Deciphering the evolutionary mechanisms shaping the regulatory circuitry underlying the novel brooding organ in syngnathids (pipefishes & seahorses) using cutting-edge single-cell multiomics

生命的多样性反映了有机体为适应不断变化的环境而进化出的多方面适应能力。新的表型性状可以通过对现有性状的逐渐改变而进化(使它们成为“同源”)，或者它们可以在没有任何表型前体的情况下作为全新的特征出现。这种“从头开始”的性状可能仍然在基因调控水平上表现出同源性(“深度同源性”)：新性状可能与其他先前存在的性状的调控回路并存，这可以加速新颖性和复杂性的进化。海豚和海马科鱼类表现出这样一种新奇的特征：雄性通过专门的孵化器官怀孕。在不同物种中，孵化器官可能是简单的(在笛鱼中，孵化器官仅用于将卵附着在父亲的腹部，在那里它们仍然暴露在海水中)、复杂(在海马直立体中，鱼尾巴上的孵化器官形成一个袋状结构，在一个受监管的微环境中容纳卵，不受海水的影响)，或中等复杂的(在笛鱼中，Syngnatus typhle和Doryrhamphus exisus)。这个项目旨在研究孵化器官背后的分子和进化机制，以加深我们对进化新结构如何产生的理解。为此，将在孵化器官发育过程中收集上述四种合并体的组织样本。在三个子项目中(i.)器官的形态发育将通过组织学和免疫组织学进行比较描述，(Ii)孵化器官的转录组基因表达模式将在不同发育阶段进行比较分析，以及(Iii)强大的调控基因网络将通过单细胞多重组学产生，整合来自相同个体细胞的RNA-seq和atac-seq，以及选定的个体。随后，形态特征将与基因表达特征相关联，细胞类型将按发育阶段和物种进行鉴定，特定性状的基因调控网络将被确定，并确定编排这些基因调控网络的上游转录调控网络。通过对孵化器官阶段和物种之间网络的比较分析，可以确定独特的基因/调控元件和调控相互作用。例如，这些分析将揭示汇聚的孵化器官表型是否通过平行的调控进化而进化，以及更复杂的表型的发展是否真的如形态观察所表明的那样在调控水平上概括了其进化。因此，这项研究将提供有价值的洞察力，揭示联体类新孵化器官背后的发育生物学、分子基础和进化机制，并更广泛地讨论新性状从从头进化到深度同源进化的进化机制。

The diversity of life reflects the multifaceted adaptations organisms evolved to adjust to an everchanging environment. New phenotypic traits can evolve due to gradual change to already existing traits (making them “homolog”), or they can arise as entirely new characteristics without any phenotypic precursor. Such “de novo” traits may still show homology on the level of gene regulation though (“deep homology”): novel traits may coopt regulatory circuitry of other preexisting traits, which can accelerate the evolution of novelty and complexity. Syngnathids (pipefish and seahorse) show such a novel trait: male pregnancy via specialized brooding organs. Across species, brooding organs may be simple (in the pipefish Nerophis ophidion the brooding organ merely serves to attach the eggs to the father’s belly, where they remain exposed to seawater), complex (in the seahorse Hippocampus erectus the brooding organ on the fish’s tail forms a pouch-like structure that holds the eggs in a regulated micro-environment shielded from seawater), or of intermediate complexity (in the pipefishes Syngnathus typhle and Doryrhamphus excisus). This project aims to investigate the molecular and evolutionary mechanisms underlying brooding organs to further our understanding of how evolutionary novel structures can arise. For this purpose, tissue samples of the four aforementioned syngnathids will be collected across brooding organ development. In three sub-projects (i.) the organs’ morphological developments will be comparatively described via histology and immunohistology, (ii.) the brooding organs’ transcriptome-wide gene expression patterns will be comparatively analyses across developmental stages, and (iii.) robust regulatory gene networks will be generated via single-cell multiomics, integrating RNA-seq and ATAC-seq from the same individual cells, of selected individuals. Subsequently, morphological characteristics will be associated to gene expression characteristics, cell types will be identified per developmental stage and species, trait-specific gene regulatory networks will be identified, and upstream transcription regulators orchestrating these gene regulatory networks determined. By comparative analyses of networks among brooding organ stages and between species, unique genes/regulatory elements and regulatory interactions can be identified. These analyses will reveal, for instance, if convergent brooding organ phenotypes evolved via parallel regulatory evolution and if the development of more complex phenotypes indeed recapitulates its evolution on a regulatory level, as suggested by morphological observations. Thus, this study will provide valuable insights into the developmental biology, molecular basis and evolutionary mechanisms underlying the syngnathids’ novel brooding organs and discuss more broadly the evolutionary mechanism underlying the evolution of novel traits, ranging from de novo evolution to deep homology.