| Part I Stat3 expression and function in vertebrate central nervous system development and in COS7 cellsSignal transducer and activator of transcription 3 (Stat3) plays a central role in mediating cell growth, differentiation, survival and movement signals. Target disruption of mouse Stat3 gene leads to early embryonic lethality, implicating its critical role in embryonic development. Here, we showed that Stat3 protein localized in different regions of mouse brain, neural tube and eye from embryonic day 10.5 to postnatal day 1. The distribution of active form of Stat3, tyrosine phosphorylated Stat3Y705, was more restricted in developing neural tube, compared with Stat3 protein. Western blots showed that both Stat3 and Stat3Y705 existed in the developing neural tube, cerebrum, cerebellum, lens and retina from E10.5 to P20. The DNA binding activity of Stat3 from developing central nervous system (CNS) and eye was tested by electrophoretic mobility shift assay, which showed that Stat3 could bind consensus sis-inducible element (SIE) to form DNA-protein complexes. In ovo electroporation of chick embryos reveals that endogenous chick Stat3 could drive SIE-sequence directed reporter gene expression, while dominant negative mouse Stat3 could block this activity. These results demonstrate that active Stat3 is present in the developing CNS and eye and participates in the formation of transcription complexes, and suggest that Stat3 might play some crucial roles during the development of CNS and eye. Using immunocytochemistry and western blot, we have showed that active form of Stat3 protein existed in COS7 cells. The endogenous Stat3 protein could induce the expression of m67-sequence directed reporter genes. Transient transfection of Stat3 cDNA into COS7 cells increased Stat3 protein level and also the reporter gene expression. Overexpression of Stat3 protein in COS7 cells caused prominent morphological changes. These cells had a much larger cell body, extended long processes with branches, lamellipodia and filopodia. Introduction of Stat3F, a dominant negative form of Stat3 protein, into COS7 cells could block Stat3 activation, but still led to similar morphological changes. These results suggest that Stat3 protein may play important roles in cytoskeleton reorganization,cell adhension and migration. Key words: Stat3; Stat3Y705; central nervous system; eye; development; in ovo electroporation; COS7 cell; overexpression; cell morphology; Stat3FPart II Expression and function analysis of mouse nestin cDNA Nestin, a member of type VI intermediate filament protein, is a specific marker for neural stem cells. After rat, human and hamster nestin gene were isolated, we have recently cloned mouse nestin cDNA. The complete cDNA comprised 5983 base pairs and encoded 1821 amino acids. The expression vector containing mouse nestin cDNA was transfected into NIH/3T3 cells to establish stable cell lines. Nestin mRNA could be detected in these cells by RT-PCR, while the nestin protein could not be detected by western blot. We deleted the 5' UTR and 3' UTR sequences from mouse nestin cDNA, and inserted mouse nestin cDNA ORF into EGFP-C1 vector to make an EGFP-nestin fusion proteinexpression vector. When transfected into COS7 and NIH/3T3 cells, nestin protein could be expressed and appeared in the filamentous cytoskeletal network. These results indicate that the mouse nestin cDNA could be expressed as active intermediate filament protein in transfected mammalian cells and could be integrated into cytoskeleton. The neuronal growth cone, a highly motile structure at the distal tip of growing axons, mediated neurite outgrowth and axon guidance. Filamentous actin and microtubules are its main cytoskeletal components. Using immunocytochemistry, we observed that nestin protein appears to be strongly expressed in neurites and growth cones of neurons differentiating from P19 embryonic carcinoma cells in vitro. Double-staining of nestin and microtubule-associated protein 2 as well as nestin and grow...
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