Fig. 3: Generation of multiple type II Cdh protein null mutant mice.

a Exonic regions encoding the translation start site of each Cdh are targeted by CRISPR/Cas9 system to introduce small deletions for the immediate frameshift. Primers for genotyping are indicated by arrows. Positions of the beta-galactosidase (LacZ) reporter modified Cdh6-5′ BAC and Cdh8-5′ BAC constructs are shown as black bars. Position of the EGFP knock-in at the Cdh11 locus is shown as bright green bar. Arrowheads, single guide (sg) RNA target sites. b Results from western blotting analyses for each Cdh and β-actin are arranged. Note that Cdh single KO mice generated are all protein null. c, d Staining pattern from the Cdh6-5′ BAC-Tg completely recapitulates Cdh6 mRNA profiles in the early forebrain (Fb) compartment at E8.5. c, d dorsal view; c′, d′ ventral view. The ratio of embryos exhibiting the Fb-specific expression pattern at E8.5 over the total number of transgenic founders is noted at the bottom left corner of c. e Pax6 whole-mount immunostaining pattern is also identical to Cdh6 mRNA profiles in the early Fb. f A Cdh8-5′ BAC-Tg embryo at the 22-somite stage (s) harbors strong enhancer activity at the Mb. The ratio of embryos exhibiting the Mb-specific expression pattern at this stage over the total number of transgenic founders is noted at the bottom left corner of the f. Section levels for g–l are indicated by white dashed lines g–l, respectively. Scale bar: 250 μm. g–l Expression patterns of Cdh8 mRNA k, l are recapitulated by LacZ staining from Cdh8-5′ BAC-Tg embryo at the 22s g–j. A white arrowhead indicates Cdh8 mRNA or LacZ negative zone in the roof plate. p, prosomere; rp, roof plate. Scale bar: 100 μm. m–q Cdh8-5′ BAC-Tg mice show progressive activation of the reporter correlated with neural tube closure. Scale bar: 250 μm. n′, En face view of the 9s embryo reveals the LacZ expression in the Mb respecting the Fb/Mb compartment boundary (white dotted line). r A section level for s–u are indicated by the black line in a whole-mount stained embryo by Pax6. s, t A Cdh8-5′ BAC-Tg embryo at the 9s is immunostained with Pax6 and LacZ. u Enlarged views of the Fb/Mb compartment boundary outlined by the white box u in the s, t are arranged. Note that Pax6 and Cdh8::LacZ expressions are mutually exclusive at the Fb/Mb boundary. v Cdh6 and Cdh8 enhancer activities at the nascent Fb/Mb compartment boundary are summarized. Noticeably, these two Cdh enhancer activities oppose each other at the dorsal portion of the compartment boundary.