Sp-Eve (Speve, even-skipped) is the purple sea urchin orthologue of the even-skipped class of homeodomain transcription factors (297 aa, homeodomain at residues 143-203). It is one of the earliest zygotic regulatory genes of the vegetal half of the embryo: transcription begins in the vegetal plate at sixth cleavage across the macromere (veg1 and veg2) descendants, runs at the leading edge of the outward expanding wave of Wnt8/beta-catenin-Tcf-dependent expression, and by 15 h after fertilization is confined to the veg1 lineage, the precursor of veg1 (hindgut) endoderm and veg1 ectoderm. Its cis-regulatory module is activated by beta-catenin/Tcf, repressed by Tcf/Groucho outside the Wnt8 range, and repressed in veg2 cells by Hox11/13b; eve also represses its own gene, which clears its expression from veg2 endoderm. Up to about 18 h Eve has no detectable target other than itself, but from 21-24 h it activates the regulatory genes of the veg1 endoderm gene regulatory network (hox11/13b, and through it brachyury and hnf1) that specify future hindgut fate, and it activates wnt1, wnt4, wnt5 and wnt16 in veg1 cells while repressing animal-ectoderm genes (e.g. fgf9, myc) there, thereby also establishing the veg1 ectoderm regulatory state. Later in development eve mRNA is a direct target of miR-31, and de-repression or overexpression of Eve disturbs skeletogenic mesenchyme patterning.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO from the homeobox signature. Sp-Eve is an even-skipped-class homeodomain protein and a direct regulator in the endomesoderm GRN, acting as an activator of hox11/13b and the veg1 wnt genes and as a repressor of its own gene and of animal-ectoderm genes. Reason: Correct and core. The cis-regulatory and perturbation literature treats Eve as a sequence-specific Pol II transcription factor with both activator and repressor outputs (see NEW GO:0001228 and GO:0001227). Direct DNA binding of the sea urchin protein has not been assayed, so the term rests on the homeodomain plus in vivo regulatory function. Supporting Evidence: PMID:12027439 Speve, an evenskipped orthologue expressed very early in all vegetal blastomeres and then gradually shifting to veg(1) derivatives by the mesenchyme blastula stage PMID:21623371 Hox11/13b expression is activated by Eve in descendants of veg1 cells at 24 h, because injection of eve MASO reduces hox11/13b expression only after 24 h (Fig. 3b). PMID:19895806 This result indicates that eve is controlled by auto-repression. |
| GO:0003677 DNA binding | IEA GO_REF:0000120 | ACCEPT | Summary: Generic DNA binding from the homeodomain InterPro entries and UniRule. True but uninformative relative to the Pol II-specific transcription factor terms; no EMSA or ChIP exists for the sea urchin protein. Reason: Not wrong, and the homeodomain is the only DNA-binding domain in the protein. Retained as the general parent of GO:0000981; the specific activator/repressor terms carry the functional content. Supporting Evidence: PMID:12027439 Speve, an evenskipped orthologue expressed very early in all vegetal blastomeres and then gradually shifting to veg(1) derivatives by the mesenchyme blastula stage |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: UniRule/subcellular-location mapping for a homeodomain transcription factor. No localization data exist for the sea urchin protein, but every documented function of Eve is transcriptional control of target genes through the GRN. Reason: Consistent with the in vivo function (activation of hox11/13b and wnt genes, autorepression); core location. Supporting Evidence: PMID:21623371 Hox11/13b expression is activated by Eve in descendants of veg1 cells at 24 h, because injection of eve MASO reduces hox11/13b expression only after 24 h (Fig. 3b). PMID:19895806 This result indicates that eve is controlled by auto-repression. |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000120 | MODIFY | Summary: Generic transcription-regulation process from ARBA/InterPro. Correct but too general; the sea urchin data show Pol II-specific positive regulation (hox11/13b, wnt1/4/5/16) and negative regulation (eve itself, animal-ectoderm genes in veg1 cells). Reason: The essence is right, but the GOA already carries the Pol II-specific parent GO:0006357 and the literature supports the sign-specific children, which are proposed as NEW rows. Proposed replacements: positive regulation of transcription by RNA polymerase II negative regulation of transcription by RNA polymerase II Supporting Evidence: PMID:21623371 Hox11/13b expression is activated by Eve in descendants of veg1 cells at 24 h, because injection of eve MASO reduces hox11/13b expression only after 24 h (Fig. 3b). PMID:19895806 This result indicates that eve is controlled by auto-repression. |
| GO:0006357 regulation of transcription by RNA polymerase II | IEA GO_REF:0000108 | ACCEPT | Summary: Inter-ontology inference from GO:0000981. Exactly what Eve does in the endomesoderm GRN, in both directions. Reason: Confirmed by eve MASO perturbation of hox11/13b, wnt1/4/5/16 and of eve itself. Retained as the parent of the proposed positive- and negative-regulation rows. Core function. Supporting Evidence: PMID:21623371 Hox11/13b expression is activated by Eve in descendants of veg1 cells at 24 h, because injection of eve MASO reduces hox11/13b expression only after 24 h (Fig. 3b). PMID:19895806 This result indicates that eve is controlled by auto-repression. PMID:25385617 Perturbation of the pan-veg1 transcription factor Eve by injection of Eve MASO resulted in decreased expression of wnt1 , wnt4 , wnt5 , and wnt16 |
| GO:0001228 DNA-binding transcription activator activity, RNA polymerase II-specific | IMP PMID:21623371 A gene regulatory network controlling the embryonic specific... | NEW | Summary: Proposed. Eve is the spatial activator of the veg1 endoderm GRN, turning on hox11/13b in veg1 descendants from 21-24 h, and it activates wnt1, wnt4, wnt5 and wnt16 in veg1 cells. Reason: eve MASO reduces hox11/13b only after 24 h (PMID:21623371) and reduces wnt1, wnt4, wnt5 and wnt16 (PMID:25385617); within the GRN these links are treated as direct inputs of Eve. IMP because the evidence is morpholino perturbation with QPCR/WMISH readout; no Eve target site has been mutated and no binding assay exists for the sea urchin protein. Supporting Evidence: PMID:21623371 Hox11/13b expression is activated by Eve in descendants of veg1 cells at 24 h, because injection of eve MASO reduces hox11/13b expression only after 24 h (Fig. 3b). PMID:25385617 Perturbation of the pan-veg1 transcription factor Eve by injection of Eve MASO resulted in decreased expression of wnt1 , wnt4 , wnt5 , and wnt16 PMID:25385617 Thus Eve plays a dual role in the specification of veg1 ectoderm: It directly represses genes of the animal ectoderm in these cells, and it activates the expression of wnt4 in veg1 endoderm and veg1 ectoderm, leading to the activation of genes of the veg1 ectodermal GRN. |
| GO:0045944 positive regulation of transcription by RNA polymerase II | IMP PMID:21623371 A gene regulatory network controlling the embryonic specific... | NEW | Summary: Proposed. The process counterpart of the activator activity; Eve-dependent activation of hox11/13b (and through it brachyury and hnf1) and of the veg1 wnt genes. Reason: eve MASO decreases hox11/13b after 24 h and the veg1 wnt genes; Eve is the transcription factor performing the activation step within the veg1 GRN. Supporting Evidence: PMID:21623371 Hox11/13b expression is activated by Eve in descendants of veg1 cells at 24 h, because injection of eve MASO reduces hox11/13b expression only after 24 h (Fig. 3b). PMID:25385617 Perturbation of the pan-veg1 transcription factor Eve by injection of Eve MASO resulted in decreased expression of wnt1 , wnt4 , wnt5 , and wnt16 PMID:25385617 In veg1 endoderm cells, expression of hox11/13b initiates at 21β24 h under the control of Eve, and transcription of wnt1 and wnt16 , activated by Hox11/13b, starts at 24 h. |
| GO:0001227 DNA-binding transcription repressor activity, RNA polymerase II-specific | IMP PMID:19895806 The endoderm gene regulatory network in sea urchin embryos u... | NEW | Summary: Proposed. Eve represses its own gene (eve MASO raises eve transcript; autorepression clears eve from veg2 endoderm) and represses animal-ectoderm genes such as fgf9 and myc in veg1 ectoderm. Reason: Injection of eve MASO is the only perturbation that changes eve levels, upward, indicating autorepression (PMID:19895806); in veg1 ectoderm Eve directly represses animal-ectoderm genes (PMID:25385617), including fgf9 and myc (PMID:25655703). IMP from morpholino perturbation. Supporting Evidence: PMID:19895806 This result indicates that eve is controlled by auto-repression. PMID:19895806 It follows that in normal embryos eve auto-repression is required to clear eve expression from veg2 cells, despite the presence of levels of Tcf/Ξ²-catenin sufficient to support expression of the veg2 endoderm genes. PMID:25385617 Thus Eve plays a dual role in the specification of veg1 ectoderm: It directly represses genes of the animal ectoderm in these cells, and it activates the expression of wnt4 in veg1 endoderm and veg1 ectoderm, leading to the activation of genes of the veg1 ectodermal GRN. PMID:25655703 fgf9 expression is excluded from subdomains 3 and 4 (Veg1 ectoderm) by Eve. |
| GO:0000122 negative regulation of transcription by RNA polymerase II | IMP PMID:19895806 The endoderm gene regulatory network in sea urchin embryos u... | NEW | Summary: Proposed. Process counterpart of the repressor activity; autorepression of eve and repression of animal-ectoderm genes in veg1 cells. Reason: Same evidence as GO:0001227; Eve is the factor performing the repression. Supporting Evidence: PMID:19895806 This result indicates that eve is controlled by auto-repression. PMID:19895806 It follows that in normal embryos eve auto-repression is required to clear eve expression from veg2 cells, despite the presence of levels of Tcf/Ξ²-catenin sufficient to support expression of the veg2 endoderm genes. PMID:25655703 fgf9 expression is excluded from subdomains 3 and 4 (Veg1 ectoderm) by Eve. |
| GO:0001714 endodermal cell fate specification | IMP PMID:21623371 A gene regulatory network controlling the embryonic specific... | NEW | Summary: Proposed. Eve is the pan-veg1 transcription factor whose activation of hox11/13b installs the veg1 endoderm GRN that specifies future hindgut fate; eve MASO removes this activation. Reason: Participation test passed. Eve itself performs the regulatory step (activation of the hindgut regulatory genes) that specifies the veg1 endoderm regulatory state; this is not merely a downstream phenotype. Comparator check with sibling GRN factors (Blimp1, FoxA, GataE, Otx) that carry the same term in this project. Hindgut-development terms are not proposed because morphogenesis of the hindgut is a downstream outcome of the GRN Eve initiates. Supporting Evidence: PMID:21623371 Hox11/13b expression is activated by Eve in descendants of veg1 cells at 24 h, because injection of eve MASO reduces hox11/13b expression only after 24 h (Fig. 3b). PMID:21623371 The assembly of the veg1 endoderm GRN, which is spatially activated by Eve, is temporally motivated by a predicted signal (V1) expressed under the control of the veg2 endoderm GRN. PMID:21623371 These result in the recruitment of two canonical hindgut regulatory genes into the veg1 endoderm GRN, which specifies future hindgut cell fate. PMID:25385617 The earliest specification of the veg1 cell lineage, the precursor of veg1 endoderm and veg1 ectoderm, is controlled by eve |
| GO:0001715 ectodermal cell fate specification | IMP PMID:25385617 Specific functions of the Wnt signaling system in gene regul... | NEW | Summary: Proposed. Eve specifies veg1 ectoderm by repressing animal-ectoderm genes in veg1 cells and activating wnt4, which in turn activates the veg1 ectodermal GRN. Reason: The authors explicitly assign Eve a dual role in the specification of veg1 ectoderm; both arms (direct repression of animal-ectoderm genes and activation of wnt4) are transcriptional steps performed by Eve. Restricted to the veg1 ectoderm sub-lineage, not ectoderm generally. Supporting Evidence: PMID:25385617 Thus Eve plays a dual role in the specification of veg1 ectoderm: It directly represses genes of the animal ectoderm in these cells, and it activates the expression of wnt4 in veg1 endoderm and veg1 ectoderm, leading to the activation of genes of the veg1 ectodermal GRN. PMID:25385617 The earliest specification of the veg1 cell lineage, the precursor of veg1 endoderm and veg1 ectoderm, is controlled by eve PMID:25655703 fgf9 expression is excluded from subdomains 3 and 4 (Veg1 ectoderm) by Eve. |
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Download this section (compressed HTML)Q: Does Sp-Eve bind the hox11/13b, wnt4 or eve cis-regulatory modules directly? All Eve outputs rest on morpholino perturbation within the GRN framework; no Eve target site has been mutated and no EMSA or ChIP has been reported for the sea urchin protein.
Q: Is the Blimp1 input into the eve cis-regulatory module (site mutation reduced Eve-GFP to 14% of control) a reporter artefact, given that blimp1 MASO does not change endogenous eve transcript levels and the two expression domains do not overlap at 18 h?
Q: Should Eve's control of the veg1 wnt ligand genes be represented as regulation of Wnt signaling, or (as here) only as transcriptional regulation with the wnt genes as inputs? Eve acts one transcriptional step upstream of the ligands.
Experiment: Mutagenesis of candidate Eve (TAAT-core homeodomain) sites in the hox11/13b and wnt4 cis-regulatory modules in BAC-GFP reporters, and EMSA/ChIP-qPCR with the Sp-Eve homeodomain, to convert the IMP activator links into direct cis-regulatory evidence.
Experiment: Autorepression site mapping in the eve cis-regulatory module (Eve-site mutation in the 2.5 kb Eve-GFP construct), to test whether veg2 clearance of eve is a direct cis effect.
Experiment: Immunolocalization or Eve-GFP fusion to document nuclear localization and protein persistence relative to the veg2-to-veg1 shift of eve transcription.
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The nature of the Eve-dependent signal V2 to veg2 endoderm and the mechanism by which Eve restricts animal-ectoderm gene expression in veg1 ectoderm remain undefined.
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