OBJECTIVETo investigate the transcription of cytoskeleton protein genes in differentiation of neurons from mouse embryonic stem (ES) cells induced by all-trans retinoic acid (RA), and to explore the possibility of setting up a method to screen small molecules with promoting or inhibiting effect.

METHODSThe hanging drop method was employed for embryonic body formation to mimic embryo development in vivo. Reverse transcriptase PCR (RT-PCR) was performed to investigate mRNA expression of the neuron-specific cytoskeleton proteins including Mtap2, Nefm and beta-tubulin III which were regarded as the inducing effect indexes of RA. Morphological evaluation and immunocytochemistry staining were conducted to identify the neural derivatives. Moreover, the inducing effects of six synthetic molecules were further evaluated.

RESULTRA up-regulated the mRNA expression of Mtap2 and Nefm, especially Mtap2 increased by 1.27 times, which was consistent with the morphological alteration. However, there was no significant changes of beta-tubulin III expression. With addition of the six synthetic molecules, the transcription of Mtap2 was inhibited, while the Nefm mRNA expression was up-regulated in some degree, especially for molecule 1 and 3 that was increased by 1.4 and 1.2 times, which, however, was not parallel to the morphological changes.

CONCLUSIONThe transcriptional levels of Mtap2 and Nefm are both up-regulated in the RA-induced differentiation of ES cells towards neurons. The up-regulation of Mtap2 is consistent with the morphological alteration, which might be the key landmark in the RA-induced differentiation of ES cells into neurons.