Specification And Patterning of the Lymphatic System

我们对斑马鱼淋巴管系统的鉴定和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课题，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。我们证明了斑马鱼的淋巴系统具有许多其他脊椎动物(包括人类)中发现的淋巴管的形态、分子和功能特征，为淋巴发育的成像和研究提供了一个极好的新模型。我们还使用转基因斑马鱼的双光子时间推移成像来追踪淋巴祖细胞的起源，提供了第一个体内确凿证据，证明早期淋巴管内皮细胞来自原始静脉血管。我们还使用了鱼类中可用的遗传和实验工具来表明，幼虫躯干淋巴网络的组装和模式是由来自邻近组织的趋化因子信号协调的，生长的淋巴芽沿着这些组织迁移。 我们目前正通过一些正在进行的项目进一步研究淋巴系统的形成： 1.我们创造了新的转基因株系，可以直接、特异地显示发育中的淋巴管系统。我们正在使用这些转基因动物的延时双光子成像来进一步表征淋巴管系统在发育过程中是如何生长和组装的。 2.我们正在利用这些新的淋巴管报告转基因品系进行正向遗传ENU突变筛选，以寻找在淋巴管系统发育中存在缺陷的新突变体，并使用外显子组测序来寻找在这些血管发育中发挥重要作用的新基因。 3.我们正在使用新开发的转基因工具来分析淋巴基因和microRNA表达的全球模式，这些工具促进了淋巴特异的基因图谱。 4.我们正在鉴定和研究在淋巴管内皮细胞中表达的新的microRNA，目的是揭示这些小的调节RNA如何影响淋巴管系统中的基因表达。 5.我们正在研究一种来自静脉-淋巴管内皮细胞的不寻常的脑血管周围细胞群，我们已经确定了这种细胞群。 我们的研究结果，结合了斑马鱼可用的遗传和实验工具，以及对活动物正在发育的血管结构进行高分辨率显微成像的能力，将继续导致对淋巴系统的起源和生长以及在淋巴发育和淋巴病理中至关重要的分子机制的重要新见解。

Our identification and 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 a 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 demonstrated 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. We also used two-photon time-lapse imaging of transgenic zebrafish to trace the origins of lymphatic progenitors, providing the first conclusive in vivo evidence that early lymphatic endothelial cells are derived from primitive venous blood vessels. We have also used the genetic and experimental tools available in the fish to show that the assembly and patterning of the larval trunk lymphatic network is orchestrated by chemokine signaling from adjacent tissues along which growing lymphatic sprouts migrate. We are currently pursuing further study of the formation of the lymphatic system through a number of ongoing projects: 1. We have generated new transgenic lines that permit direct, specific visualization of the developing lymphatic vasculature. We are using time-lapse two-photon imaging of these transgenic animals to to further characterize how the lymphatic vascular system grows and assembles during development. 2. We are carrying out forward-genetic ENU mutagenesis screens using these new lymphatic reporter transgenic lines to identify new mutants with defects in the development of the lympahtic vascualr system, and using exome sequencing to identify novel genes playing important roles in the development of these vessels. 3. We are analyzing global patterns of lymphatic gene and microRNA expression using newly developed transgenic tools facilitating lymphatic-specific gene profiling. 4. We are characterizing and studying novel microRNAs expressed in the lymphatic endothelium, with the goal of uncovering how these small regulatory RNAs influence gene expression in the lymphatic vasculature. 5. We are studying an unusual brain perivascular cell population derived from venous-lymphatic endothelium that we have identified. 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, will continue to lead to important new insights into the origins and growth of the lymphatic system and molecular mechanisms that are critical in lymphatic development and lymphatic pathologies.