The Proliferation And Differentiation Of SVZa Neural Stem Cells In Response To BMP4

One of the most concentrated region of adult NSCs, known as the SVZ, surrounds the postnatal ventricle. NSCs are located within a discrete region of the anterior part of the neonatal and postnatal subventricular zone, ie, the SVZa, and are the source of an immense number of neurons which are destined to be interneurons of the granule cell and glomerular layers of the OB. These SVZa-derived progenitors migrate to the OB as a network of tangentially oriented chains that converge in a highly restricted pathway called the rostral migratory stream (RMS). The SVZa-derived progenitors have been well characterized and contain an essentially pure population of neuronal progenitors, in contrast to the posterior subventricular zone (SVZp), where mainly astrocytes are produced. Hence, SVZa NSCs are a reliable model for neural development and regeneration.Bone morphogenetic proteins (BMPs), the largest group of ligands in the transforming growth factor β superfamily, play important roles in vertebrate neurogenesis. Many studies have demonstrated that BMPs and their antagonists including Noggin, Chordin and Follistatin induce neurogenesis, neurotrophism, neural apoptosis and neurospecification both in the peripheral and central nervous system. BMPs may alternatively activate or inhibit neuronal differentiation. Various and sometimes conflicting results have been reported depending on the stage of development, the distance from the source, or the expression of particular transcription factors in target cells. These different results suggest that modulation of the differentiation of NSCs by BMPs may involve multiple mechanisms. While SVZa-derived cells provide a nice model to explore the proliferation and differentiation of NSCs, the response to BMPs remains not fully understood in the SVZa.In the study, the expression of BMP4 was detected continuously in living SVZa, RMS and OB cells by using a reporter vector, in which the BMP4 promotor was conjugated with the red fluorescent protein (RFP). Different Noggin sensitive effects were observed when SVZa NSCs were exposed to various concentrations of BMP4 at different stages. The differentiation of SVZa NSCs was dynamically observed by using reporter vectors of theNestin enhancer and the promoters of TH and GFAP. Previous study in our lab demonstrated that Mash1 is a key gene for neuron fate decision in SVZa, so here we explored the relationship between BMP4 and Mash1. The results are as following:1. Expression of BMP4 mRNA in neonatal mouse brainAlong the migratory pathway from the SVZa to the OB, BMP4 mRNA was highly expressed in the peripheral cells of the OB, especially in the mitral cell layer and, to a lesser extent, in the periglomerular and granular cell layers. However, in the central area of the OB, the expression of BMP4 mRNA was low. BMP4 mRNA expressed moderately with sporadic distribution in the cells of the SVZa, but high in the astrocyte sheath enveloping SVZa. In the RMS, the expression of BMP4 mRNA was relatively low. In addition, the expression of BMP4 mRNA in the SVZp region, adjacent to the SVZa, was rather higher.2. Analysis of BMP4 promotor activityA small number of red fluorescence cells with strong fluorescent intensity and distingishable cell processes were seen 12 hours after the pDsRed-BMP4pro plasmid was introduced into SVZa NSCs. After 24-48 hours, the number of positive cells apparently increased, and the expression of fluorescent protein reached a maximum at 48 h, after which fluorescence decreased and was almost absent after 5 days. RFP was mainly expressed in neurons within 48 h with thick and long processes, but in astrocytes at later periods.RFP expression during the differentiation of progenitors derived from SVZa, RMS and OB was similar to that of SVZa NSCs. There also existed significant regional differences in BMP4 promotor activity. There were clearly more positive cells in the OB than in the SVZa and fluorescence appeared mainly in the neuron-like cells. There were relatively less fluorescence cells in the RMS. The results of spectrofluorometry showed that the fluorescence intensities in the OB, SVZa and RMS were 39.72±2.65, 33.93±2.99 and 23.16±0.14 respectively.3. Proliferation of SVZa NSCs in response to BMP4Cell counting showed that the proliferation of SVZa NSCs was activated by BMP4 at concentrations of 1, 2 or 5 ng/ml, with maximal BMP4 effects at 2 ng/ml. However, BMP4 at higher concentrations (10, 20, 50 or 100 ng/ml) inhibited the proliferation of SVZa NSCs. Cell-cycle analysis with FCM showed decreased cells at phase G1/G0 inresponse to BMP4 at 1, 2 or 5 ng/ml, but increased number of cells at phase S and an increased G2+M/G0 ratio. BMP4 at 2 ng/ml again produced the maximum effect. BMP4 at concentrations of 10, 20, 50 or 100 ng/ml, caused a marked increase in the number of cells at phase G1/G0 while decreasing sharply those at phases M and S.4. The neuron commitment of SVZa NSCs exposed to BMP4MAP-2 and NSE immunofluorescence stainings showed that the number of positive cells increased 2 days after the addition of BMP4 at concentrations of 1, 2, 5, 10, 20, 50 or 100 ng/ml. The FCM measurements revealed that the increase in neurons in response to BMP4 at concentrations of 1-10 ng/ml was gradual and reached a plateau level when the BMP4 was at concentrations of 10-100 ng/ml on day 2. But neuron number reduced on day 7, which was confirmed by MAP-2 and NSE immunofluorescence staining as well as FCM. We also added BMP4 at 10 ng/ml to observe the differentiation of SVZa NSCs at different time points, and the results of FCM analysis showed that BMP4 increased the number of neurons in the early stage, and this reached a peak at day 2 (at day 4 in the control), thereafter reduced.5. The antagonism of NogginThe Noggin-GFP vector was constructed and transfected into SVZa NSCs cultured in media containing various concentrations of BMP4. The results showed that Noggin could block the effects of different concentrations of BMP4. The number of neurons in the early stage was reduced, while the number of neurons was increased in the late stage. This was also validated by the FCM analysis.6. The activities of the Nestin enhancer and the promotors of TH and GFAP in response to BMP4After transfection with Nestin-GFP, many fluorescent cells appeared at 8 h, in general, without obvious processes although a few positive cells with obvious processes. The number of positive cells reached a maximum at 24 h and thereafter declined. With the addition of BMP4 (10 ng/ml), the number of fluorescence cells decreased markedly. There was a significant difference in the number of fluorescent cells in the presence of 10ng/ml BMP4 compared with the control group at early stage, especially at 24-28 h. The spectrofluorometry that was performed at day 1, 2, 3, 4, 5 ,6 or 7 showed that, with the addition of BMP4, Nestin enhancer activity was obviously reduced within 3days, butinsignificant after 4days.After transfection of SVZa NSCs with TH-GFP, only a few positive cells emerged at 8 h. Later, the number of positive cells with obvious processes gradually increased and were distributed diffusely. The maximum number of positive cells appeared at 48-72 h. After BMP4 (10 ng/ml) was added, the number of positive cells increased and after 24-48 hours incubation with BMP4, there was a significant increase compared with the control group. The spectrofluorometry showed a significant difference between BMP4 group and the control group at days 1-3, but this difference was insignificant after 4 days. Spectrofluorometry results showed a continuous enhancement of GFAP promotor activity, even after 4 days, when the Nestin enhancer and TH promotor activity remained unchanged with addition of BMP4 at 10ng/ml.7. Analysis of Mash 1 promotor activity exposed to BMP4X-gal dyeing was made out after transfection of SVZa NSCs with Mash1-LacZ. Some blue cells presented at day 1, with obvious blue color in cell body but faint in processes, and mostly in neuron-like cells. The number of positive cells increased with the addition of BMP4 (10 ng/ml)and after 24-48 hours incubation with BMP4, there was a significant increase compared with the control group appeared at 24-48 h. Mash1 promotor activity was enhanced at day 1 and day 2 according to the cell counting, and reduced after 4 days but insignificant in Statistical analysis.Several conclusions are drawn according the study, and lists as following:1 . In this study, conjugating the BMP4 promotor with red fluorescent protein represents the activity of the BMP4 promotor, and can not only indicate the internal protein expression to some extent but also allow one to continuously trace expression in the living cells. At the same time, GFP under the control of the Nestin enhancer, the promoters of TH and GFAP traced the differentiation of SVZa NSCs, which indicates it a good way to trace internal protein expression in the living cells.2. Differential expression of BMP4 exists in different locations and stages of SVZa NSCs development, and suggests that BMPs play distinct roles in the development of SVZa NSCs depending on the location and stage of development.3. Though the intervening of cell cycle, BMPs can promote or inhibit the proliferation of NSCs depending on their concentration, and BMP4 at low concentrations (1-5 ng/ml)promotes the proliferation of NSCs but BMP4 at high concentrations (10-100 ng/ml) inhibits the proliferation.4. BMP4 promotes neuron commitment in the early stage (at days 1-3) but inhibits it later. Both actions are absent after the Noggin plasmids are introduced.5. BMP4 promotes the exit from the cell cycle and triggers the differentiation of neuron progenitors in the OB, and enhances the proliferation of the committed neuron progenitors in the RMS, but in the SVZa, BMP4 may facilitate the commitment of NSCs into astrocytes as well as the exit from the cell cycle.6. BMP4 facilitates neuron commitment at the early stage though Mash1 transcription factor.