Potential roles of primitive secretin-like and insulin-like peptides in embryogenesis of a cephalochordate, the amphioxus

Abstract

Secretin and insulin peptide families are two well-studied hormone families which have pleiotropic actions on both the digestive tract and the brain in vertebrate. Their involvements in vertebrate embryogenesis have been abundantly reported. Defects in these proteins will lead to various diseases such as sterility, diabetes, growth retardation. But it’s always hard to fully understand the physiological roles of these genes when researches are performed on complex systems of vertebrate. Amphioxus belongs to the cephalochordata lineage, it has a unique evolutionarily position where is at the transition of invertebrate to vertebrate, it keeps most features of the ancient vertebrates and the embryo development is much simpler. Thus, in this project, We use amphioxus as animal model, studying the molecular and functional evolution of secretin- and insulin-like peptides in embryogenesis, providing insights into some of the complex issues in vertebrate embryo development. Amphioxus CRH-like peptides and receptors were identified in this study, functional analyses revealed that two of the cloned receptors (Bflo_CRHRa and Bbel_CRHRa) can be activated by amphioxus CRH peptide, demonstrating the existence of functional CRH peptide-receptor pair before the origin of vertebrates. By qRT‐PCR and immunofluorescence staining, it was found that CRH peptide and receptor possessed a similar distribution pattern with the highest expression level in the nerve cord. The characterization of CRH peptide-receptor pair means all five subfamilies of secretin-like GPCRs and peptides have been identified in amphioxus. Phylogenetic analysis placed amphioxus peptides/receptors at the base of the clade comprising vertebrate corresponding peptides/receptors, indicating that members in vertebrate secretin-like ligands and GPCR family were evolved from one pre-existing ancestral gene of Chordate. Studies of amphioxus transcriptome data revealed that both secretin and insulin family peptides have high expression in neurula stage during embryogenesis. In order to understand their potential roles during different embryo development stages, whole mount in situ hybridization was performed to show the spatio-temporal expression pattern. All tested 8 secretin family peptides were expressed in the developing nerve cord while ILP and IGFBP had distinct expressions in the gut, implying important and different roles of these two peptide families in amphioxus embryogenesis. Taking the advantage of biotechnology in generating amphioxus transgenic lines, seven amphioxus mutant lines have been generating including CTFP2, CRH, PACAP/Gluc-a, PACAP/GlucR, PTHR, ILP and IGFBP. Studies of ILP and IGFBP mutant lines were performed. We found IGFBP-/- amphioxus could survive to adulthood and no significant phenotypic change was detected during embryo development. ILP-/- amphioxus had severe growth retardation and could only reach two-gill-slit stage with less than one month’s life. Whole mount in situ hybridization study showed that ILP deficiency caused decreased expression of Pax1/9, Six1/2, Eya, Tbx1/10 in pharynx region and PTH2 in pre-oral pit in larva stage. No significant differences were detected for the checked anterior development markers. These results indicate that ILP is essential for amphioxus survival and it plays important roles in amphioxus embryogenesis especially in the gill development. And the involvement of insulin/IGF signaling in head and neural induction may be an innovation in vertebrate linage.