There were increased numbers of scattered subpallial Nkx2-1+, Som+, and SOX6+ cells, particularly in caudal regions of the basal ganglia (arrowheads, Figures 2F and 2F′, 2O and 2O′, S2, and S3). Furthermore, many PLAP+ cells failed to migrate from the MGE; these formed a large collection of cells in the SVZ of the dorsal MGE (ectopia [E]; Figures 3F

and 2F′). The cells in the ectopia expressed Dlx1 and Gad1 and did not express Nkx2-1, Calbindin, and SOX6, suggesting that they had properties of the LGE/dCGE rather than the MGE ( Figure S3). The Lhx6PLAP/PLAP;Lhx8−/− MGE ectopia was much more prominent than in Lhx6PLAP/PLAP mutant ( Zhao et al., 2008). Like the Lhx6PLAP/PLAP mutant, the double mutant continued to have tangentially migrating interneurons expressing Arx, Dlx1, and Gad1 ( Figures S2 and S3) presumably ALK inhibitor originating from the LGE/dCGE. Normally, Nkx2-1 expression is maintained only in interneurons migrating to the striatum and projection neurons of the basal telencephalon and septum ( Marín et al., 2000, Marín and Rubenstein, 2001 and Nóbrega-Pereira et al., 2008). GDC-0449 However, at E14.5 there were ectopic Nkx2-1+ cells in the caudal regions of

the external capsule (arrow) and ventrolateral cortex in the Lhx6PLAP/PLAP and Lhx6PLAP/PLAP;Lhx8−/− mutant and increased numbers in the striatum (arrowhead, Figures 3C, 3C′, and S2). By E18.5 there were ectopic NKX2-1+ cells in the cortical SVZ and the hippocampus of the Lhx6PLAP/PLAP, Lhx6PLAP/PLAP;Lhx8+/−, and Lhx6PLAP/PLAP;Lhx8−/− mutant ( Figures 3G, 3G′, and S3). They were most prevalent in the Lhx6PLAP/PLAP;Lhx8+/− mutant, which too also had increased numbers of NKX2-1+ cells in the SVZ of the LGE (data not shown), suggesting that these cells were in transit along their migration from the MGE to the cortex. Thus, Lhx6 and Lhx8 are required to prevent NKX2-1 expression in pallial interneurons. The ectopic NKX2-1+ cells accumulated in stratum radiatum of the hippocampus; this region also accumulated cells

expressing Lhx6-PLAP, Calbindin, Dlx1, Gad1, and Npas1, but did not express SOX6 ( Figures 3G, 3G′, and S3). Because the mutants die at P0, we have not identified their identity at maturity. The phenotype of the Lhx6PLAP/PLAP;Lhx8−/− mutant is probably the combination of cell autonomous defects in cells lacking these transcription factors and cell nonautonomous effects due to the loss of Shh expression in the MZ of the MGE ( Figure 1). While the effect of removing Shh expression from the VZ of the MGE/POA has been established to alter properties of MGE progenitors ( Gulacsi and Anderson, 2006, Xu et al., 2005 and Xu et al., 2010), the function of Shh in postmitotic neurons of the MGE is unknown. Shh is transiently expressed in most MGE neurons from ∼E10–E12 ( Figures 1A–1C; Sussel et al., 1999 and Flandin et al., 2010).