Figure 6. The involvement of Tbx24 (A–F), Notch signaling (G–L), and retinoic acid (RA) signaling (M–R) in endogenous medaka mesp-b expression and in transgene expression driven by both wild-type and mutant 2.8 kb mesp-b enhancer fragments. Dorsal views of the medaka embryos at stage 22 are shown with the anterior towards the top. In the case of the transgenic animals, the enhancer that drives EGFP reporter expression is indicated at the top right corner. (A–F) The effect of tbx24 gene knockdown. Control embryos injected with control morpholino oligonucleotide (MO) (A,C,E) and embryos injected with MO-tbx24 (B,D,F) are shown. Injection of MO-tbx24 abolishes both endogenous mesp-b expression and transgene expression. Morphologically, MO-tbx24 also completely inhibits somite segmentation (D,F). (G–L) The effects of N-(N-(3,5-difluorophenacetyl-L-alanyl))-S-phenylglycine t-butyl ester (DAPT; an inhibitor of Notch signaling) treatment upon mesp-b expression. Embryos treated with dimethylsulfoxide (DMSO; control; G,I,K) and with DAPT (H,J,L) are shown. DAPT treatment reduces the expression levels of both endogenous and transgene expression of mesp-b. Note that DAPT treatment also causes the formation of a salt-and-pepper pattern for both endogenous and transgene expression, driven by both the 2.8 kb wild-type and dN1+2 mesp-b enhancer fragment lacking both RBPJκ binding sites. In contrast, GFP expression driven by the dN1+2 enhancer maintains a segmental pattern (compare K′ with L′). DAPT treatment disturbs somite boundary formation, particularly in the posterior trunk region (J,L). (J′,K′,L′) show enlarged images taken after longer exposures. (M–R) The effect of disulfiram (an inhibitor of RA synthesis) treatment on mesp-b expression in the medaka embryo. Embryos treated with DMSO (control; M,O,Q) and with disulfiram (N,P,R) are shown. Disulfiram treatment was found to reduce both endogenous and transgene mesp-b expression. Note that the dR enhancer lacking the RARE motif still responds to disulfiram treatment (R′). Disulfiram treatment also occasionally induces fusion of the somites (R) in addition to left–right asymmetric somitogenesis (P). 2.8 indicates the embryo carrying the 2.8 kb mesp-b enhancer construct, T1±2 indicates the embryo with dT1+2 enhancer which lack both the two T-box binding sites and N1+2 indicates the embryo carrying the dN1+2 enhancer which lacks both the two RBPJκ binding sites. Bars, (A,B,G,H,M,N) 0.1 mm; (C–F,I–L,O–R) 0.1 mm; (J′,K′,L′) 0.05 mm.
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