Discovery Of Neuropeptides In Grass Carp Brain And Pituitary And Functional Characterization Of Tachykinins

In vertebrates,the brain and pituitary are extremely important organs.The brain could regulate the synthesis and secretion of pituitary hormones by synthesizing and releasing neuropeptides,thereby regulating various physiological processes,such as growth,reproduction,stress and immunity.Unlike mammals,the hypothalamus-pituitary axis of teleost lacks the portal system,which leads to larger and more complex neuropeptides upstream of the regulation of synthesis and secretion for pituitary hormones.Based on these information,using grass carp（Ctenpharyngodon idellus）as a model.Firstly,the morphology and microstructure of distinct brain subregions and pituitary of grass carp were analyzed through tissue section.The function and potential functional connections of various brain subregions and pituitary were studied via combing with bioinformatics analysis method.Then,on the basis of grass carp genome database and the transcriptome data of different brain subreigons and pituitary of grass carp,the neuropeptide encoding genes and their related receptors were deeply discovered among grass carp brain and pituitary.Tissue distributions of these genes amongst different brain subregions and pituitary of grass carp were also analyzed.Finally,regrading the tachykinin family that we discovered as the entry point,the function and mechanisms of TAC3 gene encoding products NKB and NKBRP,TAC4 gene products HK1 and HK2 were further studied in grass carp pituitary,respectively.The main findings were as follows:1. The morphology and histological H.E.staining sections of the grass carp brain and pituitary showed that,the grass carp brain was mainly composed of 6 subregions,including the olfactory bulb,telencephalon,optic tectum,hypothalamus,cerebellum,and medulla oblongata.Unlike cartilage fish and mammals,there was no hypothalamic-pituitary portal system between the grass carp brain and pituitary,and neurons of the hypothalamus directly extended into and terminated in the anterior pituitary.By analyzing the transcriptome data of different brain subregions and pituitary of grass carp,the results indicated the olfactory bulb might be related to reproduction and immunity,and the telencephalon was deemed to be involved in the regulation of appetite and reproduction.The optic tectum not only played an important role in the visual system,but also might be involved in the regulation of feeding behavior.The hypothalamus could play an important role in the regulation process of food intake and reproduction.The medulla oblongata was related to the auditory system.The pituitary gland was seemed to play a key role in energy metabolism,organ development and reproduction.Correlation analysis results showed that the hypothalamus were highly related to the telencephalonnot only by their structures,but also the overlapping functions,indicating that the telencephalon and hypothalamus might be the regulation centers for feeding and reproduction in teleost fish.2. Combining the grass carp genome database and the transcriptome data of eachsubregion of the grass carp brain and pituitary,our present study discovered a total of 91neuropeptide encoding genes from 23 families,as well as 112 corresponding receptor genes.Based on the RNA-seq data,the transcript expression levels of these genes were analysed among six distinct brain subregions and pituitary gland of grass carp.Combined with the results of past researches and our study,the main synthetic regions and functional regions of these neuropeptides were further predicted in the grass carp brain and pituitary.3. Using grass carp pituitary cells as model,transcriptome analysis showed that NKBcould induce differential expression of 729 genes in pituitary,which were mainly involved in hormone activity,food intake,signal transduction and metabolism.Based on in vitro cell culture and RT-q PCR method,the results suggested that NKB could induce four anorectic peptides（UTS1,CART,POMCb and NMB）m RNA expression in time-and dose-dependent manners in grass carp pituitary cells.Receptor selecitivity and post-receptor signaling pathway experimental results suggested that these stimulating effects of NKB on UTS1,CART,POMCb and NMB could be mediated by NK3R.These induction activities were coupled to c AMP/PKA,PLC/IP3/PKC and Ca²⁺/Ca M/Ca MK-II signal pathways.Furthermore,intraperitoneal injection of NKBa could also significantly induce the four anorectic peptides m RNA expression in grass carp pituitary.Finally,the present study found that food intake could significantly induce hypothalamus TAC3a and TAC3b m RNA expression.These results,taken together,suggested that NKB was a satiety factor,and could be involved in the regulation of food intake via regulating the expression of pituitary anorectic peptides in teleost.4. Using grass carp as model,TAC4 gene and its receptors NK1Rb and NK3Ra1 were cloned from grass carp brain and pituitary.Sequence analysis showed that the grass carp TAC4 gene could encode mature peptides HK1 and HK2.Analysis of tissue expression indicated that TAC4 was highly expressed among hypothalamus,olfactory bulb and medulla oblongata,and NK1Rb,NK3Ra2 and NK3Rb also showed considerable expression in the pituitary,indicating that TAC4 might play an important role in the pituitary.Through NKRs expressing on the membrane of HEK293T cells,HK2 showed a high activation efficiency for 6 NKRs,in which the NK2R migth be the specifc receptor for HK2.While HK1 only exerted a weak affinity for NK2R,NK3Ra2 and NK3Rb.Transcriptome and bioinformatics analysis showed that HK2 could induce differential expression of 1051genes in pituitary cells.These genes were mainly involved in the negative regulation of appetite,hormonal activity,receptor ligand binding,and G-protein couple receptor signaling pathway.In primary cell culture of grass carp pituitary,HK2 could induce the synthesis of PRL,SLα,UTS1,NMB1,CART2 and SN2 in a time-dependent and dose-dependent manner,while HK1 showed no significant effects on the above genes.In order to clarify the reason of HK1 inactivation in grass carp pituitary,HK1 containing VFGLM motif was modified to HK1 mutant（HK1-M）with FFGLM motif.Receptor-ligand activity showed that,unlike HK1,HK1-M exhibited higher activation efficiency for 6 NKRs.Meanwhile,HK1-M could significantly induce UTS1,PRL and SLαm RNA expression in grass carp pituitary cells.Furthermore,co-treatment of HK1 and HK2 on pituitary cells showed that HK1 couldn’t block the effects of HK2 in pituitary,suggesting that HK1 was not the endogenous antagonist for HK2.These results,taken together,suggested that HK2could directly interact with grass carp pituitary cells,and promoted the synthesis of PRL,SLα,UTS1,NMB1,CART2 and SN2 in the pituitary mediated by NKRs.HK1 lost its biological function due to the mutation of Phe to Val in the FXGLM motif.In summary,our study established anatomical results and transcriptome database of different brain subregions and pituitary of grass carp,and made a preliminary analysis of the structure and function of different brain regions and pituitary of grass carp.The neuropeptide precursors and their receptor genes were deeply mined.With the tachykinin family as an entry point,the function and mechanisms of the products encoded by the TAC3gene and TAC4 gene were studied in the pituitary.This study will help fill us enrich the understanding of microstructure and function in fish brain,and lay a foundation for further studies on the function of fish neuropeptides.