[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].
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.
Gao
Qian-ying
QY
Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China.
Ge
Jian
J
Wang
Zhi-chong
ZC
Chen
Hui-yi
HY
Huang
Dan-ping
DP
Yuan
Zhao-hui
ZH
D000818:Animals; D002460:Cell Line; D004627:Embryonic Induction; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D009414:Nerve Growth Factors / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics; D000075388:Netrin-1; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics; D051571:Protein Kinase C-alpha / Q000494:pharmacology*; D012097:Repressor Proteins / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics; D000098552:Slit Homolog 2 Protein
D005136:Eye Proteins; D018398:Homeodomain Proteins; D036341:Intercellular Signaling Peptides and Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; C000617353:Ntn1 protein, mouse; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; D025521:Tumor Suppressor Proteins; D000075388:Netrin-1; D051571:Protein Kinase C-alpha; D000098552:Slit Homolog 2 Protein