The Expression Of Id2 In Adult Rat Brain And Its Effects On The Development And Differentiation Of SVZa Neural Stem Cells

It is believed that the anterior subventricular zone(SVZa) and hippocampus are the most numerous areas of neural stem cells (NSCs) in adult mammal brain. The SVZa NSCs continuously migrate from SVZa to olfactory bulb (OB) by the mammal's whole life, and form the rostral migratory stream (RMS). So the SVZa NSCs are the renewing resource of OB interneurons. Besides having the general characteristics of NSCs, the SVZa NSCs have their unique features themselves. The SVZa NSCs can keep undifferentiated all along in RMS. After arriving at the center of OB, the NSCs then migrate around and differentiate into neurons and neural glia cells.The differentiation of NSCs are affected not only by inherit factors such as thyroid hormone receptors in the cells, but also by extracellular signals such as mitogenic factors (EGF), thyroid hormone(T3) ,glucocorticoids , retinoic acid(RA), serum. What kind of the mechanism it is that why the SVZa NSCs keep undifferentiated all along in RMS is still unclear now. The colleagues in our lab had observed that transcription factors such as BMP2,Mashl,Pax6,Wnt can regulate the proliferation,migration and differentiation of SVZa NSCs, but all of these factors,of whose expressions were low in the RMS are positively promoting the NSCs differentiation.The differentiation of NSCs are regulated by positive and negative factors. We had observed some positive factors which promote their differentiation of SVZa NSCs. But what about the negative factors? We select inhibitor of DNA binding-2(Id2) as the research object because of its negative regulating features (as follows), hoping to provide some basic data for the mechanism of proliferation , migration and differentiation of SVZa NSCs.Helix-loop-helix(HLH) transcrition factors include inhibitor of DNA binding (Id) and basic HLH (bHLH). bHLH contains class A and class B protein. Ubiquitously expressed bHLH class A protein as a kind of positive protein can combine with positive Class B protein.and negative Id protein. Id containing Id1,Id2, Id3 and Id4, as a factor which inhibitbHLH protein lacks the DNA binding basic sequence.The heterodimer or homodimer with bHLH class A protein and bHLH class B protein can combine with E-box protein can form complexes which can promote downstream genes transcription.Id can bind to bHLH class A protein and then interfer the complexes to form.Id2 is generally synthesized by myogenic precursor cells. Id2 regulates the development and differentiation in diverse cell types. Id2 is functioned as the blocks of cell differentiation and is reduced during the course of cell differentiation. Id2 participates in the development of T cells, tumour cell proliferation, cell cycle and muscle genesis and so on.In recent years, many evidences provide mat Id2 plays an important role in nervous system. In Id2 null mice, there are many abnormities such as immature neuralblast. It is reported that Id2 is expressed in ependymal cell, dorsal root ganlion sensory neural pregenitor in PNS by in situ hybridization. All of these evidences show that Id2 plays an important role in nervous system. But what are the relationships between Id2 and neural differentiation? We had observed that transcription factors such as BMP2, Mashl, Pax6, Wnt which were lowly expressed in the RMS, what about the negative Id2 in RMS during the SVZa NSCs migration?We have observed at first the expression mapping of Id2 protein in adult rat brains by immunofluorescent staining and then the temporal and spatial expression patterns of Id2 in SVZa-RMS-OB pathway by RT-PCR, Western blot and immunofluorescent staining. At last, we analyzed the expression changes of Id2 after being affected by T₃ during the course of SVZa NSCs differentiation in vitro..The results are as follows:1. Id2 is expressed extensively in brain. There are three types: (1) The intense signal can be seen in the prepiriform cortex,subiculum,caudate putamen, thalamic nucleus, optic tract,suprachiasmatic nucleus,asomedial nucleus of the amygdala,kolliker-fuse nucleus and so on . (2) The moderate staining is detected in the olfactory bulb,dentate gyrus,accumbens nucleus,globus pallidus, substantia nigra,red nucleus , pineal gland. (3) The weak signal is detected in field CA1, CA2 and CA3 of ammon's horn, arcuate hypothalamic nucleus, locus ceruleus, and so on.. Including caudate putamen, dentate gyrus and SVZ, Id2 expressions distributed optic tract ,Kolliker-Fuse nucleus,red nucleus ,pineal gland, thalamic nucleus, asomedial nucleus of the amygdala and so on,suggested Id2 maybe involve in various function of brain, such as smell, vision, sense, motor and neuroendocrine.2. The temporal and spatial expression patterns of Id2 in SVZa-RMS-OB pathway: (l)in different locations: the expressions of Id2 are highest in RMS, higher in SVZa, lowest in OB. (2)In different time points: the expressions of Id2 are highest in embryonic day 14(E14) in RMS and OB, and then the Id2 level reduced in newborn.There are higher expressions in postnatal day 30 (P30) and adult. The expressions of Id2 in SVZa gradually decrease from E14 to P30, then increase in adult. These results suggest that Id2 may be can promote the proliferation .migration and sustain SVZa NSCs undifferentiated. We had observed that transcription factors such as BMP2, Mashl, Pax6, Wnt can positively regulate the differentiation of SVZa NSCs. Id2 as a negative factor regulates the proliferation ,migration,differentiation of SVZa NSCs with them from each other.3. Id2 can promote the proliferation and inhibit differentiation of SVZa NSCs T₃ can promote this functon.because T₃ can promote the differentiation of SVZa NSCs, also Id2 expressed increasely after SVZa NSCs are affected by T₃ during the course of differentiation in vitro Id2 maybe regulate the proliferation , differentiation of SVZa NSCs on the mechanism which T₃R mediated.In summary, we have observed the expression mapping of Id2 in adult rat brains.Besides expressed in caudate putamen, cortex, dentate gyrus and so on which other researches have reported, Id2 also expressed thalamic nucleus,optic tract ,kolliker-fuse nucleus, globus pallidus, substantia nigra,red nucleus and so on.We also analysed the temporal and spatial expression patterns of Id2 in SVZa-RMS-OB pathway,Id2 can promote the proliferation,migration and sustain SVZa NSCs undifferentiated. We had observed that transcription factors such as BMP2, Mashl, Pax6, Wnt can positively regulate the differentiation of SVZa NSCs. Id2 as a negative factor regulates the proliferation ,migration,differentiation of SVZa NSCs with them from each other.And then discussed the expression changes of Id2 after SVZa NSCs being affected by T₃ during the course of differentiation in vitro. Id2 can promote the proliferation and inhibit the differentiation of SVZa NSCs, Id2 maybe regulate the proliferation, differentiation of SVZa NSCs on the mechanism which T3R mediated This study provides some basic data for illustrating the mechanism of SVZa NSCs proliferation,migration and differentiation.