Getting Organized: Lungfish Reveal the Evolutionary Origins of Organized Mucosal Lymphoid Tissue

每种动物都有与环境接触的地方。这些接触点是抵御病原体和外部入侵的防御屏障，并配备了形成粘膜免疫系统的专门细胞和组织。在人类和鸟类中，粘膜免疫系统包括组织良好的免疫结构，如鼻咽通道中的扁桃体。目前，有组织的粘膜免疫结构被认为只存在于现代脊椎动物中。研究人员首次提出原始扁桃体首先出现在叶状鳍（sarcopterygian）鱼类，如非洲肺鱼。非洲肺鱼代表了脊椎动物从水到陆地的过渡，是所有四足动物最接近的活祖先。调查免疫细胞和分子，驱动组织淋巴（免疫）结构的形成在粘膜表面的肺鱼将揭示组织粘膜免疫系统在脊椎动物的原始保守的功能。该项目不仅将非洲肺鱼作为实验室的动物模型，而且还将其作为教育工具，让学童了解进化、适应、生理和免疫。脊椎动物适应性免疫的进化说明了一些关键的创新如何产生更有效的机制来对抗病原体。在这些创新过程中，淋巴细胞在特定解剖区域，特别是粘膜表面的组织，已被证明是哺乳动物产生高亲和力类别转换抗体的非常有效的方法。在哺乳动物扁桃体或派尔集合淋巴结中发现的高度组织化，一定是从某种原始的有组织的淋巴结构进化而来的。目前，许多研究者认为两栖动物肠道中的淋巴聚集体是哺乳动物组织化粘膜相关淋巴组织（MALT）的前身。然而，在两栖动物之前，食肉鱼类揭示了有组织的MALT的进化起源实际上更古老，并且与脊椎动物从水中到陆地的过渡相吻合。因此，肺鱼，所有四足动物的最接近的祖先，可能是第一个通过将淋巴细胞分组在特定的淋巴结构中进行创新的动物。已经提出，肿瘤坏死因子（TNF）家族基因从硬骨鱼到哺乳动物的多样化有助于在高等脊椎动物中获得有组织的淋巴组织。为了验证这一假设，研究者提出了新发现的淋巴聚集体（LA）存在于非洲肺鱼（Protopterus sp.）粘膜的研究。该项目的目标是1）使用Irene Salinas博士（PI）和Chris Amemiya博士生成的深度测序转录组数据库来表征非洲肺鱼中的TNF家族成员并完成该超家族的进化2）利用组织学、激光捕获显微切割（LCM）、RT-PCR和FISH技术在分子、细胞和功能水平上表征肺鱼LA; 3）确定TNF基因和微生物暴露在肺鱼LA的形成和组织中的作用。

Every animal has points of contact with the environment. Those points of contact are defensive barriers against pathogens and external aggressions and are equipped with specialized cells and tissues that form the mucosal immune system. In humans and birds, mucosal immune systems include well-organized immune structures such as the tonsils in our nasopharyngeal passages. Currently, organized mucosal immune structures are thought to be present in modern vertebrates only. The investigator proposes for the first time that primitive tonsils first appeared in lobe-finned (sarcopterygian) fishes such as the African lungfish. African lungfish represent the transition of vertebrates from water to land and are the closest living ancestor to all tetrapods. Investigating the immune cells and molecules that drive the formation of organized lymphoid (immune) structures in the mucosal surfaces of lungfish will reveal the primordially conserved features of organized mucosal immune systems in vertebrates. The project will not only use African lungfish as an animal model in the laboratory but also as an educational tool for school children to understand evolution, adaptation, physiology and immunity. The evolution of adaptive immunity in vertebrates illustrates how some key innovations have resulted in more efficient mechanisms to fight pathogens. Amongst these innovative processes, the organization of lymphocytes in specific anatomical areas, particularly mucosal surfaces, has proven a very effective way for mammals to produce high affinity class-switched antibodies. The high level of organization found in mammalian tonsils or Peyer's patches must have evolved from some sort of primitive organized lymphoid structure. Currently, many investigators see the lymphoid aggregates in the gut of amphibians as the forerunners of mammalian organized mucosa-associated lymphoid tissues (MALT). However, prior to amphibians, the sarcopterygian fishes reveal that the evolutionary origin of organized MALT is actually older and it coincides with the transition of vertebrates from water to land. Thus, lungfishes, the closest ancestors of all tetrapods, may be the first animals to have innovated by grouping lymphocytes in specific lymphoid structures. It has been proposed that the diversification of the tumor necrosis factor (TNF) family of genes from bony fish to mammals has been instrumental for the acquisition of organized lymphoid tissues in higher vertebrates. To test this hypothesis, the investigator proposes the study of the newly discovered lymphoid aggregates (LAs) present in mucosa of the African lungfish (Protopterus sp.). The goals of this project are 1) to characterize the TNF family members in African lungfish and complete the phylogeny of this superfamily using deep sequencing transcriptome databases generated by Dr. Irene Salinas (PI) and Dr. Chris Amemiya (Senior Personnel); 2) to characterize lungfish LAs at the molecular, cellular and functional levels using histology, laser capture microdissection (LCM), RT-PCR and FISH; 3) to establish the role of TNF genes and microbial exposure in the formation and organization of lungfish LAs.