Lgr5 Marks Stem/Progenitor Cells Contributing to Epithelial and Muscle Development in the Mouse Esophagus

The existence and function of Lgr5+ cells within the developing esophagus remains unknown. Here, we document multiple discrete Lgr5+ populations in the developing mouse esophagus, predominantly within nascent epithelial and external muscle layers. Lgr5 expression initially emerges in the developing proximal embryonic epithelium, but progressively extends distally and persists within the distal epithelium of neonates. Fate mapping and ablation analyses reveal a long-term contribution of epithelial Lgr5+ cells to esophageal organogenesis. Additionally, Lgr5-expressing cells are present in the developing external muscle layer, particularly during the development of the striated component. Fate mapping reveals a significant contribution of these embryonic Lgr5+ cells to the adult muscle layer. Embryonic Lgr5+ epithelial cells are also found to be important for regulating epithelial development, serving as a key source of Wnt6, among other ligands, to promote epithelial cell proliferation and formation of epithelial layers. These findings significantly enhance our understanding of esophageal development and shed light on the involvement of Lgr5+ stem/progenitor cells during organogenesis. Importantly, this study lays the foundation for investigating esophageal diseases related to the Lgr5+ stem/progenitor cell pool. Embryos were harvested from WT females at E13.5 and E16.5, and esophagi were dissected from P0 neonates. Samples were fresh frozen in Tissue Freezing Medium and kept in -80 freezer before sectioning. Sections were palced on Visium Spatial Gene Expression Slide and processed according to manufacturer instructions.发育中的食管内Lgr5阳性（Lgr5+）细胞的存在及其功能至今仍不明晰。本研究报道，发育中的小鼠食管内存在多个离散的Lgr5阳性细胞群，主要分布于新生上皮层与外肌层。Lgr5的表达最初出现于发育中的胚胎近端上皮，随后逐渐向远端扩展，并持续存在于新生小鼠的远端上皮中。命运图谱分析与细胞消融实验证实，上皮源性Lgr5阳性细胞对食管器官发生具有长期贡献。此外，发育中的外肌层中亦存在表达Lgr5的细胞，尤其在横纹肌组分发育阶段。命运图谱分析显示，这些胚胎期Lgr5阳性细胞对成年小鼠食管肌层具有显著贡献。研究同时发现，胚胎期Lgr5阳性上皮细胞对调控上皮发育至关重要，其作为包括Wnt6在内的多种配体的关键来源，可促进上皮细胞增殖与上皮层形成。上述发现极大深化了我们对食管发育过程的认知，并阐明了Lgr5阳性干/祖细胞在器官发生中的参与机制。尤为重要的是，本研究为探究与Lgr5阳性干/祖细胞池相关的食管疾病奠定了研究基础。实验样本取自胚胎第13.5天（E13.5）与胚胎第16.5天（E16.5）的野生型（WT）雌性小鼠胚胎，以及出生后0天（P0）新生小鼠的解剖食管。样本经组织冷冻包埋剂（Tissue Freezing Medium）速冻后，置于-80℃冰箱保存以待切片。切片被铺于Visium空间基因表达玻片上，并按照制造商提供的操作流程进行处理。