Discovering missing links in neuropeptide evolution and function
Lead Research Organisation:
University College London
Department Name: Genetics Evolution and Environment
Abstract
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Technical Summary
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.
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.
