Khadka A , Martínez-Bartolomé M , Burr SD , Range RC .

R15 HD088272 NICHD NIH HHS

	Fig. 1. Spatiotemporal expression of sFRP-1 during ANE restriction. Aa–c sfrp-1 expression is first detected at the 32- to 60-cell stage and is present throughout the embryo until mid-blastula stage. Ad, Ae Between mesenchyme blastula stage and early gastrula, sfrp-1 is restricted to anterior and posterior blastomeres. Af–j foxq2 expression is first detected at 32- to 60-cells stage in the anterior blastomeres and is progressively downregulated from posterior ectoderm cells from the 60-cell to mesenchyme blastula stages as previously shown [21]. Ak–o Diagram showing a model for the areas of Wnt/β-catenin, Fzl5/8–JNK, and Fzl1/2/7–PKC signaling during ANE restriction consistent with the data from [20]. Each individual diagram corresponds to the developmental stages depicted in the panels directly above. B Functional protein domain organization for the CRD containing proteins including sea urchin sFRP3/4 (a) and secreted Frizzled-related protein 1 (b). Scale bar = 20 μm
	Fig. 2. sFRP-1 is necessary for ANE specification. Aa–e six3, foxq2, dkk3, nkx3.2, and sfrp-1/5 expression in mesenchyme blastula stage control embryos (24 hpf). Ag–k Embryos injected with sFRP-1 morpholino 1 show complete elimination of ANE markers. B qPCR measurements from three different cultures of embryos showing the downregulation of ANE regulatory genes at mesenchyme blastula stage (24hpf) in sFRP-1 morphants. The y axis shows the fold change in gene expression for sFRP-1 morphants versus controls. Scale bar = 20 μm. MO, morpholino
	Fig. 3. Function of maternal and zygotic sFRP-1 during ANE restriction. A Compared to control embryos (a), embryos injected with sfrp-1 mRNA show expanded foxq2 expression (b). B In the absence of sFRP-1, foxq2 expression is completely eliminated from ANE territory (Bb), whereas sFRP-1 morphants co-injected with ΔFzl5/8 show expanded foxq2 expression (Bc). The number of embryos examined that show the representative phenotypes depicted is indicated in each panel. C A model diagram for ANE restriction during late blastula stages showing Wnt1/Wnt8–Fzl5/8–JNK signaling downregulates the transcription of fzl5/8 in posterior ectoderm, restricting its expression to the ANE territory. In the ANE, Fzl5/8 signaling activates dkk1 expression, antagonizing Wnt1/Wnt8–Fzl5/8–JNK signaling and establishing the ANE territory. Maternal and/or zygotic sFRP-1 is also necessary to antagonize Fzl5/8 signaling at this stage of development. The model is based data presented in this figure and from [20]. D Expression of foxq2 from 60-cell stage to mesenchyme blastula stage in control embryos (Da–e), in translation-blocking sFRP-1 morphants (Df–j) and splice-blocking sFRP-1 morphants (Dk–o). MO, morpholino. Scale bar = 20 μm
	Fig. 4. Control of sfrp-1 expression by the Wnt signaling network governing ANE restriction. A Fzl5/8–JNK signal-mediated downregulation of ANE gene expression is enhanced in the absence of Fzl1/2/7. Control embryos showing sfrp-1 expression at 60-cell (a), blastula (b) and mesenchyme blastula stage (c). Ad–f In Fzl1/2/7 MO injected embryos, sfrp-1 is expressed at the 60-cell stage (d), but downregulated in blastula (e) and mesenchyme blastula embryos (f). Ba–c sfrp-1 expression in Fzl1/2/7 morphants is rescued by overexpressing dkk1 mRNA. The number of embryos examined that show the representative phenotypes depicted is indicated in each panel. C A model diagram, showing that maternal sFRP-1 works together with, but independently from, Fzl1/2/7 signaling during the early stages of ANE restriction (Ca) and that zygotic sfrp-1 is part of the ANE GRN downregulated by Wnt/JNK signaling during later stages of the process (Cb). The model also shows that during later stages of ANE restriction the Fzl5/8 receptor is a member of the ANE GRN and Fzl5/8–JNK signaling downregulates fzl5/8 expression in the lateral ectoderm. The model is based on the data presented in this and the preceding figures, as well as data from [20]. MO, morpholino. Scale bar = 20 μm
	Fig. 5. sFRP-1 and Dkk1 antagonize Wnt signaling during final phase of ANE restriction. A qPCR measurements showing the temporal expression of dkk1 (a) and sfrp-1 (b) transcripts per embryo from egg to mesenchyme blastula stage of development. The inset in b shows the number of sfrp-1 transcripts per embryo in fertilized eggs and 60-cell embryos. B Comparing foxq2 expression between Dkk1 morphants (Bb, Be) and sFRP-1 morphants (Bc, Bf) during early and late stages of the ANE restriction. C Beginning at blastula stages, sfrp-1 is downregulated in embryos in the absence of functional Fzl5/8 signaling (e–h). D A model diagram, showing that both zygotic sfrp-1 and dkk1 are activated by Fzl5/8 signaling in the ANE and that they work together in a negative feedback loop to antagonize Wnt1/Wnt8–Fzl5/8 signaling in the ANE territory. The model is based on the data presented in this and the preceding figures, as well as data from [20]. FE, fertilized egg; EB, early blastula; B, blastula; MB, mesenchyme blastula. MO, morpholino. Scale bar = 20 μm
	Fig. 6. A new model for the Wnt signaling network governing ANE restriction in the sea urchin embryo and the evolution of sFRP3/4 and sFRP-1 in deuterostomes. Aa Early downregulation of the ANE GRN from posterior blastomeres. (Posterior half of the embryo) A broad, maternal regulatory mechanism is able to activate the ANE GRN throughout the embryo by the 32-cell stage, but nβ-catenin signaling prevents the expression of the ANE GRN in the posterior half of the 32-cell embryo through an unknown mechanism and activates Wnt1 and Wnt8 expression at the 32-cell stage. In addition, around the 32- to 60-cell stage sFRP-1 and Fzl1/2/7 signaling antagonize the downregulation of the ANE GRN in the anterior hemisphere [20]. Ab Downregulation of the ANE GRN from cells in the lateral ectoderm domain and the establishment of the ANE around the anterior pole. (Anterior half of the embryo) Around the 32- to 60-cell stage (7–9 hpf), secreted Wnt1 and Wnt8 diffuse into more anterior ectodermal blastomeres (Wnt8 expression is activated throughout the equitorial ectoderm territory) and signal through the Fzl5/8 receptor, activating the JNK pathway. This Wnt/JNK pathway progressively downregulates ANE GRN expression from the central ectodermal territory during the early to late blastula stages (12–18 hpf). Within the same ectodermal cells, sFRP-1 and Fzl1/2/7 signaling antagonize Wnt1/Wnt8–Fzl5/8-JNK-mediated downregulation of the ANE GRN. Around the mid-blastula stage (16 hpf), an anterior signaling center activated by the cardinal ANE transcriptional regulator FoxQ2 secretes the Wnt modulators Dkk3 and sFRP-1/5 within the regressing ANE territory. Dkk3 and low levels of sFRP-1/5 are necessary to stimulate the Fzl5/8–JNK signaling during the later stages of ANE restriction. During the final phase of the process from early to late blastula stages (18–24 hpf), Fzl5/8 signaling activates the expression of the secreted Wnt antagonists sFRP-1 and Dkk1 in the anterior-most cells around the anterior pole. These antagonists establish a correctly sized ANE territory by preventing the downregulation of ANE factors by antagonizing Fzl5/8 signaling through a negative feedback mechanism [20]. B Schematic representation of the expansion of sFRP3/4 paralogues and sFRP-1 during deuterostome evolution

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