FoxN2/3 is a Forkhead (Fox) family transcription factor with a single winged-helix Fork-head DNA-binding domain; the sea urchin gene is equally related to the vertebrate FOXN2 and FOXN3 genes and is one of only two FoxN subfamily members in the genome (the other being FoxN1/4). In the embryo it is expressed in a dynamic, sequential pattern: transcripts first appear in the large micromere descendants (the precursors of the skeletogenic primary mesenchyme cells, PMCs) at the hatched blastula stage, are switched off in these cells as they ingress at the mesenchyme blastula stage, and then appear in the non-skeletogenic mesoderm and later in the endoderm of the mid- and hindgut. In the micromere lineage the gene lies downstream of maternal beta-catenin and the Pmar1/HesC double-negative gate and is activated by Ets1 and Tbr (Tbr being necessary but not sufficient), but not by Alx1; the later mesodermal and endodermal phases are driven independently of the micromere phase and of Delta/Notch signalling. During its brief window in the PMC precursors FoxN2/3 is an early transcriptional input into the skeletogenic effector gene battery: loss of function abolishes the early expression of the skeletal matrix genes (sm30, sm50, msp130) and of the VEGF receptor in PMCs, delays or blocks PMC ingression (epithelial-mesenchymal transition), and renders PMCs unable to join the syncytium that deposits the calcite larval skeleton, so that morphant PMCs remain in the blastocoel and the skeleton is malformed or absent. Target expression recovers at later stages, indicating that FoxN2/3 initiates rather than maintains the skeletogenic program. The orthologue is expressed in the prospective mesoderm of all echinoderms examined, including sea stars that lack skeletogenic mesenchyme, so the gene is thought to belong to an ancestral echinoderm mesoderm regulatory program.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003677 DNA binding | IEA GO_REF:0000104 | MODIFY | Summary: UniRule-derived generic DNA binding from the Fork-head domain. True but uninformative; Forkhead domains bind DNA in a sequence-specific manner and the more specific term already exists as a separate GOA row. No direct DNA-binding assay has been done on the sea urchin protein. Reason: Replace the generic term with the sequence-specific child (GO:0043565), which is already present in GOA and accepted below; the generic parent adds nothing. Proposed replacements: sequence-specific DNA binding |
| GO:0003700 DNA-binding transcription factor activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the Fork-head domain signatures. Consistent with the functional record: FoxN2/3 is a Forkhead transcription factor whose knockdown in the micromere lineage abolishes the early expression of a battery of skeletogenic effector genes (sm30, sm50, msp130, VEGFR). Direct binding of FoxN2/3 to any target cis-regulatory module has not been shown, and the only sign demonstrated is activation (loss of target expression on knockdown), so neither the RNA polymerase II-specific nor the activator/repressor child is proposed; the UniProt ARBA note that the protein acts as a transcriptional repressor is a vertebrate FOXN3 transfer with no sea urchin support. Reason: Core molecular function of the gene product, supported by loss-of-function evidence on downstream target genes (Lytechinus variegatus orthologue). Supporting Evidence: PMID:21303847 In addition, FoxN2/3 is necessary for activation of the early expression of many PMC specific genes. |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: Nuclear localization inferred from the Fork-head domain. No immunolocalization of the sea urchin protein is cached, but the location is not in doubt for a Forkhead transcription factor whose function is the activation of target gene expression. Reason: Expected and functionally required location of a sequence-specific transcription factor. |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Generic InterPro2GO process term. Correct by parentage of the demonstrated function, but redundant with the more informative positive-regulation term proposed as NEW below. Reason: True but generic; the informative process terms are the positive regulation of RNA polymerase II transcription and the skeletogenic developmental processes. Supporting Evidence: PMID:21303847 Early expression of genes for the skeletal matrix is dependent on FoxN2/3, but only until the mesenchyme blastula stage as foxN2/3 mRNA disappears from PMCs at that time and we assume that the protein is not abnormally long-lived. |
| GO:0043565 sequence-specific DNA binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the Fork-head domain and its conserved signature. Sequence-specific DNA binding is the defining property of the winged-helix Forkhead domain, which is intact in Q2V887 (residues 86-173). No EMSA or ChIP data exist for the sea urchin protein, so this remains a domain-based inference. Reason: Sound domain-based inference and the appropriate level of specificity given that no cis-regulatory target has been mapped; proposed as the replacement for the generic DNA binding row. |
| GO:0045944 positive regulation of transcription by RNA polymerase II | ISS PMID:21303847 The control of foxN2/3 expression in sea urchin embryos and ... | NEW | Summary: FoxN2/3 knockdown (two independent morpholinos, mRNA-rescued) abolishes the early expression of the skeletal matrix genes sm30, sm50 and msp130 and of the VEGF receptor in PMCs, and foxN2/3 is one of the transcription factors that provide the parallel inputs activating the skeletogenic effector battery. The sign is activation; whether the inputs are direct is not known. Evidence is from the Lytechinus variegatus orthologue. Reason: Loss-of-function evidence that FoxN2/3 is required for activation of multiple protein-coding (RNA polymerase II) target genes; the gene product is the transcription factor providing the input, so it performs the regulatory step. The sign-neutral GOA parent GO:0006355 is kept and this child adds the demonstrated direction. The evidence is cross-species and loss-of-function only, so no activator MF child is proposed. Evidence code: the functional data are from the Lytechinus variegatus orthologue, not from S. purpuratus, so the row is coded ISS (sequence-orthology transfer) rather than IMP, following the convention used for Snail in this batch. Supporting Evidence: PMID:21303847 When we examined other skeletogenic genes by QPCR in FoxN2/3 KD embryos, sm30 and sm50 expression levels were also strongly reduced, consistent with the hypothesis that many skeletogenic genes require FoxN2/3 input PMID:21303847 Thus, although FoxN2/3 is part of the GRN that activates skeletogenic genes and the VEGFR , it is not likely to be involved in long-term maintenance of that expression. |
| GO:0010718 positive regulation of epithelial to mesenchymal transition | ISS PMID:21303847 The control of foxN2/3 expression in sea urchin embryos and ... | NEW | Summary: FoxN2/3 is required for the timely ingression of the PMCs, the epithelial-mesenchymal transition of the sea urchin embryo. Two independent studies in Lytechinus variegatus show that foxN2/3 morphants fail to ingress on schedule; in a 13-factor comparison foxn2/3 knockdown gave the most pronounced defect in apical constriction and cell-shape change, and transplanted morphant micromeres show that the requirement is cell-autonomous. FoxN2/3 is a transcription factor acting on the effector program, hence the regulation term rather than GO:0001837. Reason: Regulatory (not executional) role in EMT supported by morpholino phenotypes in two independent studies. Comparator check: the upstream PMC regulators Alx1 and Ets1 in this directory carry the same term for the same role. Evidence is from L. variegatus. Evidence code: the functional data are from the Lytechinus variegatus orthologue, not from S. purpuratus, so the row is coded ISS (sequence-orthology transfer) rather than IMP, following the convention used for Snail in this batch. Term choice note: Snail in this directory carries the executional term GO:0001837 epithelial to mesenchymal transition because it directly silences cadherin, whereas Twist and FoxN2/3 carry the regulatory term GO:0010718 because their inputs into the de-adhesion sub-circuit are indirect. Supporting Evidence: PMID:21303847 Knockdown of FoxN2/3 inhibits normal PMC ingression and foxN2/3 morphant PMCs do not organize in the blastocoel and fail to join the PMC syncytium. PMID:24598159 Foxn2/3 was the only knockdown with a non-apically constricted shape significantly different from the mean shape for the duration of the time-course. |
| GO:0070169 positive regulation of biomineral tissue development | ISS PMID:21303847 The control of foxN2/3 expression in sea urchin embryos and ... | NEW | Summary: FoxN2/3 is an early transcriptional input into the biomineralization gene battery of the PMCs (sm30, sm50, msp130) and is required for PMCs to fuse into the skeletogenic syncytium; micromeres carrying foxN2/3 morpholino, transplanted into micromere-less hosts, give rise to PMCs that remain in the blastocoel and do not contribute to the larval calcite skeleton, and whole-embryo morphants have delayed, reduced or absent spicules. FoxN2/3 regulates the skeletogenic effector genes rather than depositing mineral itself, so the positive-regulation term is used rather than GO:0031214. Reason: Cell-autonomous loss-of-function phenotype on skeleton formation plus loss of biomineralization gene expression; the transcription factor performs the regulatory step. Comparator check: Alx1 in this directory carries the same term for the same kind of role. Evidence is from the L. variegatus orthologue. Evidence code: the functional data are from the Lytechinus variegatus orthologue, not from S. purpuratus, so the row is coded ISS (sequence-orthology transfer) rather than IMP, following the convention used for Snail in this batch. Supporting Evidence: PMID:21303847 FoxN2/3 is necessary for several steps in the formation of the larval skeleton. PMID:21303847 PMCs originating from foxN2/3 MASO-injected micromeres remained in the blastocoel without contributing to the larval skeleton PMID:21303847 Thus, knockdown of FoxN2/3 in PMCs resulted in a failure of these cells to be incorporated into the syncytium, explaining at least one of the reasons that the larval skeleton is malformed in FoxN2/3 KD embryos. |
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Download this section (compressed HTML)Q: Does FoxN2/3 bind directly to the cis-regulatory modules of the skeletal matrix genes and VEGFR in S. purpuratus (which would justify RNA polymerase II-specific and activator MF terms), or are its inputs relayed through other PMC transcription factors?
Q: Is the FoxN2/3-dependent step in PMC syncytium formation a transcriptional output (e.g. a fusogen or adhesion gene) that would support a specific cell-cell fusion annotation?
Experiment: ChIP-seq or CUT&RUN for tagged Sp-FoxN2/3 in hatched blastula-stage S. purpuratus embryos, followed by GFP reporter analysis of candidate CRMs of sm50, msp130 and vegfr, to establish direct targets in the native species.
Experiment: S. purpuratus foxN2/3 morpholino or CRISPR knockdown with time-resolved RNA-seq of isolated PMCs, to define the complete early target set and separate the PMC-autonomous role from the later mesoderm and endoderm phases.
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