrl and Ptbp1-KO samples.

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