Caihong Xia | PubMed Health

PubMed2006

Cell aggregation-induced FGF8 elevation is essential for P19 cell neural differentiation.

FGF8, a member of the fibroblast growth factor (FGF) family, has been shown to play important roles in different developing systems. Mouse embryonic carcinoma P19 cells could be induced by retinoic acid (RA) to differentiate into neuroectodermal cell lineages, and this process is cell aggregation dependent. In this report, we show that FGF8 expression is transiently up-regulated upon P19 cell aggregation, and the aggregation-dependent FGF8 elevation is pluripotent stem cell related. Overexpressing FGF8 promotes RA-induced monolayer P19 cell neural differentiation. Inhibition of FGF8 expression by RNA interference or blocking FGF signaling by the FGF receptor inhibitor, SU5402, attenuates neural differentiation of the P19 cell. Blocking the bone morphogenetic protein (BMP) pathway by overexpressing Smad6 in P19 cells, we also show that FGF signaling plays a BMP inhibition-independent role in P19 cell neural differentiation.

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PubMed2006

Visualization of bHLH transcription factor interactions in living mammalian cell nuclei and developing chicken neural tube by FRET.

Members of the basic helix-loop-helix (bHLH) gene family play important roles in vertebrate neurogenesis. In this study, confocal microscopy-based fluorescence resonance energy transfer (FRET) is used to monitor bHLH protein-protein interactions under various physiological conditions. Tissue-specific bHLH activators, NeuroD1, Mash1, Neurogenin1 (Ngn1), Neurogenin2 (Ngn2), and ubiquitous expressed E47 protein are tagged with enhanced yellow fluorescence protein (EYFP) and enhanced cyan fluorescence protein (ECFP), respectively. The subcellular localization and mobility of bHLH fusion proteins are examined in HEK293 cells. By transient transfection and in ovo electroporation, four pairs of tissue-specific bHLH activators and E47 protein are over-expressed in HEK293 cells and developing chick embryo neural tube. With the acceptor photobleaching method, FRET could be detected between these bHLH protein pairs in the nuclei of transfected cells and developing neural tubes. Mash1/E47 and Ngn2/E47 FRET pairs show higher FRET efficiencies in the medial and the lateral half of chick embryo neural tube, respectively. It suggests that these bHLH protein pairs formed functional DNA-protein complexes with regulatory elements of their downstream target genes in the specific regions. This work will help one understand the behaviours of bHLH factors in vivo.

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