Regulation Of The Transcription Factor ISL-1 By Bone Morphogenetic Proteins In Telencephalon Development

Cholinergic neurons serve a variety of functions includinglearning,memory and motion processes. Many behavioral andfunctional messes in organism are involved in disease ofcholinergic system. But the investigation on the generational ofcholinergic neurons is limited. The relationship betweenextracellular factors and homeodomain transcription factor thatinduce cholinergic neurons generation is the key of understandingthe mechanisms of it.Radial glia in ventricle zone (VZ) is a progenitor ofcholinergic neurons in neurogenesis period. Cholineacetyltransferase(ChAT)is a marker for cholinergic neuronsand it can show the generation,transplantation and distributionof cholinergic neurons. The cholinergic precursors' generation isfrom E12 in mice. The extracellular factors BMPs play criticalroles in the regulation of neurulation, and the dorsoventralpatterning during a gastrulation and of the neurogenesis.BMP4 express along VZ in neurogenesis, and there are manydomains where BMP4 and cholinergic neurons co-exist. It wasreported that in cultured neurons, BMP-9 directly induced theexpression of the cholinergic gene locus encoding cholineacetyltransferase and up-regulated acetylcholine synthesis. Itindicates that BMP4 can induce the generation of cholinergicneurons. BMPs, as an extracellular factors, cooperate withhomeodomain transcription factor in cell to regulate specialgenes expressions of cholinergic neurons, and promot theirproliferation and differentiation.From the data collected, it is supposed that ISL-1 isprobably the main homeodomain transcription factor which cancontrol the development of cholinergic neurons. ISL1 could beinvolved in the initial determination of motor neuron identity.The previous investigation shows that motor neurondifferentiation does not occur in the absence of ISL1. ISL1express in the neonatal cholinergic neurons in telencephalondevelopment. The regional specificity of the spatial correlationbetween ISL1 and ChAT expression increase the possibility thatISL1 may be involved in regulating cholinergic phenotypedevelopment. The expression of ISL1 in cholinergic cells mayplay an important part in developmental regulation of thecholinergic phenotype.Hence, the distribution of BMPs and ISL-1 expression areboth superposed with cholinergic neurons. BMPs might act onISL-1 to induce Radial glia to generate cholinergic neurons. Theintention of our investigation is to observe if ISL-1 expressioncan be changed with the changing of BMP4 concentration inculture of embryonic telencephalon cells and to confirm ISL-1 isa significant transcription factor that regulates the generation ofcholinergic neurons.To investigate BMP4 influence to embryonic telencephaloncells we treated primary cells derived from the dorsaltelencephlon of E11-E18 rat brain with BMP4 and performedImmunofluorescence staining and immunostaining. RT-PCRtechnique was performed to detect whether cholinergicprecursors ISL-1 mRNA expression varies with theconcentration of BMP4 in medium.In vitro, low level BMP4(10ng/ml,20ng/ml,40ng/ml)have little morphological influence to E11 and E12 rattelencephlon cells. Contrarily, it can promote cells of E14 andE18 rat to grow in characteristic round clusters and extend longand numerous processes. And it is found that High level BMP4(80ng/ml,100ng/ml) restrain growth of telencephlon cells.ChAT expression in the neuronal clusters in E12 and E14 rattelencephlon show that cholinergic neurons generation is fromE12. But the immunofluorescence staining and immunostainingresult show that there is little difference in morphology betweenthe experimental groups with serum and without serum. Theeffect on ISL-1 expression by different BMP4 concentrationwas detected by RT-PCR. The result shows that 10ng/ml BMP4bring little effect on ISL-1 expression and 20ng/ml BMP4enhance ISL-1 expression remarkably when 40 ng/ml BMP4decrease expression of ISL-1 in vitro. The result substantiateISL-1 is a transcription factor which is associated with BMP4inducement to cholinergic phenotype. Moreover, it is foundthat 20ng/ml is the optimum concentration of BMP4 which canbe used for reference in the future.From the result of the experiment, we find that BMP4can promote cells to grow in characteristic round clusters andextend processes in vitro. It also can increase expression oftranscription factor ISL-1.We found out that 20ng/ml is theoptimum concentration in which BMP4 can action. In ourinvestigation, ISL-1 is only proved to be one of thehomeodomain transcription factor to regulate cholinergicneurons development cooperated with BMP4. The exact roleof ISL1 and BMP4 cooperation in this process remains unclearand we need some further researches.