Class B GPCR ligands' expression patterns in developing amphioxus; their role embryogenesis and their evolutionary significance

Abstract

Due to its unique position in the evolutionary time scale at the interface of vertebrates and invertebrates Amphioxus, a cephalochordate in phylum chordate, has served as a model organism to study evolution for over a century. In our lab, we have previously reported the presence of an ortholog to Pituitary adenylate cyclase-activating peptide (PACAP), Glucagon, Parathyroid hormone (PTH) and their receptors in amphioxus. In this study, we analysed the expression pattern of class B guanine nucleotide-binding proteins (G protein)–coupled receptors (GPCRs) ligands which include Calcitonin-like, Corticotropin releasing hormone-like, PACAP-Glucagon-like and PTH-like peptides during the development of amphioxus embryo. Taking advantage of the openly available transcriptome data from Branchiostoma floridae (Bf) and Branchiostoma Belcheri (Bb), two of the widely studied Amphioxus species; we explored the expression pattern and possible functions of class B ligands in the development of amphioxus. The differential expression across different developmental stages from un-fertilized egg through mature adult were analysed from the available data. We compared the expression pattern of the class B ligands across five other species; Danio rerio (Dr-zebrafish), Xenopus tropicalis (Xt- Western clawed frog), Pelodiscus sinensis (Ps-Chinese softshell turtle), Gallus gallus (Gg-Chicken) and Mus musculus (Mm-mouse). These data helped us to focus the study further on specific stages in development from early neurula through three gill-slit stage in amphioxus. The hour-glass model of developmental evolution suggests that the constrains on evolution can induce stringency at the phylotypic stages (late neurula/early larvae) in amphioxus or early organogenesis in vertebrates. It is also important to note that the analysis on the transcriptomic data revealed that most class B GPCRs and ligands are expressed in these developmental stages. Although, there has been a constant debate on the hour-glass model, there is a general agreement that there are certain constrains like check-points in embryonic development which confer evolutionary stringency. In this study, we hope to understand how conserved the expression patterns during embryonic development is for the various orthologs of known vertebrate class B members in amphioxus. We performed whole mount in-situ hybridization experiments on these set of genes from early neurula to three gill-slit stage larvae. The results might help in further understanding the neuroendocrine system which operates in basal chordates. With only about 20,000 neurons, amphioxus is also a useful tool in helping us to understand the development of the nervous system which is extremely complex among mammals. Our investigation into the class B ligands revealed striking similarities in expression pattern in late neurula and larval stages with vertebrates. All the ligands’ transcripts were found to express in the late neurula and larval stages studied in the developing nerve cord. More interesting, when comparing the results with available information on vertebrate ortholog expression pattern, we identified similarities in the spaciotemporal expression of transcripts in PACAP-Glucagon subfamily in the developing embryo. This significant level of conservation in the expression pattern observed, has led us to propose that the class B GPCR ligands might have historically evolved as key signals in neuronal pattern formation during embryogenesis. Looking at non-chordate ortholog like DH44 (CRH-like) and DH31 (calcitonin-like) in Drosophila melanogaster, we speculate this might not be an implausible argument.