Figure 6. Directing Prmt2 to β-catenin target gene promoters is sufficient to drive dorsal specification.

(A) Schematic of the Prmt2:ΔNLef1 chimeric construct. To direct Prmt2 to Tcf/Lef DNA binding sites, the DNA-binding HMG domain of mouse Lef1 was fused to the C-terminus of mouse Myc-Prmt2. (B) 4-cell embryos were injected with either wild-type (WT) or catalytically inactive SAM-binding mutant G159,161R (GG) Prmt2:ΔNLef1 into two ventral blastomeres. Phenotypes (left panel) were scored at late-neurula / early tailbud stages. Mean frequency of secondary axis formation (both fully and partially extended) resulting from expression of wild-type or GG mutant Prmt2:ΔNLef1 is plotted on the right. N=197, 172, and 81 embryos for control, WT, and GG mutant, respectively. Error bars are SEM. P=0.017 (two-tailed Student’s T-Test) for the 4 independent trials where WT and mutant were compared directly. Equal expression of wild type and GG mutant Prmt2:ΔNLef1 was verified by western blot for the myc tag (inset). (C) Embryos were depleted for β-catenin (β-MO, panels ii-iv) and subsequently injected with 500pg of either Prmt2:ΔNLef1 (iii) or Prmt5:ΔNLef1 (iv) mRNA. Rescue of β-MO-induced ventralization (ii) was measured at tadpole stages. Note the rescue of the anterior-most, dorsally derived cement gland and eye in panel iii, compared to control, non-injected embryos (i). The percentages in the upper right corner of each panel indicate the frequency at which the phenotypes shown were observed. (D) Embryos were depleted for β-catenin (β-MO) and subsequently injected with Prmt2:ΔNLef1 or ΔNLef1 mRNA as in (C). Expression of Siamois and Xnr3 was measured by RT-PCR at stage 10. EF1α expression is shown as a loading control. (E) Prmt2:ΔNLef1 and Carm1:ΔNLef1 mRNAs (500pg) were injected and RT-PCR was performed as described for C and D.