ECB-ART-46101Evodevo January 22, 2018; 9 1.
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A novel gene''s role in an ancient mechanism: secreted Frizzled-related protein 1 is a critical component in the anterior-posterior Wnt signaling network that governs the establishment of the anterior neuroectoderm in sea urchin embryos.
The anterior neuroectoderm (ANE) in many deuterostome embryos (echinoderms, hemichordates, urochordates, cephalochordates, and vertebrates) is progressively restricted along the anterior-posterior axis to a domain around the anterior pole. In the sea urchin embryo, three integrated Wnt signaling branches (Wnt/β-catenin, Wnt/JNK, and Wnt/PKC) govern this progressive restriction process, which begins around the 32- to 60-cell stage and terminates by the early gastrula stage. We previously have established that several secreted Wnt modulators of the Dickkopf and secreted Frizzled-related protein families (Dkk1, Dkk3, and sFRP-1/5) are expressed within the ANE and play important roles in modulating the Wnt signaling network during this process. In this study, we use morpholino and dominant-negative interference approaches to characterize the function of a novel Frizzled-related protein, secreted Frizzled-related protein 1 (sFRP-1), during ANE restriction. sFRP-1 appears to be related to a secreted Wnt modulator, sFRP3/4, that is essential to block Wnt signaling and establish the ANE in vertebrates. Here, we show that the sea urchin sFRP3/4 orthologue is not expressed during ANE restriction in the sea urchin embryo. Instead, our results indicate that ubiquitously expressed maternal sFRP-1 and Fzl1/2/7 signaling act together as early as the 32- to 60-cell stage to antagonize the ANE restriction mechanism mediated by Wnt/β-catenin and Wnt/JNK signaling. Then, starting from the blastula stage, Fzl5/8 signaling activates zygotic sFRP-1 within the ANE territory, where it works with the secreted Wnt antagonist Dkk1 (also activated by Fzl5/8 signaling) to antagonize Wnt1/Wnt8-Fzl5/8-JNK signaling in a negative feedback mechanism that defines the outer ANE territory boundary. Together, these data indicate that maternal and zygotic sFRP-1 protects the ANE territory by antagonizing the Wnt1/Wnt8-Fzl5/8-JNK signaling pathway throughout ANE restriction, providing precise spatiotemporal control of the mechanism responsible for the establishment of the ANE territory around the anterior pole of the sea urchin embryo.
PubMed ID: 29387332
PMC ID: PMC5778778
Article link: Evodevo
Species referenced: Echinodermata
Genes referenced: dkk foxe3l LOC100887844 LOC115919910 LOC115919915 LOC575170 LOC582802 LOC590297 LOC594353 mapk9 pole sfrpl six6 wnt1 wnt8a
Morpholinos: dkk MO1 fzd2 MO1 sfrp5 MO3 sfrp5 MO4
Article Images: [+] show captions
|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 . 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 . 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 . 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 . 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 . 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 . 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 . B Schematic representation of the expansion of sFRP3/4 paralogues and sFRP-1 during deuterostome evolution|
References [+] :
Adamska, Wnt and TGF-beta expression in the sponge Amphimedon queenslandica and the origin of metazoan embryonic patterning. 2008, Pubmed