Sp-FoxA is the purple sea urchin orthologue of the FoxA/HNF3 class of forkhead-box transcription factors, with a winged-helix Fork-head DNA-binding domain, a Forkhead N-terminal region and a FoxA-type C-terminal region. It is a canonical endoderm regulatory gene: zygotic transcription begins at about 11 h post-fertilization in the whole veg2 endomesoderm lineage, is switched off in the prospective non-skeletogenic mesoderm at 18-20 h so that only the veg2 endoderm keeps expressing it, and later extends to veg1 endoderm, the gut after gastrulation and a patch of oral ectoderm where the mouth forms. Four cis-regulatory modules integrate a Tcf/beta-catenin toggle switch (spatial restriction to the endoderm), Su(H), Hox11/13b, Otx and Brachyury activating inputs, and a single FoxA site through which the protein represses its own gene. In the endoderm FoxA represses the mesoderm regulator gcm and thereby excludes mesodermal (pigment cell) fate from endodermal cells, helps maintain the endoderm-mesoderm boundary and provides regulatory functions essential for specification of the foregut endoderm; it also activates hedgehog. Loss of FoxA yields excess pigment and other mesenchymal cells, failure of gut formation and absence of the mouth.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of RNA polymerase II cis-regulatory sequence-specific DNA binding for the forkhead family node (PTN000930906; mouse/rat Foxa1-3, human FOXA proteins, worm PHA-4, fly fork head, yeast FKH1/2). Sea urchin FoxA is shown to act through a defined genomic target site in its own module I. Reason: Family-wide winged-helix DNA binding is robust, and the cis-regulatory dissection of the foxa locus shows that a single FoxA site in module I mediates autorepression, i.e. the protein binds a cis-regulatory region of an RNA polymerase II gene. Supporting Evidence: PMID:20479235 A mutation of a single putative FoxA site in module I increased the level of the FIJ:GFP reporter transcript at 20–24 hpf ( Fig. 3 I ). This result verifies that FoxA is an autorepressor that reduces, but does not eliminate, its gene product level. |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of RNA polymerase II-specific DNA-binding transcription factor activity from the forkhead family node. This is the core molecular function of Sp-FoxA, which is an integral node of the endoderm specification GRN with cis-regulatory-verified inputs and outputs. Reason: Sea urchin FoxA is a documented sequence-specific transcriptional regulator (autorepressor of its own gene, repressor of gcm, activator of hedgehog), so the phylogenetic transfer is directly supported on the target. Supporting Evidence: PMID:17038513 The foxa gene is an integral component of the endoderm specification subcircuit PMID:20479235 A mutation of a single putative FoxA site in module I increased the level of the FIJ:GFP reporter transcript at 20–24 hpf ( Fig. 3 I ). This result verifies that FoxA is an autorepressor that reduces, but does not eliminate, its gene product level. |
| GO:0003677 DNA binding | IEA GO_REF:0000104 | MODIFY | Summary: Generic DNA binding from the UniRule forkhead rule. Correct but uninformative; the IBA row for GO:0000978 captures the specific cis-regulatory binding. Reason: Too general to be useful. The forkhead domain binds cis-regulatory sites of RNA polymerase II genes, demonstrated for Sp-FoxA by the autorepression site in module I of its own locus, so GO:0000978 is the informative term. Proposed replacements: RNA polymerase II cis-regulatory region sequence-specific DNA binding Supporting Evidence: PMID:20479235 A mutation of a single putative FoxA site in module I increased the level of the FIJ:GFP reporter transcript at 20–24 hpf ( Fig. 3 I ). This result verifies that FoxA is an autorepressor that reduces, but does not eliminate, its gene product level. |
| GO:0003700 DNA-binding transcription factor activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping of the Fork_head domain to generic DNA-binding transcription factor activity. Correct at the family level; the RNA polymerase II-specific child (GO:0000981) is the standard term for a metazoan developmental transcription factor. Reason: Sound but redundant with, and less specific than, the IBA row GO:0000981; the RNA polymerase II-specific term should be used. Proposed replacements: DNA-binding transcription factor activity, RNA polymerase II-specific Supporting Evidence: PMID:17038513 The foxa gene is an integral component of the endoderm specification subcircuit |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of nuclear localization across the forkhead family. Expected for a transcription factor whose activity is demonstrated by cis-regulatory target-site mutagenesis. Reason: Nuclear activity is implied by the sequence-specific transcriptional autorepression and gcm repression documented in sea urchin; no contrary evidence. Supporting Evidence: PMID:20479235 This result verifies that FoxA is an autorepressor that reduces, but does not eliminate, its gene product level. |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: UniRule/subcellular-location mapping to nucleus. Same claim as the IBA row. Reason: Consistent with the transcription factor function documented for Sp-FoxA. Supporting Evidence: PMID:20479235 This result verifies that FoxA is an autorepressor that reduces, but does not eliminate, its gene product level. |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping of the Fork_head domain to generic regulation of DNA-templated transcription. Correct but uninformative; the documented direction of Sp-FoxA regulation is repression of its own gene and of gcm. Reason: Too general. Perturbation and cis-regulatory data show FoxA lowering transcription of its own gene (FoxA site mutation or foxa MO doubles reporter output) and of gcm, so the specific term GO:0000122 is preferred (see NEW row); the IBA row GO:0006357 already gives the RNA polymerase II-level parent. Proposed replacements: negative regulation of transcription by RNA polymerase II Supporting Evidence: PMID:20479235 At 24 hpf the injection of Foxa MO increased the level of the construct FIJ:GFP by 2-fold, similar to the increase of the level of the endogenous foxa gene in the same injections. PMID:17038513 pregastrular regulatory system of foxa, and Foxa represses its own |
| GO:0006357 regulation of transcription by RNA polymerase II | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of regulation of transcription by RNA polymerase II from the forkhead family node. Sp-FoxA is a verified transcriptional regulator in the endoderm GRN. Reason: Supported on the target by autorepression through a FoxA site in module I and by MO-based repression of gcm; the more specific negative-regulation term is proposed as a NEW row. Supporting Evidence: PMID:20479235 A mutation of a single putative FoxA site in module I increased the level of the FIJ:GFP reporter transcript at 20–24 hpf ( Fig. 3 I ). This result verifies that FoxA is an autorepressor that reduces, but does not eliminate, its gene product level. |
| GO:0009653 anatomical structure morphogenesis | IBA GO_REF:0000033 | MODIFY | Summary: Generic morphogenesis term propagated from the forkhead family node (many vertebrate Fox genes with organ morphogenesis phenotypes). In sea urchin, foxa knockdown causes failure of gut formation and absence of the mouth, so the family-level inference holds but is uninformative. Reason: The inference is sound but the root-level term says nothing gene-specific. The documented sea urchin phenotype is failure of archenteron/gut formation (cell intercalation and epithelial organisation) on foxa MASO, plus absence of the stomodaeum, for which GO:0048546 digestive tract morphogenesis is the appropriate descendant. This is a downstream consequence of the endoderm regulatory state FoxA drives, so it is not a core function. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000930906 SUPPORTS TRANSFER Forkhead family node; morphogenesis roles are documented for many descendants (vertebrate Foxa/Foxc/Foxd genes, fly fork head, worm pha-4), and the inference holds for sea urchin FoxA (gut and mouth formation fail on knockdown), but the root-level term is uninformative and the specific process is digestive tract morphogenesis. Proposed replacements: digestive tract morphogenesis Supporting Evidence: PMID:21130759 When foxa is downregulated by the injection of morpholino antisense oligonucleotides these processes do not occur, and there is a failure of gut formation (Oliveri et al., 2006). PMID:17038513 endomesoderm cells become pigment and other mesenchymal cell types, less gut is |
| GO:0019904 protein domain specific binding | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO mapping from the Forkhead N-terminal region (IPR013638) to protein domain specific binding. There is no sea urchin evidence for any specific protein-domain interaction, and the term conveys no information about FoxA function. Reason: A bare binding term inferred from a short N-terminal region annotation; no partner or domain is identified for Sp-FoxA and the term adds nothing beyond the transcription factor activity already captured. Not a core function. |
| GO:0030154 cell differentiation | IBA GO_REF:0000033 | MODIFY | Summary: Generic cell differentiation propagated from the forkhead family node. The sea urchin gene is specifically an endoderm regulatory gene that excludes mesodermal fate from endoderm cells and is essential for foregut endoderm specification. Reason: Correct at the family level but uninformative for this gene. foxa MASO converts endomesoderm cells to pigment and other mesenchymal fates and reduces gut specification, and FoxA provides regulatory functions essential to specification of the foregut endoderm, so the specific descendant GO:0001714 endodermal cell fate specification is the better term (see NEW row). Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000930906 SUPPORTS TRANSFER Forkhead family node; the generic differentiation inference is sound for the family and for sea urchin FoxA, but the lineage-specific term (endodermal cell fate specification) is what the target evidence supports. Proposed replacements: endodermal cell fate specification Supporting Evidence: PMID:17038513 endomesoderm cells become pigment and other mesenchymal cell types, less gut is PMID:20479235 For these endoderm cells, foxa later provides canonical regulatory functions that are essential to specification of the foregut endoderm ( 7 ). |
| GO:0043565 sequence-specific DNA binding | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated mapping (ARBA plus InterPro Fork_head signatures) to sequence-specific DNA binding. Correct; the FoxA site in module I of the foxa locus is a defined sequence-specific target. Reason: Sequence-specific DNA binding is the defining property of the forkhead domain and is supported on the target by site-directed mutagenesis of a FoxA site in a foxa reporter construct. The IBA row GO:0000978 is the more specific form. Supporting Evidence: PMID:20479235 A mutation of a single putative FoxA site in module I increased the level of the FIJ:GFP reporter transcript at 20–24 hpf ( Fig. 3 I ). This result verifies that FoxA is an autorepressor that reduces, but does not eliminate, its gene product level. |
| GO:0001227 DNA-binding transcription repressor activity, RNA polymerase II-specific | IDA PMID:20479235 Information processing at the foxa node of the sea urchin en... | NEW | Summary: Sequence-specific transcriptional repressor activity demonstrated on the foxa locus itself. Mutation of the single FoxA site in cis-regulatory module I raises reporter output, matching the 2-fold rise in endogenous and reporter foxa transcripts after foxa MO injection; in the endoderm FoxA also represses gcm, the top mesoderm regulator. Reason: Site-directed mutagenesis of a FoxA target site in a reporter construct is IDA-grade evidence that FoxA acts as a DNA-binding repressor on an RNA polymerase II gene. The gcm repression (IMP) is the biologically decisive output. Not an ancestor or descendant of any term already on the gene except the accepted parent GO:0000981. Supporting Evidence: PMID:20479235 A mutation of a single putative FoxA site in module I increased the level of the FIJ:GFP reporter transcript at 20–24 hpf ( Fig. 3 I ). This result verifies that FoxA is an autorepressor that reduces, but does not eliminate, its gene product level. PMID:20479235 At 24 hpf the injection of Foxa MO increased the level of the construct FIJ:GFP by 2-fold, similar to the increase of the level of the endogenous foxa gene in the same injections. PMID:17038513 the normal endoderm, a crucial role of Foxa is to repress gcm expression in response |
| GO:0000122 negative regulation of transcription by RNA polymerase II | IMP PMID:17038513 Repression of mesodermal fate by foxa, a key endoderm regula... | NEW | Summary: FoxA lowers transcription of its own gene (autorepression yielding an oscillatory profile) and of gcm in endodermal cells; foxa MASO increases foxa transcript levels and allows gcm expression and mesodermal fate in endoderm. Reason: Both perturbation (MO) and cis-regulatory (site mutation) evidence show FoxA reducing RNA polymerase II transcription of defined target genes. Specific child of the accepted IBA GO:0006357 and the informative replacement for the generic IEA GO:0006355. Supporting Evidence: PMID:17038513 pregastrular regulatory system of foxa, and Foxa represses its own PMID:17038513 the normal endoderm, a crucial role of Foxa is to repress gcm expression in response PMID:20479235 At 24 hpf the injection of Foxa MO increased the level of the construct FIJ:GFP by 2-fold, similar to the increase of the level of the endogenous foxa gene in the same injections. |
| GO:0042662 negative regulation of mesodermal cell fate specification | IMP PMID:17038513 Repression of mesodermal fate by foxa, a key endoderm regula... | NEW | Summary: In the veg2 endomesoderm, FoxA represses gcm, the Delta/Notch target that sits at the top of the mesoderm GRN, and so excludes mesodermal fate from endoderm cells; foxa MASO makes more endomesoderm cells become pigment and other mesenchymal cell types, and blastomere transplantation combined with MASO localizes this function to the normal endoderm. Reason: Participation test: FoxA itself performs the regulatory step (repression of gcm) that prevents mesodermal specification in endoderm; this is the central conclusion of Oliveri et al. 2006 and is restated as boundary maintenance by de-Leon and Davidson. Comparator check: GO:0042662 is carried by transcription factors that repress mesodermal fate in other systems (mouse/human MESP1, zebrafish klf2a/b), so the term is conventional for a fate-repressing transcription factor. Supporting Evidence: PMID:17038513 mesodermal fate in the veg2 endomesoderm; it is required in postgastrular PMID:17038513 to a Notch signal, and hence to repress mesodermal fate. PMID:20479235 In Xenopus ( 6 ) and the sea urchin ( 1 ) a major function of the foxa gene during embryonic development is maintenance of the endoderm–mesoderm boundary, by repression of mesoderm fate in the endoderm. PMID:21130759 The suppression of mesodermal fate in the endoderm is mediated, at least in part, by Foxa repression of the gene that encodes the transcription factor GCM (Oliveri et al., 2006), a key regulator of mesodermal fate in the sea urchin embryo (Ransick and Davidson, 2006). |
| GO:0001714 endodermal cell fate specification | IMP PMID:17038513 Repression of mesodermal fate by foxa, a key endoderm regula... | NEW | Summary: FoxA is an integral node of the veg2 endoderm specification subcircuit. It is the endoderm regulatory gene retained by the peripheral veg2 cells when endoderm and mesoderm regulatory states separate, it excludes the mesoderm program from those cells, and it provides regulatory functions essential to specification of the foregut endoderm; knockdown reduces the amount of gut specified. Reason: Participation test: FoxA is a transcription factor acting within the endoderm cells on endoderm-GRN targets (gcm repression, hedgehog activation, autorepression), not merely a required upstream input. Comparator check: GO:0001714 is the term carried by endoderm-specifying transcription factors in other organisms (worm END-1, MED-1/2, zebrafish foxh1, mouse Eomes), so it is the conventional term. Replaces the generic IBA GO:0030154. Supporting Evidence: PMID:17038513 The foxa gene is an integral component of the endoderm specification subcircuit PMID:17038513 endomesoderm cells become pigment and other mesenchymal cell types, less gut is PMID:20479235 For these endoderm cells, foxa later provides canonical regulatory functions that are essential to specification of the foregut endoderm ( 7 ). PMID:21623371 whereas the peripheral cells of the veg2 lineage (the presumptive endoderm) express foxa alone (Fig. 2a) |
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Download this section (compressed HTML)Q: Is FoxA repression of gcm direct (FoxA sites in the gcm cis-regulatory system), or mediated through the Notch/Su(H) input that activates gcm? The transplantation experiments establish the functional relationship but not the cis-regulatory mechanism.
Q: Should FoxA also be annotated as a transcriptional activator (GO:0001228)? Activation of hedgehog and the post-gastrular requirement for gut-specific gene expression are reported, but the cached primary evidence is abstract-level or second-hand, so no activator row was proposed.
Q: Does the oral-ectoderm (stomodaeum) function of foxa warrant a separate mouth development annotation, given that it is a distinct, non-endodermal regulatory network?
Experiment: Cis-regulatory analysis of the gcm locus in the endoderm, mutating candidate FoxA sites in a gcm reporter and combining with foxa MASO, to test whether gcm repression by FoxA is direct.
Type: cis-regulatory analysis
Experiment: ChIP-seq or DAP-seq for Sp-FoxA at 24-30 hpf to define its direct target set in the endoderm (gut differentiation genes, hedgehog, adhesion and intercalation genes) and separate direct activation from indirect requirements.
Type: genomic binding profiling
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