Blythe SA , Cha SW , Tadjuidje E , Heasman J , Klein PS .

R01-GM76621 NIGMS NIH HHS , T32-GM007229 NIGMS NIH HHS , T32-HD007516 NICHD NIH HHS , R01 GM076621-01A1 NIGMS NIH HHS , R01 GM076621-02 NIGMS NIH HHS , R01 GM076621-03 NIGMS NIH HHS , R01 GM076621-04 NIGMS NIH HHS , R01 GM076621 NIGMS NIH HHS , T32 HD007516 NICHD NIH HHS , T32 GM007229 NIGMS NIH HHS

	Figure 1. b-Catenin Target Genes Are Poised for Expression before the MBT (A) Onset of expression of maternal b-catenin target genes (Siamois, Xnr3, Xnr5, and Xnr6). Maternally expressed Ornithine decarboxylase (Odc) is shown as a control for loading. Odc (-RT) indicates no reverse transcriptase, as a control for genomic DNA contamination. (B) Embryos were injected at the two-cell stage with the b-catenin morpholino (Biiiv) and subse- quently at the four-cell stage (two dorsal blasto- meres) with 2 pg siamois (Biii) or 300 pg b-catenin (Biv) mRNAs. The frequency of each representa- tive phenotype is indicated. (C) Embryos were injected into two dorsal blasto- meres at the four-cell stage with 500 pg DNTcf3- GR mRNA. Wnt/b-catenin activity was inhibited by addition of dexamethasone (Dex) to the culture medium at the indicated stages. Siamois, Xnr3, and ODC mRNA expression was measured by RT-PCR at stage 10. (D) Left panel: Occupancy of initiating (CTD pSer5) RNA Pol II in the promoter-proximal regions of the Siamois and Xnr3 loci before (1000-cell stage) or after (Stage 9) the onset of expression as measured by ChIP-QPCR. Right panel: Elongating (CTD pSer2) RNA Pol II associated with the 30 CDS of the same panel of genes at the same time points. Binding of RNA Pol II at Xnr6, which is expressed at both stages, is a positive control. Pooled data from three independent experiments are presented as a percentage of input chromatin to facilitate comparison between the 1000-cell stage and Stage 9. The average signal (0.02% input) from a negative control (IgG) ChIP is marked as a dotted line. Error bars are SEM from three independent experiments. (E) PreMBT occupancy of maternal b-catenin target promoters (Siamois, Xnr3, and Xnr5) or a zygotic b-catenin target promoter (Myf5) by either H3K9/14ac or b-catenin was observed by ChIP on 1000-cell stage embryos. Myosin light chain 2 (Mlc2): negative control locus. Input indicates chromatin prior to ChIP. (F) Promoter occupancy by b-catenin or H3K4me3 measured by ChIP on 1000-cell embryos. See also Figure S1.
	Figure 5. Maternal Prmt2 Is Necessary for Dorsal Specification (A and B) Transplantation and fertilization of maternal Prmt2-depleted (prmt2-) oocytes results in a range of ventralized tadpole-stage phenotypes (A, lower panel) compared with controls, which develop normally (A, top panel). The mean frequency of phenotypes arising from maternal Prmt2 depletion is plotted in B (see text for details). The frequency of ventralized embryos (both partial and complete) is reduced by co-injection of Prmt2 mRNA (prmt2-/+mRNA). (C) Blastula stage (stage 9) siamois and xnr3 expression was measured in prmt2- and rescued (prmt2- (+mRNA)) host transfer embryos as compared with control (nondepleted) and Prmt2 mRNA injected embryos. As a control for the efficiency of knockdown, prmt2 was measured. Note that the rescuing mRNA (1 ng of mouse Prmt2, injected into oocytes) is not amplified by the Xenopus prmt2 primers used here. Expression of rescuing mRNA was confirmed by western blot (not shown). Embryos expressing Prmt2 mRNA alone developed identically to controls, with no dorsoventral defects (not shown). See also Figure S3.
	Figure 6. Directing Prmt2 to b-Catenin Target Gene Promoters Is Sufficient to Drive Dorsal Specification (A) Schematic of the Prmt2:DNLef1 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) Four-cell embryos were injected with either wild-type (WT) or catalytically inactive SAM-binding mutant G159,161R (GG) Prmt2:DNLef1 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:DNLef1 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 t test) for the four independent trials where WT and mutant were compared directly. Equal expression of wild-type and GG mutant Prmt2:DNLef1 was verified by western blot for the myc tag (inset). (C) Embryos were depleted for b-catenin (b-MO, Ciiiv) and subsequently injected with 500 pg of either Prmt2:DNLef1 (Ciii) or Prmt5:DNLef1 (Civ) mRNA. Rescue of b-MO-induced ventralization (Cii) was measured at tadpole stages. Note the rescue of the anterior-most, dorsally derived cement gland and eye in (Ciii, compared with control, noninjected embryos (Ci). The percent- ages in the upper right corner of each panel indicate the frequency at which the phenotypes shown were observed. (D) Embryos were depleted for b-catenin (b-MO) and subsequently injected with Prmt2:DNLef1 or DNLef1 mRNA as in (C). Expression of Siamois and Xnr3 was measured by RT-PCR at stage 10. EF1a expression is shown as a loading control.

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