Developmental gene expression provides clues to relationships between sponge and eumetazoan body plans

May combine two claims.

The S. ciliatum larva also contains four cruciform cells around its equator and a few maternal-origin cells inside the larval cavity.

“The larva also contains four cruciform cells distributed around its equator and a few cells of maternal origin inside of the larval cavity (Fig. 1g,j).”

May combine two claims.

Eight Wnt sequences were recovered from the transcriptome of Oscarella sp., two of them orthologous to previously published Oscarella lobularis genes.

“We have also recovered eight Wnt sequences from the transcriptome of a homoscleromorph sponge, Oscarella sp 43 , two of them orthologous to previously published Oscarella lobularis genes 44 .”

• cites [43] Hemmrich, G. & Bosch, T. C. G. Compagen, a comparative genomics platform for early branching metazoan animals reveals early origins of genes regulating stem cell differentiation. BioEssays 30, 1010–1018 (2008). sourceNot checked yet

  Not checked in the free run.

• cites [44] Lapebie, P. et al. WNT/β-catenin signalling and epithelial patterning in the homoscleromorph sponge Oscarella. PLoS ONE 4, e5823 (2009). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The number of Wnt genes in Sycon is surprisingly large compared with Amphimedon queenslandica, which has only three Wnt genes.

“The surprisingly large number of Wnt genes stands in contrast to the situation in A. queenslandica, where only three Wnt genes are present 42 .”

• cites [42] Adamska, M. et al. Structure and expression of conserved Wnt pathway components in the demosponge Amphimedon queenslandica. Evol. Dev. 12, 494–518 (2010). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Nine Sycon Wnts form a well-supported clade affiliated with a single Oscarella sequence, and smaller clades contain either Sycon or Oscarella genes, suggesting lineage-specific expansion events and obscuring the number of Wnt ligands in the last common ancestor.

“Nine of the Sycon Wnts form a well-supported clade affiliated with a single Oscarella sequence; smaller clades encompassing either Sycon or Oscarella genes are also recovered (Supplementary Fig. 1, Fig. 2a), suggesting lineage-specific expansion events and obscuring the number of Wnt ligands in the last common ancestor.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Adult Sycon ciliatum specimens are syconoid, with choanocyte-lined radial chambers surrounding an atrial cavity and a single osculum at the apical end.

“The adult specimens are syconoid, with choanocyte-lined radial chambers surrounding an atrial cavity and a single osculum (exhalant opening) at the apical end (Fig. 1a-c).”

May combine two claims.

Radial chambers bud radially around the original choanocyte chamber, which becomes an endopinacocyte-lined atrial chamber.

“Radial chambers bud in a grossly radially symmetrical manner around the original choanocyte chamber, which becomes an endopinacocyte-lined atrial chamber (Fig. 1b,c).”

May combine two claims.

Development in S. ciliatum is synchronous, and during the reproductive period oocytes and embryos constitute a significant fraction of sponge tissue.

“Development is synchronous, and during the reproductive period (Table 1) oocytes and embryos constitute a significant fraction of the sponge tissue (Fig. 1d-f).”

May combine two claims.

Bilaterian and cnidarian Wnts are confidently placed into previously described subfamilies, while sponge Wnt sequences (except Amphimedon WntB and WntC loosely affiliated with Wnt8) fall outside recognized eumetazoan subfamilies.

“While our analysis confidently places bilaterian and cnidarian Wnts into previously described subfamilies, sponge sequences (with exception of Amphimedon WntB and WntC loosely affiliated with Wnt8 subfamily) fall outside of the recognized eumetazoan subfamilies.”

May combine two claims.

Sycon has 21 Wnt genes, multiple downstream Wnt pathway components, 22 Tgfb ligands, 6 Smad genes, two Vasa and two Pl10 genes, and single Nanos, Gata, Gli and Hedgling genes.

“However, we have recovered 21 Wnt genes, multiple downstream components of the Wnt pathway, 22 Tgfb ligands, 6 Smad genes, two each of Vasa and Pl10 genes, and single Nanos, Gata, Gli and Hedgling genes.”

May combine two claims.

Only two Sycon Frizzleds contain the KTxxxW motif involved in canonical signalling, and these two (FzdB and FzdD) are closely related to eumetazoan and Amphimedon Fzds that also contain the motif.

“Only two Fzds contain the KTxxxW motif, playing a role in transduction of the canonical signalling 45 , and these two (FzdB and FzdD) appear closely related to the eumetazoan and Amphimedon Fzds that also contain this motif.”

• cites [45] Umbhauer, M. et al. The C-terminal cytoplasmic Lys-thr-X-X-X-Trp motif in frizzled receptors mediates Wnt/β-catenin signalling. EMBO J. 19, 4944–4954 (2000). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

FzdC and FzdD are expressed in choanocytes and a fraction of mesohyl cells, and FzdD additionally in a ring of mesohyl cells surrounding the osculum.

“FzdC and FzdD are expressed in choanocytes and a fraction of mesohyl cells, and FzdD additionally in a ring of mesohyl cells surrounding the osculum (Fig. 4m,q).”

May combine two claims.

WntA, WntB and WntR are expressed in the posterior half of larvae: WntB in macromeres, WntR in posterior-most micromeres, and WntA in macromeres and posterior-most micromeres.

“WntA, WntB and WntR are expressed in the posterior half of the larvae: WntB in the macromeres, WntR in posterior most micromeres and WntA in macromeres and posterior most micromeres (Fig. 3j-l).”

May combine two claims.

SciNanos is expressed in macromeres and cross cells during preinversion and is undetectable in somatic cells of adult sponges.

“SciNanos is expressed in the macromeres and cross cells during preinversion (Fig. 9j) and is undetectable in the somatic cells of the adult sponges (Fig. 9k-l).”

May combine two claims.

No true Hedgehog gene was found in Sycon, and none is present in Amphimedon.

“We have not found a true Hedgehog gene in Sycon (none is present in Amphimedon 57 ), and it is yet to be known whether Hedgling signals through Gli.”

• cites [57] Adamska, M. et al. The evolutionary origin of hedgehog proteins. Curr. Biol. 17, R836–R837 (2007). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Pl10A expression decreases during cleavage, while Pl10B expression remains strong and uniform in embryos, larvae and then choanocytes.

“Expression of Pl10A decreases during cleavage (Fig. 9b-d, top), but Pl10B expression remains strong and uniform in embryos, larvae and then choanocytes (Fig. 9b-d, bottom).”

May combine two claims.

The majority of Wnt genes are expressed in oocytes and uniformly in blastomeres during cleavage, but none was detected in preinversion stage embryos.

“The majority of Wnt genes are expressed in oocytes and uniformly in blastomeres during cleavage, but none was detected in preinversion stage embryos (for example, Fig. 3a-f).”

May combine two claims.

In adults, Gata is expressed in choanocytes and upregulated in choanocyte-derived cells of the fertilization complex and accessory cells.

“In adults, Gata is expressed in choanocytes (Fig. 9p), and is additionally upregulated in choanocyte-derived cells of the fertilization complex and the accessory cells (Fig. 9m-n).”

May combine two claims.

Smad4 expression is uniform in oocytes, throughout cleavage and preinversion embryos, then detectable only in maternal cells of early postinversion embryos, and strong in micromeres of later postinversion embryos.

“Expression of Smad4 was uniform in oocytes, throughout the cleavage and in preinversion stage embryos, then detectable only in the maternal cells of early postinversion stage embryos, and strong in the micromeres of later postinversion embryos (Fig. 7g-k).”

May combine two claims.

Bra2 is expressed strongly in oocytes, weaker during cleavage, and slightly stronger in micromeres than macromeres during preinversion.

“Bra2 is expressed strongly in the oocytes, weaker during cleavage and slightly stronger in the micromeres than macromeres during preinversion (Fig. 8f-i).”

May combine two claims.

Two Sycon Smads are affiliated with inhibitory Smads6/7, one with co-Smad4, one with Smads1/5, and two fall within receptor Smads but not clearly into Smad2/3 or Smad1/5.

“Two Sycon Smads are affiliated with the inhibitory Smads6/7, one with co-Smad4 (a co-factor for receptor-specific Smads), one with Smads1/5, and two are included within the receptor Smads group, but do not fall clearly into Smad2/3 or Smad1/5 family (Supplementary Fig. 4; Fig. 2d).”

May combine two claims.

Both Vasa genes are expressed throughout embryos, with elevated expression in cruciform cells of preinversion embryos and micromeres of postinversion embryos.

“Both Vasa genes are expressed throughout the embryos, with expression elevated in the cruciform cells of preinversion stage embryos and micromeres of postinversion stage embryos (Fig. 8f,g).”

May combine two claims.

Both b-catenin genes are dynamically expressed: bcatA in young oocytes, not in mature oocytes undergoing fertilization nor during cleavage, upregulated in micromeres of preinversion embryos and decreasing after inversion; bcatB in oocytes of all sizes and early embryos (higher in cruciform cells) and then in maternal cells.

“Both b-catenin genes are dynamically expressed: bcatA in young oocytes, but not in mature oocytes undergoing fertilization nor during cleavage, upregulated in the micromeres of preinversion stage embryos and decreasing after inversion (Fig. 5m-p); bcatB in oocytes of all sizes and in early embryos (higher in the cruciform cells) and then in the maternal cells (Fig. 5r-t).”

May combine two claims.

Almost all analyzed genes are expressed in somatic cells, as shown by transcripts in non-reproductive specimens, although expression of some genes (WntB, TgfbD, Hedgling, TboxB) appears negligible in adult tissue compared with developmental samples.

“Almost all of the analyzed genes are expressed in somatic cells, as demonstrated by the presence of transcripts in samples derived from non-reproductive specimens, although expression of some genes (for example, WntB, TgfbD, Hedgling and TboxB) appears negligible in adult tissue as compared with developmental samples.”

May combine two claims.

Both Dvl homologues are strongly expressed in oocytes; their expression decreases during cleavage in most cells but remains high in cruciform cells.

“Both Dvl homologues are expressed strongly in the oocytes; their expression decreases during cleavage in most cells, remaining high in the cruciform cells (Fig. 4r,s,v,w).”

May combine two claims.

Multiple genes including WntA, WntB, WntR, FzdA, TgfbD and Hedgling are strongly upregulated in late embryogenesis and highly expressed in larvae, while WntI, WntJ, TcfA, TgfbS and Nanos have highest expression during cleavage.

“Multiple genes including WntA, WntB, WntR, FzdA, TgfbD and Hedgling are strongly upregulated in late embryogenesis and highly expressed in the larvae, while WntI, WntJ, TcfA, TgfbS and Nanos have highest expression during cleavage.”

May combine two claims.

The single SciGata sequence clusters with AmqGata, and the two sequences fall between Gata1/2/3 and Gata4/5/6 subfamilies, indicating GATA family diversification occurred after sponge divergence.

“The single identified SciGata sequence clusters with AmqGata, and the two sequences fall between Gata1/2/3 and Gata4/5/6 subfamilies, indicating that the diversification of GATA family occurred after divergence of sponges (Supplementary Fig. 5 and Supplementary Fig. 6).”

May combine two claims.

Both Tcf genes are expressed throughout choanocytes, upregulated in oocytes and ubiquitous in embryos, with TcfB stronger in cruciform cells.

“Both Tcf genes are expressed throughout the choanocytes, upregulated in oocytes and ubiquitously expressed in the embryos, with TcfB stronger in the cruciform cells (Fig. 5e-l).”

May combine two claims.

SmadRa is expressed weakly in oocytes and throughout cleavage, and prominently in maternal cells in postinversion embryos.

“SmadRa was expressed weakly in the oocytes and throughout cleavage, and prominent in the maternal cells in postinversion stage embryos (Fig. 7m-q).”

May combine two claims.

FzdB, FzdC and FzdD are expressed uniformly in oocytes and cleavage-stage embryos, while FzdA and FzdD are expressed in embryonic and larval micromeres.

“FzdB, FzdC and FzdD are expressed uniformly in the oocytes and cleavage stage embryos, while FzdA and FzdD in the embryonic and larval micromeres (Fig. 4a-p).”

May combine two claims.

Smad1/5 expression is strong in oocytes, restricted to cruciform cells and macromeres of preinversion and early postinversion embryos, and confined to micromeres of late postinversion embryos.

“Among them, Smad1/5 displayed the most dynamic expression: strong in the oocytes, restricted to the cruciform cells and the macromeres of preinversion and early postinversion stage embryos, and confined to the micromeres of late postinversion stage embryos (Fig. 7a-e).”

May combine two claims.

The most prominent region of Tgfb expression is the top/apical region, with strong expression of TgfbN (also in entries of radial chambers), TgfbH, TgfbE, TgfbU and TgfbJ in various cell types around the osculum.

“The most prominent region of Tgfb expression is the top/apical region, with strong expression of TgfbN (also expressed in the entries of the radial chambers), TgfbH, TgfbE, TgfbU and TgfbJ detected in a variety of cell types around the osculum (Fig. 6l-q).”

May combine two claims.

TboxA is not expressed embryonically but marks accessory cells derived from choanocytes associated with pre- and postinversion embryos.

“One of Eomes-class Sycon Tboxes, TboxA, is not expressed embryonically, but instead marks accessory cells (derived from choanocytes) associated with pre and postinversion embryos (Fig. 8k-n).”

May combine two claims.

WntC is also expressed in endopinacocytes of the atrial cavity and, together with WntF, in cells surrounding entries to radial chambers, consistent with homology between proximal-distal axes of chambers and the main body axis.

“WntC is also expressed in the endopinacocytes of the atrial cavity, and, together with WntF, in cells surrounding the entries to the radial chambers (Fig. 3u-v), consistent with the notion that proximal-distal axes of chambers are homologous to the main body axis.”

May combine two claims.

TboxC belongs to the newly defined Tbox7 subfamily and is expressed in oocytes, throughout cleavage, and in preinversion embryos, stronger in micromeres.

“TboxC, which belongs to the newly defined Tbox7 subfamily 32 , is expressed in the oocytes, throughout cleavage, and in preinversion stage embryos (stronger in the micromeres) (Fig. 8r-u).”

• cites [32] Sinigaglia, C., Busengdal, H., Leclère, L., Technau, U. & Rentzsch, F. The bilaterian head patterning gene six3/6 controls aboral domain development in a cnidarian. PLoS Biol. 11, e1001488 (2013). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

SciGata expression is undetectable in oocytes but strong in central/anterior micromeres.

“SciGata expression is undetectable in the oocytes (Fig. 9m), but is strong in the central/anterior micromeres (Fig. 9n-o).”

May combine two claims.

WntJ, WntG, WntR and WntC are expressed in staggered domains around the osculum of adult sponges: WntJ in apical-most outer epithelial cells of the oscular sphincter; WntG in a narrower ring surrounding WntJ; WntR in a ring of mesohyl cells around the osculum and rare mesohyl cells throughout the body column; and WntC in a ring of choanocytes at the border between radial chambers and asconoid区域.

“WntJ, WntG, WntR and WntC are expressed in staggered domains around the osculum of adult sponges: WntJ in the apical-most outer epithelial cells of the oscular sphincter; WntG in a narrower ring of cells surrounding the WntJ domain; WntR in a ring of mesohyl cells around the osculum (and in rare mesohyl cells throughout the body column); and WntC in the ring of choanocytes on the border between”

May combine two claims.

Among 21 Wnt and 22 Tgfb genes, only WntF is expressed significantly lower in the apical than basal part, while nine Wnt and seven Tgfb genes are expressed significantly higher in the apical than middle part.

“Strikingly, among 21 Wnt genes and 22 TGFb genes, only one (WntF) was expressed significantly lower in the apical than the basal part, while nine Wnt genes and seven Tgfb genes were expressed significantly higher in the apical than the middle part.”

May combine two claims.

In postinversion embryos, TgfbF is expressed in macromeres and, along with TgfbG and TgfbB, is detectable in maternal cells.

“In postinversion stage embryos, TgfbF is expressed in the macromeres, and, along with TgfbG and TgfbB, is detectable in the maternal cells (Fig. 6g-i).”

May combine two claims.

In adults, TgfbB expression is uniform in choanocytes and high in a few cells within the oscular sphincter.

“In adults, expression of TgfbB is uniform in the choanocytes and high in a few cells within the oscular sphincter (Fig. 6j-k).”

May combine two claims.

Downstream components of Wnt and TGFb pathways and other genes potentially involved in choanoderm specification or maintenance are expressed more uniformly along the axis, except bcatA (higher top than middle) and DvlB (highest top).

“In contrast to ligands of the Wnt and TGFb pathways, downstream components of these pathways, as well as other genes potentially involved in specification or maintenance of the choanoderm, were expressed more uniformly along the axis, with exceptions of bcatA (higher in the top than in the middle) and DvlB (highest in the top).”

May combine two claims.

SciBra1 is expressed weakly in oocytes and is undetectable in early preinversion embryos.

“SciBra1 is expressed weakly in the oocytes and is undetectable in early preinversion stage embryos (Fig. 8a).”

May combine two claims.

The Tbox family shows the highest diversity of expression profiles, with TboxA highest in the top, TboxC higher in the top than middle, and Bra2 lowest in the top.

“The Tbox family displayed highest diversity of expression profiles, with TboxA highest in the top, TboxC higher in the top than in the middle and Bra2 lowest in the top.”

May combine two claims.

In Sycon embryos and larvae, key components of the canonical Wnt pathway (FzdA and FzdD, DvlA, bcatA) are specifically expressed in the micromeres, or are expressed broadly including the micromeres (Lrp5/6, Tcf).

“Here we demonstrated that the key components of the canonical Wnt pathway (FzdA and FzdD, DvlA, bcatA) are specifically expressed in the micromeres (Fig. 10a), or are expressed broadly including the micromeres (Lrp5/6, Tcf) of Sycon embryos and larvae.”

May combine two claims.

At least one gene representing each component class of the canonical Wnt pathway is also expressed in choanocytes, and some in mesohyl cells of adults.

“At least one gene representing each component class is also expressed in choanocytes, and some in mesohyl cells of adults, suggesting they continue to specify these cell types during growth of the sponge (Fig. 10f).”

May combine two claims.

This expression is not limited to a specific cell type; macromeres, posterior-most micromeres and maternal cells express these genes.

“This expression is not limited to a specific cell type, as macromeres, posterior-most micromeres and maternal cells express these genes.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The radial chambers were suggested to correspond to complete olynthus-grade bodies attached with exhalant openings to the atrium, so the juvenile apical-basal axis is reflected in proximal-distal axes of the radial chambers.

“The radial chambers were suggested to correspond to complete olynthusgrade bodies attached with exhalant openings to the atrium, so that the apical-basal axis of polarity of the juvenile is reflected in proximal-distal axes of the radial chambers 40 .”

• cites [40] Manuel, M. Origine et évolution des mécanismes moléculaires contrôlant la morphogenèse chez les Métazoaires: un nouveau modèle spongiaire, Sycon raphanus (Calcispongia, Calcaronea). PhD Thesis (Paris-Sud University, (2001).Not checked yet

  Not checked in the free run.

May combine two claims.

Wnt pathway expression and function pattern the main body axis in cnidarians and bilaterians, with expression in the larval posterior and adult oral/posterior ends.

“Expression and function of Wnt genes in the larval posterior and the adult oral end of cnidarians, as well as the posterior end of multiple bilaterians, demonstrate that the main body axis is patterned by the Wnt pathway in both lineages [20][21][22][23][24] .”

• cites [20][21][22][23][24]Not checked yet

  Not checked in the free run.

May combine two claims.

The first multicellular animals had poriferan-grade body plans with choanoflagellate-like cells used for water movement, food capture and digestion, but lacked key metazoan cell types such as nerves and muscles.

“Several lines of evidence (morphology, molecular phylogenies, fossil record) suggest that the first multicellular animals had poriferan-grade body plans with choanoflagellate-like cells (choanocytes) used for water movement, food capture and digestion, but lacked key metazoan cell types, for example, nerves and muscles [7][8][9][10][11][12] .”

• cites [7][8][9]Not checked yet

  Not checked in the free run.

May combine two claims.

The developmental gene toolkit of sponges is surprisingly similar to the eumetazoan toolkit, although information on its expression remains limited.

“Importantly, the developmental gene toolkit of sponges is surprisingly similar to the eumetazoan toolkit, although information on its expression remains limited 4,7 .”

• cites [4] Adamska, M., Degnan, B. M., Green, K. & Zwafink, C. What sponges can tell us about the evolution of developmental processes. Zoology (Jena) 114, 1–10 (2011). sourceNot checked yet

  Not checked in the free run.

• cites [7] Srivastava, M. et al. The amphimedon queenslandica genome and the evolution of animal complexity. Nature 466, 720–726 (2010). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The gastrodermis of cnidarian polyps is homologous to the bilaterian endoderm and mesoderm, although their regulatory networks differ.

“Developmental gene expression studies indicate that the gastrodermis is homologous to the bilaterian endoderm and mesoderm, although differences in regulatory networks between them exist [17][18][19] .”

• cites [17][18][19]Not checked yet

  Not checked in the free run.

May combine two claims.

Among the contentious basal metazoan clades, sponges likely retained the organization of the earliest metazoans and can provide clues to the origin of complex bilaterian body plans.

“Thus, among the three contentious 'basal' clades, sponges, which likely retained the organization of the earliest metazoans, can be expected to provide significant clues towards understanding the origin of the complex bilaterian body plans 4 .”

• cites [4] Adamska, M., Degnan, B. M., Green, K. & Zwafink, C. What sponges can tell us about the evolution of developmental processes. Zoology (Jena) 114, 1–10 (2011). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

BMP ligands of the Tgfb superfamily pattern the dorsal-ventral axis in bilaterians and are asymmetrically expressed along the directive axis of anthozoan cnidarians.

“The second (dorsal-ventral) body axis in bilaterians is patterned by bone morphogenetic protein ligands of the Tgfb superfamily, which are also asymmetrically expressed along the directive axis of anthozoan cnidarians 26,27 .”

• cites [26] Hayward, D. C. et al. Localized expression of a dpp/BMP2/4 ortholog in a coral embryo. Proc. Natl Acad. Sci. USA 99, 8106–8111 (2002). sourceNot checked yet

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• cites [27] Matus, D. Q., Pang, K., Marlow, H., Dunn, C. W., Thomsen, G. H. & Martindale, M. Q. Molecular evidence for deep evolutionary roots of bilaterality in animal development. Proc. Natl Acad. Sci. USA 103, 11195–11200 (2006). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

During settlement, the anterior pole contacts the substrate; micromeres differentiate into choanocytes and spicule-producing cells; macromeres differentiate into pinacocytes; and cruciform and maternal cells degenerate.

“During settlement, the anterior pole makes contact with the substrate; the micromeres differentiate into choanocytes and spicule producing cells; the macromeres differentiate into pinacocytes; the cruciform and maternal cells degenerate 36 .”

• cites [36] Amano, S. & Hori, I. Metamorphosis of calcareous sponges. 2. cell rearrangement and differentiation in metamorphosis. Invert. Reprod. Dev. 24, 13–26 (1993). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Maternal cells that ingress into the larval cavity are not of embryonic origin and degenerate before the juvenile sponge forms.

“While maternal cell ingression superficially resembles gastrulation, these cells are not of embryonic origin and will degenerate before the juvenile sponge is formed 36 .”

• cites [36] Amano, S. & Hori, I. Metamorphosis of calcareous sponges. 2. cell rearrangement and differentiation in metamorphosis. Invert. Reprod. Dev. 24, 13–26 (1993). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

GATA transcription factors are regulators of endomesoderm development, although in Nematostella endodermal Gata expression starts after the endoderm is formed, indicating it is not part of the endomesoderm specification kernel in this cnidarian.

“GATA transcription factors are regulators of endomesoderm development, although in Nematostella endodermal Gata expression starts after the endoderm is already formed, indicating that it is not part of the endomesoderm specification kernel in this cnidarian 18,19 .”

• cites [18] Martindale, M. Q., Pang, K. & Finnerty, J. R. Investigating the origins of triploblasty: ‘mesodermal’ gene expression in a diploblastic animal, the sea anemone Nematostella vectensis (phylum, Cnidaria; class, Anthozoa). Development 131, 2463–2474 (2004). sourceNot checked yet

  Not checked in the free run.

• cites [19] Roettinger, E., Dahlin, P. & Martindale, M. Q. A framework for the establishment of a cnidarian gene regulatory network for ‘endomesoderm’ specification: the inputs of ß-Catenin/TCF signalling. PLoS Genet. 8, e1003164 (2012). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

In Nematostella embryos, germline markers Vasa, Pl10 and Nanos2 are expressed in presumptive endoderm and their expression responds to up- or downregulation of the canonical Wnt pathway, consistent with a role in endomesoderm specification.

“In Nematostella embryos, the eumetazoan germline markers Vasa, Pl10 and Nanos2 are expressed in the presumptive endoderm and their expression responds to up or downregulation of the canonical Wnt pathway, consistent with a role in endomesoderm specification 19,54 .”

• cites [19] Roettinger, E., Dahlin, P. & Martindale, M. Q. A framework for the establishment of a cnidarian gene regulatory network for ‘endomesoderm’ specification: the inputs of ß-Catenin/TCF signalling. PLoS Genet. 8, e1003164 (2012). sourceNot checked yet

  Not checked in the free run.

• cites [54] Extavour, C. G., Pang, K., Matus, D. Q. & Martindale, M. Q. vasa and nanos expression patterns in a sea anemone and the evolution of bilaterian germ cell specification mechanisms. Evol. Dev. 7, 201–215 (2005). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Hedgehog signalling is involved in endomesoderm formation in bilaterians, and Gli is expressed in the presumptive endoderm of Nematostella.

“Hedgehog signalling is involved in endomesoderm formation in bilaterians, and the key transcription factor of the pathway, Gli, is expressed in the presumptive endoderm of Nematostella 19,56 .”

• cites [19] Roettinger, E., Dahlin, P. & Martindale, M. Q. A framework for the establishment of a cnidarian gene regulatory network for ‘endomesoderm’ specification: the inputs of ß-Catenin/TCF signalling. PLoS Genet. 8, e1003164 (2012). sourceNot checked yet

  Not checked in the free run.

• cites [56] Matus, D. Q., Magie, C. R., Pang, K., Martindale, M. Q. & Thomsen, G. H. The Hedgehog gene family of the cnidarian, Nematostella vectensis, and implications for understanding metazoan Hedgehog pathway evolution. Dev. Biol. 313, 501–518 (2008). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Two Brachyury homologues with eumetazoan-like properties, and Eomes and basal class Tboxes, are present in Sycon.

“We have recently reported the presence and eumetazoan-like properties of two Brachyury homologues, and the presence of Eomes and basal class Tboxes in Sycon 41 .”

• cites [41] Sebé-Pedrós, A. et al. Early evolution of the T-box transcription factor family. Proc Natl Acad Sci USA 110, 16050–16055 (2013). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

RNA-seq analyses showed expected similarities within replicates and distinctiveness of samples from different stages, except indistinguishable cleavage samples.

“These analyses indicated expected similarities within replicates and distinctiveness of the samples isolated at different stages (except indistinguishable cleavage samples; Supplementary Figs 7 and 8).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Several other Tbox family genes, including the Eomes class, are involved in endomesoderm formation.

“Several other Tbox family genes, including the Eomes class, are also involved in formation of the endomesoderm 53 .”

• cites [53] Arnold, S. J., Hofmann, U. K., Bikoff, E. K. & Robertson, E. J. Pivotal roles for eomesodermin during axis formation, epithelium-to-mesenchyme transition and endoderm specification in the mouse. Development 135, 501–511 (2008). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

NvNanos1 is expressed in nematocyst precursors, consistent with neuronal expression of Nanos genes in other cnidarians and bilaterians.

“In contrast, NvNanos1 is expressed in nematocyst precursors, consistent with the neuronal expression of Nanos genes in other cnidarians and bilaterians 54,55 .”

• cites [54] Extavour, C. G., Pang, K., Matus, D. Q. & Martindale, M. Q. vasa and nanos expression patterns in a sea anemone and the evolution of bilaterian germ cell specification mechanisms. Evol. Dev. 7, 201–215 (2005). sourceNot checked yet

  Not checked in the free run.

• cites [55] Kanska, J. & Frank, U. New roles for Nanos in neural cell fate determination revealed by studies in a cnidarian. J. Cell Sci. 126, 3192–3203 (2013). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Brachyury is essential for endomesoderm formation and in bilaterians and cnidarians is expressed in future endomesodermal cells as they move through the blastopore.

“The Tbox transcription factor Brachyury is essential for formation of endomesoderm, and in a variety of bilaterians and cnidarians is expressed in future endomesodermal cells as they move through the blastopore 17 .”

• cites [17] Technau, U. & Scholz, C. B. Origin and evolution of endoderm and mesoderm. Int. J. Dev. Biol. 47, 531–539 (2003).Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

Pools of samples derived from sponges containing the same developmental stages were used to generate Illumina RNA-Seq libraries, with biological replicates obtained wherever possible.

“Pools of samples derived from sponges containing the same developmental stages were used to generate Illumina RNA-Seq libraries, with biological replicates obtained wherever possible (Table1).”

May combine two claims.

Transplantation experiments in Ephydatia mulleri demonstrated organizer properties of the osculum, and this activity is likely conveyed by canonical Wnt signalling.

“Nothing is known about the expression of relevant genes in juvenile or adult demosponges, but transplantation experiments performed with Ephydatia mulleri demonstrated organizer properties of the osculum, and that this activity is likely conveyed by canonical Wnt signalling 59 .”

• cites [59] Windsor, P. J. & Leys, S. P. Wnt signalling and induction in the sponge aquiferous system: evidence for an ancient origin of the organizer. Evol. Dev. 12, 481–590 (2010). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

While sponges contain only a limited number of direct orthologues of genes involved in body plan patterning in eumetazoans, all of them are expressed in patterns supporting Haeckel's view that body plans of sponges and cnidarians are homologous: choanoderm corresponds to the gastrodermis and osculum to the mouth of the polyp.

“Thus, while sponges contain only a limited number of direct orthologues of genes involved in body plan patterning in the eumetazoans, all of them are expressed in patterns supporting Haeckel's view that body plans of sponges and cnidarians are homologous: choanoderm corresponds to the gastrodermis and osculum to the mouth of the polyp.”

• AssumptionRests on an assumption: Sponges contain only a limited number of direct orthologues of genes involved in body plan patterning in eumetazoans.

Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

Expression analysis of putative network components is the first step to identifying interaction partners.

“Expression analysis of the putative network components is the first step to identification of the interaction partners.”

Classified as probably not a substantial claim.

The described expression patterns of developmental regulatory genes, while highly suggestive, do not prove homology of the body plans.

“Clearly, the described expression patterns of the developmental regulatory genes, while highly suggestive, do not prove homology of the body plans.”

Classified as probably not a substantial claim. May combine two claims.

Only midbody fragments were used to investigate expression changes during oogenesis and embryonic development because differential expression across the body axis was expected.

“As we expected differential expression of some of the studied genes across the body axis (see below), only midbody fragments were used to investigate expression changes during oogenesis and embryonic development.”

Classified as probably not a substantial claim. May combine two claims.

Each individual sponge was dissected and part subjected to histological analysis to determine developmental stage, and part used for total RNA isolation.

“Each individual sponge was dissected and part of it subjected to histological analysis to determine the developmental stage, and part used for total RNA isolation using the AllPrep DNA/RNA Mini Kit (Qiagen).”

May combine two claims.

Tgfb ligand expression, especially BMP family members, marks the perpendicular axis in bilaterians and anthozoan cnidarians, while it is symmetrical around the hypostome of radial cnidarian polyps like Hydra.

“Expression of Tgfb ligands, especially of the BMP family, marks the perpendicular axis in bilaterians and anthozoan cnidarians, while it is symmetrical around the hypostome of radial cnidarian polyps like Hydra 26,27,58 .”

• cites [26] Hayward, D. C. et al. Localized expression of a dpp/BMP2/4 ortholog in a coral embryo. Proc. Natl Acad. Sci. USA 99, 8106–8111 (2002). sourceNot checked yet

  Not checked in the free run.

• cites [27] Matus, D. Q., Pang, K., Marlow, H., Dunn, C. W., Thomsen, G. H. & Martindale, M. Q. Molecular evidence for deep evolutionary roots of bilaterality in animal development. Proc. Natl Acad. Sci. USA 103, 11195–11200 (2006). sourceNot checked yet

  Not checked in the free run.

• cites [58] Reinhardt, B., Broun, M., Blitz, I. L. & Bode, H. R. HyBMP5-8b, a BMP5-8 orthologue, acts during axial patterning and tentacle formation in hydra. Dev. Biol. 267, 43–59 (2004). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Reproducibility of the results was assessed by Principal Component Analysis and sample-to-sample distances.

“Reproducibility of the results was assessed by Principal Component Analysis and sample-to-sample distances 50 (see supplementary materials for details).”

• cites [50] Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Genomic scaffolds and cDNA contigs were identified by aligning them with juvenile-derived genomic sequences, which are devoid of eukaryotic contaminations.

“After de-novo assemblies of the genome and transcriptome, S. ciliatum genomic scaffolds and complementary DNA contigs were identified by aligning them with juvenile-derived (and therefore devoid of eukaryotic contaminations) genomic sequences.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The failure to replicate may be because Ephydatia produces multiple oscula as it grows, while Sycon evolved mechanisms preserving a single body axis throughout its life.

“We have not been able to replicate this result in Sycon, possibly because Ephydatia, like the overwhelming majority of sponges, produces multiple oscula as it grows, while Sycon evolved mechanisms preserving a single body axis throughout its life.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Haeckel proposed that sponge and coral body plans are basically the same: inner layer digestive and source of gametes, outer layer protective and sensory.

“Almost 150 years ago, Haeckel proposed that the body plans of sponges and corals are basically the same: the inner layer serves the digestive function and is the source of gametes, and the outer layer serves the protective and sensory functions 28 .”

• cites [28] Haeckel, E. On the organization of sponges and their relationship to the corals. Ann. Mag. Nat. Hist. 5, 1–13 (1870). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

No other hypothesis on the relationship of adult body plans between sponges and eumetazoans has been put forward since Haeckel, and the prevailing view is that these body plans are incomparable.

“Since then, no other hypothesis on the relationship of the adult body plans between sponges and the eumetazoans has been put forward, and the prevailing view appears to be that these body plans are incomparable.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Phylogenetic gene trees were built using MrBayes with the LG substitution model, model parameters estimated with ProtTest3, and simulations stopped after at least 1M iterations passed after split frequency between runs reached below 0.01.

“Phylogenetic gene trees were built using the MrBayes package (Bayesian inference) 61 modified to incorporate the LG substitution model.Model parameters were estimated using the ProtTest3 package 62 .MrBayes was set to run two sets of four Markov chains.The simulations were stopped once, at least 1 M iterations passed after split frequency between the two runs reached level lower than 0.01.”

• cites [61] Huelsenbeck, J. P. & Ronquist, F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755 (2001). sourceNot checked yet

  Not checked in the free run.

• cites [62] Darriba, D., Taboada, G. L., Doallo, R. & Posada, D. ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 27, 1164–1165 (2011). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

In the Amphimedon parenchymella, presumptive choanoderm cells assume a peripheral position in the larvae by non-polarized cell migration, forming an outer layer of small, ciliated cells excluded from the posterior pole.

“In the Amphimedon parenchymella, this process occurs by non-polarized cell migration, resulting in formation of the outer layer composed of small, ciliated cells, excluded from the posterior pole 60 .”

• cites [60] Leys, S. P. & Degnan, B. M. Embryogenesis and metamorphosis in a haplosclerid demosponge: gastrulation and transdifferentiation of larval ciliated cells to choanocytes. Inv. Biol. 121, 171–189 (2002). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

In both Amphimedon and Sycon, during metamorphosis, the ciliated cells move to the inside, transdifferentiate into choanocytes and organize into the choanocyte chamber or a cluster of chambers.

“In both cases, during metamorphosis, these ciliated cells then move to the inside, transdifferentiate into choanocytes and organize into the choanocyte chamber or a cluster of chambers.”

Classified as probably not a substantial claim. May combine two claims.

Statistical significance of differential expression was based on negative binomial distribution, with P-values adjusted for multiple testing using Benjamini-Hochberg procedure and cutoff 0.1.

“Calculation of statistical significance of gene expression being different (higher) in the apical part of the sponge was based on analysis of the negative binomial distribution of the expression levels 50 .We have used the DESeq 'padj', P-value adjusted for multiple testing with the Benjamini-Hochberg procedure with cutoff r0.1 for calling the expression significantly different.”

• cites [50] Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

One of the three Amphimedon Wnt ligands is specifically expressed in the posterior pole of embryos and larvae, and one of the eight Amphimedon Tgfb ligands is expressed in the outer embryonic layer and then restricted to the posterior pole.

“One of the three Amphimedon Wnt ligands is specifically expressed in the posterior pole of the embryos and larvae, and one of the eight Amphimedon Tgfb ligands is expressed in the outer embryonic layer, and then restricted to the posterior pole 7,48 .”

• cites [7] Srivastava, M. et al. The amphimedon queenslandica genome and the evolution of animal complexity. Nature 466, 720–726 (2010). sourceNot checked yet

  Not checked in the free run.

• cites [48] Adamska, M. et al. Wnt and Tgfβ expression in the sponge Amphimedon queenslandica and the origin of metazoan embryonic patterning. PLoS ONE 2, e1031 (2007). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

For the adult body plan experiment, samples were collected in triplicates for top and bottom parts, as well as two sets of adjacent middle samples (six middle samples in total).

“In this experiment, samples were collected in triplicates for top and bottom parts of the sponges, as well as two sets of adjacent middle samples (six middle samples in total; Table 1).”

Probably not a substantial claim: may not pass S4 (based).

To test whether body axes and germ layers are homologous, one needs to know whether conserved gene regulatory networks are involved in their determination.

“To test whether the body axes and the germ layers are homologous, we need to know whether conserved gene regulatory networks are involved in their determination.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Finally, the osculum opens at the apical end, completing metamorphosis.

“Finally, the osculum opens at the apical end, completing the process of metamorphosis.”