Fig. 3.
Tbx6 protein suppresses ntl-dependent functions. (A) Suppression of notochord development. collagen type 2A (col2A) expression in the floorplate and hypochord is used to outline the notochord in lateral views of the trunk/tail region of day-1 embryos. ntl mutant embryos lack differentiated notochord. Embryos that were injected at the one- to two-cell stage (+RNA) with 100 pg of ntl-EnR-myc or 100–200 pg of tbx6-myc RNA have reduced notochord development. The tail region is greatly reduced and either entirely lacks or contains isolated islands of col2A-expressing cells. The fraction of affected embryos depended on the amount of injected tbx6-myc RNA: 100 pg of tbx6-myc RNA yielded 37% (n = 46) with truncated notochord, 200 pg of tbx6-myc RNA yielded 57% (n = 46), and 400 pg of tbx6-myc RNA yielded 80% (n = 46). (B) Tbx6 suppresses MyoD expression in adaxial cells. WT MyoD expression in adaxial cells bordering the presumptive notochord of 80% epiboly embryos is absent in ntl mutant embryos. Adaxial MyoD expression (purple staining) is absent in embryos that express Ntl-EnR-myc or Tbx6-myc (brown staining) dorsally. Ectopic expression of WT Tbx6 also suppresses MyoD expression. Ninety-four percent (n = 66) of embryos injected with 100 pg of tbx6-myc RNA and 86% (n = 102) of embryos injected with 200 pg of tbx6 RNA exhibited suppression of MyoD expression. (C) Tbx6 blocks No Tail-dependent activation of MyoD transcription in animal caps. One- to two-cell embryos were injected with 50 pg of ntl-myc RNA along with either ntl-EnR-myc RNA or tbx6-myc RNA. Data indicate the amount of MyoD transcription relative to that caused by 50 pg of ntl-myc RNA. Replicate experiments yielded similar results. (D) Overexpression of No Tail counteracts the ability of Tbx6 to suppress MyoD expression in adaxial cells. Adaxial MyoD expression in an embryo injected at the one-cell stage with 100 pg of ntl-myc RNA and 200 pg of tbx6-myc RNA. Brown immunostaining indicates that ectopic proteins are expressed widely, including the dorsal mesoderm. (E) Transcriptionally inactive forms of Tbx6 can antagonize ntl. Ectopic expression of Tbx6DBD-myc or Tbx6-EnR-myc protein on the dorsal side of the embryo suppresses MyoD expression in adaxial cells. Suppression was observed in 18 of 20 embryos injected with 100 pg of tbx6DBD-myc RNA, and 42 of 46 embryos injected with 100 pg of tbx6-EnR-myc RNA. Dotted lines indicate blastoderm margins. Solid lines indicate dorsal midlines.