Elkouby YM , Elias S , Casey ES , Blythe SA , Tsabar N , Klein PS , Root H , Frank D .

R01 GM076621-02 NIGMS NIH HHS , R01 GM076621-03 NIGMS NIH HHS , BB/E013872/1 Biotechnology and Biological Sciences Research Council , R01 GM076621 NIGMS NIH HHS , BB_BB/E013872/1 Biotechnology and Biological Sciences Research Council

	Fig. 1. The canonical Wnt pathway is necessary and sufficient for activating Meis3 gene expression. (A) In situ hybridization of late gastrula/early neurula embryos injected at the one-cell stage with either THVGR mRNA (10-20 pg, b) or mWnt3 DNA (100 pg, c). THVGR was activated by 1 μM DEX at the onset of gastrulation. Meis3 expression is anteriorly expanded in embryos injected with THVGR (113/121 embryos) or with mWnt3 (40/56 embryos). (B) sqRT-PCR of pools of 18 mid-gastrula stage AC explants dissected from embryos injected with THVGR mRNA (20, 40 pg) at the one-cell stage. THVGR was activated by 1 μM DEX at the onset of gastrulation. Ef1α was used as a loading control. No reverse transcriptase (–RT) control PCR was performed on RNA isolated from control embryos in all shown experiments. Posterior markers were not induced in THVGR DEX-untreated ACs (lanes 4, 6; n=5 experiments). (C) In situ hybridization of late gastrula/early neurula embryos injected at the one-cell stage with either Dkk1 mRNA (25 pg, b) or Wnt3a-MO (30 ng, c). Meis3 expression is eliminated in Dkk1 (98/98 embryos) and Wnt3a-morphant (37/37 embryos) embryos. (D) sqRT-PCR of pools of ten late-gastrula embryos injected with Wnt3a-MO either marginally or animally (10, 20, 40, 80 ng) at the one-cell stage. XHis4 was used as a loading control. Mesoderm marker expression is unaltered in Wnt3a-morphant embryos (n=5 experiments).
	Fig. 2. Meis3 restores posterior neural cell fates in the absence of canonical Wnt signaling. (A) In situ hybridization of mid-late neurula embryos injected at the one-cell stage with Dkk1 mRNA (35 pg; b,e,h,k,n) or, additionally, with Meis3 (0.25 ng) and β-gal (25 pg) mRNAs into one blastomere at the two-cell stage (c,f,i,l,o). All embryos are viewed dorsally, and oriented with anterior at the top, posterior at the bottom. The Meis3-injected side is on the right, as traced by the light blue/bright pink X-Gal staining. Dashed lines indicate the dorsal midline; bars indicate the posterior limits of gene expression on either side of the embryo. c′, i′, l′ and o′, show more clearly the signal in embryos treated as in c, i, l and o, respectively, but with no β-gal. XAG1 and XAnf1 expression undergoes robust posterior expansion in Dkk1 embryos (b, 113/115 embryos; e, 30/30 embryos), which is inhibited by Meis3 co-expression (c,c′, 73/81 embryos; f, 32/39 embryos). Posterior neural marker expression, Krox20, N-Tub and FoxD3, is inhibited in Dkk1 embryos (h, 78/80 embryos; k, 40/41 embryos; n, 51/52 embryos), but is strongly rescued by Meis3 co-expression (i,i′, 90/106 embryos; l,l′, 43/62 embryos; o,o′, 36/63 embryos). (B) In situ hybridization of mid-late neurula embryos injected at the one-cell stage with either Wnt3a-MO (50-60 ng; b,e,h,k,n), or, separately, with both Wnt3a-MO (50-60 ng) and Meis3 mRNA (0.5 ng; c,f,i,l,o). XAnf1 expression is posteriorly expanded in Wnt3a-morphants (b, 31/31 embryos), and suppressed by Meis3 co-expression (c, 55/55 embryos). Expression of the posterior neural markers Krox20, HoxB3, N-Tub and FoxD3 is inhibited in Wnt3a-morphants (e, 31/33 embryos; h, 31/31 embryos; k, 51/52 embryos; n, 50/51 embryos), but rescued by Meis3 co-expression (f, 29/33 embryos; i, 10/29 embryos; l, 22/39 embryos; o, 11/38 embryos). (C) In situ hybridization of late gastrula-early neurula siblings of embryos in A. All embryos are viewed dorsally, and oriented with anterior at the top, posterior at the bottom. The Meis3 injected side is on the right (bright pink X-Gal staining). Dashed lines and bars are as in A. Panels c′, f′, i′, embryos treated as in c, f, i, respectively, but with no β-gal. Otx2 expression undergoes robust posterior expansion in Dkk1 embryos (b, 49/51 embryos), which is inhibited by Meis3 co-expression (c,c′, 48/64 embryos). Expression of the posterior neural markers HoxD1 and Gbx2 is inhibited in Dkk1 embryos (e, 53/53 embryos; h, 51/53 embryos), but is strongly rescued by Meis3 co-expression (f,f′, 58/72 embryos; i,i′, 41/64 embryos). (D) Morphology of mid-late neurula embryos injected at the one-cell stage with either Wnt3a-MO (b, 70 ng), or Meis3 mRNA (c, 0.7 ng), or both (d). Neural folding and convergence extension is inhibited in Wnt3a morphants (b, 60/64 embryos); Meis3 co-expression rescued this phenotype (d, 48/49 embryos).
	Fig. 3. Wnt signaling cannot caudalize in the absence of Meis3 protein activity. (A) In situ hybridization of mid-late neurula embryos injected separately at the one-cell stage with either Meis3 MO (20 ng; b,f,j,n), or mWnt3 DNA (70 pg; d,h,l,p), or both (c,g,k,o). Posterior neural marker expression of HoxB3, Krox20, N-Tub and FoxD3 is highly inhibited in Meis3 morphants (b, 25/25 embryos; f, 40/40 embryos; j, 36/36 embryos; n, 16/16 embryos). mWnt3 co-expression did not rescue the expression of these markers (c, 40/41 embryos; g, 71/75 embryos; k, 63/63 embryos; o, 29/29 embryos). Ectopic mWnt3a levels alone expand posterior marker expression (d, 20/20 embryos; h, 48/49 embryos; l, 33/36 embryos; p, 18/18 embryos). (B) sqRT-PCR of pools of 18 mid-late gastrula AC explants dissected from embryos injected at the one-cell stage with mWnt3 DNA (200 pg) and BMP DNR mRNA (100 pg), and/or separately with Meis3 MO (33 ng) (n=5 experiments). (C) sqRT-PCR of pools of 18 mid-late neurula AC explants from siblings of embryos in B (n=5 experiments).
	Fig. 6. Meis3 is a direct target of β-catenin. (A) CHX AC assay (Materials and methods). sqRT-PCR of pools of 18 mid-late gastrula ACs were dissected from embryos injected with 20 pg THVGR mRNA at the one-cell stage. ACs were treated with CHX, DEX or CHX+DEX. Ef1α expression serves as a positive control for CHX activity and ODC as a loading control (n=2 experiments). (B) The XMeis3 genomic locus. Green ovals indicate β-catenin/TCF-binding sites, the blue line indicates the ChIP enriched fragment detected in C; the pink line indicates the 2.7 kb used for the constructs in D and E. (C) ChIP for β-catenin in late-gastrula embryos. A region 1.8 kb upstream of the Meis3 transcriptional start site is specifically enriched. Data are plotted as mean fold enrichment by β-catenin IP (black bars) over control serum IP (gray bars) for Meis3 (–1813) or a negative control locus XMLC2 (–118). Error bars show s.e.m. (n=3). (D) Reporter constructs used in E. The 2.7-luc construct contains wild-type β-catenin/TCF-binding sites (green ovals), whereas these are mutated in the 2.7δTCF-luc construct (red ovals). (E) Meis3 promoter activity is dependent on two β-catenin/TCF-binding sites. In situ hybridization of early- and mid-neurula 2.7-luc (b,e) and 2.7δTCF-luc (c, f) transgenic embryos, and of wild-type embryos (a,d).

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