Single cell analysis of developing Merkel cells reveals the emergence of non-coding RNA biotypes as a hallmark of terminal differentiation

Figures

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  Fig. 1. MC terminal differentiation is accompanied by emergence of non-protein coding transcript biotypes associated with genes related to cell specific function. A Monocle-generated trajectory plot representing differentiation pseudotime ordering of single MCs. B Scaled and centered expression heatmap of transcripts with dynamic expression over the MC differentiation pseudotime. Transcripts are ordered by row and cells by column. Transcripts are clustered by their expression patterns over pseudotime. The number of transcripts per cluster is shown. C Bar chart showing the relative proportion of transcript biotypes in each pseudotime-dependent transcript clusters from (B) and all other transcripts. All others: 13,600 detected transcripts with TPM > 10 in more than 58 MCs and not included among the dynamically expressed transcripts. Chi-squared test: p < 2.2 × 10−16 for protein-coding vs. non-coding transcript numbers across all clusters, including “All others”. D Top gene ontology terms associated with genes encoding non-coding transcript biotypes in transcript cluster 4 and 5 from (B) (highest expression in late differentiation/terminally differentiated MCs). Bar magnitude, enrichment…

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  Fig. 2. Retained intron transcript Aspa-202 is co-expressed with Aspa mRNA transcripts in single MCs and forms nuclear condensates. A Scatter plot indicating the expression levels of Aspa-201: protein-coding (mRNA) and Aspa-202: retained intron (RI) transcripts in single MCs along the pseudotime of MC differentiation. B Upper: Schematic illustration of the RI sequence between exon 3 and exon 4 in Aspa-202 transcript. Lower: IGV-generated plot of sequencing reads aligned to the Aspa gene locus indicating RI transcript in select MCs. C Boxplot showing the smFISH relative signal intensity of the target transcripts per cell in inner (less differentiated) and outer (more differentiated) neonatal touch dome MCs. Boxplots show median, interquartile range (IQR), and whiskers extending to 1.5 x IQR. Data were collected from 9 touch domes across 3 mice. N (Inner) = 88; N (Outer) = 92. D Middle: Confocal z-projection of whole mount smFISH displaying the expression of Aspa mRNA (red) and Aspa RI (cyan) transcripts in neonatal mouse epidermis. MCs, K8 immunostaining (green). Cell nuclei, DAPI (blue). Left and right: optical sections of representative MCs. Arrowhead indicates colocalization of Aspa…

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  Fig. 3. Retained intron transcripts downregulate cognate gene expression. A Confocal optical sections of smFISH displaying the expression of Aspa mRNA (red) and Aspa RI (cyan) transcripts in undifferentiated (upper) and in vitro differentiated (lower) HT-22 cells. Cell nuclei, DAPI (blue). Scale bar, 10 μm. B Confocal optical sections of smFISH displaying the expression of Aspa mRNA (red) and Aspa RI (cyan) transcripts in vector control transfected (upper) and Aspa RI transfected (lower) undifferentiated HT-22 cells. Cell nuclei, DAPI (blue). Scale bar, 10 μm. A’, B’ Boxplot showing the smFISH average relative signal intensity per cell in undifferentiated (undiff) and differentiated (diff) (A’), and in vector and Aspa RI transfected (B’) HT-22 cells. Each dot represents the average signal intensity per cell within a high-power field (range 6-28 cells). Results include cells from 3 independent experiments. N = 12 fields. A”, B” Boxplot showing the cytoplasmic fraction per cell of Aspa mRNA smFISH signal intensity in undifferentiated and differentiated (A”), and in vector and Aspa RI transfected (B”) HT-22 cells. Each dot represents an individual cell. Results are combined from 3…

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  Fig. 4. Emergence of non-coding transcript biotypes is a general feature of terminal differentiation. A–D Analyses of scRNA-seq data on mouse lung alveolar type II (AT2) cell development. E–H Analyses of scRNA-seq data on in vitro differentiation of human neural precursor cells (NPCs) to neurons. A, E Monocle-generated trajectory plots representing differentiation pseudotime ordering of mouse AT2 cells (A) and human neurons (E). E, embryonic day; P, postnatal day; D, day. B, F Scaled and centered expression heatmaps of transcripts with dynamic expression over the mouse AT2 cells (B) and human neurons (F) differentiation pseudotimes. Transcripts are ordered by row and cells are by column. Transcripts are clustered by their expression patterns over pseudotime. Number of transcripts per cluster is shown. C, G Bar charts showing the relative proportions of transcript biotypes in each pseudotime-dependent transcript clusters and in all cells (C, mouse AT2 cells; G, human neurons). All others: 11,052 all other detected transcripts with TPM > 10 in more than 9 AT2 cells (C), and 22,291 all other detected transcripts with TPM > 10 in more than 49 neurons. Chi-squared test: p < 2.2 × 10−16 for…

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  Fig. 5. Emergence of non-coding transcript biotypes is diminished in differentiating Down syndrome (DS) neurons. A Top: schematic illustration showing the in vitro differentiation of induced pluripotent stem cells (iPSCs) established from healthy control (Ctrl) and Down syndrome (DS) embryos into neural precursor cells (NPCs) and neurons. Middle: schematic illustration showing the identification of differentially expressed (DE) transcripts upregulated in neurons relative to NPCs derived from control (left) and DS (right) iPSCs. Lower: Venn diagram showing the intersection of DE upregulated transcripts identified in control and DS neuronal differentiation. B Bar chart showing the relative proportion of transcript biotypes for differentially upregulated and all other transcripts uniquely identified in control or DS neuronal differentiation. All others, expressed transcripts not upregulated in neuron samples (N for Ctrl = 8798; N for DS = 9104). Comparison of protein-coding and non-coding proportions for transcripts upregulated in Ctrl neurons versus DS neurons: p = 7.63 × 10−11. C, D Top gene ontology terms associated with genes encoding differentially upregulated non-coding transcripts…

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  Fig. 6. Graphical summary of key study findings. Full-length single cell RNA sequencing identified an enrichment of non-coding transcripts that emerge during the terminal differentiation of Merkel cells. Enriched transcripts included retained intron RNAs capable of forming extrachromosomal nuclear condensates that sequestered cognate mRNAs and decreased protein expression. The emergence of non-coding transcripts was observed during the terminal differentiation of multiple cell types and was blunted in neurons differentiated from patients with Down syndrome.