A single-cell transcriptome atlas of pig skin characterizes anatomical positional heterogeneity
Curation statements for this article:-
Curated by eLife
eLife assessment
This valuable manuscript provides a single-cell RNA sequencing analysis of adult pig skin from different species and anatomical regions. The evidence supporting the conclusions is compelling, with identification molecular and cellular differences in pig skin, yet the analyses are incomplete in the analysis of regional- or species-based differences.
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- Cell Biology (eLife)
Abstract
Different anatomical locations of the body skin show differences in their gene expression patterns depending on different origins, and the inherent heterogeneous information can be maintained in adults. However, highly resolvable cellular specialization is less well characterized in different anatomical regions of the skin. Pig is regarded as an excellent model animal for human skin research in view of its similar physiology to human. In this study, single-cell RNA sequencing was performed on pig skin tissues from six different anatomical regions of Chenghua (CH) pigs, with a superior skin thickness trait, and the back site of large white (LW) pigs. We obtained 233,715 cells, representing seven cell types, among which we primarily characterized the heterogeneity of the top three cell types, including smooth muscle cells (SMCs), endothelial cells (ECs), and fibroblasts (FBs). Then, we further identified several subtypes of SMCs, ECs, and FBs, and discovered the expression patterns of site-specific genes involved in some important pathways such as the immune response and extracellular matrix (ECM) synthesis in different anatomical regions. By comparing differentially expressed genes of skin FBs among different anatomical regions, we considered TNN, COL11A1, and INHBA as candidate genes for facilitating ECM accumulation. These findings of heterogeneity in the main skin cell types from different anatomical sites will contribute to a better understanding of inherent skin information and place the potential focus on skin generation, transmission, and transplantation, paving the foundation for human skin priming.
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Author Response
Reviewer #1 (Public Review):
This manuscript is interesting because of the exploration of a novel model organisms utilizing next-generation sequencing approaches, such as single-cell-RNA-seq. Despite the authors' efforts the manuscript lacks a cohesive narrative and suffers from being extremely preliminary in nature. For example, most of the figures are cut and pasted directly from the computational programs with very little formatting or thought to creating new knowledge from the data generated. Essentially the manuscript consists of 2-3 experiments where the authors performed single-cell-RNA-seq on different anatomical locations in the pig and also on a couple of different pig types (The Chenghua and Large White). The authors used standard computational pipelines consisting of Seurat, Monocle, Cell Chat, and others …
Author Response
Reviewer #1 (Public Review):
This manuscript is interesting because of the exploration of a novel model organisms utilizing next-generation sequencing approaches, such as single-cell-RNA-seq. Despite the authors' efforts the manuscript lacks a cohesive narrative and suffers from being extremely preliminary in nature. For example, most of the figures are cut and pasted directly from the computational programs with very little formatting or thought to creating new knowledge from the data generated. Essentially the manuscript consists of 2-3 experiments where the authors performed single-cell-RNA-seq on different anatomical locations in the pig and also on a couple of different pig types (The Chenghua and Large White). The authors used standard computational pipelines consisting of Seurat, Monocle, Cell Chat, and others to characterize differences in their data.
There is potential in this manuscript but the authors should improve upon the manuscript by mining the data better and generating a better understanding of anatomical positions of pig skin by evaluating the Hox genes.
(1) Thanks for the reviewer's positive evaluation for our article and providing valuable feedback to improve the quality of our manuscript. To provide a more cohesive narrative, we have edited throughout the manuscript.
(2) Meanwhile, we also modified and formatted some figures including Figures 2-6, Figure 4—figure supplement 1 and 2, Figure 5—figure supplement 1 and 2, and Figure 6—figure supplement 1.
(3) We have analyzed these data of regional- or species-based differences more extensively, and the added content are in Result Section of “Heterogeneity of skin FBs in different anatomic sites” and “Heterogeneity of skin cells in different pig populations”.
(4) However, in our study, we did not identify any Hox gene among these differentially expressed genes in skin fibroblasts from both different anatomical sites and different pig populations. The differences of Hox code expression patterns might come from the heterogeneity of different species.
Reviewer #2 (Public Review):
The authors aimed to analyze different dermal compositions of various skin regions, focusing on fibroblast, endothelium and smooth muscle cells. They collect skin samples from six different skin regions of adult pig skin including the head, ear, shoulder, back, abdomen, and leg skins. After dissociating the tissues into single cells, they perform single-cell RNA analyses. A total of 215 thousand cells were analyzed. The authors identified distinct cell clusters, enriched molecules within each cell cluster, and the dynamic of cell cluster transition and interactions. Based on their findings, they conclude that tenascin N, collagen 11A1, and inhibin A are candidate genes for facilitating extracellular matrix accumulation.
Strength:
The methodology they used to prepare scRNA data is appropriate. Bioinformatic analyses are solid. The authors emphasize the heterogeneous phenotypes and composition ratios of smooth muscle cells, endothelial cells and fibroblasts in each skin region. They identify potential cell communication pathways among cell clusters. Expression of selective molecules on tissue sections were done.
Weakness:
While tenascin, collagen and inhibin are highlighted as genes important for ECM accumulation, there is no functional evaluation data. The discussion section is a compilation of comparisons, and is somewhat fragmentary. More significance from this dataset could have been extracted.
(1) We appreciate the reviewer's suggestions for evaluating the functional significance further. In our next research, we will perform some experiments in vitro and in vivo to explore the functions of these identified key genes.
(2) The discussion section have been greatly modified and it shall be more logical and readable.
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eLife assessment
This valuable manuscript provides a single-cell RNA sequencing analysis of adult pig skin from different species and anatomical regions. The evidence supporting the conclusions is compelling, with identification molecular and cellular differences in pig skin, yet the analyses are incomplete in the analysis of regional- or species-based differences.
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Reviewer #1 (Public Review):
This manuscript is interesting because of the exploration of a novel model organisms utilizing next-generation sequencing approaches, such as single-cell-RNA-seq. Despite the authors' efforts the manuscript lacks a cohesive narrative and suffers from being extremely preliminary in nature. For example, most of the figures are cut and pasted directly from the computational programs with very little formatting or thought to creating new knowledge from the data generated. Essentially the manuscript consists of 2-3 experiments where the authors performed single-cell-RNA-seq on different anatomical locations in the pig and also on a couple of different pig types (The Chenghua and Large White). The authors used standard computational pipelines consisting of Seurat, Monocle, Cell Chat, and others to characterize …
Reviewer #1 (Public Review):
This manuscript is interesting because of the exploration of a novel model organisms utilizing next-generation sequencing approaches, such as single-cell-RNA-seq. Despite the authors' efforts the manuscript lacks a cohesive narrative and suffers from being extremely preliminary in nature. For example, most of the figures are cut and pasted directly from the computational programs with very little formatting or thought to creating new knowledge from the data generated. Essentially the manuscript consists of 2-3 experiments where the authors performed single-cell-RNA-seq on different anatomical locations in the pig and also on a couple of different pig types (The Chenghua and Large White). The authors used standard computational pipelines consisting of Seurat, Monocle, Cell Chat, and others to characterize differences in their data.
There is potential in this manuscript but the authors should improve upon the manuscript by mining the data better and generating a better understanding of anatomical positions of pig skin by evaluating the Hox genes.
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Reviewer #2 (Public Review):
The authors aimed to analyze different dermal compositions of various skin regions, focusing on fibroblast, endothelium and smooth muscle cells. They collect skin samples from six different skin regions of adult pig skin including the head, ear, shoulder, back, abdomen, and leg skins. After dissociating the tissues into single cells, they perform single-cell RNA analyses. A total of 215 thousand cells were analyzed. The authors identified distinct cell clusters, enriched molecules within each cell cluster, and the dynamic of cell cluster transition and interactions. Based on their findings, they conclude that tenascin N, collagen 11A1, and inhibin A are candidate genes for facilitating extracellular matrix accumulation.
Strength:
The methodology they used to prepare scRNA data is appropriate. Bioinformatic …
Reviewer #2 (Public Review):
The authors aimed to analyze different dermal compositions of various skin regions, focusing on fibroblast, endothelium and smooth muscle cells. They collect skin samples from six different skin regions of adult pig skin including the head, ear, shoulder, back, abdomen, and leg skins. After dissociating the tissues into single cells, they perform single-cell RNA analyses. A total of 215 thousand cells were analyzed. The authors identified distinct cell clusters, enriched molecules within each cell cluster, and the dynamic of cell cluster transition and interactions. Based on their findings, they conclude that tenascin N, collagen 11A1, and inhibin A are candidate genes for facilitating extracellular matrix accumulation.
Strength:
The methodology they used to prepare scRNA data is appropriate. Bioinformatic analyses are solid. The authors emphasize the heterogeneous phenotypes and composition ratios of smooth muscle cells, endothelial cells and fibroblasts in each skin region. They identify potential cell communication pathways among cell clusters. Expression of selective molecules on tissue sections were done.
Weakness:
While tenascin, collagen and inhibin are highlighted as genes important for ECM accumulation, there is no functional evaluation data. The discussion section is a compilation of comparisons, and is somewhat fragmentary. More significance from this dataset could have been extracted.
Summary:
The manuscript has the potential to be a useful cellular atlas. The direct impact of this paper on skin biology is limited because of the lack of evaluation data. But the database can be useful to many future studies using the pig skin model.
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