Sonam S et al. (2022), Cellular and molecular profiles of larval and a...

XB-ART-59307

Dev Biol 2022 Nov 01;491:13-30. doi: 10.1016/j.ydbio.2022.08.007.

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Cellular and molecular profiles of larval and adult Xenopus corneal epithelia resolved at the single-cell level.

Sonam S , Bangru S , Perry KJ , Chembazhi UV , Kalsotra A , Henry JJ .

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Corneal Epithelial Stem Cells (CESCs) and their proliferative progeny, the Transit Amplifying Cells (TACs), are responsible for homeostasis and maintaining corneal transparency. Owing to our limited knowledge of cell fates and gene activity within the cornea, the search for unique markers to identify and isolate these cells remains crucial for ocular surface reconstruction. We performed single-cell RNA sequencing of corneal cells from larval and adult stages of Xenopus. Our results indicate that as the cornea develops and matures, there is an increase in cellular diversity, which is accompanied by a substantial shift in transcriptional profile, gene regulatory network and cell-cell communication dynamics. Our data also reveals several novel genes expressed in corneal cells and changes in gene expression during corneal differentiation at both developmental time-points. Importantly, we identify specific basal cell clusters in both the larval and adult cornea that comprise a relatively undifferentiated cell type and express distinct stem cell markers, which we propose are the putative larval and adult CESCs, respectively. This study offers a detailed atlas of single-cell transcriptomes in the frog cornea. In the future, this work will be useful to elucidate the function of novel genes in corneal epithelial homeostasis, wound healing and regeneration.

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Species referenced: Xenopus laevis
Genes referenced: arhgdib atoh1 cnfn.1 col11a1 col14a1 col1a2 crp.4 dan4l itgb4 kit krt12.2 ly6e mitf mki67 mmp7 msmb.3 pcna synm tgm2 tspan1 vim znf750
GO keywords: epithelial cell development [+]

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	Graphical Abstract.
	Fig. 1. Overview depicting workflow for isolation of corneal cells from Xenopus for single-cell RNA sequencing (scRNA-seq). (A) Isolation of corneal epithelial cells from Xenopus larvae and adults. Corneal epithelia were dissected, pooled and dissociated to isolate single cells. After determining high cell viability, a single-cell library was prepared using the 10X Chromium Single Cell 3′ Reagent Kit (V3). (B) Computational workflow demonstrating data processing and analysis pipeline for scRNA-seq data. Cell Ranger was used to align raw reads and generate feature-barcode matrices. Seurat v3.1 was used to perform basic quality check (QC) and normalization, followed by use of Harmony to remove batch-specific effects.
	Fig. 2. Identification of cell types in scRNA-seq dataset of corneas from Xenopus larvae and adults. (A) Unbiased clustering of 10,659 high-quality cells from larval corneas after QC cutoffs, visualized by Uniform Manifold Approximation and Projection (UMAP). (B) Clustering of 12,155 filtered cells from adult frog corneas, visualized by UMAP. Each dot represents a single cell, and cells from the same cluster are similarly colored. Color codes in A, B represent unique clusters in each data set, and are not homologous. (C, D) Dot plot showing expression of known cell-type marker genes for each cluster. Dot diameter depicts the percentage of cluster cells expressing that marker and intensity encodes average expression of a gene among cells within that cluster. Abbreviations: Ap-L: Apical Larval; Ba-L: Basal Larval; Im-L: Immunocytes Larval; Ke-L: Keratocytes Larval; Un-L: Unknown Larval, and Ap-A: Apical Adult; Ba-A: Basal Adult; En-A: Endothelial Adult; Im-A: Immunocytes Adult; Ke-A: Keratocytes Adult; Ne-A: Neuronal Adult.
	Fig. 3. Proliferative cells are distributed throughout the corneal epithelium in Xenopus. (A, B) Gene expression UMAP overlay with proliferation genes mki67.L, mki67.S, pcna.L, and pcna.S in larval (A) and adult (B) corneas. Color intensity correlates with the relative transcript level for the gene. (C, D) UMAP plot showing the cell cycle status of each cell in (C) larval, and (D) adult clusters, determined using the “CellCycleScoring” module in Seurat v3.1. Color key indicates the cell cycle state. (E–F) Confocal images showing immunofluorescent staining for Proliferating Cell Nuclear Antigen (PCNA) (green) in the tadpole corneal epithelium. (E′-F′) Merged images for E-F with Hoechst labeled nuclei (magenta). (E, E′) Nuclear staining for PCNA is detected in some apical epithelial cells. (F, F′) PCNA labeled nuclei are detected in the basal epithelium. (G–H) Confocal images showing anti-phospho-Histone H3 (H3S10 P) (green) in the tadpole corneal epithelium. (G′-H′) Merged images for G-H with Hoechst labeled nuclei (magenta). (G, G′) Mitotic nuclei (H3S10 P) are present in a few apical epithelial cells. (H, H′) H3S10 P labeled nuclei are detected in the basal epithelium. Scale bar in H′ equals 50 μm for E-H’.
	Fig. 4. Identification of new marker genes for different cell types at two developmental time-points. (A) Violin plots showing the cluster-specific expression of the top-ranking candidate marker genes for each identified cell type in larvae. (B) Violin plots with cluster-specific expression of the top-ranking candidate marker genes for each identified cell type in the adult frog. The highlighted (grey) marker genes are differentially enriched in basal cell clusters of larvae and adults.
	Fig. 5. Pseudo-temporal ordering of epithelial cells reconstructs the larval corneal differentiation process. (A) Pseudo-time plot indicating the cellular trajectory of all larval corneal basal and apical cells (Clusters Ap-L, Ba_I-L, Ba_II-L, Ba_III-L and Ba_IV-L). Single-cell trajectories were constructed, and pseudo-time values were calculated using Monocle 2. (B) Trajectories are colored by cluster identity that corresponds to the key in Fig. 2A. (C) Heatmap depicting the classes of genes that vary along the pseudo-time plot during larval corneal differentiation. Pseudo-time is indicated by the color key similar to that shown in (A). Gene Ontology (GO) analysis was performed using DAVID and the enriched GO terms (P < 0.05) in each class are listed. Relative expression is indicated by the color key. (D) Gene expression kinetics along the pseudo-time progression of representative genes belonging to different pathways and processes, as indicated. Genes shown belong to the Retinoic Acid pathway (RA pathway), the Fibroblast Growth Factor pathway (FGF pathway), the Wnt pathway, Transforming Growth Factor (TGF) beta pathway, Epithelial-to-Mesenchymal Transition (EMT), Extracellular Matrix (ECM) deposition, and the Thyroid Hormone pathway (TH pathway).
	Fig. 6. Differentiation trajectory in adult corneal epithelium involves key cell path decisions.(A) Pseudo-temporal ordering of all cells from the basal epithelium present in the adult cornea (Ba_I-A, Ba_II-A and Ba_III-A). The trajectory has two different paths distributed around branch point 1, namely, cell paths 1 and 2. The arrows indicate the developmental directionality of the cell clusters, based on our inference. (B) Distribution of cells from three basal clusters along the pseudo-time shown in A. While cells of Ba_I-A are present towards the end of path 1, cells of Ba_II-A are distributed throughout the trajectory, and cells of Ba_III-A are located at start of trajectory. (C) Heat map showing bifurcation of gene expression programs executed along the pseudo-time after branching. Three distinct classes of genes (with top GO terms enriched in each class) were identified. (D) Pseudo-time plot to further resolve the cellular trajectories from basal clusters (Ba_I-A and Ba_II-A) to apical clusters (Ap_I-A and Ap_II-A). The branch point 3, which is under evaluation has two different paths, namely cell paths 3 and 4, around it. (E) Pseudo-time plots showing distribution of each of the four clusters along combined cellular trajectories shown in D. (F) Heatmap of gene expression along the pseudo-time plot in D, with the top GO terms enriched in each class of genes, as indicated.
	Fig. 7. Distinct gene regulatory networks are active in basal epithelial cells of the larval and adult frog corneas. (A) UMAP projection of all basal cells based on the AUC scores for each regulon calculated using SCENIC. Cells belonging to the same regulon are colored, according to the key. (B) AUC score based UMAP projection, grouped according to the origin of basal cells. Regulons of basal cells (Ba_I-L, Ba_II-L, Ba_III-L and Ba_IV-L) belonging to larval frogs group together and overlap, whereas regulons from adult basal cells (Ba_I-A, Ba_II-A and Ba_III-A) are separated from larval regulons. (C, D) Dot plot showing unique and shared regulons that are active in different basal cell states. Dot diameter depicts the percentage of cluster cells expressing that regulon and color intensity encodes average expression of a regulon among cells within that cluster, as indicated in the keys.
	Fig. 8. Dynamics of cell-cell communication networks active in the larval and adult frog corneas. (A, B) Network diagrams depicting significant cell-cell interactions marked by arrows (edges) pointing in the source-to-target direction. Thickness of arrows shows the sum of weighted paths between cell types, and the color of arrows corresponds to the source. Network diagrams for (A) larval and (B) adult frog corneas are shown. (C, D) Dot plot of representative outbound signals to different cell types in (C) larval and (D) adult frog corneas. Size of each dot signifies the weight of the corresponding ligand-receptor interaction, and the color indicates negative log10 P-value of the source-to-target interaction, as indicated in the keys. The ligands of the source cell are in black and the receptor present on the target cell follows in red. Empirical P-values were calculated and Benjamini-Hochberg correction was performed. The detailed ligand-receptor interactions are shown in Fig. S7.
	Fig. S1. Quality control (QC) metrics of Xenopus cornea scRNA-seq data. (A) Table showing the QC cutoffs applied on raw reads from scRNA-seq data prior to analysis. (B) Violin plots showing the distribution of gene counts, UMI counts and % mitochondrial content in larval and adult samples, after applying QC cutoffs.
	Fig. S2. (A, B) tSNE and PHATE projections of all larval corneal cells. (C, D) tSNE and PHATE projections of all adult corneal cells. Cells are colored by annotated cell types, as shown in Fig. 2.
	Fig. S3. The absence of skin and lens cell types in (A) larval and (B) adult frog cornea was verified based on the expression of marker genes. Feature plots of cell-type-specific marker genes. Skin cell marker genes (highlighted in blue): melanocyte inducing transcription factor (mitf.L and mitf.S), tyrosinase (tyr.S), melan-A (mlana.L), dopachrome tautomerase (dct.L). Lens cell marker genes (highlighted in red): crystallin alpha A (cryaa.S), crystallin alpha B (cryab.L and cryab.S).
	Fig. S4. Heatmap demonstrating the cluster-specific expression of the top 5 ranking novel candidate marker genes in each cluster of (A) larvae and (B) adult cornea cells identified in the scRNA-seq datasets. Cells are colored by annotated cell types, as shown in Fig. 2.
	Fig. S5. Proportion of cell populations in each cluster for (A) larval and (B) adult scRNA-seq datasets. Numbers on each bar indicate the number of cells.
	Fig. S6. (A) Pseudo-temporal ordering of all cells from basal cluster (Ba_I-A) and apical clusters (Ap_I-A and Ap_II-A) in the adult cornea to resolve the path of transition basal cells (Ba_I-A) to apical cells. (B) Distribution of clusters along the cellular trajectory identified in (A).
	Fig. S7. Dot plot representing all outbound signals to different cell types in (A) larval and (B) adult frog corneas. Size of each dot signifies the weight of the corresponding ligand-receptor interactions, and the color indicates negative log10 P-value of the source-to-target interaction, as indicated in the keys.

References [+] :

Adachi, Serial analysis of gene expression (SAGE) in the rat limbal and central corneal epithelium. 2006, Pubmed