IN SITU DIFFERENTIATION OF IRIDOPHORE CRYSTALLOTYPES UNDERLIES ZEBRAFISH STRIPING. IN SITU DIFFERENTIATION OF IRIDOPHORE CRYSTALLOTYPES UNDERLIES ZEBRAFISH STRIPING

Skin color patterns are ubiquitous in nature, evolve rapidly, and impact social behavior, predator avoidance, and protection from ultraviolet irradiation. A leading model system for vertebrate skin patterning is the zebrafish; its alternating blue stripes and yellow interstripes depend on guanine crystal-containing cells called iridophores that reflect light. It was suggested that the zebrafish’s alternating color pattern arises from a single type of iridophore migrating differentially to stripes and interstripes. When we tracked iridophores, however, we found they did not migrate between stripes and interstripes but instead differentiated and proliferated in place based on their micro-environment. RNA-sequencing analysis further revealed that stripe and interstripe iridophores had different transcriptomic states with many differences in gene expression and pathway enrichment, while cryogenic scanning electron microscopy and micro-X-ray diffraction identified different guanine crystal organizations and responsiveness to norepinephrine, all indicating that stripe and interstripe iridophores are different cell types. Based on these results, we present a new model of skin patterning in zebrafish in which distinct iridophore crystallotypes containing specialized, physiologically responsive, subcellular organelles arise in stripe and interstripe zones by in situ differentiation. In this model, pattern phenotype depends not only on interactions among pigment cells that affect their arrangements, but also on factors that specify subcellular organization and physiological responsiveness of specialized organelles. Overall design: Unbiased classification of iridophores, based solely on their RNA profiles obtained by single-cell RNA-sequencing

皮肤颜色图案在自然界中广泛存在，演化速率迅捷，同时对社会行为、天敌规避以及紫外线辐射防护均具有关键作用。脊椎动物皮肤图案研究的经典模式系统为斑马鱼，其交替排布的蓝色条纹与黄色间纹，依赖于一类可反射光线、含有鸟嘌呤晶体的虹彩细胞(iridophores)。此前有研究假说认为，斑马鱼的交替颜色图案源于单一类型的虹彩细胞，它们会差异化迁移至条纹区与间纹区。然而我们在追踪虹彩细胞时发现，此类细胞并不会在条纹区与间纹区之间发生迁移，而是会根据所处微环境在原位完成分化与增殖。RNA测序(RNA-sequencing)分析进一步显示，条纹区与间纹区的虹彩细胞具有截然不同的转录组状态，在基因表达与通路富集层面存在大量差异；而低温扫描电子显微镜与微区X射线衍射技术则鉴定出二者的鸟嘌呤晶体结构以及对去甲肾上腺素的响应性均存在显著区别，所有上述结果均表明，条纹区与间纹区的虹彩细胞属于两种不同的细胞类型。基于上述研究结果，我们提出了一种全新的斑马鱼皮肤图案形成模型：在该模型中，含有特化且具有生理响应性亚细胞细胞器的不同虹彩细胞晶型(crystallotypes)，会通过原位分化过程在条纹区与间纹区中产生。在该模型中，图案表型不仅取决于影响色素细胞排布的细胞间互作，还依赖于调控特化细胞器亚细胞结构与生理响应性的相关因子。实验整体设计：仅基于单细胞RNA测序获得的转录谱对虹彩细胞进行无偏分类。