The NK homeobox gene cluster predates the origin of Hox genes.

“Amphimedon lacks Hox genes and some other transcription factor subfamilies that are involved in specifying and patterning bilaterian nervous systems and body plans.(13,14,27,36,37) Signaling cascades, such as the Wnt, TGF-beta, Notch, and Hedgehog pathways, pattern embryos by specifying cellular identity and coordinat…”

“…homeodomains than most vertebrates, including humans, and contains representatives of both ancient (NK, Hox) and young (Rhox, Obox) homeodomain families, encompassing striking examples of both purifying and diversifying selection (Jackson et al., 2006; Larroux et al., 2007; Rajkovic et al., 2002).”

“Beginnings of an NK cluster with several NK genes were found in the sponge Amphimedon queenslandica, with no evidence of HOXL genes (Larroux et al. 2007).”

“Wnt predates the ‘‘Hox code’’ (Larroux et al., 2007; Martindale, 2005) and other developmental patterning signals, and thus appears to constitute the original ancestral program for primary body axis specification.”

“…of the complete genome sequence of the sponge Amphimedon queenslandica, which branches prior to the common ancestor of Cnidaria and Bilateria (Eumetazoa), shows that within the ANTP class, A. queenslandica possesses several NK-like genes, but no Hox, ParaHox or EHGbox genes (Larroux et al. 2007).”

“Along with the diversification signalling pathways in eumetazoans was the origination of Hox genes [24] and expansion of a range of developmental transcription factor gene families [23, 28, unpublished], which enabled further elaboration of ancestral gene regulatory networks.”

“We also point out that the regulatory gene family mentioned here, Hox/ParaHox-like genes, seems to be absent in sponges [ 49 ].”

“…clusters of Antennapedia (ANTP)-like homeobox genes (Hox, ParaHox, EHGBox, and NK clusters) that encode key regulators of animal development and seem to be, to a varying degree, conserved in all animals (Butts et al., 2008, Chourrout et al., 2006, Ferrier and Holland, 2001, Larroux et al., 2007).”

“The sponge genome still maintains an NK gene cluster with six genes [98], but there are no Hox or ParaHox genes in this genome.”

“Origins of other developmental signalling pathways, for example Hox and hedgehog, are also being investigated using phylogenomic approaches [60,61].”

“TF binding sites span 13 classes of TFs, including binding motifs for basic leucine zipper factors (bZIP), motifs for AP-1 and C/EBP, and NK homeodomain proteins; these TF families are present in Amphimedon (37, 38).”

“Studies have shown that the ancestor of the ParaHoxozoa had at least one Hox/ParaHox gene [26,54] and that these genes are missing from both ctenophores and sponges ([55] and this study).”

“The msh/msx gene, a candidate regulator of neurogenesis: The msh/msx homeogene family is highly conserved from Porifera to bilaterians (Larroux et al., 2007).”

“Gene trees for both ANTP genes (Larroux et al. 2007; Peterson and Sperling at U niversity of C alifornia, San D iego on D ecem ber 16, 2013 http://m be.oxfordjournals.org/ D ow nloaded from 2007); Sox, Fox, and T-box genes (Larroux et al. 2008); and basic helix-loop-helix genes (Simionato et al.…”

“Hughes TFs within each family, suggesting that multiple independent expansion events occurred after the branching off of demosponges [26, 126].”

“Most notably, homeobox transcription factors belonging to the extended Hox, Parahox, and NK families are also highly clustered in at least some species (Brooke et al. 1998; Larroux et al. 2007) as are the Irx, Runx, GATA, wnt, and fox genes (Wotton and Shimeld 2006; Bao and Friedrich 2008; Bolognesi et al.”

“However, early branching metazoans, such as sponges and ctenophores, lack Hox genes altogether (Larroux et al., 2007; Moroz et al., 2014; Ryan et al., 2010, 2013; Srivastava et al., 2010), and only a single Hox/ ParaHox gene has been reported in placozoans (Jakob et al.”

“…and since ParaHox is the supposed Hox sister cluster [78], the absence of Hox in Trichoplax could well be secondary Hox and ParaHox genes are absent from the Amphimedon demosponge genome [110] and attempts to recover them from other demosponge and calcisponge taxa [119,167,178] have failed .”

“In contrast, in the earlier-branching sponges, neither ParaHox nor extended Hox cluster genes exist, and only theNKclusterispresentwithasingle NK2/3/4 gene,two NK5/6/7 genes, an Msx ortholog, as well as possible Hex and Tlx orthologs 54 , (Fig.”

“This Nk gene cluster is thought to be quite ancient, possibly predating the evolutionary divergence of sponges, bilaterians and cnidarians [26].”

“queenslandica homologues (orthologues and gene family members) to cnidarian Nematostella vectensis endomesodermal genes (listed in [29]) based on previously published data [28,53-58] and sequence similarity searches in NCBI databases (via BLASTp).”

“In some cases, an in-house trace assembly pipeline was used to derive missing sequence information for local gaps or breaks in the genomic contigs (Larroux et al. 2007).”

“Thus a matrix was built with 117 cnidarian and 94 bilaterian Antp homeodomain sequences and was completed by 5 sequences from the placozoan Trichoplax adherens [46], the 8 available sequences from the demosponge Amphimedon queenslandica [47] and the 4 available sequences from the ctenophore Mnemiopsis leidyi [48].”

“Among them at least five (NK5-7, Tlx, Hex, Msx and NK2-4) might have composed a hypothetic ‘protoNK’ cluster.((23)) Nevertheless, expression data remain scarce and”

“Since the origin of these gene families predates the Bilateria [73,74], a discussion of their role in cnidarian and ctenophore development is useful as it relates to the origin of bilaterian larval apical organs.”

“2006), whereas in the sponge, Amphimedon queenslandica, there are only 31 (Larroux et al. 2007; Larroux et al. 2008), which raises the question of whether this fourfold disparity reflects a primitive simplicity in poriferans or gene loss that resulted in a secondary simplification (Peterson and Sperling 2007).”

“…sponges have a strong affinity with their more complex metazoan relatives, in terms of both their genome and their life cycle (Adell & Müller 2005; Larroux et al. 2006, 2007, 2008; Nichols et al. 2006; Adamska et al. 2007a,b; Sakarya et al. 2007; Simionato et al. 2007; Fahey et al. 2008;…”

“teleta, orthologs of NK2 homeobox genes are found in a sponge and two cnidarians [86-89], goosecoid is also found in a cnidarian [90], and both genes have been identified in the genomes of many bilaterians (see [51]) including ecdysozoans [48,49,53] spiralians [50,55,56] and chordates [45,47,52].”

“…et al. 2009) and ⁄ or gene discovery, developmental biology, novelty acquisition analysis and functional genetics (e.g. Nichols et al. 2006; Adamska et al. 2007, 2010; Larroux et al. 2007; Sullivan et al. 2008; Funayama et al. 2010; Harcet et al. 2010; Rivera et al. 2011; Leys & Riesgo in press).”

“Sponges have several NK homeobox genes but not definite Hox or ParaHox genes [35], and it has been suggested that Hox and ParaHox genes were generated from an ancestral NK-like cluster by tandem gene duplication (Figure 1).”

“…is available for sponges, the initial data demonstrate that these developmental gene toolkits play a role in sponge development and morphogenesis (Larroux et al. 2006, 2007, 2008; Adamska et al. 2007a, b, 2010, 2011; Fahey et al. 2008; Gauthier and Degnan 2008; Gazave et al. 2008; Degnan et al.…”

“queenslandica traces were assembled using an in-house assembly pipeline (Larroux et al. 2007).”

“Note the relative timing of the duplications to generate the full complement of ProtoHox, HoxL, NK, and NKL genes is still debated and some components may well have arisen before others (e.g., NK expansion before Hox; Larroux et al. 2007).”

“Hox clusters arose near the origins of the animal kingdom (Larroux et al. 2007; Ryan et al. 2007).”

“Evidence from the sequenced genome of the demosponge Amphimedon suggests that Hox genes emerged after the metazoan crown ancestor [33].”

“This gene group was fundamental to the origin of metazoan life on earth [91].”

“Transcription factors, G-protein coupled receptors, and kinase genes were obtained from previous studies [23,56,57].”

“Hox genes are well characterised in cnidarians and, given their importance in embryonic development, it is not surprising that molecular evidence from the Cnidaria reveal that the genetic origins of Hox genes predate the cnidarian-bilaterian divergence [83-85] yet had evolved after divergence of the sponge and eumetaz…”

“The recent availability of raw sequencing reads from the Amphimedon queenslandica sequencing project provided evidence for the existence of even more genes in sponges—most notably the homeobox (Wiens, Batel et al. 2003; Wiens, Mangoni et al. 2003; Larroux et al. 2007), Wnt (Adamska et al.”

“Background Gene clusters have been crucially important in the evolution of animals because close physical linkage affects genetic recombination, molecular evolution and gene regulation [1-7].”

“Homeobox genes are found across eukaryotes but are most diverse in animal genomes, following an evolutionary expansion of this group of genes in the early evolution of Metazoa [2].”

“But how can a single gene exert such global pan-ontogenic function?”

“Wnt signalling is thought to predate the “Hox patterning system” and is possibly therefore the original symmetry breaking mechanism and primary axis organizer (Larroux et al. 2007; Martindale 2005).”

“Furthermore Amphimedon has the smallest ANTP-class gene repertoire with 8 (all NK related) genes [16], while Trichoplax harbors 14 and Nematostella close to 80 ANTP class genes [13].”

“[21] surveyed the complete genome of a demosponge, finding only a cluster of NK-type genes among the ANTP-like homeoboxes.”

“…in insect heart D E V E LO P M E N T 3156 Drosophila and Tribolium are sister groups, whereas Apis is the outgroup of this comparison (Savard et al., 2006), and the gene duplication event that gave rise to ladybird and C15 occurred before the protostome/deuterostome split (Larroux et al., 2007).”

“The various families of ANTP genes, distributed into four subclasses (Hox, Parahox, EHGbox, and NKL; Larroux et al. 2007), are found in the three major clades of Bilateria (deuterostomes, lophotrochozoans, and ecdysozoans) and with a few exceptions, they are also present in cnidarian genomes (Chourrout et al.”

“Indeed, the NK gene cluster is believed to be ancient, with aspects predating the divergence of sponges, cnidarians and bilaterians [5].”

“Several recent whole genome scale studies [6,7,38] suggest that ancient animal genomes may have been more complicated than originally thought, with previous misconceptions arising from limited taxon sampling that was largely restricted to a small handful of (often derived) model organisms.”

“More specifically, the presence/absence pattern of NK genes in demosponges would seem to demand, according to the hypothesis of Larroux et al. (2007), that the Hox family was derived from the eumetazoan Msx gene, given that this is the only near relative found in the demosponge (see Fig.”

“The NK family appears to have arisen in the last common ancestor of sponges (the most primitive metazoans) and eumetazoa (cnidarians and bilaterians) after the spongeeumetazoan divergence (Larroux et al. 2007, 2008), while an eye-pathway related Pax gene is present even in sponges (Kozmik 2008).”

“This possibility is suggested by the presence of Msx, Emx, Tlx, and NK genes and the absence of true Hox and ParaHox genes in the sponges [8].”

“Being likely absent from poriferans (Larroux et al., 2007), we assume that H/P genes arose early in eumetazoan evolution to be recruited into regulatory networks that allowed the differentiation of more complex anatomies.”

“Larroux et al. (2007) found different genes belonging to the NKL cluster in Amphimedon, but no Hox or ParaHox genes, which made them suggest that the NKL cluster pre-dated the appearance of the other ANTP clusters in metazoans.”

“, that are typical of eumetazoan development, in agreement with the general lack of sponge othologous genes involved in these regionalizations (Larroux et al. 2007; Leininger et al. 2014; Riesgo et al. 2014).”

“One of the first steps of eumetazoan development is segregation of germ layers, which is regulated by the highly conserved endomesoderm specification network including bcatenin, TCF, Brachyury, Otx, FoxA, Blimp and GATA genes, although position of some of these components within the network differs between bilaterians…”

“Amphimedon development and developmental gene structure and expression have been studied extensively (Leys and Degnan 2001, 2002; Degnan et al. 2005; Larroux et al. 2006, 2007, 2008a; Adamska et al. 2007a,b; Sakarya et al. 2007; Fahey et al. 2008; Gauthier and Degnan 2008a,b).”

“Due to the lack of Hox and ParaHox genes in both Amphimedon and the ctenophoreMnemiopsis leidyi the origin of Hox and ParaHox genes have been proposed to have occurred after the divergences of sponges and ctenophores from all other animals (Larroux et al., 2007; Ryan et al., 2010).”

“It is notable that ANTP, PRD, LIM and POU classes (and the SINE class) have been considered metazoanspecific [35], consistent with a model in which homeobox genes were recruited for spatial patterning specifically in metazoan evolution.”

“Three NK6 paralogs were identified, Nkx6.”

“11(4):1250–1257. doi:10.1093/gbe/evz052 Advance Access publication March 11, 2019 determine the true phylogeny (Penny et al. 1992).”

“Hox genes are found in all metazoan phyla except poriferans (Larroux et al. 2007).”

“This gene belongs to the NK gene cluster, which is probably the most ancient homeobox gene cluster, dating at least from the base of Metazoa (Larroux et al., 2007), and NK4 gene homologues have been identified in numerous species (Harvey, 1996; Elliott et al.”