How to stay attached: Evolution and development of the plug, a transient novel tissue in ricefishes (Beloniformes: Adrianichthyidae)

新奇的起源是进化生物学中的一个重要问题。根据压力诱导进化创新（SIEI）模型，新的结构成分可以从祖先的压力反应中产生，例如改良的炎症。鲸类动物（鲸鱼和海豚）极厚的表皮和胎盘哺乳动物的蜕膜间质细胞是这种创新的罕见例子。另一个例子可能是苏拉威西岛米鱼的“塞”。雌性米鱼通过从生殖孔伸出的长长的附着细丝将受精卵附着在身体上。虽然大多数物种在产卵后不久就会产卵（转移孵卵），但在一些物种中，它们一直附着在一起直到幼鱼孵化（盆腔孵卵）。卵塞是盆腔育雏米鱼雌性生殖道中附着丝周围形成的一种新的、短暂的组织，对保持卵的附着起着重要作用。我们自己的数据显示，盆腔育雏物种Oryzias eversi的转录组主要由炎症信号控制。进一步，我们发现，基于组织学，异物多核巨细胞，异物肉芽肿的标志，存在于塞。在拟议的项目中，我们将(1)基于形态学比较不同的解决方案来延长米鱼的产卵时间，(2)在单细胞水平上研究米鱼的塞子形成。在第一部分中，我们将结合微计算机断层扫描、组织学和免疫细胞染色来比较雌性米鱼（三种盆腔育成米鱼和两种盆腔育成米鱼，以及两种密切相关的转移育成米鱼）在五个不同时间点的生殖道宏观和微观解剖结构。在第二部分中，我们将结合O. everi（在孵育期间的不同时间点）的plug- invasion和周围细胞的单细胞转录组学和空间转录组学。获得的数据将用于绘制和了解塞子中细胞类型的分布和起源，以及确定与鱼类肉芽肿形成和人类肉芽肿疾病（例如结核病）的异同。拟议项目的结果将为解释苏拉威西米鱼盆腔产卵的进化途径提供新的见解。此外，它还将揭示Oryzias谱系中栓子的发育是否是炎症诱导的新组织形成的结果，以及栓子的形成是否类似于异物肉芽肿的形成。如果是这样，米鱼塞将是第一个在非疾病情况下表现出功能改变的肉芽肿的例子。

The origin of novelty is a substantial question in evolutionary biology. According to the Stress Induced Evolutionary Innovation (SIEI) model, novel structural components can arise from ancestral stress responses, such as modified inflammation. The extremely thick epidermis of cetaceans (whales and dolphins) and the decidual stromal cells of placental mammals are rare examples of such innovations. Another example might be the "plug" in Sulawesi ricefishes (Beloniformes: Adrianichthyidae). Female ricefishes carry fertilized eggs attached to their body by long attaching filaments that protrude from the genital pore. While most species deposit their eggs shortly after spawning (transfer brooder), in some species they stay attached until the juveniles hatch (pelvic brooder). The plug is a novel, transient tissue that forms in the female reproductive tract of pelvic brooding ricefish species around the attaching filaments and plays an important role in keeping the eggs attached. Our own data revealed that the transcriptome of the plug of the pelvic brooding species Oryzias eversi is dominated by inflammatory signaling. Further we found, based on histology, that foreign body multinucleated giant cells, a hallmark of foreign body granulomas, are present in the plug. In the proposed project we will (1) compare the different solutions towards prolonged egg carrying in ricefishes based on morphology and (2) study plug formation in O. eversi at single cell level. For the first part, we will combine µ-computed tomography, histology and immune cell staining to compare the macro- and micro-anatomy of the reproductive tract of female ricefishes (three pelvic brooding Oryzias species and two pelvic brooding Adrianichthys species at five different time points as well as two closely related transfer brooding Oryzias species). For the second part, we will combine single-cell transcriptomics and spatial transcriptomics of plug-invading and surrounding cells in O. eversi (at different time points during brooding). The data obtained will be used to map and understand the distribution and origin of cell types in the plug as well as to identify similarities and differences with granuloma formation in fish and granuloma disease in humans (e.g., tuberculosis). The results of the proposed project will provide new insights into the evolutionary pathways that explain pelvic brooding within Sulawesi ricefishes. In addition, it will reveal if the development of the plug in the Oryzias lineage is a result of inflammation-induced formation of a novel tissue and if plug formation resembles foreign body granuloma formation. If so, the ricefish plug would be the first example of a granuloma exhibiting an altered function in a non-disease context.