Expression Patterns Of Ndrg2 In Developing And Adult Mouse Brain

Ndrg2 is a member of the N-myc downstream-regulated gene(NDRG) family. Thus far, the full-length cDNA of Ndrg2 encodes 2 different protein variants of 371 aa(41kDa) and 357aa, respectively. It is a cytoplasmic protein involved in cell differentiation, development and apoptosis (Yao et al., 2008) (Choi et al., 2003). At embryonic day 9.5(E9.5), Ndrg2 mRNA was already detected in the brain by Northern blot (Okuda and Kondoh, 1999). In embryos, however, the detailed distribution of NDRG2 expression sites and the function of the Ndrg2 remain unclear. Here, in order to determine the role of Ndrg2 gene in the mouse nervous system, we preformed in situ hybridization (ISH) and immunocytochemistry on mice aged E10.5, E11.5, E12.5, E14.5, E16.5, E18.5, P0,P7,P14,adult to study the expression of the Ndrg2 through mouse embryogenesis and into adulthood. Our ISH results showed that during brain development Ndrg2 was highly expressed in cells found in places rich in neural progenitors such as the VZ , the SVZ and SGZ. In situ hybridization analysis on the embryonic brain sections indicated that at RNA levels Ndrg2 is mainly expressed in frontal cortex, DT(dorsal thalamus), ganglionic eminence and medulla, particularly in the ventricular zones. However, no clear data are available on the expression of Ndrg2 in proliferating precursor cells of the SVZ.MethodsTo address these questions we examined Ndrg2 mRNA expression in proliferating precursor cells of the SVZ by using a double labeling procedure, combining in situ hybridization for Ndrg2 mRNA with immunohistochemistry for bromodeoxyuridine (BrdU),Ki67 and Nestin, a marker for proliferating precursor cells . Further phenotypic studies of Ndrg2 expressing cells revealed that in the SVZ the majority of Ndrg2-positive cells express glial fibrillary acid protein(GFAP),and also colocalize with cells expressing doublecortin,a marker of differentiating and migrating neuroblasts. Our results demonstrate that Ndrg2 is expressed in neural progenitor cells ,which suggests that Ndrg2 might play an important role in the development of murine central nervous system.Results1. We further investigated the expression pattern of Ndrg2 in the developing embryonic brain. Ndrg2 mRNA signals were found in early the embryos of the majority of neuroepithelial cells(Fig.1A for E11.5).The examination of the hybridization signals with the antisense Ndrg2 riboprobe shows a widespread expression(Fig1A). Ndrg2 is highly expressed in regions of active neurogenesis in the developing brain.Throughout the study, adjacent sections were hybridized with both the sense and antisense probes to confirm the specificity of Ndrg2 mRNA hybridization signals. No signal was observed in sections hybridized with the sense probe(Fig.1B for E11.5,not shown for other ages). At E14.5, the expression was generally more intense than the previous ages throughout the central and peripheral nervous system, Interestingly, by E14.5, A large number of cells were showing strong positive reaction for the gene in the neopallial cortex, which is composed of an inner broad nuclear layer(cortical plate),with a narrow outer relatively annuclear layer (marginal zone; Fig.2E) the differential expression in the cortex shifts from the deep layers (SVZ and VZ) to the upper layers (CP and MZ).Strong expression can be seen in CP , moderate expression in M intermediate zone(IZ) and low exresssion in the subplate (SP)(Fig.2E)2. An important finding of our study is the demonstration of the enriched Ndrg2 expression in the subventricular zone of the anterior lateral ventricle (SVZa), the rostral migratory stream and the olfactory bulb. Ndrg2 is expressed in close proximity to the subgranular zone(SGZ), our findings have suggested the existence in the subventricular zone(SVZ)from saggittal sections of adult rat brain of a trophic mechanism, mediated by Ndrg2. To address these questions we examined Ndrg2 mRNA in proliferating precursor cells of the SVZ by using a double-labeling procedure, Double immunfluorescence characterization of Ndrg2 expressing cells in adult neurogenic regions(by confocal microscopy). Observations at higher power revealed that the localization of Ndrg2 mRNA signal in the dentate gyrus was restricted to the innermost layer of the granular cell layer of the dentate gyrus and pyramidal cell layer of CA1-CA3.3. Ndrg2 mRNA was also present in many non-neural tissues. Ndrg2 expression was restricted to a ventral region of the ventricular zone from E11.5 (Fig.5A). Ndrg2 expression was detected weakly in scattered cells in the ventral spinal cord at P0. At P7,cells in the ventral grey horn strongly expressed Ndrg2 mRNA. Ndrg2 expression in the cerebellar anlagen and ependymal layer of the IVth ventricle in a E14.5 mouse. At E16.5, my colleague and I showed that Ndrg2 is highly expressed in the retinal ganglion cell layer of the developing retina.Conclusion1. Ndrg2 transcripts were detected in the developing CNS, such as the ventricular zones lining the lateral ventricle(LV), ganglionic eminence, mesencephalic vesicle, fourth ventricle(IVV), epithalamus (ET),the dorsal thalamus(DT) and spinal cord. Strong expression can be seen in CP .2. Ndrg2 expression in the subventricular zone of the anterior lateral ventricle (SVZa), the rostral migratory stream and the olfactory bulb. Ndrg2 is expressed in close proximity to the subgranular zone(SGZ).3. We found that strong Ndrg2 expression level was also observed in the postmitotic neurons in postnatal cerebellum and embryonic retina and spinal cord.