Zebrafish Hes6 (Her13.2) is a nuclear Hairy/Enhancer-of-split-type basic helix-loop-helix (bHLH)-Orange protein with a C-terminal WRPW motif, a member of the vertebrate HES6 subfamily (co-orthologous, with zebrafish her13, to mammalian HES6). Unlike the Notch-driven cyclic genes her1 and her7, hes6 is not transcriptionally oscillating; it is induced by FGF signalling and expressed in a posterior-to-anterior gradient in the tailbud and posterior presomitic mesoderm, although its protein level oscillates post-transcriptionally. Hes6 is a dimerization partner within the segmentation-clock core circuit: it forms heterodimers with Her1 and Her7 and a homodimer. Its short HLH loop prevents DNA binding by the homodimer, but as a Her7:Hes6 heterodimer it binds N/E-box-type sites in the her1, her7 and deltaC promoters and represses their transcription, while Her1:Hes6 dimers are largely non-DNA-binding. By shortening the effective half-lives of Her1 and Her7, Hes6 sets the clock period: hes6 mutants segment more slowly and form fewer somites, and loss of hes6 together with her1 abolishes somite boundaries.
Existing Annotations Review
GO Term
Evidence
Action
Reason
GO:0000122 negative regulation of transcription by RNA polymerase II
IBA GO_REF:0000033
ACCEPT
Summary: Negative regulation of RNA polymerase II transcription is central to the Hes6-containing segmentation-clock circuit. Target-specific reporter and DNA-binding experiments support the IBA independently of general Hairy-family naming.
Reason: PAINT node PTN004213585 (Hairy/E(spl) bHLH-Orange repressors) is the right clade, and the zebrafish protein has direct evidence for the function: Her13.2/Hes6 augments her1 autorepression in reporter assays [PMID:15905406] and Her7:Hes6 heterodimers bind cyclic-gene promoter sites [PMID:22911291]. Core process.
Her13.2 augments autorepression of her1 in association with Her1 protein.
GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding
IBA GO_REF:0000033
ACCEPT
Summary: Her7:Hes6 heterodimers bind specific sites in cyclic-gene promoters directly. This supports the term in a heteromeric context; it does not assert that isolated Hes6 or its homodimer binds those sites. A contributes_to qualifier best expresses the subunit contribution.
Reason: Sequence-specific promoter binding is shown for the Her7:Hes6 heterodimer, not for Hes6 alone [PMID:22911291]; the IBA is correct for the gene product as a subunit of that complex, which is why the core function records it as contributes_to. Accepted.
Summary: Hes6 contributes to a DNA-binding repressor with Her7, coupling promoter recognition to cyclic transcription. The IBA function is supported in that complex context; an autonomous Hes6 homodimeric transcription factor is not established.
Reason: Hes6 provides half of a promoter-binding, transcription-repressing bHLH heterodimer with Her7 [PMID:22911291] and enhances Her1-mediated repression [PMID:15905406]; the family-node placement is sound. Accepted, with the caveat that isolated Hes6 shows no strong DNA binding.
IGI PMID:16545363 her1 and her13.2 are jointly required for somitic border spe...
ACCEPT
Summary: The cited study explicitly reports loss of somitic borders after combined her1/her13.2 inactivation, directly supporting the core segmentation role.
Reason: Abstract-only cache, but the abstract itself states that joint her1/her13.2 inactivation abolishes somitic borders. Hes6 does work in this process as a segmentation-clock repressor subunit, so it passes the participation test. Core.
IGI PMID:20637625 Segment number and axial identity in a segmentation clock pe...
ACCEPT
Summary: The clock-period mutant study and independently documented hes6 identity support a genetic contribution to somite timing and number. Full-text follow-up explicitly identifies this mutant as hes6.
Reason: Abstract-only cache for the period-mutant paper; the later full-text study identifies the mutant as a hes6 allele and re-tests her1;hes6 combinations [PMID:29624170]. Clock-component role, core.
IMP PMID:15905406 Zebrafish hairy/enhancer of split protein links FGF signalin...
ACCEPT
Summary: The original Her13.2 perturbation study directly establishes its requirement for periodic gene repression and proper somite segmentation.
Reason: Full text read: her13.2 morphants lose periodic repression of her1/her7 and proper somite segmentation. Hes6 is a clock repressor subunit, so the process annotation reflects participation, not a downstream phenotype. Core.
IMP PMID:16545363 her1 and her13.2 are jointly required for somitic border spe...
ACCEPT
Summary: The study directly tests her13.2 loss of function, including the enhanced phenotype in combination with her1. The abstract establishes the target and somitic phenotype; no unsupported exact assay detail is inferred.
Reason: Abstract states the single and combined her13.2 loss-of-function somite phenotypes; deferring to ZFIN for the assay details of this second morpholino record. Core.
IMP PMID:20637625 Segment number and axial identity in a segmentation clock pe...
ACCEPT
Summary: Slowing the Hes6-dependent segmentation clock reduces segment number, fitting the core function of the gene in somitogenesis.
Reason: The period mutant (later shown to be hes6) forms fewer segments because the clock runs slower; Hes6 is part of the oscillator itself [PMID:22911291]. Core; abstract-only cache, deferring to ZFIN for genotype details.
IMP PMID:20637625 Segment number and axial identity in a segmentation clock pe...
ACCEPT
Summary: This separate GOA perturbation row represents the same experimentally grounded core somitogenesis function. The biological conclusion is supported without collapsing distinct curator-recorded reagents or genotypes.
Reason: The period mutant (later shown to be hes6) forms fewer segments because the clock runs slower; Hes6 is part of the oscillator itself [PMID:22911291]. Core; abstract-only cache, deferring to ZFIN for genotype details.
IMP PMID:29624170 Segmentation of the zebrafish axial skeleton relies on notoc...
ACCEPT
Summary: The full paper generates a new hes6 TALEN allele and tests clock-mutant combinations, including her1;hes6 embryos with disordered myotome boundaries. Its conclusion that centra can segment independently of the clock does not contradict Hes6 function in somite formation.
Reason: Full text read: a new hes6 TALEN allele is used, and her1;hes6 mutants show disordered myotome boundaries, while the paper's conclusion concerns centra rather than somites. Somitogenesis role stands. Core.
Summary: Generic DNA binding is supported in the Her7:Hes6 complex, but promoter-sequence-specific DNA binding is more informative. The target contributes as a heterodimeric subunit rather than an independently DNA-binding homodimer.
Reason: InterPro bHLH-Orange mapping; DNA binding is demonstrated only for the Her7:Hes6 heterodimer and at specific promoter sites [PMID:22911291], so modify to the sequence-specific cis-regulatory term already carried by IBA.
IPI PMID:22911291 Topology and dynamics of the zebrafish segmentation clock co...
MODIFY
Summary: The Her1 interaction is a defined bHLH heterodimerization event in the clock network, better represented by protein heterodimerization than generic protein binding. Her1:Hes6 does not share the strong DNA binding of Her7:Hes6.
Reason: The Her1 (Q90463) interaction is bHLH heterodimerisation within the clock circuit [PMID:22911291, PMID:15905406]; the informative, evidence-backed MF is protein heterodimerization activity, which the gene already carries by IPI with the same partner. Modify per the protein-binding policy.
IPI PMID:22911291 Topology and dynamics of the zebrafish segmentation clock co...
REMOVE
Summary: The donor is Myod1, and the full paper actually tests this interaction as an example of promiscuous bHLH association. Bare protein binding is removed under the protein-binding policy; the observed co-purification does not establish a physiological myogenic function for Hes6, and no specific MF is supported for this pair.
Reason: The partner is Myod1 (Q90477); in the paper MyoD is co-purified by Her1, Her7 and Hes6 alike as a demonstration of promiscuous bHLH association, with a non-bHLH negative control, and no function is assigned to a Hes6:MyoD dimer [PMID:22911291]. Generic protein binding adds no functional information and there is no evidence-backed specific MF for this pair, so remove; removal does not deny that the interaction was observed.
even the bHLH containing factor MyoD, but not the negative control non-bHLH protein PPARΞ³, was co-purified by Her1, Her7, and Hes6 to a similar extent
IPI PMID:22911291 Topology and dynamics of the zebrafish segmentation clock co...
MODIFY
Summary: The Her7 interaction forms the directly characterized DNA-binding Her7:Hes6 heterodimer. Protein heterodimerization describes this molecular event more precisely than generic protein binding.
Reason: The Her7 (Q9I9K1) interaction forms the DNA-binding Her7:Hes6 heterodimer, the functional unit of Hes6 in the clock [PMID:22911291]. Modify to protein heterodimerization activity per the protein-binding policy.
Summary: The conserved nuclear-location inference is confirmed for zebrafish Hes6 by immunostaining that is lost in hes6 mutants, and is consistent with its promoter binding and transcriptional repression.
Reason: Family node PTN004213585 (Hairy/E(spl) bHLH-Orange repressors), inside which Hes6 (PTHR10985:SF11) sits. Target-specific evidence exists as well, which the earlier review missed; a monoclonal antibody raised against full-length zebrafish Hes6 stains a nuclear antigen in wild-type PSM that is absent in hes6 mutants [PMID:22911291], and the protein's promoter-binding and repression activities require nuclear action. Accepted.
Summary: Nuclear localization is directly observed for zebrafish Hes6 by antibody staining that is absent in hes6 mutants, so the electronic SubCell/UniRule mapping is correct.
Reason: SubCell/UniRule mapping agrees with direct immunostaining of a nuclear Hes6 antigen in the PSM that is lost in hes6 mutants [PMID:22911291], and with the nuclear transcriptional-repressor function [PMID:15905406]. Accepted; same claim as the IBA nucleus row.
Summary: The broad transcription association is valid, but Hes6 regulates transcription negatively rather than catalyzing RNA synthesis. Use the specific negative-regulation term.
Reason: UniRule-transferred 'DNA-templated transcription' is the wrong branch for a repressor: Hes6 regulates transcription negatively rather than participating in RNA synthesis [PMID:15905406]. Modify to the negative-regulation term.
only Her1 homodimers and Her7:Hes6 heterodimers have strong DNA binding activity and target similar DNA sites.
GO:0006355 regulation of DNA-templated transcription
IEA GO_REF:0000120
MODIFY
Summary: The transcriptional role is directly supported and can be specified as negative regulation of RNA polymerase II transcription in the segmentation clock.
Reason: Correct but generic; the direction (negative) and polymerase (RNA Pol II) are established [PMID:15905406, PMID:22911291], so modify to GO:0000122.
Summary: In GO, somitogenesis (GO:0001756) is a direct is_a child of anterior/posterior pattern specification, so this IBA is a correct but general parent of the somitogenesis role the gene already carries with strong zebrafish evidence. Refine to somitogenesis; the period-mutant result argues against a separate role in Hox-associated axial identity.
Reason: The IBD at node PTN000105428 is seeded largely by vertebrate segmentation-clock Hes/Her genes, zebrafish hes6 (ZDB-GENE-030828-5) among them, so the target is inside the clade and supplies its own experimental grounding. The only a/p patterning supported for zebrafish Hes6 is segmentation itself, and GO:0001756 somitogenesis is_a GO:0009952 (verified with OLS parents). The period mutant changes segment number without changing Hox-associated regional identity [PMID:20637625], so there is no separate axial-identity role to keep. The term is true but less specific than the evidence, so MODIFY to somitogenesis, which this gene already carries (earlier review had KEEP_AS_NON_CORE, which the general-term rule does not allow).
Propagation Review
Root cause:NO FAILURE CORE
Failure modes:GRANULARITY MISMATCH
Sources checked:
PANTHER:PTN000105428SUPPORTS TRANSFER
Node seeded by vertebrate segmentation-clock Hes/Her genes including zebrafish hes6 itself; the inherited a/p patterning is segmentation, captured more precisely by somitogenesis.
IC PMID:15905406 Zebrafish hairy/enhancer of split protein links FGF signalin...
ACCEPT
Summary: The inferred repressor complex is supported by the original interaction/repression assays and the directly characterized Her7:Hes6 DNA-binding heterodimer. Different possible dimers need not have equivalent repressor activity.
Reason: Inferred by the curator from the repression (GO:0045892) and heterodimerisation (GO:0046982) evidence in the same paper; the Her7:Hes6 DNA-binding heterodimer [PMID:22911291] later confirmed a repressor-complex context. Accepted.
IPI PMID:22911291 Topology and dynamics of the zebrafish segmentation clock co...
MODIFY
Summary: Direct assays show Hes6 self-association, so protein homodimerization is more informative than identical protein binding. The resulting homodimer lacks the strong promoter DNA binding of Her7:Hes6.
Reason: Self-association of Hes6 is directly assayed [PMID:22911291]; protein homodimerization activity is the informative child term. Note that the homodimer lacks the strong DNA binding of Her7:Hes6, so the homodimer is not the active repressor.
only Her1 homodimers and Her7:Hes6 heterodimers have strong DNA binding activity and target similar DNA sites.
GO:0045892 negative regulation of DNA-templated transcription
IDA PMID:15905406 Zebrafish hairy/enhancer of split protein links FGF signalin...
MODIFY
Summary: The reporter experiments directly establish enhanced her1 autorepression in association with Her13.2/Hes6. The repressed target is the RNA polymerase II promoter of the protein-coding her1 gene, so the RNA Pol II-specific child term is supported.
Reason: Full text read: a luciferase reporter driven by the 8.6-kb her1 promoter is not repressed by Her13.2 alone, but Her13.2 enhances Her1 self-repression [PMID:15905406]; Her7:Hes6 heterodimers bind her1/her7/dlc promoter sites [PMID:22911291]. The repressed promoters are RNA Pol II protein-coding gene promoters, so refine to GO:0000122, which the gene already carries by IBA. Core.
This promoter activity was repressed by Her1, but not affected by Her13.2, in 293T cells
GO:0045892 negative regulation of DNA-templated transcription
IGI PMID:15905406 Zebrafish hairy/enhancer of split protein links FGF signalin...
MODIFY
Summary: The genetic and expression experiments place Hes6 upstream of periodic repression of her1 and her7 transcription (nascent her1 intron signal is derepressed in morphants). These are RNA polymerase II-transcribed protein-coding genes, so the RNA Pol II-specific term is supported.
Reason: Genetic interaction with her1 (ZDB-GENE-980526-125): her13.2 is required for periodic repression of her1/her7 transcription [PMID:15905406], and Her7:Hes6 directly binds their promoters [PMID:22911291], so this is direct participation in repression, not a downstream phenotype. Refine to GO:0000122 as for the IDA row. Core.
IPI PMID:15905406 Zebrafish hairy/enhancer of split protein links FGF signalin...
ACCEPT
Summary: Her13.2/Hes6 association with Her1 is directly tested, and broader biophysical experiments establish heterodimer formation with clock components.
Reason: Direct interaction with Her1 [PMID:15905406] and systematic dimerisation assays showing all Her1/Her7/Hes6 pairs form [PMID:22911291]. This is the defining molecular activity of Hes6 in the clock; core.
Summary: Both homo- and heterodimer formation are experimentally demonstrated, so the broad InterPro-derived dimerization term is true but less specific than the evidence; refine to the two specific dimerization activities already carried by this gene.
Reason: InterPro bHLH/Orange mapping. Heterodimers with Her1 and Her7, and a Hes6 homodimer, are all experimentally shown [PMID:22911291; PMID:15905406]. Both specific descendants are supported, so both are listed (the earlier ACCEPT argued the term was broad, which the action rules do not allow). The heterodimerization activity is the functionally important one.
Summary: The IBA is placed at a deep Hairy/E(spl) node. Within that node, Hes6-subfamily (PTHR10985:SF11) orthologs in Xenopus and mouse promote neurogenesis, so the term is phylogenetically plausible. Zebrafish has two SF11 co-orthologs of HES6 (hes6/her13.2 and her13), and the published zebrafish hes6 work tests only the presomitic-mesoderm clock. Which paralog, if either, kept the neural role is untested.
Reason: Node PTN000105428 is seeded by Drosophila E(spl)/hairy, mouse Hes1/Hes3/Hes5-type genes and zebrafish her genes. No Hes6-subfamily gene is among the seeds, although mouse Hes6 (MGI:1859852) is a seed for other PTHR10985 IBDs that this gene receives. Zebrafish hes6 is inside the node, and its subfamily has neurogenesis evidence (Hes6 promotes neurogenesis in Xenopus gain-of-function [PMID:10976052]), so there is no phylogenetic ground to call the propagation wrong. However, the Hes6 lineage acts in the opposite direction from the ancestral E(spl) inhibition. Zebrafish also retains two SF11 genes, equidistant from mammalian HES6 (0.58 identity each; file:DANRE/hes6/hes6-bioinformatics/RESULTS-rereview.md), which leaves room for subfunctionalization. The only zebrafish hes6 functional data are mesodermal [PMID:15905406, PMID:22911291]. With neither target-specific support nor evidence of loss, this stays UNDECIDED.
Propagation Review
Root cause:UNRESOLVED
Failure modes:CONTEXT OR TISSUE MISMATCH
Sources checked:
PANTHER:PTN000105428UNRESOLVED
Deep Hairy/E(spl) node seeded by fly E(spl)/hairy and vertebrate Hes1/Hes5-type genes, with no HES6-subfamily seed. Hes6 orthologs promote rather than inhibit neurogenesis, and zebrafish retains two SF11 paralogs, so whether hes6 itself kept a neural role is untested.
FB:FBgn0002609SUPPORTS SOURCE BUT NOT TARGET
Drosophila donor with neurogenesis evidence; not informative about the zebrafish hes6 tissue context.
MGI:MGI:104853SUPPORTS SOURCE BUT NOT TARGET
Mouse Hes donor; mammalian neural roles are established but zebrafish hes6 is studied only in the segmentation clock.
Q: Does zebrafish Hes6 have a neural role distinct from its experimentally established presomitic segmentation-clock function?
Suggested Experiments
Experiment: Whole-mount in situ hybridisation and hes6 mutant analysis of neural markers (e.g. neurog1, elavl3) in the developing hindbrain and spinal cord to test whether zebrafish hes6 has any role in neurogenesis, as the family-level IBA implies.
Hypothesis: Zebrafish hes6 expression and function are confined to the presomitic mesoderm segmentation clock, and neurogenesis proceeds normally in hes6 mutants.
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
DNA binding, transcriptional regulation and dimerization are experimentally grounded in zebrafish Hes6, but all three predicted terms are broad parents of more specific, experimentally supported annotations the gene already carries (Pol II cis-regulatory sequence-specific DNA binding as a Her7:Hes6 heterodimer, negative regulation of Pol II transcription, and protein heterodimerization activity), so each is less precise than existing curation.
Review rationale: Direct assays establish sequence-specific promoter binding by the Her7:Hes6 heterodimer. The predicted term is already in GOA (InterPro IEA) and is an ancestor (verified in OLS) of RNA polymerase II cis-regulatory region sequence-specific DNA binding (GO:0000978), which the gene carries by IBA and the gene review accepts. Generic DNA binding is valid for the complex contribution but must not be interpreted as autonomous Hes6 homodimer binding.
Supporting Evidence:
PMID:22911291: "only Her1 homodimers and Her7:Hes6 heterodimers have strong DNA binding activity and target similar DNA sites."
GO:0006355 regulation of DNA-templated transcriptionGO_BP
LSP β Less precise than existing annotation Review score: 2/2
Review rationale: Hes6-dependent negative transcriptional regulation is established by reporter, genetic and biochemical experiments. The predicted term is already in GOA (IEA) and is an ancestor (verified in OLS) of negative regulation of transcription by RNA polymerase II (GO:0000122), which the gene carries by IBA and the gene review accepts as core, so the prediction is correct but less specific.
Supporting Evidence:
PMID:15905406: "Her13.2 augments autorepression of her1 in association with Her1 protein."
PMID:15905406: "her13.2 is required for periodic repression of the Notch-regulated genes her1 and her7, and for proper somite segmentation."
Review rationale: Both homo- and heterodimerization are directly demonstrated for zebrafish Hes6, which acts as the hub of the Her/Hes dimer network. The predicted term is already present in GOA (InterPro IEA) and is the parent (verified in OLS) of protein heterodimerization activity (GO:0046982), which the gene carries by IPI and which the gene review treats as its core molecular activity, and of protein homodimerization activity. The prediction is therefore correct but materially less specific than existing supported curation.
Supporting Evidence:
PMID:22911291: "all of the possible dimers between Her1, Her7 and Hes6 form"
PMID:22278920: "Dimerization is specific, with Hes6 serving as the hub of the network."
PMID:22911291: "only Her1 homodimers and Her7:Hes6 heterodimers have strong DNA binding activity and target similar DNA sites."