Hex is the single sea urchin orthologue of the vertebrate hematopoietically-expressed homeobox protein HHEX, a 317-residue transcription factor with one homeodomain flanked by disordered N- and C-terminal regions. In the embryo it is zygotically activated in the large micromere-derived skeletogenic mesenchyme lineage (the primary mesenchyme cells), downstream of nuclear beta-catenin and pmar1 and of the lineage-founding regulators ets1 and alx1. There it is the hub of a dynamic state-stabilization subcircuit, forming interlocking positive feedback loops with the ETS factor Erg (erg-hex) and the TALE homeobox factor Tgif (hex-tgif), so that knockdown of hex depresses erg and tgif transcripts and vice versa; this transcriptional lockdown converts the transient initiating inputs into a stable skeletogenic regulatory state. Hex outputs also go to foxo and, as a feed-forward driver together with Alx1, Ets1, Tbr, Tel, Erg, FoxB and Dri, to the battery of differentiation genes that build the larval calcite endoskeleton. Morpholino knockdown of hex yields embryos with a full complement of ingressed skeletogenic cells that nevertheless cannot form the biomineral skeleton. After skeletogenic cell ingression hex is switched on a second time in the oral non-skeletogenic mesoderm, together with erg and ets1/2. In Lytechinus variegatus the hex orthologue is additionally required for the apical constriction and motility steps of the skeletogenic epithelial-mesenchymal transition.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference for the HHEX homeobox family node (PTN001963821), seeded by human HHEX and by yeast homeodomain factors. Consistent with the S. purpuratus data; Hex has a canonical homeodomain and, in the skeletogenic micromere GRN, its knockdown depresses transcripts of its loop partners erg and tgif and abolishes skeletogenic differentiation, which is the expected consequence of losing a sequence-specific cis-regulatory DNA-binding activator. No direct binding assay of Sp-Hex is cached, so the term rests on the phylogenetic placement plus the perturbation data. Reason: Core molecular function; the phylogenetic placement is sound for a homeodomain factor and is corroborated by perturbation evidence in the reviewed organism. Supporting Evidence: PMID:18413610 These genes are engaged in interlocking positive double feedback loops ( erg with hex ; and hex with tgif ). Interference with expression of any of these genes severely depresses transcript levels of the others ( Fig. S3 ). Thus they dynamically stabilize all aspects of the regulatory state downstream of themselves. |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the homeobox conserved-site signature (IPR017970). Correct: Hex is a transcription factor of the skeletogenic micromere GRN whose perturbation alters transcript levels of the regulatory genes erg and tgif and of the differentiation genes, and it is itself among the drivers of the biomineralization gene battery. The sign of regulation is inferred from perturbation only, so the sign-neutral term is appropriate. Reason: Core molecular function, consistent with the GRN perturbation evidence. Supporting Evidence: PMID:18413610 three additional genes are activated: one encoding the Ets family factor Erg ( 9 ), and the other two, hex and tgif , encoding homeobox factors ( 5 ) PMID:18413610 these differentiation genes require as drivers products of all of the now familiar components of the skeletogenic regulatory state ( alx1 , ets1 , tbr , tel , erg , hex , foxb , dri ) |
| GO:0003677 DNA binding | IEA GO_REF:0000120 | MODIFY | Summary: UniRule/InterPro-derived generic DNA binding from the homeodomain signatures. True but uninformative; the homeodomain is a sequence-specific helix-turn-helix DNA-binding domain, and the more specific cis-regulatory binding term is already present from PAINT. Reason: Replace the generic term with sequence-specific DNA binding (GO:0043565), which is what the homeodomain confers; this is a family-level inference since no direct DNA-binding assay of Sp-Hex is cached. Proposed replacements: sequence-specific DNA binding |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: UniRule/UniProt subcellular-location inference (Nucleus) from the homeodomain. Expected and functionally required for a homeodomain transcription factor; no immunolocalization of Sp-Hex is cached, but the location of a homeodomain factor that regulates transcription of GRN target genes is not in doubt. Reason: Correct location for the demonstrated transcriptional regulatory role. Supporting Evidence: PMID:18413610 In addition to tgif , the outputs of hex go to erg , to differentiation genes as we see below, and to foxo , a late activated regulatory gene the functions of which occur beyond the time frame of this GRN. |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Generic InterPro2GO regulation-of-transcription term from the homeobox signature. Correct by parentage of the demonstrated function but redundant with the RNA polymerase II-specific IBA row and with the positive-regulation term proposed as NEW. Reason: True but uninformative parent term; the informative annotations are the RNA polymerase II-specific child and the lineage-level process terms. |
| GO:0006357 regulation of transcription by RNA polymerase II | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference for the HHEX family node. Supported in the reviewed organism: hex knockdown severely depresses erg and tgif transcript levels (hex-erg and hex-tgif positive feedback loops), hex outputs go to foxo and to the differentiation genes, and the skeletogenic differentiation genes require Hex among their feed-forward drivers. Reason: Core process, corroborated by morpholino perturbation and QPCR in S. purpuratus; a positive-regulation child is proposed as NEW. Supporting Evidence: PMID:18413610 These genes are engaged in interlocking positive double feedback loops ( erg with hex ; and hex with tgif ). Interference with expression of any of these genes severely depresses transcript levels of the others ( Fig. S3 ). Thus they dynamically stabilize all aspects of the regulatory state downstream of themselves. PMID:30264451 One prominent example is a set of mutual regulatory interactions among erg, hex, and tgif that reinforces the expression of all three genes. |
| GO:0030154 cell differentiation | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT inference of a role in cell differentiation, seeded by vertebrate Hhex (hematopoietic, hepatic and vascular differentiation) and worm/fish homologues. Hex does act in differentiation in the sea urchin embryo (it locks down the skeletogenic mesenchyme regulatory state and is required for the ingressed skeletogenic cells to build the biomineral skeleton), so the term is not wrong, but it is too generic to be informative. Reason: Generic; the specific processes (mesodermal cell fate commitment, positive regulation of biomineral tissue development) are proposed as NEW. Supporting Evidence: PMID:18413610 This is not the case for tbr , hex , or tgif : interference with their expression results in embryos with a full complement of ingressed cells of the micromere lineage but these cells are unable to create the biomineral skeleton. |
| GO:0045944 positive regulation of transcription by RNA polymerase II | IMP PMID:18413610 Global regulatory logic for specification of an embryonic ce... | NEW | Summary: In the skeletogenic micromere GRN, hex is the hub of two interlocking positive feedback loops (erg-hex and hex-tgif): interference with hex expression severely depresses erg and tgif transcript levels (QPCR after morpholino perturbation, Fig. S3). Hex outputs also go to foxo and to the differentiation genes, and Hex is one of the transcription factors required as a feed-forward driver of the skeletogenic differentiation gene battery. Reason: Perturbation evidence (morpholino knockdown, QPCR of target transcripts) in S. purpuratus shows a positive input of Hex into erg, tgif, foxo and the differentiation gene battery, so the positive-regulation child of the accepted GO:0006357 is warranted; IMP because no direct cis-regulatory assay of a Hex-dependent module is available, and the activator MF (GO:0001228) is therefore not proposed. Supporting Evidence: PMID:18413610 These genes are engaged in interlocking positive double feedback loops ( erg with hex ; and hex with tgif ). Interference with expression of any of these genes severely depresses transcript levels of the others ( Fig. S3 ). Thus they dynamically stabilize all aspects of the regulatory state downstream of themselves. PMID:18413610 In addition to tgif , the outputs of hex go to erg , to differentiation genes as we see below, and to foxo , a late activated regulatory gene the functions of which occur beyond the time frame of this GRN. PMID:18413610 these differentiation genes require as drivers products of all of the now familiar components of the skeletogenic regulatory state ( alx1 , ets1 , tbr , tel , erg , hex , foxb , dri ) |
| GO:0001710 mesodermal cell fate commitment | IMP PMID:18413610 Global regulatory logic for specification of an embryonic ce... | NEW | Summary: Hex is the central node of the dynamic state-stabilization subcircuit (erg-hex and hex-tgif positive feedback loops) that locks down the skeletogenic mesenchyme (primary mesenchyme cell) regulatory state after the transient initiating inputs from the double-negative gate have faded. Its outputs drive erg and tgif, feed other nodes of the GRN (foxo) and the differentiation genes, so Hex performs the regulatory step that commits the lineage rather than merely being required for it. It is expressed selectively in the large micromere-PMC lineage during the relevant period. Reason: Participation test passed: Hex is a transcription factor whose activity maintains the lineage regulatory state; the commitment term (parent of specification and determination) is chosen because hex is activated after the specification genes alx1/ets1/tbr/tel and its documented role is stabilization/lockdown of the already-specified skeletogenic mesodermal fate. IMP from the morpholino/QPCR perturbation data. Same term and reasoning as the sibling Erg review. Supporting Evidence: PMID:18413610 three additional genes are activated: one encoding the Ets family factor Erg ( 9 ), and the other two, hex and tgif , encoding homeobox factors ( 5 ) PMID:18413610 These genes are engaged in interlocking positive double feedback loops ( erg with hex ; and hex with tgif ). Interference with expression of any of these genes severely depresses transcript levels of the others ( Fig. S3 ). Thus they dynamically stabilize all aspects of the regulatory state downstream of themselves. PMID:18413610 The tgif-hex and erg-hex loops in the skeletogenic micromere GRN are canonical examples. Outputs of these genes not only serve as drivers for one another but also feed multiple other nodes of the GRN, so that their activity ensures the persistence of surrounding activities. PMID:30264451 Such feedback loops may convert the transient expression of pmar1 into a more stable regulatory state in the skeletogenic lineage and buffer against initial variation in the level of expression of these genes. |
| GO:0070169 positive regulation of biomineral tissue development | IMP PMID:18413610 Global regulatory logic for specification of an embryonic ce... | NEW | Summary: Hex is one of the transcriptional drivers of the skeletogenic differentiation genes that constitute the larval calcite biomineral, and morpholino knockdown of hex produces a skeleton-minus phenotype in which a full complement of skeletogenic cells ingresses but cannot build the biomineral skeleton (Fig. 3C-E). Hex regulates the transcription of the biomineralization genes rather than executing mineral deposition, hence the regulation term. Reason: Loss-of-function morphology (skeleton-minus with normal ingression) plus the requirement of hex for differentiation-gene transcript levels in S. purpuratus give a specific, non-pleiotropic positive input of Hex into biomineral (spicule) formation; recorded as IMP. Supporting Evidence: PMID:18413610 interference with their expression produces a skeleton-minus phenotype, as illustrated in Fig. 3 B–G for tbr , hex , tgif , ets1 , and alx1 PMID:18413610 This is not the case for tbr , hex , or tgif : interference with their expression results in embryos with a full complement of ingressed cells of the micromere lineage but these cells are unable to create the biomineral skeleton. PMID:18413610 these differentiation genes require as drivers products of all of the now familiar components of the skeletogenic regulatory state ( alx1 , ets1 , tbr , tel , erg , hex , foxb , dri ) |
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Download this section (compressed HTML)Q: Is Hex a direct activator of erg, tgif and of the skeletogenic differentiation genes (which would justify GO:0001228 / GO:0000978 with IDA), or does it act through intermediates? No cis-regulatory dissection of a Hex-dependent module has been reported for S. purpuratus.
Q: Why do hex morphants ingress normally in S. purpuratus (Oliveri 2008) but fail apical constriction and motility during EMT in L. variegatus (Saunders and McClay 2014)? Is this a species difference, a difference in knockdown efficiency, or a difference in scoring?
Q: Does Ets1 provide a direct input into hex? Oliveri et al. 2008 reported loss of hex expression after ets1 knockdown, whereas Rafiq et al. 2014 did not detect such a change.
Q: What is the function of the later oral non-skeletogenic mesoderm expression of hex (with erg and ets1/2), and does the same hex-erg loop operate there, as the vertebrate hematopoietic parallels suggest?
Experiment: ChIP-qPCR/ChIP-seq of tagged Sp-Hex in primary mesenchyme cells, combined with GFP reporter analysis of the erg and tgif cis-regulatory regions with mutated homeodomain (TAAT-core) sites, to test direct Hex input into its feedback-loop partners and into the differentiation genes.
Experiment: Side-by-side morpholino and CRISPR knockdown of hex in S. purpuratus and L. variegatus with time-lapse imaging of ingression, and QPCR of erg, tgif and biomineralization genes at matched time points, to resolve the ingression-phenotype discrepancy.
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