Twist is the single sea urchin member of the Twist family of basic helix-loop-helix (bHLH) transcription factors, with a TWIST1-type bHLH domain and the C-terminal WR motif characteristic of the family. In the sea urchin embryo twist is transcribed in the skeletogenic mesoderm lineage (the large micromere-derived primary mesenchyme cells, PMCs) from the late blastula, is maintained in PMCs through gastrulation, and is later also expressed in the non-skeletogenic (secondary) mesenchyme. It is activated downstream of the PMC specifier Alx1 and feeds back positively on alx1 expression, forming a positive-feedback loop that maintains the PMC regulatory state after the transient initiating inputs have faded. Twist knockdown in the congener Lytechinus variegatus delays PMC ingression (epithelial-mesenchymal transition), with a specific failure of the de-adhesion step, disrupts PMC migration and syncytial fusion, lowers transcripts of the biomineralization genes msp130, sm50 and sm30, and abolishes the larval calcite skeleton; overexpression produces supernumerary and mis-positioned spicules. Later roles in secondary mesenchyme derivatives (pigment and muscle cells) are also reported. The protein is the echinoderm orthologue of the mesoderm and EMT regulators Twist1/Twist2 of vertebrates and Drosophila Twist.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000977 RNA polymerase II transcription regulatory region sequence-specific DNA binding | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference from the Twist-family node (PTN002360678), seeded by experimentally characterized mammalian and Drosophila Twist proteins that bind E-box sequences in RNA polymerase II regulatory regions. No DNA-binding assay has been performed on the sea urchin protein, but its bHLH basic region is conserved with the family and its knockdown lowers transcripts of alx1 and of PMC biomineralization genes, consistent with regulatory-region binding. Reason: Sound phylogenetic inference of the family-wide DNA-binding function to a protein with an intact Twist-type bHLH domain; no sea urchin evidence contradicts it. Supporting Evidence: PMID:18495103 Multiple sequence alignment of Twist proteins shows that LvTwist and other Twist family members are highly conserved within the bHLH domain ( Fig. 1A ). PMID:18495103 Downstream of Twist, the mRNA expression level of Lvmsp130, Lvsm50 , and Lvsm30 were examined by QPCR, and were all significantly reduced in Twi morphants when compared to MB controls |
| 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 Twist family node. This is the core molecular function of the gene product: Twist is a bHLH transcription factor that, in the sea urchin skeletogenic GRN, is required for normal transcript levels of alx1 and of the PMC differentiation genes msp130, sm50 and sm30. Whether these inputs are direct has not been tested by cis-regulatory analysis, and no activator/repressor sign is asserted. Note that all functional data on sea urchin Twist come from Lytechinus variegatus (LvTwist); the S. purpuratus protein was identified in silico by the same authors and is 92% identical. Reason: Core function; family-level inference supported by knockdown effects on target-gene transcription in the congener. Supporting Evidence: PMID:18495103 Downstream of Twist, the mRNA expression level of Lvmsp130, Lvsm50 , and Lvsm30 were examined by QPCR, and were all significantly reduced in Twi morphants when compared to MB controls PMID:18495103 In Twi morphants, Lvalx1 expression level is reduced at MB stage, but not at earlier stages, while Lvets1 expression is unaffected in Twi morphants. PMID:18495103 However, whether this mutually positive regulation is due to direct intergenic inputs will requires further examination to confirm the relationship, such as cis-regulatoy analyses. |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the TWIST1-type bHLH signature (IPR047093). Redundant with the IBA row for the same term and equally consistent with the knockdown data from L. variegatus. Reason: Core molecular function of a Twist-family bHLH transcription factor; same evidence as the IBA row. Supporting Evidence: PMID:18495103 Multiple sequence alignment of Twist proteins shows that LvTwist and other Twist family members are highly conserved within the bHLH domain ( Fig. 1A ). PMID:18495103 These data show that Lvtwist functions upstream of the PMC skeletogenic differentiation program. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of nuclear activity from the Twist family. No subcellular localization has been reported for the sea urchin protein, but the expected location of a bHLH transcription factor whose loss alters transcript levels of GRN genes is the nucleus. Reason: Expected and functionally required location of a DNA-binding transcription factor; not contradicted. Supporting Evidence: PMID:18495103 In Twi morphants, Lvalx1 expression level is reduced at MB stage, but not at earlier stages, while Lvets1 expression is unaffected in Twi morphants. |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: UniRule/subcellular-location rule inference from the bHLH domain (PROSITE PRU00981). Same conclusion as the IBA row. Reason: Consistent with the transcription factor function; redundant with the IBA nucleus row. |
| GO:0006357 regulation of transcription by RNA polymerase II | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of regulation of RNA polymerase II transcription from the Twist family. In the sea urchin, Twist knockdown reduces alx1 transcripts at mesenchyme blastula and strongly reduces msp130, sm50 and sm30 transcripts, and Twist is modelled as a positive input into alx1 in the PMC GRN; the sign-neutral parent term is appropriate because directness and sign have not been shown at the cis-regulatory level. Note that all functional data on sea urchin Twist come from Lytechinus variegatus (LvTwist); the S. purpuratus protein was identified in silico by the same authors and is 92% identical. Reason: Core process of the gene product; phylogenetic inference supported by morpholino/QPCR perturbation data in the congener. Supporting Evidence: PMID:18495103 In Twi morphants, Lvalx1 expression level is reduced at MB stage, but not at earlier stages, while Lvets1 expression is unaffected in Twi morphants. PMID:18495103 Downstream of Twist, the mRNA expression level of Lvmsp130, Lvsm50 , and Lvsm30 were examined by QPCR, and were all significantly reduced in Twi morphants when compared to MB controls PMID:18495103 Alx1 appears necessary for twist expression, and later Twist appears to feed back to positively regulate alx1 expression ( Fig. 8 ). PMID:18495103 However, whether this mutually positive regulation is due to direct intergenic inputs will requires further examination to confirm the relationship, such as cis-regulatoy analyses. |
| GO:0032502 developmental process | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT inference of the very general term developmental process, because Twist proteins across animals act in mesoderm development. True for the sea urchin protein (knockdown perturbs PMC ingression, skeletogenesis and secondary mesenchyme derivatives) but uninformative; the specific processes are proposed as NEW rows below. Reason: Correct but too general to describe the function; retained as a non-core ancestor of the specific lineage and EMT terms. Supporting Evidence: PMID:18495103 Perturbations of Twist either by morpholino knockdown or by overexpression result in defects in progressive phases of PMC development, including specification, ingression/EMT, differentiation and skeletogenesis. |
| GO:0046983 protein dimerization activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro mapping of the bHLH domain (IPR011598/IPR036638) to protein dimerization activity. Twist proteins bind DNA as bHLH homo- or heterodimers (with E proteins) in other animals, and the sea urchin protein has an intact HLH dimerization motif, but no dimerization partner or assay has been reported for the sea urchin protein. Reason: Structural inference from the bHLH fold; a generic binding term that does not describe the gene's role in the GRN. Retained as a non-core, structurally implied property. Supporting Evidence: PMID:18495103 Multiple sequence alignment of Twist proteins shows that LvTwist and other Twist family members are highly conserved within the bHLH domain ( Fig. 1A ). |
| GO:0010718 positive regulation of epithelial to mesenchymal transition | ISS PMID:18495103 Twist is an essential regulator of the skeletogenic gene reg... | NEW | Summary: Twist is one of the transcription factors that control the ingression (EMT) of the skeletogenic PMCs. Morpholino knockdown (two non-overlapping morpholinos) delays ingression of >80% of embryos; micromere-transplant chimeras show the requirement is cell-autonomous in the micromere lineage; and a five-assay dissection of EMT shows that twist morphant PMCs remodel the basement membrane, move, constrict and keep polarity but fail the de-adhesion step, placing twist with alx1 and snail in a positive-feedback de-adhesion sub-circuit. Twist regulates the EMT programme transcriptionally rather than executing it, hence the regulation term. Note that all functional data on sea urchin Twist come from Lytechinus variegatus (LvTwist); the S. purpuratus protein was identified in silico by the same authors and is 92% identical. Reason: Participation test passed for a regulatory role: the gene product is a transcription factor in the GRN sub-circuit that drives de-adhesion, and its loss delays/blocks completion of EMT. Morpholino knockdown and chimera experiments in L. variegatus, there being no S. purpuratus functional data; kept as a regulation term because no EMT effector target of Twist has been identified. 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:18495103 PMCs of Twi morphants failed to ingress (>80%, Fig. 3E ) until after a significant delay. PMID:18495103 The chimeras show that Twist is essential in micromeres for these cells to ingress punctually and migrate properly into the blastocoel as PMCs. PMID:24598159 Our analysis of both snail and twist knockdowns showed a failure of PMCs to complete EMT in spite of four other successful cell state changes PMID:24598159 Therefore, alx1, twist and snail display control over adhesive state change and make a distinct sub-circuit of the GRN, a positive-feedback lockdown, that governs the de-adhesion process ( Fig. 7E ). |
| GO:0070169 positive regulation of biomineral tissue development | ISS PMID:18495103 Twist is an essential regulator of the skeletogenic gene reg... | NEW | Summary: Twist knockdown abolishes the larval calcite skeleton even after prolonged culture, strongly reduces transcripts of the biomineralization genes msp130, sm50 and sm30, delays appearance of the PMC-specific MSP130 (1d5) antigen, and cell-autonomously prevents the syncytial PMC fusion that precedes spicule deposition; overexpression gives extra and mis-positioned spicules. Twist is a transcriptional regulator upstream of the skeletogenic differentiation programme, not an executor of mineral deposition, hence the regulation term. Note that all functional data on sea urchin Twist come from Lytechinus variegatus (LvTwist); the S. purpuratus protein was identified in silico by the same authors and is 92% identical. Reason: Specific, non-pleiotropic positive input of Twist into biomineral (spicule) formation, shown by loss-of-function morphology, QPCR of biomineralization genes and PMC-fusion chimeras in L. variegatus. Same term as used for the sibling PMC regulators Alx1, Hex and FoxB. 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:18495103 Twi morphants failed to form a skeleton ( Fig. 3I ). PMID:18495103 Downstream of Twist, the mRNA expression level of Lvmsp130, Lvsm50 , and Lvsm30 were examined by QPCR, and were all significantly reduced in Twi morphants when compared to MB controls PMID:18495103 These data show that Lvtwist functions upstream of the PMC skeletogenic differentiation program. PMID:18495103 Taken together, these data support the hypothesis that Twist is required for PMC fusion, and therefore for developmental events upstream of skeletogenesis. |
| GO:0001710 mesodermal cell fate commitment | ISS PMID:18495103 Twist is an essential regulator of the skeletogenic gene reg... | NEW | Summary: Twist is activated in the skeletogenic (PMC) lineage downstream of Alx1 and feeds back to maintain alx1 expression after ingression, forming an Alx1-Twist positive-feedback loop that the authors show is required for maintenance of the PMC specification state; without Twist, PMC differentiation and skeletogenesis progressively fail. Because twist comes on after the specification genes alx1/ets1 and acts in maintenance rather than initiation, the commitment term (parent of specification and determination) is used, as for the sibling state-stabilization regulators Hex and Erg. Note that all functional data on sea urchin Twist come from Lytechinus variegatus (LvTwist); the S. purpuratus protein was identified in silico by the same authors and is 92% identical. Reason: Participation test passed: Twist is a transcription factor whose activity locks down the already-specified skeletogenic mesodermal regulatory state (positive feedback onto alx1). Morpholino/QPCR perturbation in L. variegatus. Directness of the Twist-to-alx1 input is untested, so no transcription-activator term 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:18495103 Evidence is presented that Twist expression is required for the maintenance of the PMC specification state, and a reciprocal regulation between Alx1 and Twist offers stability for the subsequent processes, such as PMC differentiation and skeletogenesis. PMID:18495103 In Twi morphants, Lvalx1 expression level is reduced at MB stage, but not at earlier stages, while Lvets1 expression is unaffected in Twi morphants. PMID:18495103 At LHB and EMB stage, the expression of Lvtwist is reduced in Alx1 morphants, but not in either Ets1 or Sna morphants. PMID:18495103 Alx1 appears necessary for twist expression, and later Twist appears to feed back to positively regulate alx1 expression ( Fig. 8 ). PMID:24598159 In the sea urchin de-adhesion sub-circuit, alx1 is upstream of both twist and snail, with twist also positively regulating alx1. |
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Download this section (compressed HTML)Q: Is Twist a direct activator of alx1 (and of sm50/msp130/sm30) in PMCs, which would justify a GO:0001228 activator annotation, or does it act through intermediate regulators?
Q: Does Sp-Twist show the same PMC-restricted, post-ingression expression and knockdown phenotype as LvTwist, so that the L. variegatus-based annotations can be confirmed in S. purpuratus?
Q: Which bHLH partner (an E-protein homo/heterodimer?) does sea urchin Twist use, and does it also regulate the proposed conserved targets hex and fgfr1?
Experiment: Whole-mount in situ hybridization time course and morpholino/CRISPR knockdown of Sp-twist in S. purpuratus, scored for ingression timing, PMC fusion and skeleton formation, to transfer the L. variegatus phenotype to the species of the UniProt record.
Experiment: Twist ChIP-seq or reporter analysis of the alx1 and sm50 cis-regulatory modules in PMCs to test whether the positive Twist inputs modelled in the GRN are direct and activating.
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