Wnt8 is a secreted, lipid-modified Wnt-family ligand (358 aa, single Wnt domain with the family-conserved cysteine pattern) of the purple sea urchin embryo. It is the earliest zygotic intercellular signal of the vegetal pole: transcription begins at the 16-cell stage in the four micromeres, under control of the maternally nuclearized beta-catenin/Tcf system, and then spreads at the 60-cell stage to the adjacent veg2 tier of macromere descendants. Reception of Wnt8 by neighbouring cells drives beta-catenin into their nuclei, and because the wnt8 cis-regulatory system itself depends on a beta-catenin/Tcf input (together, in one module, with the zinc-finger factor Blimp1), each Wnt8-producing cell recruits its neighbours to produce Wnt8 in turn. This intercellular positive feedback, coupled to blimp1 autorepression that later extinguishes expression at the centre, generates an expanding torus of wnt8 and blimp1 expression that sweeps outward from the skeletogenic micromere lineage through the non-skeletogenic mesoderm and into the veg2 endoderm, carrying with it the beta-catenin/Tcf input on which most endomesodermal regulatory genes depend, and setting where the complementary Delta/Notch signal is deployed. Blocking Wnt8 translation prevents specification of both the mesoderm (primary mesenchyme, pigment and muscle cells) and the endoderm and abolishes gastrulation, while overexpressed Wnt8 induces ectopic endoderm and multiple invagination sites. Later, from the blastula stage, wnt8 expression moves into veg1 and posterior ectoderm, where Wnt8 together with Wnt1, acting through the Frizzled5/8 receptor and JNK rather than beta-catenin, restricts the anterior neuroectoderm to the anterior pole.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005102 signaling receptor binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping of the Wnt domain to the generic 'signaling receptor binding'. True but uninformative; Wnt8 is a secreted ligand that activates Frizzled receptors, and the specific terms (frizzled binding by IBA; receptor ligand activity, proposed below) already say this. Reason: Replace the generic binding term with GO:0048018 receptor ligand activity, which is what the sea urchin evidence shows (reception of the ligand by adjacent cells causes beta-catenin nuclearization) and which the GO definition of GO:0005102 itself points to. Proposed replacements: receptor ligand activity Supporting Evidence: PMID:16289024 The implication is that zygotic expression of wnt8 is stimulated in neighboring cells by its own gene product, since reception of the Wnt8 ligand causes beta-catenin nuclearization. |
| GO:0005109 frizzled binding | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference, on the Wnt family node, that the protein binds Frizzled receptors, from the Frizzled binding shown experimentally for many fly, mouse, rat, human and Xenopus Wnts. Wnt8 has the complete Wnt domain and behaves in vivo as a Frizzled-pathway ligand (its reception nuclearizes beta-catenin; its later ectodermal role depends on Fzl5/8), so the inherited activity is consistent with everything known, although no binding assay exists for the sea urchin protein. Reason: Core molecular function of a Wnt ligand; the phylogenetic placement is sound and nothing in the sea urchin literature suggests divergence. Direct binding evidence in this species is lacking, which is noted in suggested_experiments. Supporting Evidence: PMID:23335859 Here we show that the Wnt-dependent restriction of neuroectoderm to the anterior pole involves not only Wnt/Ξ²-catenin but also a series of linked steps mediated by Wnt/JNK signaling through Wnt1, Wnt8, and Fzl5/8, the homolog of vertebrate Fzl8. |
| GO:0005125 cytokine activity | IBA GO_REF:0000033 | MODIFY | Summary: PAINT inference of 'cytokine activity' on the Wnt family node from a few mouse and human Wnts. The GO definition (a soluble extracellular ligand that changes receptor activity to control survival, growth or differentiation) is broad enough that a Wnt fits, but the term is conventionally used for immune and haematopoietic mediators and adds nothing that the parent receptor ligand activity does not already say for a developmental morphogen-like Wnt. Reason: The essence (an extracellular receptor-activating ligand) is correct, but GO:0048018 receptor ligand activity is the accurate and less misleading term for a Wnt ligand; this is an argument about term choice, not about the phylogenetic placement. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000246517 SUPPORTS TRANSFER Wnt family node; the inference that the sea urchin protein is an extracellular receptor-activating ligand is sound, but 'cytokine activity' is a poorly fitting label for a developmental Wnt and the parent receptor ligand activity states the same function more accurately. Proposed replacements: receptor ligand activity Supporting Evidence: PMID:17975065 whereas its outward expansion follows reception of the Wnt8 ligand by adjacent cells |
| GO:0005576 extracellular region | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference that the protein is active in the extracellular region, from the secreted localization of essentially every characterized Wnt. Wnt8 is a secreted ligand whose diffusion to adjacent cells is the mechanism of torus expansion in the sea urchin, so the location is where it acts. Reason: Correct and core; the ligand acts on neighbouring cells after secretion. Supporting Evidence: PMID:19104065 Meanwhile, Wnt8 diffusion causes expansion of the subcircuit expression torus to the adjacent cells of the next domain. |
| GO:0005576 extracellular region | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping of the Wnt domain to extracellular region; the same statement as the IBA row, with a located_in qualifier. Reason: Correct for a secreted Wnt ligand. Supporting Evidence: PMID:17975065 whereas its outward expansion follows reception of the Wnt8 ligand by adjacent cells |
| GO:0016055 Wnt signaling pathway | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping of the Wnt domain to the generic Wnt signaling pathway. Correct but less specific than the IBA row for the canonical (beta-catenin) pathway, which is the branch shown in the sea urchin (Wnt8 reception nuclearizes beta-catenin; wnt8 MASO and cadherin-mediated beta-catenin sequestration give the same endomesoderm phenotype). Reason: Replace with the specific child GO:0060070 canonical Wnt signaling pathway, already present by IBA and experimentally supported in this species. Proposed replacements: canonical Wnt signaling pathway Supporting Evidence: PMID:19104065 Thus, blocking Wnt8 translation with morpholino antisense oligonucleotides (MASO), or blocking nuclearization of its downstream effector, Ξ²-catenin, by overexpression of an intracellular fragment of Cadherin ( 15 , 6 ), prevents both NSM and endoderm specification. |
| GO:0030182 neuron differentiation | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: PAINT inference of a role in neuron differentiation on the Wnt family node, from fly, worm, fish and mammalian Wnts that act in neural development. In the sea urchin, the only neural-related role documented for Wnt8 is that, with Wnt1, it restricts the anterior neuroectoderm (foxq2 domain) to the anterior pole through Fzl5/8 and JNK; that is a regionalization of the neuroectoderm along the A/P axis, not participation in the differentiation of neurons. Reason: The family-level term is too broad for this gene product; the sea urchin function is better captured by GO:0009952 anterior/posterior pattern specification, proposed below as a non-core annotation. The IBA is not contradicted, but no evidence places Wnt8 in neuron differentiation itself. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: CONTEXT OR TISSUE MISMATCH GRANULARITY MISMATCH Sources checked: PANTHER:PTN000246517 SUPPORTS TRANSFER Wnt family node seeded by fly, worm, fish and mammalian Wnts with neural-development phenotypes; the sea urchin protein has a documented ectodermal role, but it is A/P regionalization of the neuroectoderm rather than neuron differentiation, so the family-level term overstates what is known. Supporting Evidence: PMID:23335859 embryos injected with either Wnt1 or Wnt8 morpholinos failed to down-regulate foxq2 expression in posterior ectoderm. |
| GO:0045165 cell fate commitment | IBA GO_REF:0000033 | MODIFY | Summary: PAINT inference of a role in cell fate commitment on the Wnt family node, seeded by Wnts with fate phenotypes in fly, mouse, Xenopus (wnt8, P28026), worm (mom-2 and others) and zebrafish (wnt8a). For the sea urchin protein this is directly true and more can be said; wnt8 MASO abolishes specification of both the endoderm and the mesoderm (primary mesenchyme, pigment and muscle lineages), and overexpression induces ectopic endoderm. Reason: The phylogenetic placement is sound and the term is correct, but the sea urchin evidence supports the specific descendants GO:0001714 endodermal cell fate specification and GO:0007501 mesodermal cell fate specification, proposed below as NEW rows by IMP; the general term should be replaced by them rather than kept alongside. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000246517 SUPPORTS TRANSFER Wnt family node; fate-commitment roles are documented for many descendants (Xenopus wnt8, zebrafish wnt8a, worm mom-2, mouse Wnt3/Wnt3a) and the inference holds for sea urchin Wnt8, but the specific lineages (endoderm and mesoderm of the vegetal plate) are established experimentally and should replace the generic term. Proposed replacements: endodermal cell fate specification mesodermal cell fate specification Supporting Evidence: PMID:19104065 Expression of wnt8 is also essential for specification of both NSM and endoderm. PMID:15282746 SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. |
| GO:0060070 canonical Wnt signaling pathway | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference, on the Wnt family node, that the protein signals through the canonical beta-catenin pathway. This is exactly what is shown in the sea urchin embryo; Wnt8 reception causes beta-catenin nuclearization in the receiving cell, the wnt8 gene is itself a beta-catenin/Tcf target, and blocking the ligand or sequestering beta-catenin gives the same loss of endomesoderm specification. It is the pathway that defines this gene's core function. Reason: Core process, inherited from the family node and independently supported by MASO, cadherin-fragment and cis-regulatory experiments in S. purpuratus. Supporting Evidence: PMID:18413610 Wnt8, which enhances nuclearization of Ξ²-catenin in recipient cells, including themselves PMID:16289024 The implication is that zygotic expression of wnt8 is stimulated in neighboring cells by its own gene product, since reception of the Wnt8 ligand causes beta-catenin nuclearization. PMID:18061160 The wnt8 gene is functionally linked into the subcircuit in that cells receiving this ligand generate a Ξ²-catenin/Tcf input required for blimp1 expression, while the wnt8 gene in turn requires a Blimp1 input PMID:19104065 Thus, blocking Wnt8 translation with morpholino antisense oligonucleotides (MASO), or blocking nuclearization of its downstream effector, Ξ²-catenin, by overexpression of an intracellular fragment of Cadherin ( 15 , 6 ), prevents both NSM and endoderm specification. |
| GO:0048018 receptor ligand activity | IMP PMID:15282746 Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cel... | NEW | Summary: Proposed. Wnt8 is the secreted ligand whose reception by adjacent cells activates the Frizzled/beta-catenin pathway. In S. purpuratus, blocking translation of the ligand and blocking nuclearization of its downstream effector beta-catenin give the same loss of endomesoderm specification, overexpression of the ligand induces ectopic endoderm, and cis-regulatory reconstruction shows that outward expansion of the blimp1/wnt8 torus follows reception of the ligand by neighbouring cells. Reason: This is the molecular function GO uses for secreted Wnt ligands (the definition of the existing IEA 'signaling receptor binding' row itself recommends it), and it is what the sea urchin loss- and gain-of-function data establish. No direct binding assay exists for the sea urchin protein, so the more specific frizzled binding is left to the IBA row rather than asserted experimentally. Supporting Evidence: PMID:15282746 Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. PMID:15282746 Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. PMID:17975065 A cis-regulatory reconstruction experiment revealed that blimp1 autorepression accounts for progressive extinction of expression in the center of the torus, whereas its outward expansion follows reception of the Wnt8 ligand by adjacent cells. PMID:18413610 Therefore, an intercellular feedback circuit is set up, as each wnt8 -expressing cell also causes the adjacent recipient cells to drive more Ξ²-catenin into its nucleus and further express wnt8 |
| GO:0001714 endodermal cell fate specification | IMP PMID:15282746 Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cel... | NEW | Summary: Proposed. Wnt8 secreted by the micromeres and then by veg2 delivers the beta-catenin/Tcf input on which the endodermal regulatory genes depend. Blocking Wnt8 translation abolishes endoderm formation and gastrulation, and Wnt8 overexpression induces ectopic endoderm with multiple invagination sites. Reason: Participation test: the ligand itself performs the intercellular step of the specification; it is the signal, released by the micromere/veg2 cells, that nuclearizes beta-catenin in the receiving veg2 cell and thereby activates the endoderm regulatory state. Comparator check (QuickGO): the C. elegans Wnt ligand mom-2 carries GO:0001714 by IMP, so a specification term on an inductive Wnt ligand is within GO convention. Proposed as the specific replacement for the IBA cell fate commitment row, so it is not redundant with a retained ancestor; no GO-CAM covers this gene (gocams/index.tsv has no S. purpuratus entries). Supporting Evidence: PMID:15282746 Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. PMID:15282746 SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. PMID:19104065 Expression of wnt8 is also essential for specification of both NSM and endoderm. PMID:16289024 Expression of the wnt8 gene is the key transcriptional motivator of an intercellular signaling loop which drives endomesoderm specification forward early in sea urchin embryogenesis. PMID:18061160 Morpholino-substituted antisense oligonucleotide (MASO) targeting blimp1 blocks endomesoderm specification ( Livi and Davidson, 2007 ), just as does MASO directed against the wnt8 gene ( Minokawa et al., 2005 ; Wikramanayake et al., 2004 ). |
| GO:0007501 mesodermal cell fate specification | IMP PMID:15282746 Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cel... | NEW | Summary: Proposed. The same Wnt8 signal is required for the mesodermal lineages of the vegetal plate. In Wnt8 morphants the micromeres form but fail to differentiate as primary mesenchyme cells, and the secondary mesenchyme-derived pigment and muscle cells are absent; blocking the ligand or beta-catenin nuclearization prevents specification of the non-skeletogenic mesoderm, and the intercellular wnt8 feedback loop is needed for specification of the skeletogenic micromere lineage itself. Reason: Participation test: Wnt8 is the ligand that supplies the beta-catenin/Tcf input to the skeletogenic and non-skeletogenic mesoderm regulatory states (in the micromeres it acts on the very cells that produce it, and positions the Delta/Notch signal that specifies NSM). Comparator check: Xenopus wnt8 (P28026) carries mesoderm development by IDA, and Drosophila FGF ligands carry GO:0007501 by IMP. Proposed with GO:0001714 as the specific replacement for the IBA cell fate commitment row. Downstream skeletogenic differentiation and pigment cell terms are deliberately not proposed (executed by the lineage GRNs and by Delta/Notch). Supporting Evidence: PMID:15282746 Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. PMID:15282746 SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. PMID:19104065 Thus, blocking Wnt8 translation with morpholino antisense oligonucleotides (MASO), or blocking nuclearization of its downstream effector, Ξ²-catenin, by overexpression of an intracellular fragment of Cadherin ( 15 , 6 ), prevents both NSM and endoderm specification. PMID:18413610 blockade of wnt8 expression and disruption of the wnt8 intercellular feedback loop plays havoc with endomesodermal specification, including that of the skeletogenic micromere lineage |
| GO:0009952 anterior/posterior pattern specification | IMP PMID:23335859 Integration of canonical and noncanonical Wnt signaling path... | NEW | Summary: Proposed, non-core. From the blastula stage wnt8 expression moves into veg1 and posterior ectoderm, and Wnt8 together with Wnt1, signaling through Fzl5/8 and JNK, restricts the anterior neuroectoderm (foxq2 domain) to the anterior pole; Wnt8 morphants fail to down-regulate foxq2 in posterior ectoderm. Reason: The ligand itself performs the posteriorizing signaling step, and A/P regionalization of the neuroectoderm is the process the experiment measures. This replaces the over-broad family-level 'neuron differentiation' IBA as the accurate statement of Wnt8's ectodermal role. Not part of the core endomesoderm function; the pathway in this context is beta-catenin-independent (JNK), which is noted in suggested_questions rather than asserted as a non-canonical pathway term. Supporting Evidence: PMID:23335859 Here we show that the Wnt-dependent restriction of neuroectoderm to the anterior pole involves not only Wnt/Ξ²-catenin but also a series of linked steps mediated by Wnt/JNK signaling through Wnt1, Wnt8, and Fzl5/8, the homolog of vertebrate Fzl8. PMID:23335859 embryos injected with either Wnt1 or Wnt8 morpholinos failed to down-regulate foxq2 expression in posterior ectoderm. PMID:23335859 As development progressed, wnt8 expression first moved into the next most anterior tier of blastomeres (veg1) and then, during late blastula stages (18 and 24 hpf), into both veg1 and overlying posterior ectoderm cells |
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Download this section (compressed HTML)Q: Should frizzled binding remain an IBA-only statement, or is there (or should there be) direct evidence that Sp-Wnt8 binds a specific sea urchin Frizzled (Fzl1/2/7 or Fzl5/8)? The endomesoderm role is beta-catenin-dependent and the later ectodermal role runs through Fzl5/8-JNK, suggesting Wnt8 engages more than one receptor.
Q: The later A/P neuroectoderm role is beta-catenin-independent (Wnt/JNK through Fzl5/8). Should a non-canonical Wnt signaling pathway term be added for Wnt8, given that the PLoS Biol study attributes the JNK step jointly to Wnt1 and Wnt8 and does not separate their receptor engagement?
Q: Is Blimp1 a necessary in vivo input into wnt8? The reporter-module analysis says yes, but the whole-locus BAC experiment in the endoderm GRN paper says the Blimp1-site mutation has no effect and that wnt8 turns on in veg2 before blimp1b; this affects how the torus subcircuit should be modelled but not the annotations here.
Experiment: Cell-based or in vitro binding assay between recombinant Sp-Wnt8 and the extracellular cysteine-rich domains of the sea urchin Frizzled receptors (Fzl1/2/7, Fzl5/8, Fzl9/10) to convert the inherited frizzled binding into direct evidence and identify which receptor mediates the endomesodermal versus the ectodermal response.
Type: biochemical
Experiment: Tagged Wnt8 (BAC knock-in or mRNA with an epitope tag) to visualize the extracellular protein and its range of diffusion from micromeres to veg2 and veg1, testing the diffusion-based model of torus expansion.
Type: localization
Experiment: Lineage-restricted rescue (micromere-only versus veg2-only wnt8 expression in a Wnt8 morphant or CRISPR background) to separate the contributions of micromere-derived and veg2-derived Wnt8 to skeletogenic, non-skeletogenic mesoderm and endoderm specification.
Type: genetic
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