Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada.
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada.
Methods Mol Biol. 2021;2230:115-137. doi: 10.1007/978-1-0716-1028-2_8.
The biological signals that coordinate the three-dimensional outgrowth and patterning of the vertebrate limb bud have been well delineated. These include a number of vital embryonic signaling pathways, including the fibroblast growth factor, WNT, transforming growth factor, and hedgehog. Collectively these signals converge on multiple progenitor populations to drive the formation of a variety of tissues that make up the limb musculoskeletal system, such as muscle, tendon, cartilage, stroma, and bone. The basic mechanisms regulating the commitment and differentiation of diverse limb progenitor populations has been successfully modeled in vitro using high density primary limb mesenchymal or micromass cultures. However, this approach is limited in its ability to more faithfully recapitulate the assembly of progenitors into organized tissues that span the entire musculoskeletal system. Other biological systems have benefitted from the development and availability of three-dimensional organoid cultures which have transformed our understanding of tissue development, homeostasis and regeneration. Such a system does not exist that effectively models the complexity of limb development. However, limb bud organ cultures while still necessitating the use of collected embryonic tissue have proved to be a powerful model system to elucidate the molecular underpinning of musculoskeletal development. In this methods article, the derivation and use of limb bud organ cultures from murine limb buds will be described, along with strategies to manipulate signaling pathways, examine gene expression and for longitudinal lineage tracking.
协调脊椎动物肢芽三维生长和模式形成的生物信号已经得到了很好的描述。这些信号包括许多重要的胚胎信号通路，如成纤维细胞生长因子、WNT、转化生长因子和 hedgehog。这些信号共同作用于多个祖细胞群体，驱动构成肢体骨骼肌肉系统的各种组织的形成，如肌肉、肌腱、软骨、基质和骨骼。使用高密度的初级肢间充质或微团培养物，已经成功地在体外模拟了调节不同肢祖细胞群体的定向和分化的基本机制。然而，这种方法在更真实地模拟祖细胞组装成跨越整个骨骼肌肉系统的有组织组织方面的能力有限。其他生物系统受益于三维类器官培养的发展和可用性，这改变了我们对组织发育、稳态和再生的理解。虽然仍然需要收集胚胎组织，但不存在有效模拟肢体发育复杂性的此类系统。然而，肢芽器官培养已被证明是阐明骨骼肌肉发育分子基础的有力模型系统，尽管仍然需要使用收集的胚胎组织。在本方法文章中，将描述从鼠肢芽中衍生和使用肢芽器官培养物的方法，以及操纵信号通路、检查基因表达和进行纵向谱系追踪的策略。
