Lineage and cell migration in patterning the limb primordium

描述（由申请人提供）：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化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模式信号整合，为肢体形态发生奠定基础。尽管在分子水平上进行了二十年的深入研究，但肢体模式的许多关键方面仍然没有得到理解。例如，我们不知道为什么肢芽会在特定的时间和地点形成。这一过程涉及特定的局部上皮-间充质转化（EMT），但如何控制EMT启动肢芽形成还没有描述。也没有研究阐明为什么肢芽长出像桨，沿着它的背腹轴沿着变平，而不是像半球形的丘。此外，虽然肢芽内不同细胞类型之间的正常谱系关系在很大程度上是了解的，但在再生生长等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，以及使用标记的逆转录病毒载体文库进行谱系分析。在这些研究中发现的细胞行为的线索将用于设计候选基因的功能研究，我们假设这些候选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。类似地，有暗示性证据表明分泌的配体Wnt 5a的活性在引导细胞运动中起作用，导致肢芽扁平成桨状。这些基因的野生型、激活和显性阴性形式的错误表达，加上细胞迁移和谱系变化的成像和分析，将使这些假设得到直接评估。 公共卫生相关性：我们正在研究谱系和细胞迁移对肢芽模式的贡献。先天性肢体畸形是人类最常见的出生缺陷之一，其中许多（如果不是大多数）可归因于肢体模式正确建立的缺陷。此外，我们所学到的关于细胞行为和细胞外信号在组织肢体中的整合的一般经验教训，将作为理解胚胎发生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.