Lineage and cell migration in patterning the limb primordium

描述(申请人提供)：本研究的主要目的是阐明发育肢体中不同细胞类型和不同区域细胞群体之间的谱系关系，建立肢体原基内细胞运动和迁移的模式，并确定谱系和迁移如何与图案信号相结合，为肢体形态发生奠定基础。尽管在分子水平上进行了20年的密集研究，但肢体模式的许多关键方面仍然不清楚。例如，人们不知道为什么肢芽会在它形成的时间和地点形成。这一过程涉及特定的局部上皮-间充质转化(EMT)，但如何控制这一过程以启动肢芽形成尚未被描述。也没有研究澄清为什么肢芽长出时是沿着背腹轴扁平的桨，而不是半球形的丘状突起。此外，尽管肢芽内不同细胞类型之间的正常谱系关系已经被很大程度上了解，但例如在再生生长过程中细胞分化的可塑性程度仍然存在争议。为了解决这些问题，我们将利用一系列最先进的方法，包括通过双光子显微镜对细胞运动进行实时成像，以及使用逆转录病毒载体的标签库进行谱系分析。这些研究中确定的细胞行为线索将被用于设计候选基因的功能研究，我们假设这些基因可能参与调节肢芽中的细胞运动。例如，初步数据显示，不同HOX基因的动态表达与细胞从侧板迁移到肢体的时间相关。同样，有提示证据表明，分泌配体WNT5a的活性在指导细胞运动中发挥作用，导致肢芽扁平成桨。这些基因的野生型、激活和显性阴性形式的错误表达，结合细胞迁移和谱系变化的成像和分析，将使这些假说得到直接评估。 公共卫生相关性：我们正在研究血统和细胞迁移对肢芽形成的贡献。先天性肢体畸形是人类最常见的先天缺陷之一，其中许多(如果不是大多数)可归因于肢体模式的正确建立方面的缺陷。此外，我们所学到的关于在组织肢体过程中整合细胞行为和细胞外信号的一般经验教训，将作为理解胚胎发育过程中这些过程的范例。

DESCRIPTION (provided by applicant): The major objectives of this study is to elucidate the lineage relationships between different cell types and different regionalized populations of cells in the developing limb, to establish patterns of cell movement and migration within the limb primordium, and to determine how lineage and migration are integrated with patterning signals to set the stage for limb morphogenesis. In spite of two decades of intense study on a molecular level, many critical aspects of limb patterning are still not understood. For example, it is not known why the limb bud forms in the place and time that it does. This process involves specific localized epithelial- mesenchymal transition (EMT), but how this is controlled to initiate limb bud formation has not been described. There also have been no studies clarifying why the limb bud grows out as a paddle, flattened along its dorsal- ventral axis, rather than as a hemispherical mound. Moreover, although the normal lineage relationships between different cell types within the limb bud are largely understood, the degree of plasticity in cell differentiation during, for instance, regenerative growth remains controversial. To address these issues we will utilize a range of state-of-the-art approaches including live imaging of cell movements by two-photon microscopy and lineage analysis using tagged libraries of retroviral vectors. Clues to cell behavior identified in these studies will be used to design functional studies of candidate genes that we hypothesize may be involved in regulating cell movements in the limb bud. For example, preliminary data shows that the dynamic expression of different Hox genes correlate with the timing of cells migrating into the limb from the lateral plate. Similarly, there is suggestive evidence that the activity of the secreted ligand Wnt5a plays a role in directing the cell movements leading to flattening of the limb bud into a paddle. Misexpression of wild type, activated, and dominant-negative forms of these genes in conjunction with imaging and analysis of cell migration and of lineage changes, will allow these hypothesis to be directly assessed. PUBLIC HEALTH RELEVANCE: We are studying the contribution of lineage and cell migration to the pattering of the limb bud. Congenital limb malformations are among the most common human birth defects, many, if not most, of which are attributable to defects in the proper establishment of the limb pattern. Moreover, the general lessons we learn about the integration of cellular behavior and extracellular signaling in organizing the limb, will serve as paradigms for understanding these processes during embryogenesis.