Six1/2 (Strongylocentrotus purpuratus, UniProt H6WNA5) - curation notes

Identity

• UniProt H6WNA5 (H6WNA5_STRPU, unreviewed/TrEMBL, 336 aa, SubName "Six1") is the
  mRNA deposited by the Davidson lab in Dec 2011 (EMBL JQ264781 / AEZ53927.1, RefSeq
  NP_001268684.1, GeneID 576117). Its two RX/RA lines are the unpublished-at-submission
  versions of Ransick & Davidson 2012 ("Cis-regulatory logic driving glial cells
  missing") and Materna et al. ("The regulatory origin of oral and aboral mesoderm",
  published 2013 as "Diversification of oral and aboral mesodermal regulatory states").
• Both papers state that JQ264781 is the corrected coding sequence of the GRN gene
  six1 / six1/2:
  PMID:22509525
  PMID:23261933
• Conclusion: H6WNA5 IS the six1/2 gene of the Davidson endomesoderm/mesoderm GRN
  (also written six1, Six1, Sp-Six1/2, SpSix1/2, gene model SPU_017379/SPU_17397).
  Confidence: high (direct accession match in two primary papers).
• Domain architecture (UniProt FT): SIX1_SD N-terminal Six domain (Pfam PF16878) plus a
  homeobox (PROSITE PS50071, residues 120-180; DNA_BIND 122-181), followed by a long
  His-rich disordered C-terminus. PANTHER PTHR10390 / PTHR10390:SF61 (labels taken
  from the UniProt record, not written from memory). Echinoderms have a single
  Six1/2 gene, co-orthologous to vertebrate SIX1 and SIX2; the Six DNA-binding
  consensus used in cis-regulatory work is GGGTATCA
  PMID:22509525.
• The gene was catalogued in the genome-wide survey of S. purpuratus homeobox genes
  (Howard-Ashby et al. 2006, PMID:17055477, abstract only in cache).

Place in the mesoderm GRN (aboral non-skeletogenic mesoderm, pigment lineage)

Six1/2 is a late-tier regulatory gene of the aboral NSM (pigment cell precursor)
regulatory state, downstream of Delta/Notch -> Gcm -> GataE, whose main documented
output is positive feedback onto gcm.

• Lineage and timing: expressed in the aboral NSM shortly after gataE and z166, with
  its cofactor eya
  PMID:23261933.
  WMISH places six1 mRNA in gcm+ pigment cell precursors from mesenchyme blastula and
  in dispersed pigment cells
  PMID:22509525.
  It is not expressed before late hatched blastula, i.e. after primary specification
  PMID:22509525.
• Inputs: Delta/Notch (indirect) - six1/2 fails without D/N signalling
  PMID:22306924;
  Gcm and GataE
  PMID:23261933
  PMID:23261933
  PMID:22509525.
• Output (the key functional evidence): Six1 is a positive input into the gcm late
  cis-regulatory module.
• Cis: the module's two critical elements are Gcm and Six1 consensus sites
  PMID:22509525;
  deleting the GGGTAT element reduces reporter output and no compensatory Six1
  sites exist in the gcm BAC.
• Trans: translation-blocking six1-MO
  PMID:22509525
  and the cis and trans results converge
  PMID:22509525.
• Network interpretation: gcm-six1 positive intergenic feedback loop stabilising the
  aboral NSM regulatory state
  PMID:22509525
  PMID:23261933
  PMID:22238426.
• Loss-of-function phenotype is mild: Six1 depletion does NOT abolish pigment cells
  PMID:22509525.
  So Six1/2 is a maintenance/stabilising input, not a specification factor; a
  pigment-cell-differentiation BP term is not justified for this gene.

Later expression: archenteron tip, coelomic pouches and the left-right axis

• At gastrula/pluteus, six1/2 is expressed with eya, pax6, soxE and dach in the aboral
  veg2-derived cells of the archenteron tip and then the LEFT coelomic pouch
  (hydrocoel); this left-sided expression depends on BMP signalling and is excluded
  from the right side by Nodal
  PMID:23055827
  PMID:23055827
  PMID:23055827
  PMID:23055827.
  Note: six1/2 is a left-sided (BMP-dependent) marker, not a right-sided Nodal
  target; Nodal inhibition makes it bilateral. Expression-only evidence; no Six1/2
  perturbation in this paper, so no left/right BP annotation is proposed.
• Co-expression mapping at the archenteron tip (S. purpuratus) places Six1/2 with SoxE,
  FoxF, Gcm, MyoR and Mef2 in the aboral-anal (AbAn) domain, not in the FoxY/FoxC
  myoblast precursors
  PMID:24295205.
• In Lytechinus variegatus (orthologue; not S. purpuratus), Six1/2 is part of a
  Pax6/Six3/Six1/2/Eya/Dach1 retinal-determination-like subcircuit in the coelomic
  pouch mesoderm that directs homing of the small micromeres; mesoderm-targeted
  Six1/2 knockdown impairs homing, and Six1/2 is upstream of six3, eya and itself
  PMID:26402456
  PMID:26402456
  PMID:26402456.
  This is a non-cell-autonomous (regulation of migration of another cell type)
  effect in a different species; recorded here as context, not annotated.

Annotation decisions (summary)

• GOA has only electronic rows (5 IBA from PAINT node PTN000044272, 4 IEA). All are
  consistent with a SIX-family sequence-specific transcription factor; the sea urchin
  cis/trans evidence on the gcm late module supports GO:0000978, GO:0000981,
  GO:0006357 and nucleus. GO:0003677 (DNA binding) and GO:0006355 are generalisations
  of these and are flagged MODIFY to the specific Pol II terms. GO:0005667
  (transcription regulator complex, IBA) is accepted on the Six-Eya cofactor
  relationship, which is documented by co-expression and synergy in sea urchins but
  not by a biochemical complex assay in this species.
• NEW: GO:0001228 (activator activity, IMP+IDA from gcm late-module cis/trans
  perturbation) and GO:0045944 (positive regulation of transcription by RNA pol II,
  IMP). No lineage specification / pigment differentiation BP (knockdown has no
  pigment phenotype; six1 acts after specification). No left/right or cell-migration
  BP (expression-only in S. purpuratus; migration phenotype only in L. variegatus).