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 .
 the classical view of neural plate development held that it arises from the ectoderm , after its separation from the mesodermal and endodermal lineages . 
 however , recent cell lineage tracing experiments indicate that the caudal neural plate and paraxial mesoderm are generated from common bipotential axial stem cells originating from the caudal lateral epiblast ( cle)1,2 . tbx6 null mutant mouse embryos which produce ectopic neural tubes at the expense of paraxial mesoderm3 must provide a clue to the regulatory mechanism underlying this neural versus mesodermal fate choice . here 
 we demonstrate that tbx6-dependent regulation of sox2 determines the fate of axial stem cells . in wild - type embryos 
 , enhancer n1 of the neural primordial gene sox2 is activated in the cle , and the cells staying in the superficial layer sustain n1 activity and activate sox2 expression in the neural plate4 - 6 . 
 in contrast , the cells destined to become mesoderm activate tbx6 and turn off enhancer n1 before migrating into the paraxial mesoderm compartment . in tbx6 mutant embryos , however , enhancer n1 activity persists in the paraxial mesoderm compartment , eliciting ectopic sox2 activation and transforming the paraxial mesoderm into neural tubes . an enhancer n1-specific deletion mutation introduced into tbx6 mutant embryos prevented this sox2 activation in the mesodermal compartment and subsequent development of ectopic neural tubes , indicating that tbx6 regulates sox2 via enhancer n1 . 
 tbx6-dependent repression of wnt3a in the paraxial mesodermal compartment is implicated in this regulatory process . 
 paraxial mesoderm - specific misexpression of a sox2 transgene in wild type embryos resulted in ectopic neural tube development . 
 thus , tbx6 represses sox2 by inactivating enhancer n1 to inhibit neural development , and this is an essential step for the specification of paraxial mesoderm from the axial stem cells . 
Methods summary
Supplementary Material
In situ hybridization
Immunostaining
Transgenic mouse production, LacZ staining and embryo transfection
Production of enhancer N1 mutant mice
Electrophoretic mobility shift assay (EMSA)