Development and regeneration of the zebrafish maxillary barbel

描述(申请人提供)：斑马鱼是一种脊椎动物，可以再生几种成年组织类型。这些结构包括研究得很好的结构，如鳍、心脏和眼睛，但也有研究较少的结构，如胡须状的面部触须。斑马鱼上颌骨触须(ZMB)由皮肤细胞、腺体、味蕾、结缔组织、神经和血管组成。因为透明的杠铃是外部的，所以里面所有分化的细胞类型都是可见的，并且可以通过实验获得。已知触须在其他种类的鱼类中会再生，但还没有研究斑马鱼的这种能力。根据迄今的研究，我们假设Wnt信号分子和Glypcan辅助受体对ZMB的生长和损伤后的再生都是至关重要的。在我们的第一个目标中，我们将记录这个附属器在发育和再生过程中的细胞分裂和细胞死亡的模式，并确认我们第一个描述的触须淋巴管的存在和功能。在第二个目标中，我们将1)在ZMB组织中定位非规范的Wnt配体和受体(Gypicans和Frizzleds)的空间和时间分辨率，2)使用转基因FISH来测试在斑马鱼突变体中恢复GYPICA功能是否恢复ZMB的生长，3)调查ZMB的萌发和再生是否需要规范的WNT信号，以及4)使用吗啡方法测试ZMB中的候选基因。从这些研究中获得的知识将大大有助于我们理解皮肤和皮肤附属物的发育和维持所涉及的遗传相互作用。由于ZMB是一个可以再生的成年器官，这项研究还将阐明许多终末分化细胞类型对损伤的反应，包括血管、淋巴管和神经。与斑马鱼尾鳍的再生不同，ZMB的再生以异常细胞增殖和细胞外基质沉积为特征，这一过程可能与脊椎动物的伤口愈合病理有关，如瘢痕疙瘩和/或增生性瘢痕。斑马鱼触须神经将轴突再生到味蕾目标的能力，可能表明了恢复创伤组织周围感觉的方法。最后，ZMB内的毛细血管环和大的可接近的淋巴管是研究血管系统的生理和病理功能的一个有吸引力的系统，包括液体平衡和肿瘤转移。) 公共卫生相关性：这项研究将建立一种新的、容易获得且相对非侵入性的系统，用于研究斑马鱼Danio rerio的皮肤附属物的发育和再生。由于所有脊椎动物都有许多共同的基因，该项目的发现有望与其他物种(如啮齿动物或人类)成年组织中伤口愈合/疤痕形成、血管重建和神经再生的细胞机制有关。这个为期3年的项目将为我们学院的本科生提供斑马鱼遗传学、分子生物学和细胞再生方面的全面培训计划，这些都是下一代生物医学专业人员的关键技能。

DESCRIPTION (provided by applicant): The zebrafish is a vertebrate animal that can regenerate several adult tissue types. These include well-studied structures such as fins, heart and eyes, but also poorly studied ones, such as the whisker-like facial barbels. The zebrafish maxillary barbel (ZMB) contains skin cells, glands, taste buds, connective tissues, nerves, and vascular components. Because the transparent barbells are external, all of the differentiated cell types within are visible and experimentally accessible. Barbels are known to regenerate in other species of fishes, but have not been studied for this capacity in zebrafish. Based on studies to date, we hypothesize that Wnt signaling molecules and glypican co- receptors are critical for both ZMB outgrowth and regeneration after injury. In our first aim, we will document patterns of cell division and cell death during development and regeneration of this appendage, and confirm the existence and function of the barbel lymphatics, which we are the first to describe. In the second aim, we will 1) map the spatial and temporal resolution of non-canonical Wnt ligands and receptors (glypicans and frizzleds) in ZMB tissue, 2) use transgenic fish to test if restoring glypican function restores ZMB outgrowth in a zebrafish mutant, 3) survey if canonical Wnt signaling is required for ZMB budding and regeneration, and 4) test candidate genes in the ZMB using a morpholino approach. Knowledge gained from these investigations will contribute significantly to our understanding of the genetic interactions involved in the development and maintenance of skin and skin appendages. Because the ZMB is an adult organ that regenerates, this study will also illuminate the response to injury of many terminally differentiated cell types, including blood vessels, lymphatics and nerves. Unlike regeneration in the zebrafish caudal fin, regeneration in the ZMB is characterized by abnormal cell proliferation and extracellular matrix deposition, a process that may be relevant to vertebrate wound healing pathologies such as keloids and/or hypertrophic scars. The ability of zebrafish barbel nerves to regrow axons to their taste bud targets may suggest approaches to restore peripheral sensation to traumatized tissues. Finally, the availability within the ZMB of both a capillary loop and a large, accessible lymphatic is an attractive system in which to study the physiological and pathological functions of the vasculature, including fluid balance and tumor metastasis. ) PUBLIC HEALTH RELEVANCE: This study will establish the biological potential of a novel, easily accessible and relatively non-intrusive system for studying skin appendage development and regeneration in the zebrafish, Danio rerio. Because all vertebrates share many genes in common, findings from this project are expected to relate to the cellular mechanisms of wound healing/scarring, revascularization and nerve regeneration in the adult tissues of other species, such as rodents or humans. The 3-year project will provide undergraduates at our institution with a comprehensive training program in zebrafish genetics, molecular biology and cellular regeneration, all vital skills for the next generation of biomedical professionals.