twin of eyeless (toy) encodes one of the two Drosophila Pax6 transcription factors, arising from an insect-lineage duplication of the ancestral Pax6 gene; Toy is more similar to vertebrate Pax6 than its paralog Eyeless (Ey). It carries an N-terminal paired domain and a paired-type homeodomain, both DNA-binding, and a C-terminal transactivation region, and acts in the nucleus as a sequence-specific RNA polymerase II transcription factor. Toy is expressed early in the embryonic head, brain and visual primordia and sits at the top of the retinal determination gene network: it directly binds and activates the eye-specific enhancer of eyeless, so it is required to initiate ey expression, and ectopic Toy induces ectopic eyes largely through Ey. Toy also binds enhancers of sine oculis and the Clock locus. Together with ey it is required for growth and development of the entire eye-antennal imaginal disc (strong mutants are headless), and it is the principal Pax6 gene for development of the dorsal ocelli, where it initiates eyes absent and sine oculis expression with Hedgehog signalling. Toy also contributes to embryonic brain patterning and mushroom body development, and regulates Clock expression in circadian pacemaker neurons.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IDA PMID:15790965 Functional analysis of the chicken delta1-crystallin enhance... | ACCEPT | Summary: Blanco et al. 2005 assayed the chicken delta1-crystallin DC5 enhancer in Drosophila. Although D-Pax2 is the physiological DC5 regulator in cone cells, the full text (checked online; cache is abstract-only) shows that His-TOY binds DC5 in EMSA and that Toy with SoxN activates DC5 in S2 cells and in vivo, demonstrating sequence-specific DNA-binding transactivation by Toy. Reason: Toy is a paired/homeodomain Pax6 transcription factor. This IDA is consistent with independent direct evidence that Toy binds and activates the ey eye enhancer (Czerny et al. 1999) and the so10 enhancer (Punzo et al. 2002). It is the core MF of the gene. Supporting Evidence: PMID:15790965 DC5 contains PAX6 and SOX2 binding sites, and its activity depends on the cooperative binding of these two transcription factors PMID:10198632 Toy functions upstream of ey by directly regulating the eye-specific enhancer of ey PMID:11830564 both Drosophila Pax6 proteins namely EY and TOY bind and positively regulate so10 expression through different binding sites |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro homeobox-conserved-site (IPR017970) mapping to RNA Pol II DNA-binding TF activity. Reason: Correct and consistent with experimental evidence that Toy directly binds and activates Pol II-transcribed target enhancers (ey, so). Supporting Evidence: PMID:10198632 Toy functions upstream of ey by directly regulating the eye-specific enhancer of ey PMID:11830564 both Drosophila Pax6 proteins namely EY and TOY bind and positively regulate so10 expression through different binding sites |
| GO:0003677 DNA binding | IEA GO_REF:0000120 | MODIFY | Summary: Generic DNA binding from paired-domain/homeodomain InterPro and UniRule mappings. Reason: Correct but uninformative. Toy binds specific cis-regulatory enhancer sequences of Pol II target genes (ey eye enhancer, so10, Clk locus), so the more specific RNA polymerase II cis-regulatory region sequence-specific DNA binding term is better supported. Proposed replacements: RNA polymerase II cis-regulatory region sequence-specific DNA binding Supporting Evidence: PMID:10198632 Toy functions upstream of ey by directly regulating the eye-specific enhancer of ey PMID:11830564 both Drosophila Pax6 proteins namely EY and TOY bind and positively regulate so10 expression through different binding sites PMID:24916389 toy binds multiple sites at the Clk locus |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: Nuclear localization inferred from homeodomain/UniRule. Reason: As a sequence-specific DNA-binding transcription factor that directly regulates target enhancers, Toy acts in the nucleus; consistent with all Pax6 family members. Supporting Evidence: PMID:10198632 Toy functions upstream of ey by directly regulating the eye-specific enhancer of ey |
| GO:0006351 DNA-templated transcription | IEA GO_REF:0000104 | MARK AS OVER ANNOTATED | Summary: UniRule-based annotation to DNA-templated transcription. Reason: Toy is a sequence-specific transcription factor that regulates transcription of target genes; it is not part of the core transcription machinery. Its role is better captured by positive regulation of transcription by RNA polymerase II, which is already annotated. Supporting Evidence: PMID:10198632 Toy functions upstream of ey by directly regulating the eye-specific enhancer of ey |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000120 | MODIFY | Summary: Regulation of DNA-templated transcription from InterPro/UniRule mappings. Reason: Correct but too general. Toy acts as an activator of Pol II target enhancers (ey, so10), so positive regulation of transcription by RNA polymerase II is the appropriate, more specific term. Proposed replacements: positive regulation of transcription by RNA polymerase II Supporting Evidence: PMID:10198632 Toy functions upstream of ey by directly regulating the eye-specific enhancer of ey PMID:11830564 both Drosophila Pax6 proteins namely EY and TOY bind and positively regulate so10 expression through different binding sites |
| GO:0008056 ocellus development | IGI PMID:20580700 Genetic interactions of eyes absent, twin of eyeless and ort... | ACCEPT | Summary: Blanco et al. 2010 analysed genetic interactions of toy with eya and oc in so regulation during ocellar development, using an enhancer-driven rescue of a strong toy allele. Reason: toy is the principal Pax6 gene of the ocelli; it initiates eya and so expression in the ocellar primordium, and TOY binding sites in the so10 enhancer are required for ocellus development. Core developmental role. Supporting Evidence: PMID:20580700 the toy positive effect on so expression is largely eya-mediated PMID:21104743 the role of the pax6 gene toy, together with the hh signaling pathway, in the initiation of eya and so expression PMID:11830564 the EY and TOY binding sites are required for compound eye and ocellus development respectively |
| GO:0008056 ocellus development | IMP PMID:20580700 Genetic interactions of eyes absent, twin of eyeless and ort... | ACCEPT | Summary: toy loss-of-function (rescued strong allele) affects so expression in the ocellar primordium (Blanco et al. 2010). Reason: Supported by multiple independent studies (Punzo 2002; Brockmann 2011) that toy is required for ocellar specification. Core role. Supporting Evidence: PMID:20580700 the toy positive effect on so expression is largely eya-mediated PMID:21104743 the role of the pax6 gene toy, together with the hh signaling pathway, in the initiation of eya and so expression PMID:11830564 the EY and TOY binding sites are required for compound eye and ocellus development respectively |
| GO:0009791 post-embryonic development | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA machine-learning annotation to post-embryonic development. Reason: Technically true (eye-antennal disc and ocellus development are post-embryonic) but uninformative; specific terms (eye-antennal disc development, ocellus development, compound eye development) are annotated with experimental evidence. Supporting Evidence: PMID:11861484 Strong mutants of twin of eyeless or of eyeless are headless, which suggests that they are required for the development of all structures derived from eye-antennal discs |
| GO:0010628 positive regulation of gene expression | IMP PMID:20580700 Genetic interactions of eyes absent, twin of eyeless and ort... | KEEP AS NON CORE | Summary: toy positively affects sine oculis expression in ocellar precursors, though largely via eya (Blanco et al. 2010). Reason: Consistent with Toy's activator role, but the cited study found the effect on so to be marginal and largely indirect (eya-mediated). The more informative direct activity is captured by positive regulation of transcription by RNA polymerase II. Supporting Evidence: PMID:20580700 the toy positive effect on so expression is largely eya-mediated |
| GO:0016319 mushroom body development | IMP PMID:19901536 Brain patterning defects caused by mutations of the twin of ... | KEEP AS NON CORE | Summary: toy mutants show severe deformation of mushroom body lobes in pharate adults (Furukubo-Tokunaga et al. 2009). Reason: Experimentally supported brain role, but secondary to the gene's primary visual-system specification function; earlier embryonic analyses found no gross CNS defects in toy nulls. Supporting Evidence: PMID:19901536 Mutations of toy perturb brain neuromere formation in the embryonic stages, and result in severe deformation of the MB lobes in pharate adult brains |
| GO:0030154 cell differentiation | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA annotation to cell differentiation. Reason: Very general term; Toy acts in tissue/organ specification (eye-antennal disc, ocelli, compound eye) rather than in a defined cell differentiation step. Specific developmental terms are already annotated. Supporting Evidence: PMID:10198632 Toy is therefore required for initiation of ey expression in the embryo and acts through Ey to activate the eye developmental program |
| GO:0035214 eye-antennal disc development | IMP PMID:11861484 Headless flies produced by mutations in the paralogous Pax6 ... | ACCEPT | Summary: Strong toy mutants are headless, lacking all eye-antennal disc derivatives (Kronhamn et al. 2002). Reason: toy (with ey) is required for development of the entire eye-antennal disc; confirmed by CRISPR null and RNAi analyses (Zhu et al. 2017). Core role. Supporting Evidence: PMID:11861484 Strong mutants of twin of eyeless or of eyeless are headless, which suggests that they are required for the development of all structures derived from eye-antennal discs PMID:28584125 Pax6 controls eye progenitor cell survival and proliferation through the activation of teashirt (tsh) and eyg |
| GO:0045944 positive regulation of transcription by RNA polymerase II | IDA PMID:15790965 Functional analysis of the chicken delta1-crystallin enhance... | ACCEPT | Summary: Toy, with SoxN, activates the DC5 enhancer in S2 cell transfection and in vivo (Blanco et al. 2005; full text checked online). Reason: Toy is an activator of Pol II target genes; independently shown to directly activate the ey eye enhancer and the so10 enhancer in vivo. Supporting Evidence: PMID:10198632 Toy functions upstream of ey by directly regulating the eye-specific enhancer of ey PMID:11830564 both Drosophila Pax6 proteins namely EY and TOY bind and positively regulate so10 expression through different binding sites |
| GO:0045944 positive regulation of transcription by RNA polymerase II | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA annotation to positive regulation of transcription by RNA polymerase II. Reason: Consistent with experimental evidence of Toy activating ey and so enhancers. Supporting Evidence: PMID:10198632 Toy functions upstream of ey by directly regulating the eye-specific enhancer of ey PMID:11830564 both Drosophila Pax6 proteins namely EY and TOY bind and positively regulate so10 expression through different binding sites |
| GO:0048731 system development | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA annotation to system development. Reason: Extremely general; adds nothing beyond the specific experimentally supported developmental terms (compound eye, ocellus, eye-antennal disc, brain). Supporting Evidence: PMID:11861484 Strong mutants of twin of eyeless or of eyeless are headless, which suggests that they are required for the development of all structures derived from eye-antennal discs |
| GO:0048749 compound eye development | TAS PMID:11875444 Selector and signalling molecules cooperate in organ pattern... | ACCEPT | Summary: Traceable author statement from a review on selector genes (Curtiss et al. 2002, abstract-only in cache). Reason: Well supported by primary data: Toy is required for initiation of ey expression and acts through Ey to activate the eye developmental program; ectopic Toy induces ectopic eyes. Core role. Supporting Evidence: PMID:10198632 Toy is therefore required for initiation of ey expression in the embryo and acts through Ey to activate the eye developmental program |
| GO:0048854 brain morphogenesis | IMP PMID:19901536 Brain patterning defects caused by mutations of the twin of ... | KEEP AS NON CORE | Summary: toy mutations perturb embryonic brain neuromere formation and mushroom body lobe structure (Furukubo-Tokunaga et al. 2009). Reason: Experimentally supported brain patterning role, but pleiotropic/secondary to visual system specification. Supporting Evidence: PMID:19901536 Mutations of toy perturb brain neuromere formation in the embryonic stages, and result in severe deformation of the MB lobes in pharate adult brains |
| GO:0090596 sensory organ morphogenesis | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: ARBA annotation to sensory organ morphogenesis. Reason: Broadly consistent with Toy's roles in compound eye and ocellus development, but Toy acts in organ specification upstream of morphogenesis; the specific terms are preferred. Supporting Evidence: PMID:11830564 the EY and TOY binding sites are required for compound eye and ocellus development respectively |
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Download this section (compressed HTML)Q: Which direct Toy target genes (beyond ey, so and Clk) distinguish its ocellar specification role from Ey's compound-eye role, and are they bound via the paired domain or the homeodomain?
Q: Does Toy's regulation of Clock in small ventrolateral neurons represent a conserved Pax6 role in circadian neuron specification, warranting a process annotation?
Experiment: Genome-wide ChIP-seq/CUT&RUN for endogenously tagged Toy versus Ey in embryonic eye primordia and late larval eye-antennal discs to define shared and paralog-specific direct targets.
Experiment: Domain-swap knock-ins exchanging the Toy and Ey C-terminal transactivation regions at the endogenous loci to test whether transactivation differences explain ocellar versus compound-eye specialization.
twin of eyeless (toy) encodes a transcription factor that is involved in eye development as part of the retinal determination gene network, and contributes to formation of the central nervous system.
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