Martínez-Bartolomé M , Range RC .

R15 HD088272 NICHD NIH HHS

	Fig. 1. Spatiotemporal expression of the wnt16 ligand during early anterior-posterior specification and patterning. (A) Diagram showing a model for the areas of Wnt/β-catenin, Fzl5/8-JNK and Fzl1/2/7 signaling during ANE early AP patterning, consistent with previous data (Khadka et al., 2018; Range, 2018; Range and Wei, 2017; and Range et al., 2013). (B) Whole-mount in situ hybridization analysis of wnt16 expression during ANE restriction. (Ba,b) Expression of wnt16 mRNA transcripts was first detected and they were broadly expressed throughout zygotes and 32-cell-stage embryos. (Bd-j) Between the 120-cell stage and late gastrula (30†hpf), wnt16 expression was progressively downregulated from anterior and equatorial ectoderm cells, resulting in a localized expression in the posterior endoderm and mesoderm region of the embryo. (C) qPCR measurements showing the temporal expression of wnt16 transcripts from three different batches of embryos from egg to mesenchyme blastula stages (24†hpf). The number of wnt16 transcripts per embryo (y axis) is based on the Ct value of z12 transcripts. The absolute concentrations of z12 transcripts are known at each stage (Wang et al., 1995). Scale bars: 20†µm. VV, vegetal view.
	Fig. 2. Wnt16 represses endomesoderm genes at the 120-cell stage and is necessary for ANE GRN expression. (A) Expression of the endomesoderm markers gataE, foxA, wnt1 and wnt8, and three Wnt ligands (wn4, wnt5 and wnt6) in control, Wnt16 morpholino-injected and wnt16 mRNA-injected embryos at the 120-cell stage. The expression of the endomesoderm markers gataE, foxA, wnt1 and wnt8 was upregulated in Wnt16 MO1-injected embryos (Ah-j,n compared with Aa-c,g). wnt16 mRNA overexpression downregulated the expression of the endomesoderm markers gataE, foxA, wnt1 and wnt8 (Ah-j,n compared with Aa-c,g). Solid lines indicate the posterior boundaries of each endomesoderm marker. The expression of wnt4, wnt5 and wnt6 ligands was not affected in Wnt16 morpholino-injected and wnt16 mRNA-injected embryos (Ak-m,r-t compared with Ad-f). (B) The angle α shown in A was measured from three different samples of 120-cell-stage embryos using ImageJ. Volume=0.5(1-cos α/2) was used to calculate the percentage of the surface area (±s.e.m.) occupied by the endomesoderm territories in control and Wnt16 knockdowns. (C) Wnt1 and/or Wnt8 might interact with Wnt16 to repress endomesoderm genes at the 120-cell stage. The expression of the endomesoderm markers foxA and gataE was not affected in wnt1 or wnt8 mRNA-injected embryos (Cb,c and Ch,i) compared with control (Ca,g). In embryos overexpressing wnt16 mRNA, foxA and gataE, expression was severely downregulated (Cd,j). Embryos co-injected with wnt16 mRNA and either wnt1 or wnt8 mRNA showed either a strong downregulation (main panels) or a significantly reduced expression (insets) of both foxA and gataE genes (Ce,f and Ck,l). Insets show significantly reduced phenotypes of foxA and gataE expression in embryos injected with wnt1 and wnt16 mRNA (40% and 33%, respectively), and in embryos injected with wnt8 and wnt16 mRNA (33% and 43%). The remaining percentages of embryos observed that show the representative phenotypes depicted are indicated in each panel. (D) foxq2 and six3 expression at the 120-cell stage in control (Da,b) and Wnt16 morpholino-injected embryos (Dg,h). Expression of ANE makers (foxq2, six3, dkk3 and sfrp1/5) in control embryos (Dc-f) and in Wnt16 MO1-injected embryos (Di-l) at the mesenchyme blastula stage (24†hpf). MO, morpholino. Scale bars: 20†µm.
	Fig. 3. Wnt16-Fzl1/2/7 signaling antagonizes the Wnt1/Wnt8-Fzl5/8-JNK pathway during the ANE restriction mechanism. (A) The expression of the ANE marker foxq2 expanded in embryos overexpressing wnt16 mRNA at the mesenchyme blastula stage (compare Ab,d with Ab,c). (B) At mesenchyme blastula stage, the expression of the cardinal regulator foxq2 was expanded in ΔFzl5/8 mRNA-injected embryos (Bc) compared with control embryos (Ba). In the absence of Wnt16, foxq2 expression was severely downregulated in ANE (Bb), whereas Wnt16 morphants co-injected with ΔFzl5/8 rescued the expression of ANE factors, showing a normal or expanded foxq2 expression (91%) (Bd). (C) Control embryos showing foxq2 expression at the 120-cell and mesenchyme blastula stage (24†hpf) (Ca,Ce). ANE expression of foxq2 was completely eliminated in Fzl1/2/7 morpholino-injected embryos (Cb,Cf). At the 120-cell stage, foxq2 was expressed in embryos injected with wnt16 mRNA (Cc). At mesenchyme blastula stage, foxq2 expression was strongly upregulated and expanded towards the posterior pole in embryos injected with wnt16 mRNA (Cg). Overexpression of wnt16 in a Fzl1/2/7 morphant background produced a completely elimination of foxq2 expression, mimicking the Fzl1/2/7 knockdown phenotype (Cd,Ch). MO, morpholino; ΔFzl5/8, dominant negative Fzl5/8. Scale bars: 20†µm.
	Fig. 4. Gene expression patterns of wnt16, eve, foxA and gcm. (A) Double in situ hybridization showing posterior/vegetal views that combine wnt16 and either eve (Aa-c), foxA (Ae-g) or gcm (Ai-k) at late blastula stage (18†hpf). (Ad,h,l) Schematic diagrams showing spatial expression patterns in relation to cell lineage (anterior/veg1 endoderm, posterior/veg2 endoderm and veg2 mesoderm). (B) Posterior/vegetal views that combine wnt16 and either eve (Ba-c) or foxA (Ae-g) at mesenchyme blastula stage (24†hpf). (Bd,h) Schematics showing spatial patterns of gene expression in relation to cell linage. endo, endoderm precursors; mes, mesoderm precursors.
	Fig. 5. Regulation of wnt16 expression by the AP Wnt signaling network and the role of Wnt16 in activating endoderm genes. (Aa) Control embryo showing wnt16 expression in the posterior endoderm and mesoderm regions of the embryo at mesenchyme blastula stage (24†hpf). wnt16 expression was downregulated in Axin mRNA-injected embryos (Ab). wnt16 expression was downregulated in embryos injected with a dominant-negative form of Fzl5/8 (ΔFzl5/8) (Ac). wnt16 expression was unperturbed in Fzl1/2/7 morphants (Ad). (B) Wnt16 knockdown embryos at mesenchyme blastula stage showing that Wnt16 was not necessary for the expression of the endoderm genes gataE, foxA, wnt1 and wnt8 (Bh-k compared with Ba-d), but was necessary for the expression of eve, blimp1b and hox11/13b (Bl-n compared with Be-g). MO, morpholino; ΔFzl5/8, dominant negative Fzl5/8. Scale bars: 20†µm.
	Fig. 6. The role of Wnt16 in morphogenetic movements during gastrulation. Morphology of control embryos at mesenchyme blastula (24 hpf), early gastrula (30 hpf), gastrula (36 hpf), late gastrula (48 hpf), and pluteus larva (72 hpf) stages (A-E), and embryos injected with morpholino targeting wnt16 transcripts (G-K). Normal numbers of pigment cells were shown in both control and Wnt16 morphants at pluteus larva stage (F,L), whereas the arrangement of those cells was disrupted in Wnt16 knockdowns. Control=74.5±6.7 (n=21); Wnt16 MO1=71±7.7 (n=21). Data are mean±s.d. hpf, hours post-fertilization.
	Fig. 7. Wnt16 function in mesoderm morphogenesis and gastrulation. (A) 1d5 and Meso1 antibody staining at mesenchyme blastula stage (24†hpf). 1d5 (blue) stains skeletogenic mesoderm cells, and Meso1 (orange) is a general mesoderm marker. Neither 1d5 nor Meso1 staining was affected in Wnt16 knockdown embryos (Ae-h) compared with control embryos (Aa-d). (B) F-actin staining as measured by phalloidin binding. F-actin accumulation was present in the invagination of both control and Wnt16 knockdown embryos (Ba-d). MO, morpholino.
	Fig. 8. Model for two phases of Wnt16 activity during early AP axis specification, patterning and morphogenesis of the sea urchin embryo. (A) Broad maternal non-canonical Wnt16-Fzl1/2/7 signaling antagonizes Wnt/β-catenin and Wnt1/Wnt8-Fzl5/8-JNK signaling during the ANE restriction process. Illustrated is an extended model for early anterior-posterior axis patterning during sea urchin early development (see Introduction and Discussion for details). (B) Posteriorly localized wnt16 expression in the endoderm and mesoderm territories is activated by Wnt/β-catenin and Fzl5/8 signaling. In addition, the extended model indicates a role for the activation of the key endoderm GRN components, hox11/13b, blimp1 and eve. hox11/13b appears to be necessary for both gastrulation and mesoderm morphogenesis (Arenas-Mena et al., 2006), and blimp1b is necessary for gastrulation (Livi and Davidson, 2006), both similar to the Wnt16 phenotypes described.

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