HES and Mox genes are expressed during early mesoderm formation in a mollusk with putative ancestral features

The mesoderm is considered the youngest of the three germ layers. Although its morphogenesis has been studied in some metazoans, the molecular components underlying this process remain obscure for numerous phyla including the highly diverse Mollusca. Here, expression of Hairy and enhancer of split (HES), Mox, and myosin heavy chain (MHC) was investigated in Acanthochitona fascicularis, a representative of Polyplacophora with putative ancestral molluscan features. While AfaMHC is expressed throughout myogenesis, AfaMox1 is only expressed during early stages of mesodermal band formation and in the ventrolateral muscle, an autapomorphy of the polyplacophoran trochophore. Comparing our findings to previously published data across Metazoa reveals Mox expression in the mesoderm in numerous bilaterians including gastropods, polychaetes, and brachiopods. It is also involved in myogenesis in molluscs, annelids, tunicates, and craniates, suggesting a dual role of Mox in mesoderm and muscle formation in the last common bilaterian ancestor. AfaHESC2 is expressed in the ectoderm of the polyplacophoran gastrula and later in the mesodermal bands and in putative neural tissue, whereas AfaHESC7 is expressed in the trochoblasts of the gastrula and during foregut formation. This confirms the high developmental variability of HES gene expression and demonstrates that Mox and HES genes are pleiotropic.

in the cephalochordate Branchiostoma belcheri, MHC expression is found during somite formation and in the notochord 27,28 . In vertebrates, MHC is involved in the development of skeletal, cardiac, and smooth muscles 29 . In the non-bilaterian cnidarian Nematostella, MHC transcripts are present in the tentacle muscles and in retractor muscles of primary polyps 25 . They are also found in muscle progenitor cells in the tentacle root of the ctenophore Pleurobrachia pileus 26 .
Mox genes possess a conserved helix-turn-helix DNA-binding homeodomain 30 . Previous studies have suggested a sister group relationship to the homeotic gene even-skipped (Evx) 31 . In chordates, Mox expression was reported during formation and differentiation of the main mesodermal derivatives, the somites, that give rise to muscles, bones, and connective tissue 13,32 . Expression of the Drosophila Mox ortholog buttonless is restricted to dorsal median cells which play a crucial role in axon guidance. Importantly, buttonless expression was not detected in Drosophila muscle progenitor cells or muscle tissue 33 , suggesting a loss of Mox in myogenesis in this lineage.
HES genes are members of the basic helix-loop-helix superfamily and direct downstream targets of the Delta-Notch signalling pathway 34 . They possess an additional HES-specific hairy orange domain and a WRPW motif at the C-terminal end 19 . HES genes are involved in a variety of developmental processes such as mesoderm formation, maintaining stem cell potential, or partitioning of morphological territories (e.g., segmentation in annelids, arthropods, chordates, as well as budding in Hydra) 19,31,[35][36][37] . HES genes in mollusks have so far only been studied in the gastropod Crepidula fornicata, where one HES gene was found to be expressed around the mouth as well as in neurosensory cells in the early larva, while the other one shows more dynamic expression domains in the lateral ectoderm around the mouth 38 .
In order to test whether MHC, Mox, and HES are expressed during mesoderm formation in mollusks, we investigated tempo-spatial expression of MHC, Mox, and HES genes in Acanthochitona fascicularis, a member of Polyplacophora that displays several morphological characteristics thought to be ancestral for one of the two major molluscan lineages, the Aculifera 39,40 . In addition, we provide a metazoan-wide comparative survey on the tempo-spatial expression domains of these genes. By plotting these data on current phylogenies and by applying a ground pattern reconstruction approach using parsimony, we discuss scenarios concerning the emergence and loss of involvement of these genes in mesoderm formation and myogenesis across major lineages of the metazoan tree of life.

Material and methods
Animals and fixation. Adult Acanthochitona fascicularis specimens were collected in the intertidal region between the Station Biologique de Roscoff and the Île Verte in Roscoff, France (48° 43′ 44.70″ N 3° 59′ 13.53″ W). Adults and all developmental stages were maintained in glass dishes with filtered seawater at 18-21 °C. Spontaneous spawning of mature males and females generally occurred 1 to 3 days after collection. Gametes were inseminated by adding drops of sperm to the eggs. Upon the first observation of 2-cell stages (~ 80 min after fertilization), the embryos were washed multiple times with filtered sea water to prevent polyspermy and bacterial or fungal infection.
The gastrula stage was reached at around 8 h post fertilization (hpf). Trochophore larvae hatched from 18 hpf onwards. At 48-60 hpf, larvae reached the metamorphic competent stage (referred to as "late trochophore larva" herein). Early juveniles that had completed metamorphosis appeared between 60 and 90 hpf.
Signal development was stopped by washing the specimens twice for 5 min each in AP buffer and thrice for 10 min each in PBT. Then, the specimens were post-fixed in 4% PFA for 30 min each and subsequently washed twice for 5 min each and twice for 10 min each in PBT. Specimens were stored in 50% glycerol (Roth #3783.1) diluted in PBT. Prior to clearing, specimens were washed twice for 10 min each in an ascending DEPC series in PBT (20%, 40%, 60%, 80%, 100%) and afterwards twice for 10 min each in an ascending ethanol series in DEPC (20%, 40%, 60%, 80%, 100%). Specimens were mounted on glass slides and cleared in 2:1 benzyl benzoate:benzyl alcohol (Sigma-Aldrich #B9550 and #402834). Specimens were studied with an Olympus BX53 light microscope (Olympus, Tokyo, Japan) and images were taken with a DP73 camera (Olympus). Images were edited with Fiji 44 . Expression pattern schemes were designed with Inkscape (version 0.92.4; https:// inksc ape. org) and Gimp 2 (Version 2.8.22; https:// www. gimp. org).
Between 15 and 40 specimens per gene and developmental stage were investigated in detail for precise location of their expression domains. In almost all cases, 100% of the specimens showed identical expressions patterns. Exceptions to this are HES2 expression in the gastrula (consistent expression in 20 out of 25 specimens) and in the early larva (25 consistent patterns out of 35 specimens) as well as HES7 in the early larva (20 consistent expression domains out of 25 specimens). For HES7 expression experiments in the mid-trochophore stage only five specimens were available, all of which showed identical expression patterns.
Screening for genes of interest. The publicly available Acanthochitona fascicularis translated transcriptome 45 (erroneously assigned to as Acanthocithona crinita therein) was downloaded (https:// zoolo gy. univie. ac. at/ open-data/) and de-duplicated using cd-hit (Version 4.7), setting the sequence identity threshold to 0.95 46,47 . Mox and MHC sequences from other mollusks and lophotrochozoans were obtained from the NCBI GenBank database (https:// www. ncbi. nlm. nih. gov/) (Supplementary Tables 2, 3, 4) and were used for reciprocal similarity-based searches of the A. fascicularis database using the blastp tool (Version 2.8.1+) 48 with the e-value set to 1e − 6. Protein domain architecture of the resulting A. fascicularis candidate sequences was determined using the hmmscan algorithm against the Pfam A database (https:// pfam. xfam. org/). In the case of the HES genes, a hmm search (Version 3.1b2) 49 53 with the parameters -maxiterate set to 1000 and -localpair. Alignments were trimmed using BMGE (Version 1.12) 54 by setting the entropy-like value of the blosum matrix to -BLOSUM30, the maximum entropy threshold to 1, and the minimum length of selected regions to 1. The models for amino acid replacement were calculated using prottest (Version 2.1) 55,56 . All available matrices were included (-all-matrices) and models with rate variation among sites (-all-distributions) were included. The likelihood of the predicted models was assessed with the Akaike information criterion (-sort A) 57 . Selected amino acid substitution models were LG 58 for MHC and HES, and WAG 59 for Mox. Maximum likelihood trees and Bootstrap analyses (100 bootstraps, -b 100) were performed using phyml (Version 20120412) 60 . Tree topology (t), branch length (l), and rate parameters (r) were optimized (-o tlr). Visualisation and annotation of alignments was performed using aliview (Version 1.0.0.0; https:// ormbu nkar. se/ alivi ew/) 61

Identity of genes of interest. Myosin heavy chain (MHC). One AfaMHC ortholog was found in the
Acanthochitona fascicularis transcriptome 45 (see Suppl. Fig. 1A). The annotated AfaMHC sequence contains one myosin head domain and one myosin tail domain. A MHC-specific glycine (peptide sequence: idfGxdl) insertion within the myosin head domain 22 provides further confirmation of gene identity (Suppl. Fig. 1B). Phylogenetic analysis was performed with eight other members of the myosin superfamily that are commonly found in metazoans (Suppl. Fig. 1A). Myosin members which are specific to given taxa were not included in the analysis 24 . A bootstrap analysis with 100 bootstrap replicates was performed to provide statistical support. Myosin I is argued to be an ancient member of the myosin superfamily 24 and thus was used to root the tree. The annotated AfaMHC sequence clusters together with its respective metazoan orthologs. The MHC clade is well supported as are the clades of the other myosin family members.
Mox. In the Acanthochitona fascicularis transcriptome two Mox sequences, referred to as AfaMox1 and AfaMox2, were found. Mox genes possess a homeodomain with a glutamic acid site that is specific for Mox genes (Suppl. Fig. 2B). It shares a common origin with Evx, another homeotic gene. Mox and Evx together form the sister group to the Hox class genes 64 . Bootstrap analysis with 100 replicates supports the clustering of AfaMox1, AfaMox2, and AfaEvx with their orthologs (Suppl. Fig. 2A).
Hairy and enhancer of split (HES). Seven putative HES sequences, AfaHESC1 ("C" is for candidate), AfaHESC2, AfaHESC3, AfaHESC4, AfaHESC5, AfaHESC6, and AfaHESC7, were found in the Acanthochitona fascicularis transcriptome. HES proteins belong to the bHLH transcription factors and possess two domains, namely a bHLH domain that contains a HES gene-specific proline residue and a Hairy orange domain. In addition, they possess a HES-specific WRPW motif at their C-terminal end (Suppl. Fig. 3B). The phylogenetic analysis supports the monophyly of the identified HES sequences, which form a sister group relationship to Hey-class genes (Hairy and enhancer of split related with a YRPW motif, see Suppl. Fig. 3A). These possess the same two domains mentioned above, in addition to the tetrapeptide with a tyrosine instead of a tryptophan at the first position. Mox is expressed in the mesodermal bands and in a subset of the musculature. Of the two Mox sequences identified we were only able to produce expression data by in situ hybridisation for AfaMox1. Expression of this gene was first detected in the early trochophore larva ( Fig. 2A,B), where it is prominently expressed in the developing paired mesodermal band (Fig. 2C-F). In the late trochophore larva, AfaMox1 expression is confined to the ventrolateral muscle ( Fig. 2G-J). No Mox expression was detected in later stages of development.
HES genes are expressed in ectodermal and mesodermal domains. Two of the seven HES family genes identified (AfaHESC2 and AfaHESC7) yielded expression signals. Both genes start to be expressed in the late gastrula stage. Their expression is maintained in early larval stages but only AfaHESC2 is expressed in the late trochophore larva. In the gastrula, AfaHESC2 is expressed in ectodermal cells (Fig. 3A,B). In the early trochophore larva, AfaHESC2 is expressed in the mesodermal bands. A weaker expression domain extends from the anterior pole of the mesodermal bands into the apical region of the larva where it closes in an inverted U-shaped manner (Fig. 3C-F). In the late trochophore larva, AfaHESC2 expression is limited to the region of the adult buccal ganglion close to the dorsal ectoderm, where two spot-like expression domains are located ( Fig. 3G-J). Expression of AfaHESC7 first occurs in the prospective trochoblasts in the equatorial region of the gastrula (Fig. 4A,B). In the early larval stage, AfaHESC7 expression is restricted to a domain around the mouth (Fig. 4C,D). Throughout larval development, AfaHESC7 expression continues to be expressed around the mouth and in the region of the presumptive foregut. AfaHESC7 expression ceases in the late trochophore larva (Fig. 4E-H).

Myosin heavy chain: a conserved marker of metazoan myogenesis. Results from the cnidarian
Nematostella 25 suggest that myosin heavy chain (MHC) was already a key component of contractile cells in the last common ancestor of cnidarians and bilaterians. MHC has been used as a marker to study early muscle differentiation across lophotrochozoans [66][67][68] , ecdysozoans 18,69,70 , and deuterostomes 28,29,71 . Consistent with these data, MHC is expressed during the early formation of several muscle systems in Acanthochitona larvae, including the ventrolateral muscle, the enrolling muscle, and the rectus muscle. These results confirm the utility of MHC as a marker of early myogenesis in Mollusca, although further studies are needed to allow for a more detailed comparison of the initial stages and domains of muscle differentiation in this phylum. In deuterostomes, Mox expression typically begins around the time of gastrulation in early mesodermal precursors. In the hemichordate Saccoglossus kowalevskii, Mox (SkoMox) is expressed in the ventral mesoderm during formation of the paired coelomic cavities of the metasome 14 . Data are inconclusive as to whether or not SkoMox expression continues during subsequent development 14 . In the ascidian Ciona intestinalis, the Mox ortholog Meox (CinMox) is specifically expressed in muscle precursor cells in the early gastrula 77 and in the cephalochordate Branchiostoma floridae, BbeMox is expressed in the paraxial mesoderm during somite formation 13  www.nature.com/scientificreports/ dynamics and have been implicated in the early anterior-posterior patterning of the embryonic mesoderm as well as in somite specification and differentiation 32 . A reduction of limb muscle tissue in MmuMox2 null mice revealed the importance for muscle development 78 . A Mox mutation in zebrafish causes defects in bone development such as vertebral fusion, congenital scoliosis, and asymmetry of the pectoral girdle, providing evidence for the involvement of Mox in establishing mesodermal derivatives 79 . These data imply a conserved involvement of Mox in the initial specification of the deuterostome mesoderm and in the development of its derivatives. In the diverse Lophotrochozoa, Mox expression has only been studied in three species, namely the gastropod Haliotis asinina 72 , the brachiopod Terebratalia transversa 3 , and the polychaete Alitta virens 7 . For each of these, only one Mox gene has been described, while we found a second Mox sequence in the polyplacophoran Acanthochitona fascicularis. All four species start to express Mox shortly after gastrulation in lateral mesodermal bands that flank the endoderm. Accordingly, an early role for Mox in mesodermal band specification appears to be an ancestral feature of lophotrochozoans. During later stages, Mox continues to be expressed in the developing foot musculature in Haliotis 72 , in precursor cells of the future body wall and pharyngeal muscles of Alitta 7 , and in the ventrolateral muscle of late Acanthochitona trochophore larvae. Since we were not able to produce consistent expression results for AfaMox2, a putative role of this gene remains speculative. However, taken together, these data support a dual role of Mox in early mesoderm specification and in myogenesis, that is conserved among lophotrochozoans and deuterostomes. Notably, however, several lineage-specific evolutionary events have resulted in the loss of conserved roles and in co-option of Mox into novel ones. The Mox ortholog of the sea urchin Strongylocentrotus purpuratus, for example, is not expressed during mesoderm formation but in ectodermal neurons in the region of the larval apical organ 80 . This expression disappears in later stages, indicating that SpuMox plays a role in early neurogenesis rather than in mesoderm or muscle formation 80 . A similar situation is found in the fruit fly Drosophila melanogaster, where the Mox ortholog buttonless (DmeMox) is expressed in the dorsal median cells which derive from the ventral mesoderm and play a crucial role in axon guidance. Importantly, however, DmeMox is not expressed in muscle progenitors or muscular tissue 33 . In the second major   81 . Since other ecdysozoans and echinoderms are yet to be tested for Mox expression, a potential association between the loss of mesodermal Mox expression and the evolution of a neurogenesis-related role remains uncertain. In summary, the data currently available suggest that Mox was recruited into mesoderm formation in the last common bilaterian ancestor (LCBA) and may thus have played an important role in mesoderm evolution (Fig. 5). In addition, it appears that Mox was simultaneously recruited into myogenesis in the LCBA with loss of this role at least in Drosophila and putatively in both, myogenesis and mesoderm formation, in echinoderms (Fig. 5).
Variability of HES gene expression in metazoan development. HES genes are fast evolving genes that have undergone repeated species-specific, independent gene duplications 37 . The actual number of HES copies varies from one single sequence in the cnidarian Hydra 36 , the leech Helobdella 82 , the fly Drosophila 83 , and the sea urchin Strongylocentrotus 84 to up to 22 copies in the zebrafish Danio 37 . In Acanthochitona fascicularis, seven HES genes were identified, and two (AfaHESC2 and AfaHESC7) were further investigated here by in situ hybridization.
HES genes have been implicated in a wide range of developmental processes including neurogenesis as well as digestive tract and mesoderm formation. Thus, HES expression domains vary considerably between taxa. A comparative overview of the identified Mox, HES, and MHC genes and their respective expression domains across Metazoa is provided in Supplementary Table 5. In the sea anemone Nematostella, two HES genes, NveHES2 and NveHES3, are expressed in ectodermal cells of the gastrula, while NveHES3 expression expands to oral ecto-and endoderm in the planula larva 85 . In contrast, the single Hydra HES gene (HvuHES) is expressed during budding at the bud base shortly before separation from the mother animal, but was not detected in earlier stages 36 . In early embryos of the acoelomorph Symsagittifera roscoffensis, the only HES gene, SroHES, is expressed in the www.nature.com/scientificreports/ anterior-median region. In juveniles, it is expressed posterior to the statoblast, dorsally in the nerve cords, and mid-ventrally in the brain, but not in the mesoderm 86 . These data indicate that HES genes were initially involved in neurogenesis and in development of anterior ecto-and endodermal tissues and that their mesodermal expression might be a nephrozoan (or even bilaterian) novelty. Deuterostomes, such as the cephalochordate Branchiostoma, and vertebrates possess multiple HES genes that are broadly expressed across all germ layers. In Branchiostoma, four out of eight HES genes (BbeHESA-D) are expressed in the anterior endoderm, in the presumptive neural plate, and in the presomitic mesoderm of the mid-gastrula 87 . In neurula stages, expression is further found in the endoderm, in the neural tube, in the somites, as well as in the paraxial mesoderm, the foregut, the neural plate, and in the notochord 87 . In vertebrates (mouse, chicken, and Xenopus), HES genes also play a crucial role during somitogenesis, gut formation, neurogenesis, as well as in the maintenance of stem cell potential and separation of different brain areas from each other 35,88,89 . A functional study employing HES gene knockdown in Xenopus laevis resulted in a decrease of cell proliferation. This indicates anti-apoptotic functions and highlights the ability for transcriptional repression of HES genes 89 . In the sea urchin Strongylocentrotus on the other hand, no mesodermal expression of HES was observed 84 . Instead, HES is expressed from blastula to gastrula stages in the oral ectoderm and (weakly) in the archenteron 84 . This is consistent with data on SpuMox that, in contrast to Mox genes of other deuterostomes, is also absent from the mesoderm and is exclusively expressed in ectodermal neurons in the sea urchin 80 .
Interestingly, Mox and HES genes also seem to be of relatively little importance for mesoderm development and myogenesis in ecdysozoans 33 90 . In later stages, DmeHES is also expressed in the nervous system, the foregut, the tracheal primordia and surrounding mesoderm, as well as in somatic and visceral muscles 92,93 . However, DmeHES does not seem to affect myogenesis, but rather contributes to tracheal development 93 .
Lophotrochozoan HES gene expression is highly species-specific and has been described across all germ layers. The single planarian HES gene is exclusively expressed in neuronal progenitor cells 94 , whereas the three and 13 HES genes of the annelids Platynereis and Capitella, respectively, are expressed across various body regions, including the growth zone, the chaetae, the nervous system, and the digestive tract 19,37 . In the brachiopod Terebratalia, TtrHES1 is only transiently expressed in the lateral ectoderm of the gastrula 21 , while TtrHES2 is expressed in the mesoderm and in the developing chaete but not during formation of the gut 21 . This is similar to the gastropod Crepidula, where two HES genes, HESA (CfoHES1) and HESB (CfoHES2), are predominantly expressed in ectodermal cells around the blastopore (CfoHES2) and mouth (CfoHES1). In addition, CfoHES2 is expressed in ventral neurosensory cells and, during further development, in the anlage of the foot 38 . In contrast to both, the brachiopod and the gastropod, HES gene expression was absent during myogenesis in the  www.nature.com/scientificreports/ polyplacophoran Acanthocitona. A potential reason for this is that only two out of seven HES genes were detectable by in situ hybridization during Acanthochitona ontogeny. While in-situ hybridization sensitivity is high, it is possible that one or more of the remaining five HES genes are indeed involved in myogenesis but did not meet the minimum expression threshold required for detection. Although we were unable to unequivocally assign AfaHESC2 expression to distinct morphological features, it is briefly expressed in the mesodermal bands and later appears to overlap with the region of the developing buccal ganglia 95 . AfaHESC7 expression was observed in the oral ectoderm, around the foregut, and, surprisingly, in the trochoblasts. The latter are specialized founder cells that give rise to the ciliated cells of the prototroch and have so far not been reported to express a HES gene in any other lophotrochozoan. Taken together, these data show that mesodermal and muscular HES gene expression is likely an ancestral feature of bilaterians that was lost in multiple lineages including echinoderms, nematodes, planarians, and possibly also acoelomorphs and polyplacophoran mollusks. Involvement in endoderm specification, on the other hand, likely emerged in the last common ancestor of Metazoa and was also lost in several lineages, such as acoelomorphs, nematodes, planarians, and brachiopods. Altogether, ectodermal and/or neural HES gene expression appears to be particularly well conserved across metazoans. Since HES genes chiefly act in separating tissues from each other that are destined to undergo fate determination ("territorialisation"), they have been co-opted into various additional developmental processes, such as the formation of the chaete in annelids and brachiopods, segmentation in annelids and arthropods, somitogenesis in chordates, and budding in cnidarians. This enormous variability highlights their importance for the evolution of distinct ontogenetic pathways throughout the animal kingdom (Fig. 6, Supplementary Table 5). Figure 6. HES gene expression in metazoan organogenesis. Lophotrochozoa: Mollusca: Expression is in ectodermal cells of pre-larval stages and subsequently during mesoderm formation as well as in neurogenesis and development of the digestive tract. Annelida: Expression is during formation of the digestive tract, neurogenesis, segmentation, and chaetogenesis. Brachiopoda: Expression is in the ectoderm of pre-larval stages, during early mesoderm formation, and in chaetogenesis. Ecdysozoa: Hexapoda: Expression is during segment formation. Nematoda: Expression is during neurogenesis. Deuterostomia: Echinodermata: Expression is in the larval ectoderm. In the late pluteus larva, HES expression is in the region of the apical organ. Chordata: Expression is during neurogenesis, somitogenesis, and in the digestive tract. Xenacoelomorpha: Expression is during neurogenesis. Cnidaria: Expression is in the ectoderm and endoderm of early developmental stages and during budding in hydrozoans. Data from previous investigations 19

Conclusion
The present study shows that Mox and HES genes are expressed during mesoderm formation in the mollusk Acanthochitona fascicularis. Expression of Mox in the mesodermal bands and in their major derivatives, the muscles, is congruent with the situation in other lophotrochozoans, suggesting a dual role of this gene in the last common bilaterian ancestor. Mox experienced loss in myogenesis in ecdysozoans and loss in both myogenesis and mesoderm formation in echinoderms, where it is instead expressed in the ectoderm. Expression of HES occurs during early mesoderm development, neurogenesis, and digestive tract formation in a number of bilaterians as well as in ectodermal and endodermal domains in cnidarians, implying either a wide variety of roles already at the dawn of bilaterian evolution or a particularly high degree of variability (co-option) of HES genes with various independent gain-of-function events along individual bilaterian lineages.