We have used spatially and temporally controlled Tsc1 gene deletion to address how altered

thalamic development has the potential to perturb widespread neural function and behavior. To temporally and spatially control Tsc1 gene deletion, we combined three genetically modified mouse alleles (see Figure S1A available online): (1) Gbx2CreER, which targets CreER expression to thalamic cells ( Chen et al., 2009); (2) Tsc1fl, which is converted into a null allele (Tsc1Δ) by Cre-mediated recombination ( Kwiatkowski et al., 2002); PR-171 purchase and (3) either R26LacZ ( Soriano, 1999) or R26tdTomato ( Madisen et al., 2010), which produce β-galactosidase (β-gal) or red fluorescent protein (RFP), respectively, upon Cre-mediated recombination. CreER remains quiescent until it is transiently activated by tamoxifen. Subsequently, the Tsc1fl gene is permanently converted to Tsc1Δ and the conditional reporter genes

are permanently activated in the thalamus ( Figures S1B and S1C). Gbx2CreER expression has been reported in the spinal cord ( Luu et al., 2011) but, within the brain, regions outside of the thalamus had only very sparse recombination with tamoxifen at E12.5 ( Figure S1). We validated the fidelity of Tsc1fl recombination in the thalamus Fasudil in vivo compared to the neocortex ( Figures S1D and S1E). Operationally, we use Tsc1ΔE12/ΔE12 to indicate mutant animals that received tamoxifen on embryonic day (E) 12.5 and Tsc1ΔE18/ΔE18 to indicate mutants that received tamoxifen on E18.5. We first performed genetic inducible fate mapping on Gbx2CreER;R26LacZ animals to characterize the extent, spatial distribution, and molecular identity of recombined cells ( Figure 1). We administered tamoxifen to pregnant females carrying Gbx2CreER;R26LacZ embryos at E12.5 or E18.5 and determined the long-term lineage contribution

to the thalamus. Postnatal brain sections were analyzed by immunohistochemistry (IHC) for β-gal expression from the activated R26LacZ allele. E12.5 fate-mapped cells (green) were distributed widely throughout the full medial-lateral extent of the thalamus ( Figures 1A–1F). In animals that received tamoxifen at E18.5, the spatial extent of recombination was reduced ( Figures 1G–1L). Regions that underwent recombination at both E12.5 and E18.5 include the anteromedial and mediodorsal nuclei. The ventrolateral, ventromedial, Temozolomide ventrobasal, laterodorsal, and the lateral geniculate nuclei underwent recombination at E12.5 but were not marked at E18.5. Nuclei that underwent extensive recombination early (E12.5) and moderate mosaic recombination later (E18.5) include the posterior nucleus and the medial geniculate nucleus. We investigated whether recombination occurred in a particular cell type by IHC for β-gal in combination with parvalbumin (PV, red, Figures 1A–1C and 1G–1I) or calbindin (Calb, red, Figures 1D–1F and 1J–1L). Within relay nuclei, β-gal+ cells contributed to both Calb− and Calb+ cells at both E12.5 and E18.5 ( Figures 1D–1F and 1J–1L, arrowheads).