[The effect of protein kinase C alpha on expression of developmental genes during differentiation of mouse embryonic stem cells into neuron-like cells in vitro].

ABSTRACT

OBJECTIVE: To study the effect of protein kinase C alpha on the expression of developmental genes pax-6, slit-2 and netrin-1 during differentiation of mouse embryonic stem cells into neuron-like cells in vitro, in an attempt to elucidate their roles in signaling. METHODS: ES-BALB/c cells were induced to form embryoid bodies in the ES conditioned medium for 4 days, and were plated separately on coated glass coverslip into 6-well culture dishes for immunohistochemical study and into 100 mm dishes for RT-PCR assay. These cultures were collected after 1, 3, 5, 7 and 14 days in the presence of 5 x 10(-7) mol/L retinoic acid (RA). The neuron-like cells were stained with antibody to NSE and NF-200. mRNA level of the development related genes (pax-6, slit-2 and netrin-1) in undifferentiated and differentiated ES cells was assessed by RT-PCR assay. Effects of PMA and D-sphingosine on the developmental genes were also observed. RESULTS: Most of the neuron-like cells stained with the antibodies to NSE and NF-200. RT-PCR assay showed that levels of PKC alpha, pax-6 and netrin-1, but not slit-2 transcripts decreased dramatically upon induction on the 1st day, but then raised slowly and resumed to normal level on 14th day. PMA upregulated the levels of PKC alpha, pax-6 and netrin-1; while D-sphingosine downregulated their levels. CONCLUSIONS: The results of the present experiments demonstrate that the developmental genes pax-6 and netrin-1 play a very important role during differentiation of mouse ES cells into neuron-like cells through PKC alpha signaling pathway.