Discovering missing links in neuropeptide evolution and function

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.

Echinoderms (starfish, sea urchins) will be used as experimental systems to discover missing links in our knowledge of neuropeptide (NP) evolution and function.

The first aim is to identify candidate ligands for 37 NP-type 'orphan' G-protein coupled receptors (GPCRs) in the starfish A. rubens. A chromosomal assembly of the A. rubens genome will analysed to identify genes encoding candidate ligands based on: i). evolutionary conservation of NP sequences and/or NP gene structure/synteny and ii). structural analysis of the orphan NP-type GPCRs. Candidate ligands will then be synthesized and tested in luminescence-based assays in which orphan receptors are co-expressed with promiscuous G-proteins in CHO-K1 cells stably expressing the Ca2+-sensitive reporter GFP-aequorin. Discovery of NPs that act as ligands for orphan GPCRs in A. rubens will provide i). key missing links in our knowledge of the evolution of NP signalling ii). a basis for functional characterisation of NP signalling in echinoderm larvae (aim 2).

The second aim is to perform the first multi-gene analysis of NP function using echinoderm larvae as experimental systems. The sea urchin S. purpuratus will be used as the primary test species for this aim due to the all year round availability of larvae and advanced functional genomic resources. Cellular maps of NP and NP receptor expression in S. purpuratus larvae will be generated using i). single cell RNA sequencing, ii). multiplex whole-mount fluorescent in situ hybridization and immunohistochemistry and iii). fluorescent protein (FP) reporter constructs. This will provide a framework for experimental investigation of NP function, where gene knockdown (morpholino oligonucleotides) and gene knockout (CRISPR/Cas9) methods will be employed to screen for effects on larval growth, feeding and swimmining behaviour. To enable comparative analysis of NP signalling in larval echinoderms, NP expression and function will also be investigated in A. rubens larvae.