Specification And Patterning of the Lymphatic System

我们最近对斑马鱼中淋巴管系统的鉴定和初步表征使我们有可能利用这种模式生物中可用的强大的遗传学、实验胚胎学和成像工具来研究淋巴管是如何形成的，以及是什么调节它们的生长和组装。鉴于最近的证据表明，淋巴管是许多肿瘤转移的主要途径，了解如何调节和控制淋巴管的形成是临床上非常感兴趣的话题。该项目的长期目标是扩大我们的初步发现，以获得更详细的了解淋巴管如何在鱼体内组装，然后使用这个强大的模型来研究淋巴管生成是如何在体内调节的。 近年来，淋巴系统因其在正常和病理过程中的重要作用而成为人们极感兴趣的主题，但在了解淋巴系统的起源和早期发展方面的进展一直受到在体内观察淋巴细胞和在现有模型生物中对淋巴系统进行明确的遗传和实验操作的困难的阻碍。我们最近首次展示了斑马鱼拥有与其他脊椎动物(包括人类)中发现的淋巴管相同的许多形态、分子和功能特征的淋巴系统，为成像和研究淋巴管发育提供了一个极好的新模型。利用转基因斑马鱼的双光子时间推移成像，我们还追踪了单个细胞整合到淋巴管内皮细胞的迁移和谱系，首次提供了体内确凿证据，证明早期淋巴管内皮细胞来自原始静脉血管。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(向淋巴管中注入染料的成像)、组织学以及扫描和透射电子显微镜来进一步表征淋巴管系统在发育过程中如何生长和组装。我们还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哪里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和以前表征的基因。我们的目标是确定它们在淋巴管内皮细胞的指定和分化以及淋巴管的形成和生长中所起的功能作用。我们已经在遗传筛选中鉴定了几个淋巴特异性突变体，目前正在对这些突变体进行定位克隆和鉴定。我们还使用反义寡核苷酸敲除技术来检测一些基因的功能丧失表型。作为这些研究结果的一个例子，我们最近使用了这些敲除方法来表明Netrin，一种先前被证明在神经元指导中具有重要作用的分泌分子，也是淋巴组装和模式所必需的。通过对转基因动物的淋巴管成像和检测淋巴标记基因的表达，正在评估突变和反义寡核苷酸处理的胚胎的表型。 我们的研究结果，结合了斑马鱼可用的遗传和实验工具，以及对活体动物正在发育的血管结构进行高分辨率显微成像的能力，正在为淋巴系统的起源和组装带来重要的新见解。

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. As one example of the results of these studies 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. The phenotypes of mutants and antisense oligonucleotide-treated embryos are being assessed by imaging lymphatic vessels in transgenic animals and examining expression of lymphatic marker genes. 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 assembly of the lymphatic system.