2

In conclusion, we cloned the full-length sequence of chicken MC5R gene and characterized its promoter activity in DF1 chicken embryonic fibroblasts. The functional assay demonstrated that MC5R exhibited higher sensitivity to chicken ACTH/α-MSH compared to β-MSH/γ-MSH. It also showed that both MRAP1 and MRAP2 inhibited the trafficking of chicken MC5R to the plasma membrane, and that only MRAP2 significantly reduced the sensitivity of MC5R to stimulation by α-MSH. MC5R and MRAP1 mRNA were co-expressed in the liver of post-hatch chickens. We found that ACTH may increase glucose production, decrease triglyceride content, and dose-dependently downregulate the expression levels of ELOVL6 and THRSPA genes in hepatocytes, suggesting that ACTH has a unique endocrine role in regulating hepatic glucolipid metabolism. However, further studies are needed to characterize and confirm the critical role of the ACTH-MC5R/MRAP1 axis in regulating glucose and lipid metabolism in chicken liver.

In summary, we cloned NPS and NPSR1 genes from the chicken brain. Functional assays proved the cNPSR1 can be potently activated by its endogenous ligand NPS and trigger multiple downstream signaling pathways (Figure 7). qPCR assay revealed that both NPS and NPSR1 are widely expressed in chicken tissues with a relatively high expression level noted in the brain. Our data provide clear evidence that as in mammals, an NPS-NPSR1 system function in birds, which may play roles similar to those in mammals, such as central regulation of food intake in birds.

In summary, the full-length cDNA of the three chicken 5-HT receptor genes (HTR1B, HTR1E, and HTR1F) were first reported in the present study. Sequence alignment showed that the three genes showed high sequence identity with their counterparts in other vertebrates. Moreover, synteny analysis revealed that HTR1B, HTR1E, and HTR1F are highly conserved among vertebrates and supported previous findings that chicken receptors are orthologous to the known receptor groups. Functional studies demonstrated that HTR1B, HTR1E, and HTR1F are functional and responsive to 5-HT and various selective agonists CP94253, BRL54443 and LY344864, respectively. Together with the enriched expression of the three receptors in the pituitary, the observation that LY344864 can partially inhibit PRL secretion in cultured chicken pituitary cells suggest that HTR1F is likely involved in regulating the release of the chicken pituitary PRL. Our findings represent the first key step in establishing the molecular basis for the better understanding of the serotonergic modulation network in avian species.

In summary, our data sets a critical reference point to reveal the novel aspects of pituitary biology across vertebrates, and helps to figure out how the anterior pituitary, as a pivotal signal converging and output center for the brain and peripheral tissues, can orchestrate so many physiological processes (e.g., growth, reproduction, metabolism, and stress) to meet the changing physiological demands or pathological status observed in vertebrates, including birds and humans.

In addition, we found that pig MRAP2 can decrease surface expression, increase the ligand sensitivity and selectivity of pMC4RAsp/pMC4RAsn, and enhance the basal constitutive activity of pMC4RAsp.

In summary, we cloned six genes of the MC4R signaling system in a representative teleost species, Nile tilapia. Functional study proved that MC4R can interact with MRAP2b and mediate the actions of α-MSH, ACTH, and AgRP in vitro. Moreover, we demonstrated that MRAP2b cannot only inhibit the basal constitutive activity of MC4R, but also increase its sensitivity and selectivity for ACTH, thus becoming an ACTH-preferring receptor. These findings, together with evidence showing the co-expression of MC4R, POMCs, AgRP, and MRAP2b in the hypothalamus, strongly suggest that the MC4R signaling system plays a conserved role in the regulation of food intake and growth in Nile tilapia. Considering that Nile tilapia is a freshwater fish of economic importance worldwide, our data provides a theoretical basis to improve the economic traits of tilapia, such as promoting the appetite and growth via modifying MC4R signaling (e.g., gene-editing MC4R system) in aquaculture.

In summary, we proved that AVT possesses four functional receptors (AVPR1A, AVPR1B, AVPR2b and MTR), and MT has a single receptor (MTR).

In summary, we characterized two APLNRs in chickens and turtles, and three APLNRs in zebrafish.

The orphan receptor GPR25 was cloned from zebrafish, pigeons and spotted gars. GPR25 is activated by Apelin and Apela in non-mammalian vertebrates. Like Apelin receptor, GPR25 is coupled to Gi-cAMP signaling pathway. GPR25 mRNA is widely expressed in zebrafish/spotted gar/human tissues.

Collectively, our data helps to elucidate the physiological roles of NMU/NMS peptides in birds and reveal the functional conservation and changes of NMU/NMS-NMUR axis across vertebrates.