Fig. 4

Homozygous deletion of TR1 significantly reduces the expression of tbx6 and leads to abnormal expression patterns of segmentation genes similar to tbx6 null mutants at the 12-somite stage. (A) Whole-mount in situ hybridization revealed the expression patterns of endogenous tbx6 in sibling (tbx6^+/+; n=15) and tbx6^ΔTR1/ΔTR1 (n=16) embryos during embryogenesis. All of the photos were taken at the same magnification from dorsal views. (B) Immunostaining of Tbx16 (green) and Tbx6 (magenta) in the PSM of sibling (n=5) and tbx6^ΔTR1/ΔTR1 (n=6) embryos at the 12-somite stage. Dorsal views of flat-mount embryos after yolk removal. Anterior is towards the top. Around the posterior edge of the tailbud, a background signal was observed for Tbx6 signal. (C) Whole-mount in situ hybridization was performed to compare the expression patterns of genes involved in somite segmentation. Expression patterns of mesp-ba (wild type, n=11; tbx6^ΔTR1/ΔTR1, n=11; tbx6^−/−, n=9), mesp-bb (wild-type, n=11; tbx6^ΔTR1/ΔTR1, n=11; tbx6^−/−, n=9), her1 (wild-type, n=10; tbx6^ΔTR1/ΔTR1, n=11; tbx6^−/−, n=9), papc (wild-type, n=10; tbx6^ΔTR1/ΔTR1, n=11; tbx6^−/−, n=9), fgf8a (wild-type, n=11; tbx6^ΔTR1/ΔTR1, n=11; tbx6^−/−, n=9), tbx18 (wild-type, n=11; tbx6^ΔTR1/ΔTR1, n=11; tbx6^−/−, n=9), myod (wild-type, n=11; tbx6^ΔTR1/ΔTR1, n=11; tbx6^−/−, n=9) and uncx4.1 (wild-type, n=11; tbx6^ΔTR1/ΔTR1, n=11; tbx6^−/−, n=9) were analyzed at the 10- to 12-somite stage. The arrowheads indicate the expression of segmentation genes in the anterior PSM. Dorsal views. For the somite polarity marker, magnified views around the newly formed somites are shown in the inset. After in situ staining, the genotype of each embryo was determined. Scale bars: 100 µm.