The WNK-OSR1-Lhx8 pathway is involved in the specification of neural fate.

<p>(A) Western blotting analysis of phospho-SPAK/OSR1, as an indicator of WNK signaling pathway activity in differentiating Neuro2A cells by retinoic acid (RA). The value obtained from each samples was normalized to the level of SPAK or OSR1. The value of phospho-SPAK or OSR1 at 0 minutes from undifferentiated cells (lane 1) was set to 100. (B) Expression of <i>Lhx8</i> induced by RA. The value obtained from each samples was normalized to the level of <i>GAPDH</i>. The value of <i>Lhx8</i> at 24 hours after induction (lane 4) was set to 100. (C–G) Differentiation of siRNA-treated Neuro2A cells induced by RA for 24 hours; (C) Control siRNA, (D) si<i>Wnk1</i>, (E) si<i>Wnk4</i> or (F) si<i>Wnk1</i>+si<i>Wnk4</i>. (G) Gene expressions by RT-PCR or quantitative RT-PCR analysis were examined in Neuro2A cells. Cells treated with siRNA against <i>Lhx8</i> (si<i>Lhx8</i>); (lanes 1–2) undifferentiated cells, (lanes 3–4) cells differentiated by RA for 24 hours. The value obtained from each samples was normalized to the level of <i>GAPDH</i>. The value of <i>ChAT</i> or <i>Gad1</i> from differentiated cells under the treatment of control siRNA (lane 3) was set to 100. (H–I) Neuro2A cells were transfected with various combinations of siRNAs and expression plasmids and gene expressions by RT-PCR or quantitative RT-PCR analysis were examined. Cells were treated with control siRNA (Control) in lanes 1,2,9,10, with si<i>Wnk1</i>in lanes 3,4,11,12, with si<i>Wnk4</i> in lanes 5,6,13,14, and with both <i>siWnk1 and siWnk4</i> in lanes 7,8,15,16. Cells were also transfected with control expression vector pRK5 in lanes 1,3,5,7,9,11,13,15 and with mOsr1^S325D (H) or Lhx8 (I) overexpression vector in lanes 2,4,6,8,10,12,14,16. Cells were left undifferentiated in lanes 1–8 and differentiated cells by RA for 24 hours (H) or 48 hours (I) in lanes 9–16. The value obtained from each samples was normalized to the level of <i>GAPDH</i>. The value of <i>Lhx8</i>, <i>ChAT</i> or <i>Gad1</i> from differentiated cells under the treatment of control siRNA (lane 9 in both H and I) was set to 100.