Role Of Activin A In Development Of Embryonic Dorsal Root Ganglia Of The Chicken

Activin A is a multifunctional growth and differentiation factor belonging to thetransforming growth factor-beta (TGF-beta) superfamily. It is widely distributed andplays an important role in the early embryo formation, neural differentiation anddevelopment, hematopoietic cell proliferation and differentiation, apoptosis,tumorigenesis and sex hormone secretion, etc. Its receptors, composed of ⅠandⅡreceptors, belong to TGF-beta superfamily of serine/threonine kinase (Ser/Thr)receptor. Our previous studies have found that activin A can promote neuriteoutgrowth of chicken embryonic dorsal root ganglion (DRG) and maintain survival ofneuron. However, the role of activin A in development of DRG remains unclear.Therefore, first, the expressions of activin A and its receptors, as well as Smads inchicken DRG of different days were examined to analyze the relationship betweenactivin A and DRG development, and then the neurotransmitters released by neuronsof DRG were detected to further analyze the mechanism of activin A in thedevelopment of DRG.1. MethodsDRG were dissected from8d chicken embryos under a dissecting microscopeand cultured in vitro. Then the role of activin A and nerve growth factor (NGF) instimulating neurite outgrowth were analyzed. Reverse transcription PCR (RT-PCR)and immunohistochemical staining were used to examine the expressions of activin Aand its receptors, Smads in DRG of chicken of different embryonic days (8d,10d,12d,14d,16d,18d). Enzyme linked immunosorbent assay (ELISA) was used to detect theneurotransmitters of Norepinephrine (NE) and5-Hydroxytryptamine (5-HT) levels etcin the supernatant of cultured DRG cells in vitro.2. Results2.1Activin A promotes the neurite outgrowth of embryonic DRG of chickenDissect the DRG of8d chicken embryos under dissecting microscope, and then the DRG was planted in48-well culture plate. The experiment was divided intoactivin A group, activin A+anti-NGF antibody group, activin A+FS group, NGF group,NGF+anti-NGF antibody group and NGF+FS group. Neurite outgrowth of DRG wasobserved daily after treatment for5d. The results showed that activin A could promotethe neurite outgrowth of DRG, which was not influenced by anti-NGF antibody, whileanti-NGF antibody could block the neurite outgrowth of DRG induced by NGF. Itsuggests that activin A promotes the neurite outgrowth of DRG independent of NGF.2.2Correlation of activin A and its receptor, Smads with the development ofembryonic DRG of chickenIn order to further explore the role of activin A in the development of embryonicDRG, RT-PCR was used to examine the expressions of activin A and its receptors, etal. of embryonic DRG of chicken of different embryonic days (8d,10d,12d,14d,16d,18d) respectively. The results showed that the expressions of activin A, ActRⅡA andActRⅡB mRNA decreased gradually, but FS mRNA,, an activin-binding protein,mRNA increased day by day. The expression of Smad3mRNA, a downstreamsignaling molecule of activin, also decreased with the development of embryonicDRG, but Smad2mRNA didn’t change with the development of embryonic DRG. Theresults of immunohistochemical staining showed that activin A was high expressed incytoplasm of neurons of chicken embryonic DRG from8d to12d, while from14d to18d, the expression of activin A decreased gradually. The expressions of ActRⅡA andActRⅡB were similar to that of activin A. On the contrary, the expression of FSincreased with the development of DRG. The above results revealed that activin A wasinvolved in the early development of embryonic DRG.2.3The mechanism of activin A in the development of chicken embryonic DRGMonolayer-cultured cells of DRG of8d chicken embryos were cultured in vitroand neurons could be distinguished by staining Nissl bodies with toluidine blue. Theresults showed that activin A could inhibit the proliferation of glial cells effectively,while NGF can not. ELISA results showed that activin A and NGF could enhance thelevels of5-HT, NE and GABA in the supernatant of cultured embryonic DRG cellssignificantly (p<0.05, p<0.01), while the level of Glu decreased (p<0.05, p<0.01).There was no effect of Activin A and NGF on DA and ACh in the supernatant ofcultured embryonic DRG cells. RT-PCR result showed that the expressions of NGF and its receptor of cultured embryonic DRG cells were not effected by activin A.These results indicated that activin A might be involved in the development ofembryonic DRG through inhibiting the proliferation of glial cells and regulating therelease of neurotransmitters such as5-HT, NE, GABA and Glu.3. ConclusionIn summary, our study demonstrated that activin A promoted the neuriteoutgrowth of embryonic DRG, which was not dependent on endogenous NGF. ActivinA was involved in the development of embryonic DRG. Also, Activin could inhibit theproliferation of glial cells and promote the release of NE, GABA and Glu. Therefore,activin A may play an important role mainly in the early development of embryonicDRG, which is associated with inhibiting proliferation of glial cells and regulatingneurotransmitter release, such as NE, GABA and Glu. This study will contribute toreveal the molecular mechanism of neurodevelopment, explain repairmen of nervoussystem damage, and search for new therapeutic targets of nervous system damage aswell.