Discovering missing links in neuropeptide evolution and function

为了人类和其他动物的生存和繁殖，生长和生理/行为过程，如摄食和交配，需要由神经和内分泌系统控制和协调。这是通过细胞分泌“信使分子”来实现的，这些信使分子作用于其他细胞/组织/器官(例如肌肉)，在短(秒)到长(小时-天)的时间尺度上刺激或抑制它们的活动。最大和最多样的神经信使分子是神经肽，它通过与靶细胞上特定的受体蛋白结合来发挥作用。神经肽如何控制生理和行为的许多重要见解已经从对果蝇和线虫等无脊椎动物的实验研究中获得。然而，我们对神经肽信号系统的了解仍有许多空白。例如，人类和无脊椎动物中的一些神经肽之间的进化关系尚未确定，还有许多所谓的“孤儿受体”，神经肽的合作伙伴尚未被发现。重要的是，不断增长的各种动物的基因组测序为解决这些问题提供了令人兴奋的新机会。在拟议的项目中，我们将使用棘皮动物(海星、海胆)作为实验系统，以发现我们在神经肽进化和功能方面知识中缺失的环节。我们选择棘皮动物作为这个项目的实验系统的主要理由是：第一，作为后口无脊椎动物，棘皮动物在动物界占据着进化的地位，这为对果蝇和线虫等研究广泛的原生动物无脊椎动物的研究与对人类和其他脊椎动物的研究提供了重要的联系。第二，我们与桑格研究所的达尔文生命树项目合作，最近获得了常见海星Asterias Rubens的染色体水平的基因组序列组装，为英国和海外的研究人员提供了宝贵的新资源。海胆已经是成熟的实验系统，已经被广泛用于确定控制动物胚胎发育的遗传机制。这个项目的主要目的有两个：首先，我们将分析鲁本斯弧菌的基因组序列，以确定编码神经肽的基因作为该物种的孤儿受体的候选伙伴，然后我们将进行生化实验，以测试预测的神经肽-受体伙伴关系。在海星身上发现神经肽是“孤儿受体”的伙伴，这将为我们在动物界中神经肽信号的进化提供缺失的一环。其次，我们将使用最先进的基因敲除方法(CRISPR-Cas9)来研究海胆和海星幼虫中神经肽的功能，这两种幼虫的神经系统中的神经元数量比成年动物少得多，但具有类似的分子复杂性。通过首次发现多种神经肽类型在棘皮动物幼虫阶段的生理/行为作用，我们将获得对动物界神经肽功能进化的重要新见解。这将有助于推进重建神经肽信号系统进化史的更广泛目标，以了解神经肽是如何以及何时被招募来调节动物生命树的不同分支和不同环境背景下的不同生理和行为过程。这项研究的发现也将为开发针对人类神经肽受体的新型治疗剂提供帮助。

For humans and other animals to survive and reproduce, growth and physiological/behavioural processes such as feeding and mating need to be controlled and coordinated by nervous and endocrine systems. This is achieved by cells secreting "messenger molecules" that are act on other cells/tissues/organs (e.g. muscles) to stimulate or inhibit their activity over short (seconds) to long (hours-days) timescales. The largest and most diverse class of neuronal messenger molecules are "neuropeptides", which exert their effects by binding to specific receptor proteins on target cells.Many important insights into how neuropeptides control physiology and behaviour have been obtained from experimental studies on invertebrate animals such as the fruit fly Drosophila and the nematode worm C. elegans. However, there are still many gaps in our knowledge of neuropeptide signalling systems. For example, evolutionary relationships between some neuropeptides in humans and invertebrates have not been determined and there are many so-called 'orphan receptors' for which neuropeptide partners have yet to be discovered. Importantly, exciting new opportunities to address these issues have been provided by sequencing of the genomes of an ever-growing variety of animals.In the proposed project we will use echinoderms (starfish, sea urchin) as experimental systems to discover missing links in our knowledge of neuropeptide evolution and function. The main rationales for our selection of echinoderms as experimental systems for this project are:Firstly, as deuterostome invertebrates, echinoderms occupy an evolutionary position in the animal kingdom that provides an important link between research on well-studied protostome invertebrates such as Drosophila and C. elegans and research on humans and other vertebrates.Secondly, in collaboration with the Sanger Institute's Darwin Tree of Life Project we have recently obtained a chromosomal-level assembly of the genome sequence of the common starfish Asterias rubens, providing an valuable new resource for researchers in the UK and overseas.Thirdly, sea urchins are already well-established experimental systems that have been used extensively for determination of the genetic mechanisms that control embryonic development in animals.There are two main aims of this project:Firstly, we will analyse the A. rubens genome sequence to identify genes encoding neuropeptides as candidate partners for 'orphan receptors' in this species and then we will perform biochemical experiments to test predicted neuropeptide-receptor partnerships. Discovering neuropeptides that are partners for 'orphan receptors' in starfish will provide missing links in our knowledge of the evolution of neuropeptide signalling in the animal kingdom. This will have broad impact by influencing interpretation of findings from research on neuropeptide signalling in other animals, including humans.Secondly, we will use state-of-the-art gene-knockout methods (CRISPR-cas9) to investigate the functions of neuropeptides in sea urchin and starfish larvae, which have nervous systems comprising much smaller populations of neurons than adult animals, but with comparable molecular complexity. By discovering the physiological/behavioural roles of multiple neuropeptide types for the first time during the larval stage of echinoderms, we will obtain important new insights into the evolution of neuropeptide function in the animal kingdom. This will facilitate advancement of the broader aim of reconstructing the evolutionary history of neuropeptide signalling systems to gain an understanding of how and when neuropeptides were recruited to regulate diverse physiological and behavioural processes in different branches of tree of animal life and in contrasting environmental contexts. The findings of this study will also provide insights that may facilitate development of novel therapeutic agents that target neuropeptide receptors in humans.