Endogenous and ectopic expression of Mgn.

(A) Relative expression levels of Mgn and Mash1 in mutant to wild-type mice. The bar height and error bars show the mean and standard deviation, respectively. (B)Mgn mRNA was expressed at high levels in the vlMB of E12.5 mouse embryos compared with the low level of expression in the dMB determined by ISH, LacZ staining of Mgn^tLacZ mice, and IHC of wild-type embryos with the anti-MGN antibody on coronal sections. The lines indicated the borders of m3-m5 domains. MASH1 protein is also expressed at higher levels in the vlMB compared with its dorsal expression, as determined by IHC with anti-MASH1 antibody. (C) WISH was performed with Gad67 probe in ONTC of wild-type E11.5 embryos with Mgn cDNA electroporated in one of the sides (right side), whereas the other side served as a control (left side). The neural tube is opened along the ventral anterior-posterior axis. Lightning bolt icons point to the ectopic expression of Gad67 after Mgn electroporation. Subdomains are shown according to the prosomeric model [21]. (D) WISH was performed with a Gad67 probe in the ONTCs of E11.5 wild-type mouse embryos after 10 h of incubation. The red dashed line delineates the alar/basal (a/b) boundary. The black dashed lines delineate the transversal neural tube domain boundaries. (E and F) E11.5 Mash1^−/− ONTCs 24 h after micro-electroporation in the mesencephalic alar plate with the Mgn-IRES-EGFP plasmid. (E) A fluorescence signal showing the electroporated location. (F) Induction of Gad67 expression after electroporation of Mgn cDNA in Mash1^−/− background embryos. (F1) A merged profile of the fluorescence signal and Gad67 induction. Scale bars: 500 μm. Abbreviations: a/b, alar/basal plate boundary; d/m, diencephalic–mesencephalic boundary; Hy, hypothalamus; Is, isthmus; p1–p3, prosomeres 1 to 3 in diencephalon; PcP, precommissural domain in the pretectum (p1); rp, roof plate; Zli, zona limitans intrathalamica.