Specification And Patterning of the Lymphatic System

我们最近对斑马鱼淋巴管系统的鉴定和初步表征使我们能够利用这种模式生物中可用的强大遗传、实验胚胎学和成像工具来解决淋巴管如何形成以及是什么调节淋巴管的问题。它们的生长和组装。 了解如何调节和控制淋巴管的形成是一个相当大的临床兴趣的话题，最近的证据表明，淋巴管是肿瘤转移的主要途径，如果不是大多数癌症。 该项目的长期目标是扩展我们的初步发现，以便更详细地了解淋巴管如何在鱼中组装，然后使用这个强大的模型来研究淋巴管生成如何在体内调节。 淋巴系统由于其在正常和病理过程中的重要作用，近年来已成为人们非常感兴趣的主题，但在了解该系统的起源和早期发育方面的进展受到阻碍，因为难以在体内观察淋巴细胞并在目前可用的模型生物体中对淋巴系统进行定义的遗传和实验操作。 我们最近首次表明，斑马鱼拥有一个淋巴系统，该系统与其他脊椎动物（包括人类）中发现的淋巴管具有许多形态，分子和功能特征，为成像和研究淋巴发育提供了极好的新模型。 使用转基因斑马鱼的双光子延时成像，我们还追踪了掺入淋巴内皮的单个细胞的迁移和谱系，提供了第一个确凿的体内证据，证明早期淋巴内皮细胞来源于原始静脉血管。 我们正在继续研究斑马鱼淋巴系统的组装和起源。 在正在进行的研究中，我们正在使用转基因动物的延时双光子成像，淋巴管造影术（注射到淋巴管中的染料成像），组织学，扫描和透射电子显微镜，以进一步表征淋巴管系统在发育过程中如何生长和组装。 我们也在研究淋巴管基因的空间和时间表达，以更好地了解细胞在何处以及如何被指定为淋巴管内皮细胞。 此外，我们正在表征一些新的以及以前表征的基因。 我们的目标是确定它们在淋巴管内皮细胞的特化和分化以及淋巴管的形成和生长中发挥什么样的功能作用。 我们已经确定了几个淋巴特异性突变体的遗传筛选，现在正在进行定位克隆和这些突变体的特性。 我们还使用反义寡核苷酸敲除技术来检查一些基因的功能丧失表型。 我们最近使用这些敲除方法表明，netrin，一种分泌的分子，以前被证明在神经元引导中具有重要作用，也是淋巴组装和图案化所必需的。 此外，我们最近还发现趋化因子信号传导在指导躯干淋巴管网络的形成中的作用 我们的研究结果结合了斑马鱼中可用的遗传和实验工具，并能够对活体动物中正在发育的血管结构进行高分辨率显微成像，这将导致对淋巴系统起源和生长的重要新见解。

Our recent identification and preliminary characterization of a lymphatic vascular system in the zebrafish has made it possible to bring the powerful genetic, experimental embryologic,and imaging tools available in this model organism to bear on the question of how lymphatic vessels form and what regulates their growth and assembly. Understanding how to regulate and control lymphatic vessel formation is a topic of considerable clinical interest given recent evidence suggesting that lymphatics are the major route for tumor metastasis in many if not most cancers. The long-term goal of this project is to expand on our preliminary findings in order to gain a more detailed understanding of how lymphatic vessels assemble in the fish, and then use this powerful model to study how lymphangiogenesis is regulated in vivo. The lymphatic system has become the subject of great interest in recent years because of its important role in normal and pathological processes, but progress in understanding the origins and early development of this system has been hampered by difficulties in observing lymphatic cells in vivo and performing defined genetic and experimental manipulation of the lymphatic system in currently available model organisms. We recently showed for the first time that the zebrafish possesses a lymphatic system that shares many of the morphological, molecular, and functional characteristics of the lymphatic vessels found in other vertebrates (including humans), providing a superb new model for imaging and studying lymphatic development. Using two-photon time-lapse imaging of transgenic zebrafish, we also traced the migration and lineage of individual cells incorporating into the lymphatic endothelium, providing the first conclusive in vivo evidence establishing that early lymphatic endothelial cells are derived from primitive venous blood vessels. We are continuing to examine the assembly and origins of the lymphatic system of the zebrafish. In ongoing studies we are using time-lapse two-photon imaging of transgenic animals, lymphangiography (imaging of dye injected into lymphatic vessels), histology, and scanning and transmission electron microscopy to further characterize how the lymphatic vascular system grows and assembles during development. We are also examining the spatial and temporal expression of lymphatic genes to gain a better understanding of where and how cells become specified as lymphatic endothelial cells. In addition, we are characterizing a number of novel as well as previously characterized genes. Our goal is to determine what functional role they play in lymphatic endothelial specification and differentiation, and in lymphatic vessel formation and growth. We have identified several lymphatic specific mutants in genetic screens and are now carrying out positional cloning and characterization of these mutants. We are also using antisense oligonucleotide knockdown technologies to examine the loss-of-function phenotypes of a number of genes. We have recently used these knockdown methods to show that netrin, a secreted molecule previously shown to have an important role in neuronal guidance, is also required for lymphatic assembly and patterning. In addition, we have also recently identified a role for chemokine signaling in directing the patterning of the trunk lymphatic vascular network The results of our studies, combining the genetic and experimental tools available in the zebrafish with the ability to perform high-resolution microscopic imaging of developing vascular structures in living animals, are leading to important new insights into the origins and growth of the lymphatic system.