Havrilak Jamie A, Melton Kristin R, Shannon John M
Molecular and Developmental Biology Graduate Program, University of Cincinnati College of Medicine, 3333 Burnet Ave, Cincinnati, OH 45229, United States; Section of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229, United States.
Section of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229, United States.
Dev Biol. 2017 Jul 1;427(1):93-105. doi: 10.1016/j.ydbio.2017.05.003. Epub 2017 May 10.
Crosstalk between mesenchymal and epithelial cells influences organogenesis in multiple tissues, such as lung, pancreas, liver, and the nervous system. Lung mesenchyme comprises multiple cell types, however, and precise identification of the mesenchymal cell type(s) that drives early events in lung development remains unknown. Endothelial cells have been shown to be required for some aspects of lung epithelial patterning, lung stem cell differentiation, and regeneration after injury. Furthermore, endothelial cells are involved in early liver and pancreas development. From these observations we hypothesized that endothelial cells might also be required for early specification of the respiratory field and subsequent lung bud initiation. We first blocked VEGF signaling in E8.5 cultured foreguts with small molecule VEGFR inhibitors and found that lung specification and bud formation were unaltered. However, when we examined E9.5 mouse embryos carrying a mutation in the VEGFR Flk-1, which do not develop endothelial cells, we found that respiratory progenitor specification was impeded. Because the E9.5 embryos were substantially smaller than control littermates, suggesting the possibility of developmental delay, we isolated and cultured foreguts from mutant and control embryos on E8.5, when no size differences were apparent. We found that both specification of the respiratory field and lung bud formation occurred in mutant and control explants. These observations were unaffected by the presence or absence of serum. We also observed that hepatic specification and initiation occurred in the absence of endothelial cells, and that expansion of the liver epithelium in culture did not differ between mutant and control explants. Consistent with previously published results, we also found that pancreatic buds were not maintained in cultured foreguts when endothelial cells were absent. Our observations support the conclusion that endothelial cells are not required for early specification of lung progenitors and bud initiation, and that the diminished lung specification seen in E9.5 Flk embryos is likely due to developmental delay resulting from the insufficient delivery of oxygen, nutrients, and other factors in the absence of a vasculature.
间充质细胞与上皮细胞之间的相互作用影响多种组织（如肺、胰腺、肝脏和神经系统）的器官发生。然而，肺间充质包含多种细胞类型，驱动肺发育早期事件的间充质细胞类型的确切身份仍不清楚。内皮细胞已被证明在肺上皮模式形成、肺干细胞分化以及损伤后的再生等方面是必需的。此外，内皮细胞还参与肝脏和胰腺的早期发育。基于这些观察结果，我们推测内皮细胞可能也是呼吸场早期特化和随后肺芽起始所必需的。我们首先用小分子VEGFR抑制剂阻断E8.5期培养的前肠中的VEGF信号，发现肺的特化和芽形成未受影响。然而，当我们检查携带VEGFR Flk-1突变且不发育内皮细胞的E9.5期小鼠胚胎时，我们发现呼吸祖细胞的特化受到阻碍。由于E9.5期胚胎比同窝对照胚胎小得多，提示存在发育延迟的可能性，我们在E8.5期分离并培养了突变体和对照胚胎的前肠，此时未观察到大小差异。我们发现突变体和对照外植体中均发生了呼吸场的特化和肺芽形成。这些观察结果不受血清存在与否的影响。我们还观察到在没有内皮细胞的情况下肝脏特化和起始发生，并且培养物中肝脏上皮的扩增在突变体和对照外植体之间没有差异。与先前发表的结果一致，我们还发现当没有内皮细胞时，培养的前肠中胰腺芽无法维持。我们的观察结果支持以下结论：内皮细胞不是肺祖细胞早期特化和芽起始所必需的，并且在E9.5期Flk胚胎中观察到的肺特化减少可能是由于在没有脉管系统的情况下氧气、营养物质和其他因子供应不足导致的发育延迟。
