Identification and Characterization of Developmentally Regulated Components of the Stress Axis in Petromyzon Marinus

Abstract

Genes resembling elements of the Corticotropin releasing hormone (CRH) receptor-ligand system (CRH system) have been identified in invertebrate species and suggest the CRH system has existed, in some form, for approximately a billion years. It is theorized that vertebrates inherited components of the CRH system from an invertebrate ancestor. The association of the CRH system with the stress response, however, is specific to vertebrate species and theorized to have accompanied the development of hypothalamic pituitary (HP) axes, specifically the HP interrenal (HPI) axis in fish. A functional HPI axis has recently been suggested in the lamprey species Petromyzon marinus, a member of the ancient vertebrate superclass agnatha, by identification of pituitary and inter-renal components corticotrophin (ACTH) and 11- deoxycortisol respectively. This study, however, is the first to identify the hypothalamic components, specifically the CRH system, of the HPI. In P. marinus the expression of six CRH system genes, including three hormones, CRH A, CRH B and UCN III-like; two receptors, CRH Rα and CRH Rβ; and a binding protein, CRH BP, are identified by PCR and in silico methodologies. Analysis of the P. marinus CRH system genes appear to support the occurance of the Agnathan superclass prior to a theorized second vertebrate whole genome duplication (WGD) event. This is supported by the P. marinus CRH hormones appearing to represent two of the four vertebrate CRH family paralogues; CRH A and B both being orthologous to vertebrate CRH, and UCN III-like being orthologous to vertebrate UCN III. Additionally, neither CRH Rα nor CRH Rβ, while identified as distinct from one another and related to other vertebrate receptors, were phylogenetically indistinguishable as either ii type 1 or 2. This suggests, the two P. marinus CRH receptor genes identified appear type 1 or 2. This suggests, the two P. marinus CRH receptor genes identified appear to have arisen out of a lamprey specific duplication event they diverged separately from the formation of the type 1 and type 2 receptors. The P. marinus CRH BP deduced amino acid sequence was found to contain regions highly conserved and functionally significant in other vertebrates as well as invertebrate species, and occupies a unique phylogenetic branch. Expression of these genes in brain, gill, liver, kidney as measured by reverse transcription quantitative PCR (RT qPCR) over the life history of P. marinus (including pre-metamorphic larvae, each of the seven stages of metamorphosis, and juvenile parasites) indicated significant variation in gene expression both between tissues and through the life history. Differences in expression were observed for each P. marinus CRH system gene and correlate with significant physiological changes occurring in the developing P. marinus. Some of these include increases in Na+/K+ -ATPase activity in the gill, possibly relating to salt water tolerance, and lipogenic and lipolytic metabolic phases in the kidney and liver. Interestingly, comparatively high expression levels of CRH A, CRH B and CRH Rβ were observed in the JP gonad relative to other JP organs. This suggests these genes may have a paracrine role in this organ, possibly by local regulation of sex steroids, similar to that observed in mice and humans. Interestingly, CRH system mRNA expression did not vary in response to multiple successive acute stressors, including dewatering and salt water exposure, over a 24 hour period as measured by RT qPCR. This suggests that P. marinus CRH system genes may not respond to such stressors at the level of mRNA expression. Collectively, these data indicate that lamprey contain all necessary components of a complete HPI axis, and that the CRH system likely plays an important role in the normal development.