Structural and functional evolution of GnRH and its receptors in three chordate models : Branchiostoma floridae, Ciona intestinalis and Danio rerio.

Date

2010-04-08T20:04:38Z

Authors

Tello, Javier Ananda

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Abstract

Neural control of reproduction in vertebrates and invertebrates has generated considerable interest due to the presence of common neuropeptides. Gonadotropin-releasing hormone (GnRH), a neuropeptide, is the final integrator of neural regulation governing reproduction in vertebrates by controlling the release of gonadotropins. Little is known about GnRH before the origin of vertebrates or about the biological significance of multiple GnRH forms in a single species. To understand the role of GnRH in invertebrates, I selected a tunicate, Ciona intestinalis, the sister group to vertebrates and amphioxus, Branchiostoina floridae, a group more basal than tunicates. Neural control of reproduction in these chordates was compared with that in the zebrafish, Danio rerio. From the zebrafish, I isolated four GnRH receptor cDNAs that each map to a distinct chromosome and are expressed in a variety of tissues. Each receptor was functional, as shown by its response to physiological doses of native GnRH peptides. Also, two receptors showed selectivity between GnRH1 and GnRH2. Protein localization of each zebrafish GnRH receptor with specific antisera showed that all four receptors are present in the pituitary. However, the most striking localization revealed the presence of GnRH networks in a major motor control centre and fibre tract system in the hindbrain and spinal cord. Both structures are major components in the control of motor movements, such as swimming. Phylogenetic and synteny analysis segregates the four zebrafish GnRH receptors into two distinct phylogenetic groups that are separate gene lineages conserved throughout vertebrate evolution. In Ciona intestinalis, we found two GnRH genes that each encode three GnRH decapeptides in tandem, for six unique GnRH forms from this species. These genes are expressed throughout development. With an immunocytochemical approach, at least one peptide was found in the dorsal strand nerve plexus adjacent to the gonads in adults. Injection near the gonads of gravid Ciona quickly induced spawning, suggesting a novel action for control of reproduction by GnRH. My further studies identified four novel GnRH receptors encoded within the genome of this protochordate, and showed that three receptors responded to Ciona GnRHs by stimulating intracellular accumulation of cAMP. In contrast, only one receptor activated inositol phosphate turnover in response to one of the Ciona GnRHs. My final study involved identifying the GnRH signalling components in amphioxus. I found four novel GnRH receptors, with three displaying sensitivity to the highly conserved vertebrate GnRH2 and one of these showing selectivity for GnRH1. My pharmacological testing showed that the capacity to respond to GnRH1 and GnRH2 is evolutionarily conserved between amphioxus and vertebrates, and that key motifs found to be important in GnRH binding, signalling and activation are present in the amphioxus receptors. Phylogenetic analysis showed that two receptors cluster with the recently identified octopus GnRHR-like sequence; the other two receptors group at the base of the vertebrate GnRHR clade and may represent the proto-vertebrate condition, after which gene duplication and sequence divergence resulted in the four contemporary vertebrate GnRHRs. This work reveals novel and important features of the GnRH signalling axis throughout chordate evolution.

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Keywords

Luteinizing hormone releasing hormone, Chordata

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