Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA.
Immunobiology Graduate Program, Iowa State University, Ames, IA, USA.
J Anim Sci. 2023 Jan 3;101. doi: 10.1093/jas/skac318.
Understanding regional distribution and specialization of small intestinal epithelial cells is crucial for developing methods to control appetite, stress, and nutrient uptake in swine. To establish a better understanding of specific epithelial cells found across different regions of the small intestine in pigs, we utilized single-cell RNA sequencing (scRNA-seq) to recover and analyze epithelial cells from duodenum, jejunum, and ileum. Cells identified included crypt cells, enterocytes, BEST4 enterocytes, goblet cells, and enteroendocrine (EE) cells. EE cells were divided into two subsets based on the level of expression of the EE lineage commitment gene, NEUROD1. NEUROD1hi EE cells had minimal expression of hormone-encoding genes and were dissimilar to EE cells in humans and mice, indicating a subset of EE cells unique to pigs. Recently discovered BEST4 enterocytes were detected in both crypts and villi throughout the small intestine via in situ staining, unlike in humans, where BEST4 enterocytes are found only in small intestinal villi. Proximal-to-distal gradients of expression were noted for hormone-encoding genes in EE cells and nutrient transport genes in enterocytes via scRNA-seq, demonstrating regional specialization. Regional gene expression in EE cells and enterocytes was validated via quantitative PCR (qPCR) analysis of RNA isolated from epithelial cells of different small intestinal locations. Though many genes had similar patterns of regional expression when assessed by qPCR of total epithelial cells, some regional expression was only detected via scRNA-seq, highlighting advantages of scRNA-seq to deconvolute cell type-specific regional gene expression when compared to analysis of bulk samples. Overall, results provide new information on regional localization and transcriptional profiles of epithelial cells in the pig small intestine.
了解小肠上皮细胞的区域分布和特化对于开发控制猪的食欲、应激和营养吸收的方法至关重要。为了更好地了解猪小肠不同区域特有的特定上皮细胞，我们利用单细胞 RNA 测序（scRNA-seq）从十二指肠、空肠和回肠中回收和分析上皮细胞。鉴定出的细胞包括隐窝细胞、肠细胞、BEST4 肠细胞、杯状细胞和肠内分泌（EE）细胞。根据 EE 谱系决定基因 NEUROD1 的表达水平，将 EE 细胞分为两个亚群。NEUROD1hi EE 细胞激素编码基因的表达水平较低，与人和小鼠的 EE 细胞不同，表明 EE 细胞是猪特有的亚群。通过原位染色发现，最近发现的 BEST4 肠细胞存在于整个小肠的隐窝和绒毛中，而在人类中，BEST4 肠细胞仅存在于小肠绒毛中。通过 scRNA-seq 注意到 EE 细胞中激素编码基因和肠细胞中营养转运基因的近端到远端表达梯度，表明存在区域特化。通过对不同小肠部位上皮细胞分离出的 RNA 进行 qPCR 分析，验证了 EE 细胞和肠细胞的区域基因表达。尽管通过 qPCR 分析总上皮细胞时许多基因具有相似的区域表达模式，但一些区域表达仅通过 scRNA-seq 检测到，这突出了 scRNA-seq 在与分析批量样本相比时，具有解卷积细胞类型特异性区域基因表达的优势。总的来说，这些结果提供了关于猪小肠上皮细胞区域定位和转录谱的新信息。
