Inhibition of mouse GPM6A expression leads to decreased differentiation of neurons derived from mouse embryonic stem cells.
Glycoprotein M6A (GPM6A) is known as a transmembrane protein and an abundant cell surface protein on neurons in the central nervous system (CNS). However, the function of GPM6A is still unknown in the differentiation of neurons derived from embryonic stem (ES) cells. To investigate the function of GPM6A, we generated knockdown mouse ES cell lines (D3m-shM6A) using a short hairpin RNA (shRNA) expression vector driven by the U6 small nuclear RNA promoter, which can significantly suppress the expression of mouse GPM6A mRNA. Real-time polymerase chain reaction (real-time PCR) and immunocytochemical analysis showed that expression of shRNA against GPM6A markedly reduced the expression of neuroectodermal-associated genes (OTX1, Lmx1b, En1, Pax2, Pax5, Sox1, Sox2, and Wnt1), and also the number of neural stem cells (NSC) derived from D3mshM6A cells compared to control vector-transfected mouse ES cells (D3m-Mock). Moreover, our results show a decrease in both the number of neuronal markers and the number of differentiating neuronal cells (cholinergic, catecholaminergic, and GABAergic neurons) from NSC in D3m-shM6A cells. Hence, our findings suggest that expression level of GPM6A is directly or indirectly associated with the differentiation of neurons derived from undifferentiated ES cells.
Michibata
Hideo
H
Advanced Medical Research Laboratory, Mitsubishi Tanabe Pharma Corporation, 16-89 Kashima-3-chome, Yodogawa-ku, Osaka, Japan.
Okuno
Tsuyoshi
T
Konishi
Nae
N
Wakimoto
Koji
K
Kyono
Kiyoshi
K
Aoki
Kan
K
Kondo
Yasushi
Y
Takata
Kazuyuki
K
Kitamura
Yoshihisa
Y
Taniguchi
Takashi
T
D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis* / Q000235:genetics; D019556:COS Cells; D002454:Cell Differentiation / Q000502:physiology*; D002490:Central Nervous System / Q000166:cytology / Q000378:metabolism; D002522:Chlorocebus aethiops; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012342:RNA, Small Nuclear / Q000235:genetics
D000943:Antigens, Differentiation; C083480:Gpm6a protein, mouse; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA