Paired box protein Pax-3-A is the S-subgenome homeolog of Xenopus Pax3, a nuclear transcription factor of the Pax3/7 subgroup that binds DNA through a paired domain and a paired-type homeodomain. Pax3 is one of the earliest neural plate border specifiers: Wnt and FGF signals from the underlying mesoderm, acting partly through Msx1, induce it in the lateral and posterior neural plate during gastrulation, before the onset of neural crest specifier genes. Together with Zic1 it directly activates neural crest specifiers (snail1/2, foxd3, twist1, tfap2b, sox8, sox9) and positively autoregulates its own gene, and the Pax3-Zic1 pair is both necessary and sufficient to induce neural crest, including migration and differentiation of crest derivatives. Acting without Zic1, Pax3 instead promotes hatching gland fate, so the relative levels of Pax3 and Zic1 partition the border into neural crest, hatching gland and preplacodal fates. Pax3 is also expressed in the dorsal neural tube. Border expression of Pax3/7 is conserved in amphioxus, placing Pax3 in the ancestral chordate border layer of the neural crest gene regulatory network; the direct wiring to crest specifier genes is a vertebrate feature.
Definition: The formation of the neural plate border, the region of ectoderm at the boundary between the neural plate and the non-neural ectoderm that is competent to give rise to neural crest, preplacodal ectoderm, dorsal neural tube and epidermis.
Justification: GO has no term for the neural plate border. GO:0014029 neural crest formation is defined as forming this ectodermal region but is placed under epithelial to mesenchymal transition, so border specifiers end up annotated to an EMT term. The border is conserved in chordates without neural crest (amphioxus, tunicates), so it cannot be a part of neural crest formation. See projects/NEURAL_CREST_FORMATION_OBSOLETION.md.
Parent term: ectoderm development
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding | IBA GO_REF:0000033 | ACCEPT | Summary: IBA: Pol II cis-regulatory sequence-specific DNA binding, inherited from the Pax family node. Frog Pax3 binds defined cis elements in the snail2 promoter and its own upstream enhancer by EMSA with mutation and antibody-supershift controls, and occupies hundreds of sites in vivo by ChIP-seq. Reason: The paired domain plus homeodomain architecture and direct frog EMSA/ChIP-seq evidence support this inherited activity. The PAINT node placement is sound: Q645N4 sits inside the Pax3/7 clade with no evidence of divergence. Supporting Evidence: PMID:24360906 We showed that Pax3 binds efficiently to snail2 oligonucleotide but much less to the oligonucleotide mutated in the putative binding site (Figure 4D). PMID:38683994 (B) Pax3 ChIP-seq analysis identified 657 candidate direct targets, among which 475 expressed in NC. file:XENLA/pax3-a/pax3-a-deep-research-falcon.md Its paired domain and homeodomain indicate DNA recognition rather than catalysis, transport, or a structural role. |
| GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter IEA for the same DNA-binding activity as the IBA row. Reason: Same activity as the IBA row; consistent with direct binding to snail2 and pax3 regulatory elements. Supporting Evidence: PMID:24360906 We showed that Pax3 binds efficiently to snail2 oligonucleotide but much less to the oligonucleotide mutated in the putative binding site (Figure 4D). |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IBA GO_REF:0000033 | ACCEPT | Summary: IBA: DNA-binding transcription factor activity, RNA polymerase II-specific. Pax3 is a sequence-specific transcription factor whose frog targets (snail2, foxd3, twist1, tfap2b, pax3 itself) are immediate-early, translation-independent targets. Reason: Correct core molecular function at the PAINT level. The more specific activator child (GO:0001228) is proposed as NEW below, based on frog evidence for direct activation; this IBA is kept as the phylogenetically grounded parent. Supporting Evidence: PMID:24360906 We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. PMID:24360906 We showed that Pax3 binds efficiently to snail2 oligonucleotide but much less to the oligonucleotide mutated in the putative binding site (Figure 4D). |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA (InterPro homeobox + TreeGrafter) for Pol II DNA-binding transcription factor activity. Reason: Duplicates the IBA; correct. Supporting Evidence: PMID:24360906 We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. |
| GO:0003677 DNA binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO DNA binding from the paired domain and homeodomain signatures. Reason: Correct but general; the more informative cis-regulatory DNA-binding term is already present by IBA. Retained as a valid broad IEA. Supporting Evidence: PMID:24360906 We showed that Pax3 binds efficiently to snail2 oligonucleotide but much less to the oligonucleotide mutated in the putative binding site (Figure 4D). |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: IBA: active in nucleus, consistent with a DNA-binding transcription factor. Reason: Pax3 acts on chromatin; nuclear localisation is conserved across the Pax family (human PAX3 nucleus by IDA). Supporting Evidence: file:XENLA/pax3-a/pax3-a-uniprot.txt SUBCELLULAR LOCATION: Nucleus PMID:38683994 (B) Pax3 ChIP-seq analysis identified 657 candidate direct targets, among which 475 expressed in NC. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping to nucleus. Reason: Consistent with transcription factor function. Supporting Evidence: file:XENLA/pax3-a/pax3-a-uniprot.txt SUBCELLULAR LOCATION: Nucleus |
| GO:0005634 nucleus | ISS GO_REF:0000024 | ACCEPT | Summary: ISS from mouse Pax7 (P63015) for nuclear localisation. Reason: Pax3/Pax7 are close paralogs with conserved nuclear localisation; uncontroversial. Supporting Evidence: file:XENLA/pax3-a/pax3-a-uniprot.txt SUBCELLULAR LOCATION: Nucleus |
| GO:0005654 nucleoplasm | TAS Reactome:R-XLA-9835813 | ACCEPT | Summary: Reactome TAS: nucleoplasm, from the Xenopus neural-border event in which Tfap2a and Gbx2 activate pax3 expression. Reason: Nucleoplasm is a valid refinement of nucleus for a transcription factor. Note that the Reactome event describes regulation OF pax3 rather than an activity of Pax3, but the location claim stands. Supporting Evidence: Reactome:R-XLA-9835813 Tfap2a activates expression of pax3 in the neural plate border file:XENLA/pax3-a/pax3-a-uniprot.txt SUBCELLULAR LOCATION: Nucleus |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO regulation of DNA-templated transcription. Reason: Correct but general; the Pol II-specific IBA and the positive-regulation term (see MODIFY of the ISS row) are more precise. Supporting Evidence: PMID:24360906 We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. |
| GO:0006357 regulation of transcription by RNA polymerase II | IBA GO_REF:0000033 | ACCEPT | Summary: IBA: regulation of transcription by RNA polymerase II. Reason: Correct process for a Pol II transcription factor; frog evidence specifically shows positive regulation (direct activation of snail2 and pax3 itself). Supporting Evidence: PMID:24360906 We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. PMID:24360906 These data show that Pax3 exerts a positive autoregulation on its own transcription and suggest new putative regulatory elements mediating this regulation. |
| GO:0006357 regulation of transcription by RNA polymerase II | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter IEA for regulation of transcription by RNA polymerase II. Reason: Duplicates the IBA; correct. Supporting Evidence: PMID:24360906 We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. |
| GO:0007399 nervous system development | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA: nervous system development, from a broad Pax node (Pax3/7, Pax2/5/8, Drosophila paired-class genes). Reason: Pax3 is expressed in the dorsal and posterior neural plate/tube and contributes to dorsal neural tube patterning, so the term is not wrong, but it is a generic family-level process. The specific, core role of frog Pax3 is at the neural plate border (neural crest formation and fate commitment). Supporting Evidence: PMID:9169853 Pax-3 is a paired-type homeobox gene that is specifically expressed in the dorsal and posterior neural tube. |
| GO:0008543 fibroblast growth factor receptor signaling pathway | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA IEA: fibroblast growth factor receptor signaling pathway. Reason: Pax3 is a transcriptional integrator downstream of FGF8 (FGF8 acts through Msx1 and Pax3 activities), not a component of the FGF receptor signal transduction cascade. The machine-learned rule reflects the experimental IGI row below, which has the same issue. Supporting Evidence: PMID:15691759 Thus, WNT and FGF8 signals act in parallel at the neural border and converge on Pax3 activity during neural crest induction. |
| GO:0008543 fibroblast growth factor receptor signaling pathway | IGI PMID:15691759 Msx1 and Pax3 cooperate to mediate FGF8 and WNT signals duri... | MARK AS OVER ANNOTATED | Summary: IGI with fgf8 (Q8AXC5): FGF8 induces neural crest through Msx1 and Pax3 activities; Pax3 morpholino blocks FGF8-dependent crest induction. Reason: The epistasis is real and is respected here, but it places Pax3 downstream of FGF8 as a transcription factor whose expression and activity are required for the crest outcome of the signal, i.e. a target/output of the pathway. GO signaling pathway terms are reserved for gene products that transduce the signal (ligand, receptor, intracellular relays and the signal-regulated effector TF). Pax3 is induced by Msx1 rather than being directly modified by FGFR signaling. The biology is better captured by the proposed neural plate border formation term (NTR, replacing GO:0014029) and GO:0014034, which the gene carries. Supporting Evidence: PMID:15691759 Thus, WNT and FGF8 signals act in parallel at the neural border and converge on Pax3 activity during neural crest induction. PMID:15691759 Msx1 induces Pax3 and ZicR1 cell autonomously, in turn, Pax3 combined with ZicR1 activates Slug in a WNT-dependent manner. |
| GO:0014029 neural crest formation | IGI PMID:15691759 Msx1 and Pax3 cooperate to mediate FGF8 and WNT signals duri... | MODIFY | Summary: IGI with msx1 (Q3B8L1): Msx1 and Pax3 are both required for neural crest formation, and Pax3 acts downstream of Msx1. Reason: Core annotation. Pax3 is a neural plate border specifier: expressed early in the lateral neural plate border before crest specifiers, induced by Wnt/FGF via Msx1, and required for the border territory to give rise to crest. Pax3 with Zic1 also directly induces crest fate, so neural crest cell fate commitment (GO:0014034) applies as well. GO:0014029 is proposed for obsoletion (projects/NEURAL_CREST_FORMATION_OBSOLETION.md): its definition describes forming the neural plate border region while its placement is under epithelial to mesenchymal transition. This gene's role is border formation, captured by the proposed term 'neural plate border formation' (NTR). Proposed replacements: neural plate border formation neural crest cell fate commitment Supporting Evidence: PMID:15691759 Msx1 and Pax3 are both required for neural crest formation, display overlapping but nonidentical activities, and that Pax3 acts downstream of Msx1. PMID:24360906 The combined activity of the neural border specifiers establishes a robust neural border territory during gastrulation PMID:15843410 Pax3 and Zic1 are expressed in an overlapping manner in the presumptive neural crest area of the Xenopus gastrula, even prior to the onset of the expression of the early bona fide neural crest marker genes Foxd3 and Slug. |
| GO:0014029 neural crest formation | IMP PMID:17409353 The activity of Pax3 and Zic1 regulates three distinct cell ... | MODIFY | Summary: IMP (morpholino) in Hong and Saint-Jeannet 2007: Pax3 is required, with Zic1, to specify neural crest at the neural plate border, and the relative levels of Pax3 and Zic1 partition the border into crest, hatching gland and preplacodal fates. Reason: Core border-specifier role, at the level of formation of the border/crest-forming region. Same reasoning as the IGI row. GO:0014029 is proposed for obsoletion (projects/NEURAL_CREST_FORMATION_OBSOLETION.md): its definition describes forming the neural plate border region while its placement is under epithelial to mesenchymal transition. This gene's role is border formation, captured by the proposed term 'neural plate border formation' (NTR). Proposed replacements: neural plate border formation neural crest cell fate commitment Supporting Evidence: PMID:17409353 Pax3 and Zic1 are necessary and sufficient to promote hatching gland and preplacodal fates, respectively, whereas their combined activity is essential to specify the neural crest. PMID:17409353 At the end of gastrulation, Pax3 and Zic1 are coexpressed in the neural crest forming region. In addition, Pax3 is expressed in progenitors of the hatching gland |
| GO:0014034 neural crest cell fate commitment | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA IEA: neural crest cell fate commitment. Reason: Consistent with the experimental rows from PMID:15843410 and with direct activation of crest specifiers by Pax3/Zic1. Supporting Evidence: PMID:24360906 We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. |
| GO:0014034 neural crest cell fate commitment | IGI PMID:15843410 Neural crest determination by co-activation of Pax3 and Zic1... | ACCEPT | Summary: IGI with zic1 (O73689) in Sato et al. 2005: co-activation of Pax3 and Zic1 determines neural crest fate; together they induce ectopic crest in ventral ectoderm and their co-presence is essential for initiating crest differentiation. Reason: Core. Beyond forming the border, Pax3 with Zic1 directly activates the crest specifiers (snail1/2, foxd3, twist1, tfap2b; sox8 and sox9 in a second screen) and the pair is sufficient for full crest determination including migration and differentiation of derivatives. Commitment (which includes the inductive initiation of the fate) is the right level for this upstream inducer; GO:0014036 fate specification was not added because it is a part_of descendant of this term and the specification proper is executed by the downstream specifiers. Supporting Evidence: PMID:15843410 Misexpression of both Pax3 and Zic1 together efficiently induces ectopic neural crest differentiation in the ventral ectoderm, whereas overexpression of either one of them only expands the expression of neural crest markers within the dorsolateral ectoderm. PMID:15843410 Loss-of-function studies in vivo and in the animal cap show that co-presence of Pax3 and Zic1 is essential for the initiation of neural crest differentiation. PMID:24360906 We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. PMID:23509273 coactivating transcription factors Pax3 and Zic1 not only initiate neural crest specification from various early embryonic lineages in Xenopus and chicken embryos but also trigger full neural crest determination. |
| GO:0014034 neural crest cell fate commitment | IMP PMID:15843410 Neural crest determination by co-activation of Pax3 and Zic1... | ACCEPT | Summary: IMP (morpholino) in Sato et al. 2005: loss of Pax3 blocks initiation of neural crest differentiation. Reason: Core, as for the IGI row from the same paper. Supporting Evidence: PMID:15843410 Loss-of-function studies in vivo and in the animal cap show that co-presence of Pax3 and Zic1 is essential for the initiation of neural crest differentiation. PMID:24360908 Among the targets identified we found several well-characterized NC-specific genes, including snail2, foxd3, gbx2, twist, sox8 and sox9, which validate our approach. |
| GO:0016055 Wnt signaling pathway | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA IEA: Wnt signaling pathway. Reason: Pax3 expression is induced by Wnt signals and Pax3 mediates Wnt-dependent Slug induction, making it a downstream transcriptional target, not a Wnt pathway component. Mirrors the IGI row. Supporting Evidence: PMID:10433827 Pax-3 inductive signals from posterior nonaxial mesoderm are Wnt-dependent. |
| GO:0016055 Wnt signaling pathway | IGI PMID:15691759 Msx1 and Pax3 cooperate to mediate FGF8 and WNT signals duri... | MARK AS OVER ANNOTATED | Summary: IGI with wnt7b (P31289) in Monsoro-Burq et al. 2005: WNT-induced Slug requires Pax3 activity. Reason: Wnt from posterior non-axial mesoderm induces pax3 expression, and Pax3 is required for the crest output of Wnt; but Pax3 is not a transducer of the Wnt signal (that role belongs to beta-catenin/Tcf). The epistasis supports the neural crest terms rather than pathway membership. Not removed because the genetic interaction is real and the full text was not available. Supporting Evidence: PMID:10433827 Pax-3 inductive signals from posterior nonaxial mesoderm are Wnt-dependent. PMID:15691759 Msx1 induces Pax3 and ZicR1 cell autonomously, in turn, Pax3 combined with ZicR1 activates Slug in a WNT-dependent manner. |
| GO:0045893 positive regulation of DNA-templated transcription | ISS GO_REF:0000024 | MODIFY | Summary: ISS from mouse Pax3 (P24610): positive regulation of DNA-templated transcription. Reason: Correct in essence and now supported directly in frog: Pax3 activates snail2 and its own gene as immediate-early targets and binds their cis elements. The Pol II-specific child is more precise and matches the IDA annotations on human and mouse Pax3. Proposed replacements: positive regulation of transcription by RNA polymerase II Supporting Evidence: PMID:24360906 These data show that Pax3 exerts a positive autoregulation on its own transcription and suggest new putative regulatory elements mediating this regulation. PMID:24360906 We showed that Pax3 binds efficiently to snail2 oligonucleotide but much less to the oligonucleotide mutated in the putative binding site (Figure 4D). PMID:24360906 We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. |
| GO:0048785 hatching gland development | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: ARBA IEA: hatching gland development. Reason: Correct (see IMP row) but a distinct, non-crest border fate. Supporting Evidence: PMID:17409353 Pax3 and Zic1 are necessary and sufficient to promote hatching gland and preplacodal fates, respectively, whereas their combined activity is essential to specify the neural crest. |
| GO:0048785 hatching gland development | IMP PMID:17409353 The activity of Pax3 and Zic1 regulates three distinct cell ... | KEEP AS NON CORE | Summary: IMP in Hong and Saint-Jeannet 2007: Pax3 is necessary and sufficient to promote hatching gland fate, independently of Zic1. Reason: Well supported (gain and loss of function; Pax3 is expressed in hatching gland progenitors). It is another output of the same border-specifier activity, set by Pax3 acting without Zic1, but the hatching gland is an anamniote-specific border derivative and not the conserved core role used to place Pax3 in the crest network. Supporting Evidence: PMID:17409353 Pax3 and Zic1 are necessary and sufficient to promote hatching gland and preplacodal fates, respectively, whereas their combined activity is essential to specify the neural crest. PMID:17409353 At the end of gastrulation, Pax3 and Zic1 are coexpressed in the neural crest forming region. In addition, Pax3 is expressed in progenitors of the hatching gland file:XENLA/pax3-a/pax3-a-uniprot.txt Probable transcription factor. Promotes both hatching gland and neural crest cell fates, two of the cell populations that arise from the neural plate border. |
| GO:0001228 DNA-binding transcription activator activity, RNA polymerase II-specific | IDA PMID:24360906 Pax3 and Zic1 trigger the early neural crest gene regulatory... | NEW | Summary: Frog Pax3 is a sequence-specific transcriptional activator: it binds the snail2 promoter and its own upstream enhancer (EMSA with mutant and supershift controls) and activates both as immediate-early targets. Reason: Existing MF annotations stop at generic Pol II DNA-binding transcription factor activity. Direct binding plus translation-independent activation of snail2 and pax3 supports the activator child term. Comparator: human PAX3 and mouse Pax3 carry GO:0045944 by IDA. Mammalian Pax3 can also repress some targets; no frog evidence of repression was found, so the repressor term is not proposed. Supporting Evidence: PMID:24360906 We showed that Pax3 binds efficiently to snail2 oligonucleotide but much less to the oligonucleotide mutated in the putative binding site (Figure 4D). PMID:24360906 These data show that Pax3 exerts a positive autoregulation on its own transcription and suggest new putative regulatory elements mediating this regulation. PMID:24360906 We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. |
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Download this section (compressed HTML)Q: GO has no term for neural plate border formation or specification; the project proposes one and the obsoletion of GO:0014029 neural crest formation. Should the new border term's scope explicitly accommodate border-derived non-crest fates (hatching gland, preplacodal ectoderm, Rohon-Beard neurons)?
Q: All experimental Pax3 rows are duplicated on pax3-a (Q645N4, pax3.S) and pax3-b (Q0IH87, pax3.L), while the reagents were generic Pax3 morpholinos/mRNAs. Do both homeologs contribute equally at the border, or has one subfunctionalised?
Q: Mammalian Pax3 carries neural crest migration and pigmentation terms but no neural crest formation/commitment terms, while frog Pax3 carries the reverse. Should Splotch-derived crest data and frog border data be reconciled across orthologs (e.g. is mammalian Pax3 required for crest induction, given Pax7 redundancy in chick/mouse)?
Q: Is the vertebrate-specific step in the Pax3/7 border module a change in the protein (ability to co-activate crest specifier enhancers with Zic) or in the cis-regulatory elements of the targets (snail2, foxd3, sox8)?
Experiment: Test whether amphioxus Pax3/7, with amphioxus Zic, can substitute for Xenopus Pax3 in the Pax3+Zic1 animal-cap crest induction assay and bind/activate the frog snail2 Pax3 site, to separate protein from cis-regulatory evolution.
Experiment: Homeolog-specific knockdown or CRISPR F0 knockout of pax3.L vs pax3.S, scoring border markers (msx1, zic1), crest specifiers (snai2, foxd3, sox8) and hatching gland (xhe).
Experiment: CUT&RUN/ChIP for endogenous Pax3 at gastrula stage 12 to confirm in vivo occupancy of the snail2 and pax3 elements identified by EMSA, and to determine whether Pax3 and Zic1 co-occupy crest specifier enhancers.
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