in situ hybridization and immunostaining were performed to analyze expression of relevant gene transcripts and protein products , respectively . targeted knockout mice were used to analyze the consequence of loss in embryo of tbx6 and/or sox2 enhancer n1 , and transgenic mice to monitor enhancer n1 activity or to ectopically express sox2 . whole mount in situ hybridization was done as previously described by wilkinson26 with the following modifications : proteinase k treatment was done at 5 g / ml ( e8 embryos ) or 10 g / ml ( e9 embryos ) for 5min ; incubation with glycine after proteinase k treatment was omitted ; digoxigenin - labeled probes were used at 0.5 g / ml ; and anti - digoxigenin antibody was reacted in 1.5% blocking reagent ( roche ) . the stained embryos were photographed in 80% glycerol / pbt , then embedded in paraffin and sectioned . the probes used : tbx613 , pax627 , uncx4.128 , foxf129 , pax230 , foxa131 , wnt8a32 , fgf833 , fgf434 , sox1 3 utr ( stui - xhoi fragment ) , sox2 ( sacii - acci coding sequence fragment ) , sox3 ( 627 - 1128 bp , genbank nm_009237 ) , pax3 ( hindiii - psti coding sequence fragment ) , mox1 ( 1688 - 2235 bp , genbank nm_010791 ) , egfp ( full coding sequence ) , and wnt3a ( a combination of probes for wnt3a 3utr35 and the wnt3a coding sequence ) . embryos were fixed overnight with 4% pfa in pbs , immersed in 15% and 25% sucrose in pbs in sequence for 2 - 3 hr each step , and embedded in oct compound . cryosections with thickness of 10 m were prepared , and treated at 105 c for 15 min in antigen unmasking solution ( vector laboratories ) using an autoclave , which also quenched venus fluorescence . the sections were reacted with 10% normal donkey serum for 30 min , then with the primary antibodies overnight at 4 c . the primary antibodies , used at 1:200 dilutions , were : rabbit anti - laminin ( l9393 sigma ) , goat anti - sox2 ( af2018 r&d systems ) , rabbit anti - pax6 ( prb-278 covance ) , and rat anti - ha ( 1867423 roche ) . after several washes , the samples were incubated with fluorescent dye - coupled secondary antibodies for 1 hr at ambient temperature . the following donkey antibodies were used at 1:200 dilutions : alexa fluor 488-labeled anti - goat igg ( a-11055 molecular probes ) , alexa fluor 555-labeled anti - rabbit igg ( a-31572 molecular probes ) , and cf 633-labeled anti - rat igg ( 20137 biotium ) . after several washes the samples were stained with 0.5 g / ml hoechst33342 , and mounted in permafluor ( thermo scientific ) . transgene dna constructs were linearized by digestion with restriction enzyme , freed from vector sequences . injection of dna ( 4 ng/l ) into the male pronucleus of fertilized eggs and lacz enzyme staining were done using the standard procedures36 . n1-tkegfp were used to establish transgenic lines , while other transgenes were used in primary transgenic embryos . the integration of transgenes into embryo genomes was determined by pcr amplification of the lacz or egfp / venus sequences from yolk sac dna . embryo culture and mesodermal transfection with pcaggs - cnoggin4 the targeting vector used to create the enhancer n1 mutation was constructed by using a 15 kb mouse dna fragment , which including enhancer n1 , derived from the bac clone rp23 - 274p9 ( bacpac resource center , children s hospital oakland research institute , ca , usa ) . the stneob cassette38 was inserted 3 of enhancer n1 , loxp sequences were inserted to flank these sequences , and a dt - a cassette39 was inserted at the 3 terminus of the vector . the linearized vector was electroporated into r1 es cells , which had been engineered to have ires - egfp immediately downstream of the sox2 orf . after germ - line transmission from chimeras , heterozygous mice were crossed with caggs - cre mice40 to obtain the n1-deleted allele of sox2 . a schematic of the procedure full length tbx6 ( 436 a.a . ) was synthesized using a tnt system ( promega ) , and emsa analysis was performed as described previously41 using 0.1 g/l poly[di - dc ] as a non - specific competitor .