A novel function of dcf1 during the differentiation of neural stem cells in vitro

Abstract

The study of neural dendrite formation is of great significance both in theory and applications. However, the molecular mechanisms of regulation remain unclear. We previously described a novel EST, which has high homology with dentritic cell factors (DCF1), expressed differentially between undifferentiated and differentiated neural stem cells (NSCs). In this study, we cloned, expressed, and silenced the dcf1 gene and offered insight into its function in regulating dendrite formation during the differentiation of NSCs. The results indicated that dcf1 encoded a 42 kD protein and could be successfully expressed both in Escherichia coli and NSCs. In order to silence dcf1 gene, three different kinds of siRNA vectors were constructed and transformed into the NSC line C17.2 and primary NSCs, resulting in down regulation of the dcf1 mRNA. Analysis of immunofluorescence or GFP illuminated that with overexpression of the dcf1 gene, the NSCs were maintained in undifferentiated status. After the dcf1 gene was silenced, cells tended to differentiate into neurons and astrocytes.

Fig. 1

Over-expression of dcf1 gene maintains NSCs in undifferentiated status. The dcf1 fragment was inserted in pEGFP-C1-DCF1, the positive clones were digested by BamH1 and released a fragment of 972 bp (a); C17.2 cells were transfected by pEGFP-C1 vectors as a control by EGFP fluorescence observation. Long and ramose neurite can be seen (b). Over-expression of the DCF1 gene was observed by transfecting pEGFP-C1-DCF1. NSCs maintained in undifferentiated status (c). Statistical analysis of neurite length showed in (d). It indicated a significantly different

Fig. 2

Silence of dcf1 gene. Schematic diagram of RNAi vector construction (a). The dcf1 gene was effectively down-regulated by psiRNA-1 and psiRNA-2 (b, lanes 1 and 2), while the scrambled sequence psiRNA-3 or blank sample had no affect (b, lanes 3 and 4)

Fig. 3

Immunostaining analysis of DCF1 protein expression. NSCs transfected with psiRNA-2 appeared a down-regulation of DCF1 protein expression (c) compared control (g). (a–d) RNAi silence. (e–h) Control group. (a, e) Photos in the white light. (b, f) Nuclei were stained blue with 4,6-diaminodino-2-phenylindole (DAPI). (c, g) NSCs (C17.2) were stained with DCF1 antibody after incubation for 4 days (d, h) were merge images

Fig. 4

Differentiated fate after knockdown of DCF1 in cell line C17.2. After 2 weeks of selection by G418, the transfected cells were immunostained for neuron specific marker NSE with FITC (green, a–e) or astrocyte specific marker GFAP with TRITC (red, f–j). (e, j) The results of transfection by scrambled psiRNA-3 vectors. The average percent of immunoreactive cells were obtained by counting four different fields of view on each well in at least two independent cultures (k)

Fig. 5

Differentiated fate after knockdown of dcf1 in primary NSCs. When primary NSCs were transfected by psiRNA-2, they could be differentiated into astrocytes (a) and neurons (c). Photos in white light are showed in (b) and (d), respectively. Real-time PCR was carried out to quantitatively measure dcf1 gene expression. Both psiRNA-1 and psiRNA-2 effectively silence dcf1 (e)