Insights & Perspectives

Did the evolve from an

Hypotheses ancient axial muscle? The axochord hypothesis

Thibaut Brunet, Antonella Lauri and Detlev Arendt*

The origin of the notochord is one of the key remaining mysteries of our Regarding notochord evolution, we can evolutionary ancestry. Here, we present a multi-level comparison of the thus ask: what structure in the last notochord to the axochord, a paired axial muscle spanning the ventral common bilaterian ancestor gave rise to the chordate notochord? This structure midline of worms and other . At the cellular level, necessarily existed, but its nature and comparative molecular profiling in the marine P. dumerilii and C. teleta complexity – representing a simple reveals expression of similar, specific gene sets in presumptive axochordal and population of cells, a certain tissue, or notochordal cells. These cells also occupy corresponding positions in a even a distinct organ such as a specific conserved anatomical topology and undergo similar morphogenetic move- muscle – remain to be defined. Which structures in non-chordate lineages has ments. At the organ level, a detailed comparison of bilaterian musculatures it given rise to? The answers to these reveals that most phyla form axochord-like muscles, suggesting that such a questions are currently unclear [2, 3]. muscle was already present in urbilaterian ancestors. Integrating comparative The notochord has variously been pro- evidence at the cell and organ level, we propose that the notochord evolved by posed to be related to the stomochord of modification of a ventromedian muscle followed by the assembly of an axial enteropneusts [4, 5]; to the hydrocele of complex supporting swimming in ancestors. [6]; to a longitudinal stiff- ening ofthegut [7, 8];and, by one author, to a ventral midline muscle in annelid Keywords: worms [9]. However, none of these axochord; evo-devo; evolution; mesoderm; musculature; notochord; urbilateria . homology proposals has gained wide- Additional supporting information may be found in the online version of this spread acceptance. : article at the publisher’s web-site. How can homology of two structures be experimentally tested? The nature of possible supporting evidence is sum- marized by Remane’s triple homology Introduction and comparison of homologous struc- criteria [10, 11]: (i) specific quality: tures in modern forms,allowing inference similarity in structural detail [12]; (ii) The reconstruction of last common ances- of the most likely ancestral states. Homo- position: they should have the same tors of modern groups is one of the key logy refers to structures in two modern relative position within the body; (iii) challenges in evolutionary . It is that have been inherited from continuity: they should be present in possible by two methods: observation of their last common ancestor. It applies phylogenetically intermediate groups. (which, for early bilaterian evolu- at all levels of biological organization – Later, Hennig, building on these cri- tion, represent a still patchy record [1]), genes, cell types, tissues, and organs. teria [12], emphasized their importance as pre-requisites for homology (as the DOI 10.1002/bies.201500027 school did after him [13]). He extended the continuity criterion Developmental Biology Unit, European Molecular † Present address: Institute for Biological and by emphasizing the need to test for Biology Laboratory, Heidelberg, Germany Medical Imaging and Institute of Developmental absence or presence of a character along Genetics, Helmholtz Zentrum Munchen,€ Neuher- the branches of a to *Corresponding author: berg, Germany. Detlev Arendt infer ancestral states. According to the E-mail: [email protected] cladistic approach, a character is only

836 www.bioessays-journal.com Bioessays 37: 836–850, ß 2015 The Authors. Bioessays published by WILEY Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited...... Insights & Perspectives T. Brunet et al. Hypotheses

Figure 1. The bilaterian phylogenetic tree, after [15]. Levels of homology (cells and organs) are parsimoniousinterpretationaccordingto indicated, together with the taxa for which evidence is available as well as the nature of that Hennig’s cladistic approach would be evidence. The three branches known to separate annelids and are colored. The that it existed in bilaterian ancestors. As axochord hypothesis implies conservation of a ventromedian muscle along at least these three we will outline in the second part of this branches, and possibly other branches within once their phylogeny is solved. review, these comparative data make a strong case that such a muscle was considered homologous if its distributed cell type level, hence suggesting that indeed present. Future developmental presence in a clade supports its likely the notochordal cells might have arisen and genetic studies will reveal its cellular existence in the last common ancestor. from ancient contractile cells in the characteristics and will allow further (This method is called “ancestral state ventral midline. For obvious reasons, testing of the continuity criterion. reconstruction,” see below.) such detailed developmental and We recently published a detailed molecular investigations have so far comparison of cell types between only covered few species, and more remote groups: those that assemble into species need to be examined to test for Are the axochord and the a ventromedian muscle in the annelid presence/absence (iii) of these genetic notochord homologous? worms Platynereis dumerilii and Cap- and developmental traits in the bilat- itella teleta and those that form the erian tree. Molecular profiling and notochord in chordates [14]. We found However, continuity can already be developmental data support strong similarities between these cells tested at the tissue/organ level, as a vast cell type-level homology of in terms of gene expression, morpho- repertoire of anatomical data is available axochordal and notochordal genetic movements, and position in the to test for the presence of ventromedian cells in annelids and chordates bodyplan. Following the criteria of muscles in various bilaterians (Fig. 1). If a structural similarity (i) and topology ventromedian muscle were present in the Similar to the notochord, the P. dumer- (ii), our data suggest homology at the majority of bilaterian lineages, the most ilii axochord develops by convergence-

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Figure 2. The axochord in annelids. A: Development of the axochord in Platynereis dumerilii following [14]. Red cells are axochordal cells; green cells are presumptive ventral oblique muscles; cells give rise to the rest of the mesoderm; foregut is in ; dotted circle is the mouth. B: Development of the axochord in Capitella teleta following [14]. C: Ancestral state reconstruction for annelids. The tree follows [22, 23]. Only one group of known phylogenetic position, sipunculids, lacks an axochord (the other family, Sphaerodoridae, has not been included in any phylogenomic analysis).

extension of mesodermal cells towards transcription factors (brachyury, foxA, investigated in amphioxus are also the midline [14] (Fig. 2A). These cells foxD, twist, soxD, soxE) and eight general notochord markers (apart from differentiate into a rod of tissue located effector genes (colA1, colA2, chordin, soxE). Since this combined signature is between the central nervous system noggin, netrin, slit and hedgehog) that found nowhere else in the body, its co- and the axial blood vessel, serve as uniquely defines it, and also uniquely option from another expression territory an attachment band for transverse define the vertebrate notochord. is unlikely: its convergent acquisition muscles, and likely secrete a collagen- Together, these 13 genes represent the would require multiple, independent rich extracellular matrix (as suggested most complete and most evolutionarily and identical co-option events. This is by the expression of the genes colA1 and stable molecular profile for notochordal unparsimonious, since the known colA2). These histological, morphoge- cells that can be put forward after an instances of convergent cell type evo- netic and positional properties are unbiased screening of the vertebrate lution have involved the independent reminiscent of those of the chordate literature – thus avoiding arbitrary production of similar cellular pheno- notochord [16]. Moreover, the axochord “cherry-picking” of markers [17]. All types by completely different molecular expresses a specific combination of six the genes of this list that have been components [18, 19].

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It is worth noting that the molecular histological data is challenging, as converge in late development and form profile of the axochord includes a they are often of insufficient resolution a proper axochord before hatching [14] notochord-like combination of signal- to observe axochord-like structures, (Fig. 2B). Axochord development thus Hypotheses ing molecules (noggin, hedgehog, which are frequently of minute size. underwent a heterochronic shift netrin, and slit), suggesting that the Facilitating our search, however, a between Platynereis and Capitella:in annelid axochord, like the notochord, large dataset of phalloidin stainings Capitella, axochordal cells first form functions as a signaling center. This covering virtually all bilaterian phyla differentiated myofibers and then con- hypothesis, however, still awaits direct has been produced in the last 20 years, verge, while in Platynereis those events functional testing. allowing widespread testing for the happen in the opposite order presence of axochord-like structures in (Fig. 2A and B). Gene expression data bilaterians. for all axochord markers investigated Bridging the phylogenetic gap (brachyury, foxA, netrin, slit, hedgehog, between annelids and and twist2) are consistent with expres- chordates: A cladistic approach The axochord is conserved sion in the Capitella axochord [14, 28, 29]. across annelids The Capitella data thus confirm conser- Obviously, our cellular comparison is vation of at least part of the axochord/ powerful with regard to the two first The first implication of our hypothesis is notochord molecular signature within homology criteria (position and specific that the axochord must be an ancestral annelids, and provide a possible mech- quality). However, these in-depth data annelid feature. Annelids are a highly anism for the evolutionary transition covered only two annelid genera (Pla- diverse group, for which the internal between paramedian and ventromedian tynereis and Capitella) – thus leaving phylogeny has been recently clarified by configurations. Finally, in a subgroup of unsolved whether the third criterion of phylogenomics [22, 23], making it an Sedentaria (, which include continuity (i.e. presence in intermediate ideal test case. Phalloidin stainings earthworms andleeches), theentire body groups), is satisfied. Indeed, it has been have been published for 14 families, is surrounded by a continuous longitu- pointed out that our comparison leaves covering both main annelid clades dinal muscle layer [30], complicating out a large number of intermediate (Errantia and Sedentaria) and two observations. However, in earthworms branches [20]. In fact, our current families that likely diverged earlier and , a distinct ventromedian knowledge of the bilaterian phylogeny (Oweniidae [14, 24] and Mageloni- longitudinal muscle (called “epineural implies that, if such cells were present dae [25]). Axochord-like ventromedian muscle” or “capsular muscle” [31, 32]) is in the last common annelid/chordate muscles have been observed in virtually present immediately above the ventral ancestor, they should also have been all of them, and usually serve as nerve cord and below the ventral blood present in the ancestors of ambulacrar- attachment bands for transverse vessel – thus representing a bona fide ians (branch 1), chaetognaths (branch muscles. Axochords are always com- axochord. Like the Platynereis axochord, 2), and ecdysozoans (branch 3), plus a posed of a pair of longitudinal myofibers the epineural muscle is firmly embedded yet unknown number of branches in the closely flanking the midline, which within the ventral nerve cord sheath. lophotrochozoan stem-line [21] (Fig. 1). contact each other in the main part of Its contractions are thought to allow Since (with the exception of the highly the trunk, but diverge at their anterior deformation of the nerve cord in con- specialized fruit flies and ) and posterior extremities (behind the cert with body shape changes during not much molecular and developmental mouth and in front of the anus). The peristaltic motion. Molecular data on data are available for these lineages, this degree of terminal divergence is modest clitellates are scarce, but the ventrome- hypothesis remains to be tested at the cell in Platynereis and most other genera, dian myofibers of leeches have been type level. Fortunately and interestingly, but more extensive in Pomatoceros [26]. reported to express the specific inter- at the organ level, there is a rich body of In Prionospio, both myofibers closely mediate filament-encoding gene hif-3, comparative anatomical literature cover- flank the midline, but do not actually which is absent from lateral longitudi- ing virtually all bilaterian phyla. As will touch each other; in this configuration, nal muscles [33]. be outlined below, these comparative the corresponding muscle has been Only two annelid families clearly data support the ancestral presence of a called “paramedian muscle” [25]. lack an axochord: Sphaerodoridae [34] ventromedian muscle in bilaterians. We hypothesized that the parame- and Sipunculidae [35]. Future comparative studies will unravel dian configuration can be developmen- The most parsimonious ancestral how this ancient ventromedian muscle tally explained by incomplete state for annelids is the presence of a was genetically specified and how it convergence toward the midline of canonical axochord, composed of two developed. axochord-like precursor cells during adjacent longitudinal myofibers flank- early development. We tested this ing the midline, with attached trans- hypothesis by studying an annelid verse muscles (Fig. 2C). Importantly, known to possess such a paramedian conservation of a stereotypical axo- Testing the presence of an muscle at early larval stages [27]: C. chord is compatible with the huge axochord across teleta, a model species belonging to variety of annelid lifestyles and mor- Sedentaria [22]. Phalloidin stainings phologies, including sessile suspen- Assessing the presence of a ventrome- revealed that the previously docu- sion-feeders, errant bottom-dwellers, dian muscles from classical morpho- mented paramedian muscle fibers burrowers, and undulatory swimmers.

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Conclusions about other phyla face two been described in the larvae of Aplaca- sides, are not considered here, as their main limitations: for most, the internal phora (Wirenia argentea) and Polypla- strongly modified bodyplan precludes phylogeny is still under debate (apart cophora (Leptochiton asellus and comparisons. from annelids, molluscs, , Mopalia muscosa) [40] (Fig. 3A), where and chordates), and the interrelation- it serves as an attachment point for Axochords have a mosaic presence in ships of the phyla themselves (i.e. the transverse muscles. Together, Aplaco- gnathiferans (, higher-order bilaterian phylogeny) phora and Monoplacophora form a and micrognathozoans) and remain partially unresolved. While clade considered the sister-group of all rouphozoans (platyhelminthes and “Chordata,” “,” “Ecdyso- other molluscs [41, 42]. The ventrome- gastrotrichs) zoa,” and “” seem stable, their dian and transverse muscles exist only internal branching is more contentious. transitorily during mollusc develop- In interstitial phyla, paired myofibers Hypotheses Moreover, the bilaterian phylogeny is ment, and have been speculated to closely flanking the ventral midline strongly dichotomous: the general represent recapitulative instances of have also been reported – for example, structure of the phylogenetic ancient structures [40]. in the trunk of the rotifers Proales tree seems closer to successive sym- In , ventromedian myo- daphnicola [49] and Brachionus urceo- metrical bifurcations between equally fibers have been detected in the ventral laris [50], of the gastotrich Xenotrichula large groups, than to successive branch- midline of the early three-lobed larvae intermedia [51], and of the gnathosto- ing of individual phyla from one stem – of Argyrotheca and Terebratalia ([43] mulid Gnathostomula peregrina [52] hence producing a “balanced” or “sym- and Dr. Andreas Altenburger, personal (Fig. 3B). As in annelids, they diverge metrical” phylogenetic tree [36, 37]. In communication; Fig. 3A and Supp. anteriorly and posteriorly, and some such a tree, there are no strategically Fig. S1). Conserved expression of the other species display the same fibers located “basal” branches that would annelid axochord markers mox, foxD, in a more divergent, paramedian carry higher weight on the inferred and noggin has been reported in a stripe configuration. Variable degrees of ancestral states at key nodes, and of ventromedian mesoderm in Terebra- convergence (ranging all the way from conclusions can only be reached after talia, suggesting conservation of the ventromedian to paramedian) can examination of a broad sample. With axochord molecular profile between coexist within the same – for these caveats in mind, a survey of the annelids and brachiopods [44]. At very example, Proales () [49], or available data allows some insights into late larval stages, only faint phalloidin Xenotrichula () [51]. Despite musculature evolution and the possible stainings are visible in the ventral these differences, this muscle has ancestrality of ventromedian muscles. midline [44, 45], which suggests the been recognized as clearly being the Terebratalia axochord might grow at a same under both configurations (from smaller rate (or regress) compared to its position, general and The axochord is conserved other ventral muscles; its earlier pres- connections) in the descriptions of across non-annelid spiralians ence is however unambiguous ([43] and these genera. This suggests that, as Fig. S1). in annelids, the transition between Annelids are part of the superphylum In nemerteans, a ventromedian ventromedian and paramedian Spiralia, which includes both large muscle (without transverse fibers) is muscles is easily achieved by complete coelomate and small acoelomate among the first muscles to form in the versus partial convergence processes. groups of the interstitial fauna embryo of Prosorhochmus [46] (Fig. 3A). The adaptive significance for these (or “,” which are likely not One acoelomate has been varying degrees of convergence is monophyletic [21]) [38]. One additional tentatively assigned to Lophotrocho- unclear. microscopic phylum, Cycliophora, exclu- zoa [15]: the minute cycliophorans. In In maerski, the only sively as a commensal on the this group, planktonic larvae possess species of the small gnathiferan phyla mouthparts of [39]. Strikingly, a hugely expanded and vacuolized Micrognathozoa, transverse muscles axochord-like ventromedian muscles ventromedian muscle: the “chordoid are attached to a paramedian muscle, have been described in both coelomate organ” [39, 47, 48] (Fig. 3B). Its function which itself is attached to the posterior and acoelomate spiralians. For ancestral in a that moves primarily by ciliary border of the [53] (Fig. 3B) – state reconstruction, we will use a recent beating is unclear: its role might be hence displaying connection properties phylogeny of Spiralia [21], which pro- maintaining body shape (as both the similar to the ventromedian/parame- poses that this clade is composed of three axochord and the notochord do [14]), dian muscles of other gnathifers and of monophyletic groups: Lophotrochozoa and in particular bracing the midline annelids. We hypothesize here that (containing all coelomate spiralians), when ventrolateral muscles contract there is homology between those mid- and two acoelomate groups: Rouphozoa during turning. The chordoid organ line-flanking paired longitudinal and . cells contain circular myofilaments, muscles across Spiralia: while varying organized as “ring fibers” surrounding degrees of convergence can result in An axochord is present in molluscs, the vacuoles. The peculiar orientation of slightly different morphologies, their brachiopods and nemerteans these fibers might be a consequence of connection properties are conserved, vacuolization (see below). as their molecular profiles should be – In molluscs, a ventromedian muscle Ectoprocts and entoprocts, which allowing eventual testing of this composed of adjacent paired fibers has lack unambiguous dorsal and ventral hypothesis by expression profiling.

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Figure 3. The axochord in spiralians. The tree follows [21]. A: . Annelid after [14], larval axochord might persist by sheer molluscs after [40], after [43] (and personal communication of Dr. Andreas phylogenetic inertia, or it might still Altenburger), nemertean after [46]. B: Cycliophora, Rouphozoa and Gnathifera. Cycliophoran fulfill transient function, such as larval after [39], gastrotrich after [54], rotifer after [49] (note that the schematic in this paper locomotion or signaling. presents the paired ventromedian fibers slightly more distant than they are in the actual specimen; Dr. Martin V. Sørensen, personal communication), after [52], micrognathozoan after [53]. are as in Fig. 2A, and the paramedian muscle of micrognathozoans is orange. Is an axochord conserved in ? The axochord likely represents an evolutionary transitions from ancestral ancestral spiralian feature muscular systems based on antagonism The internal ecdysozoan phylogeny is between ventromedian, transverse, still unclear [48–50, 56, 57]. Three An annelid-like axochord has been and ventrolateral myofibers (possibly frequently proposed clades are Panar- reported for the majority of spiralian already surrounded by a circular thropoda (onychophorans, , phyla, and usually serves as an attach- layer [55]), to worm-shaped peristaltic and arthropods), (pria- ment band for repeated transverse forms relying exclusively on continuous pulids, kinorhynchs, and loriciferans), muscles. The fact that the axochord is longitudinal and circular layers (e.g. the and (nematodes and nem- a sometimes transient feature of early adults of some large nemerteans), or to atomorphs), and we follow this view development supports its ancestral sessile lophophorate forms (e.g. adult here. Ecdysozoans are defined by presence in Spiralia and argues for brachiopods). A temporary embryonic/ the shared presence of a moulting

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Figure 4. The axochord in ecdysozoans and . A: Ecdysozoans. Loriciferan muscles exist in the kinorhynch Anty- after [60], kinorhynch after [59]. Onychophoran reconstituted after [62]. B: Deuterostomes gomonas [59] and in the Higgins larva and chaetognaths. Chaetognath after [14]. reconstituted after after [77]. Colors of the loriciferan Armorloricus [60] are as in Fig. 2A. (Fig. 4A). Adult priapulids rely on antagonism between continuous longi- exoskeleton [58] which, in several of ecdysozoan musculature can be tudinal and circular layers around phyla, shows a tendency to become proposed. the body, as typical for burrowing increasingly rigid and to replace worms [30], and the musculature of muscles as supporting structures or Ventromedian muscles are present in embryonic/larval priapulids is still as antagonists. Some degree of Scalidophora (kinorhynchs and incompletely known (but see Ref. [61] repeated muscle loss would thus be loriciferans) for a recent description of the Priapulus unsurprising in ecdysozoans. Never- caudatus larval musculature with a theless, some phyla have an axochord, Paired ventromedian muscles serving as mention of a ventromedian retractor and hypotheses on the evolution an attachment band for transverse muscle in the first lorica larva).

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Ventromedian muscles are present in components with axochord and noto- The axochord has most likely been lost onychophorans but not in tardigrades chord (laminin) but also some that are in Nematoida (nematodes and not (collagen IV and the - nematomorphs) Hypotheses Onychophorans have a hugely devel- specific protein glutactin). Finally, the oped ventromedian muscle [62] key defining transcription factor of the Nematoids have a near-continuous lon- (Fig. 4A) – which probably acts in DM cells, the homeodomain protein gitudinal muscle layer surrounding the bracing the body, and notably in mox/buttonless [69], is also expressed body. Unlike the clitellate configura- preventing deformation of the ventral in the Platynereis axochord (but not in tion, this longitudinal muscle layer lies side (housing the ventral nerve cord) the notochord) [14]. internal to the ventral nerve cord during hydrostatic expansion/retrac- However, a number of key differ- (though the nervous ganglia seconda- tion of lateral appendages. Unlike the ences cast doubt on the homology of the rily “sink” below the muscle layer by annelid axochord, the onychophoran mesodermal midline glia to the axo- crossing it during nematomorph devel- ventromedian muscle is not an attach- chord: 1) DM cells, which have an opment [74]). Conservation of the axo- ment band for transverse muscles: elongated monopolar shape, extend chord molecular profile in nematoids is onychophoran appendage muscles long lateral processes in a transverse, unlikely, because, at least in the model attach to the tegument, on a structure rather than longitudinal, direction [68, C. elegans, several key called the ventral organ [63]. This 69] 2) DM cells coexpress paraxis axochord/notochord genes (including argues against homology of onycho- (CG12648/CG33557) [72] and engrailed brachyury, colA,andsoxE) have simply phoran appendicular muscles to the [73], which together represent a specific been lost from the genome. This makes it annelid ventral oblique muscles moving profile for the annelid ventral oblique difficult to identify any potential axo- the parapodia – consistent with the muscles – which also express mox and chord homolog, which might either have common assumption that Urbilateria netrin [14]. On the other hand, DM cells been lost or modified beyond recogni- lacked trunk appendages [64], and that express none of the specific axochordal/ tion. Ventral longitudinal muscles of C. different muscles might have been co- notochordal transcription factors (such elegans still specifically express unc130/ opted or neoformed for appendage as brachyury and foxA). 3) The lateral foxD [75] and netrin [76] (two ventral movement in different phyla. No ven- processes of DM cells are anchored at somatic muscle markers in Platynereis), tromedian muscle is known in the the attachment point of the lateral but not foxA (PHA-4) – showing that, trunk (though a minute one longitudinal muscles on the body wall while some general musculature pattern- is present in the foregut [65, 66]). (muscle 7) [68]. These connection prop- ing is recognizable in nematodes, a erties are expected if they are equivalent specific axochord homolog cannot be The special case of the arthropod to transverse muscles, which in annel- identified. The axochord might thus have mesodermal midline glia: a modified ids reach out to the ventrolateral been lost, together with transverse axochord? longitudinal muscles (Fig. 2A) – but muscles, in conjunction with the evolu- have nothing to do with those of tion of the specialized nematoid locomo- In line with the evolution of a scle- ventromedian myocytes. tion, relying on the antagonism between rotized cuticle as a supporting scaffold, By their molecular profile, orienta- ventro- and dorsolateral muscle blocks arthropods have been proposed to tion and muscular connections, DM and an elastic cuticle [30]. have undergone a massive reduction cells are more similar to annelid ventral of their ancestral onychophoran-like oblique muscles than to the axochord, The axochord has a mosaic presence in circular/longitudinal musculature, that and it can be hypothesized that they are ecdysozoans lost its ancestral bracing function [67]. modified transverse muscles. In this Any ventromedian muscle (absent hypothesis, if a ventromedian muscle Of the three ecdysozoan clades, only from all investigated arthropods) was ancestrally present in panarthro- scalidophorans can be inferred to would plausibly have been lost in this pods (as suggested by the onycho- ancestrally possess an axochord. The process. phoran situation), it would have been panarthropod ancestral state is unde- However, insects do possess a non- entirely lost in Drosophila,andformer termined: only onychophorans have a muscular mesodermal midline: the so- transverse myocytes would have come clear ventromedian muscle. Finally, called “mesodermal midline glia” or to occupy the vacant mesodermal mid- nematoids possess a simplified muscu- “DM cells” (“dorsal median,” as they are line. This homology hypothesis is test- lature – and, at least in C. elegans,a positioned immediately dorsally to the able in several ways: while, as noted simplified genome. The ancestral state central nervous system). At first sight, above, onychophorans lack annelid- for ecdysozoans therefore remains the Drosophila mesodermal midline glia like transverse muscles, they might undecided. However, the clear presence seem to display a number of similarities possess mox/en/netrin/paraxisþ DM- of an axochord in at least three to the axochord: they are present under like cells – which should attach both ecdysozoan phyla – and its inferred the form of segmentally repeated pairs to the ventromedian muscle (lost in ancestral presence in the outgroups of cells immediately below the ventral Drosophila) and to the lateral longitu- Spiralia and (see below) nerve cord [68]; are required for com- dinal muscles (as in Drosophila); the – make the hypothesis of an ancestral missural axon guidance [69], express transverse muscles of kinorhynchs ecdysozoan axochord attractive. To netrin [70], and are specialized in matrix would be interesting to investigate in this ground pattern, scalidophoran-like secretion [71] – including some common this respect. transverse muscles might be added.

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Molecular characterization of ecdyso- and , plus potentially The enteropneust trunk is almost entirely zoan ventral mesodermal cells will be xenacoelomorphs), and Chordata. surrounded by longitudinal myofibers. key in testing these hypotheses. While no enteropneust phalloidin stain- ing has been published (apart from the Ventromedian muscles have a interstitial species Meioglossus [93]), his- A ventromedian muscle is mosaic presence in tological data indicate the existence of present in Chaetognatha, a Ambulacraria (hemichordates conspicuous paired ventromedian fibers possible outgroup and echinoderms) connected to the ventral mesentery in Protoglossus (Fig. 4B) [77, 94], and of Chaetognatha is a relatively small (but Hemichordates include the worm-like smallersimilar fibersinSaccoglossus([95] very abundant) phylum (120 species) of enteropneusts and the sessile suspen- and Dr. Sabrina Kaul-Strehlow, personal Hypotheses worm-shaped swimming invertebrates, sion-feeding pterobranchs. Enterop- communication) – so small that they are which might have diverged before all neusts might be paraphyletic [82], or usually omitted from classical sche- other [78, 79]. The chaetog- both groups might be monophyletic [82, matics [96]. In Protoglossus,theydiverge nath body comprises pairs of coelomic 83]. Most authors have argued so far for behind the foregut [77]. However, the cavities surrounding the gut, separated the enteropneust morphology being ventromedian fibers display no reported along the midline by myoepithelial dorsal closer to the ancestral situation [84, morpho-anatomical feature that would and ventral mesenteries [80]. The body is 85], and we follow this view here. The readily distinguish them from ventro- almost entirely surrounded by strong most popular candidate for a notochord laterallongitudinalmuscles.Molecular longitudinal striated muscles. Flanking homolog in enteropneusts has histor- individuality might nevertheless exist, as the ventral midline, directly connected to ically been the stomochord, an anterior suggested by the specific expression of the ventral mesentery, are specialized vacuolated expansion of the pharynx the transcription factors mox and foxD in longitudinal myofibers of triangular into the proboscis [4, 5]. Its very anterior the ventral-most mesodermal cells of the cross-section, which present a unique and dorsal position argues against any developing Saccoglossus –thelocation type of striation, and are hence called affinity to the notochord; moreover, it from which the ventromedian myofibers “secondary muscles” [81]. This distin- lacks expression of key notochord/ should originate [92, 97]. Again, more guishes them from all neighboring ven- axochord markers such as brachyury, developmentalandmolecularstudieswill trolateral longitudinal muscles, as this foxA,andnoggin [86, 87] – but it does be needed to assess further the potential peculiar striation type is only present in express colA, in line with its structural existence of a hemichordate axochord two other locations in the body (laterally role [88].While the stomochord underlies homolog. and in the dorsal midline). Their nature, theinvaginating neuralcord of thecollar, Echinoderms display a highly modi- orientation, triangular shape and con- thus displaying morphological similar- fied adult bodyplan [30], and their early nection to the ventral mesentery are ities to the chordate notochord/neural larvae only possess visceral muscles reminiscent of the axochord. Moreover, tube complex [89], and expresses hedge- [98]. Electron microscopy has provided like the axochord, the chaetognath ven- hog [88], the bulk of molecular and hints to the presence of a more complex tromedian longitudinal musclebifurcates anatomical data argue against its homol- somatic muscle system in late starfish behind the foregut [14] (Fig. 4B). The ogy to the notochord. Expression of nk2.1 bippinaria larvae [99], but this system chaetognath ventromedian muscle might and foxE suggests instead affinities to still awaits characterization by phalloi- thus be an axochord homolog. part ofthe chordateforegut – possiblythe din stainings. With the data at hand, it is No transverse muscles are known. endostyle/thyroid [87, 90]. reasonable to assume that the ventro- The weak circular smooth fibers of myoe- Since morphological [91] and molec- median mesoderm has most likely been pithelial cells within the mesenteries ular data [92] suggest that hemichor- lost in echinoderms, or modified beyond provide some limited antagonism to dates, unlike chordates, are not recognition. longitudinal muscles, but they are incom- dorsoventrally inverted compared to parable in nature and position to the protostomes, a genuine notochord transverse muscles of other proto- homolog should be looked for in the What is the origin of the stomes [80]. Transverse fibers might have ventral trunk. Such a candidate struc- chordate notochord and been lost during the evolution of the ture is the pygochord, a vacuolated backbone? highly specialized chaetognath undula- thickening of the ventral mesentery of tory swimming, which is effected by dorso- ptychoderid enteropneusts. However, Did an axochord evolve into the ventral antagonism; alternatively, they the pygochord is located between the notochord? might have evolved only after chaetog- ventral blood vessel and the gut – naths branched off the protostome stem. unlike the axochord and the notochord, Finally, we propose that dorsoventral which are positioned between the axial inversion in the stem lineage of chor- blood vessel and the central nervous dates put the former axochord in a Is the axochord an ancient system. The best candidate for a noto- dorsal position. The muscular noto- feature? chord/axochord homolog would be a chord of amphioxus [100, 101] would striated paired ventromedian muscle represent a clue to this transition The deuterostome tree is a bifurcation between the ventral nerve cord and (Fig. 4B). Evolution of the notochord between Ambulacraria (echinoderms the ventral blood vessel. from a paired median muscle would be

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Figure 5. Rotation of transverse myofibers in early development of Platynereis and Xenopus. amphioxus, but is unsegmented in Axochord/notochord in red, transverse myofibers in green. A: 45˚ rotation of the developing [8]; still, its homology is transverse myofibers in Platynereis, forming ventral oblique muscles, drawn after [116]. B: undisputed. Nevertheless, it is worth 90˚ rotation of adaxial myofibers in Xenopus, forming longitudinal muscles, drawn after [113]. noting that the frog notochord has been reported to secrete extracellular matrix in a segmental fashion [108, 109]. consistent with the fact that, after it bifurcated behind the mouth, as forms by evagination from the arch- observed in modern protostomes and Did the transverse muscles evolve into enteron roof, the early amphioxus enteropneusts (see above). In chor- pioneer myocytes? notochord is composed of two adjacent dates, the notochord is also present longitudinal rows of cells, which sec- along the whole length of the trunk, but In both annelids and chordates, the ondarily intercalate into a single series does not bifurcate behind the mouth [30] axochord/notochord acts as an attach- (“stack of coins”) [102]. In conjunction – consistently with the idea that chor- ment band for lateral locomotory with increased reliance on undulatory dates secondarily evolved a new mouth, muscles that develop directly adjacent swimming, the notochord acquired non-homologous to the ancestral bilat- to it. In annelids, they are called incompressible intracellular vacuoles erian mouth [107]. The loss of the “ventral oblique muscles”. In zebrafish, [103], hence preventing shortening ancestral mouth might have allowed the only muscle cells directly contacting and making it an elastic antagonist. In some plasticity in the antero-posterior the notochord (“pioneer myocytes”) amphioxus, contraction of the myofila- extension of the notochord, which develop from the paraxial mesoderm ments fine-tunes the notochord stiffness reaches the anterior tip of the animal cells that are closest to the chordame- during locomotion [104]. The appear- in amphioxus, but is restricted to the soderm (adaxial cells). In both annelids ance of such vacuoles would have in and stops behind the and chordates, these muscles are entailed a new distribution of mechan- infundibulum in vertebrates [8]. uniquely defined by coexpression of ical constraints within the notochordal Unlike the annelid axochord, the foxd and engrailed [14, 110], suggesting cells, resulting in realignment of the notochord never presents any overt homology of the annelid ventral oblique myofilaments in a transverse direction. morphological segmentation. This muscles to the chordate pioneer Consistently, vertebrate longitudinal non-segmented character was consid- myocytes. muscles have been observed to develop ered significant enough in the early 20th The orientation of these muscles, unusually oriented filaments, perpen- century to constitute a fatal objection to however, differs between both phyla – dicular to their main axis (“ring the hypothetical derivation of chordates transverse in annelids, longitudinal in fibers”), in response to membrane from annelid-like ancestors [2]. Today, chordates. Indeed, according to one buckling due to hypercontraction [105] this objection seems less serious, as it is hypothesis, transverse muscles have or in myopathies with pathological more broadly accepted that segmenta- been lost in chordate ancestors, after vacuolization [106]. tion of a structure can be lost or gained having been replaced by the elastic The ancestral axochord was likely during evolution. For example, the notochord as antagonists to the longi- present along the whole trunk and lateral plate forms segmentally in tudinal musculature [111]. Interestingly,

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Box 1 the notochord and the neural tube [96, 121–123] (see Fig. C; but see Ref. [124] for a slightly different view). The The origin of the sclerotome amphioxus sclerocoel has been argued, from morpho- logical data, to be a sclerotome homolog [122], but this still After having shifted from muscular-contractile to chordoid- awaits molecular confirmation. The simplest vertebrae-like supporting functions, the notochord formed (together with structures are known in hagfish, as small cartilaginous the cartilaginous slits and oral cirri [117–120]) the first nodules around the dorsal aorta, on the inner side of the chordate skeleton. The notochord was later comple- dorsal mesentery [125]. Similarly, in , the first mented (and in some species, like ourselves, almost sclerotome develops as a thin line of Col2a1aþ somitic cells entirely replaced) by the backbone, in the form of first ventral to the hypochord, bordering the space in which the Hypotheses cartilaginous – and later mineralized – vertebrae. The origin future dorsal aorta will form [126]; the sclerotome then of the backbone remains mysterious, but clues might expands dorsally, as the amphioxus sclerocoel, to form an come from comparative data within deuterostomes. The extensive axial skeleton surrounding the notochord and the amphioxus sclerocoel develops as an evagination of the neural tube. Finally, in vertebrates, the sclerotome still gives coelomic lining facing the future dorsal aorta (the dorsal rise to both vertebral tissue and to smooth myocytes in mesentery), that expands and folds dorsally to surround the wall of the dorsal aorta (Fig. D) [127–129] – which are

Figure. Origin of the sclerotome from the deuterostome axial mesen- tery. A: Cross-section of the Platy- nereis trunk, showing connexion of the axochord to the ventral mesen- tery, the hollowing of which forms the ventral blood vessel. Drawn from [14] B: Cross-section of the Ptychodera trunk, which features a connexion between the ventral lon- gitudinal muscle mass and the ven- tral mesentery housing the blood vessel. Just dorsal to the blood vessel, a vacuolated expansion of the mesentery forms the pygochord. Drawn from Ref. [134] C: Develop- ment of the amphioxus sclerocoel from the early dorsal mesentery, outlining the paired dorsal aorta. Drawn from [122]. D: Development of the vertebrate sclerotome from the ventrolateral lining of the epithelial somites. The sclerotome gives rise to vertebrae and to smooth myocytes around the dorsal aorta. Note the fusion of the early paired dorsal aorta into a unique median vessel. Drawings are according to published cross-sec- tions of mammalian embryos: for the first two panels, humans [135, 136] and for the third one, pig [137].

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proposed to represent remnants of the ancestral lining of the In enteropneusts as well, the ventral mesentery dorsal aorta (the dorsal mesentery) before the vascular (homologous to the chordate dorsal mesentery) has at endothelium evolved [130]. least once been modified into a skeletal structure: the Hypotheses Taken together, these data suggest that the sclero- pygochord of ptychoderid enteropneusts is a stiffened tome evolved from a local thickening of the dorsal thickening of the ventral mesentery immediately contin- mesentery (Figure). This localized supporting structure uous with the lining of the ventral blood vessel would have ancestrally provided a ventral support to the (Fig. B) [132]. There thus seems to be a tendency in notochord, and then secondarily formed the vertebral deuterostomes for modifying the ventral mesentery (dorsal centra by expanding around the notochord; at interseg- in chordates) into a supporting organ. Pygochord and mental septa, dorsal expansion gave rise to dorsal arches sclerotome would thus illustrate an instance of parallel and ventral expansion to haemal arches, providing the evolution, i.e. of independent, similar modification of the basic groundplan for a vertebra [8]. Fusion of primitive same ancestral organ in two sister groups [133]. Future vertebral tissue from adjacent somites lying on both sides research will determine whether the ancestral ventral of the septum further stiffened the skeleton, and this mesentery already performed discrete and hitherto process is still recapitulated nowadays during vertebrate unrecognized supporting functions, paving the way for development by sclerotome resegmentation [8, 131]. its further modification into pygochord and sclerotome.

in frogs, the early slow myofibers arising Spiralia and Ecdysozoa – while the last Worsaae for insightful discussions, and from adaxial cells develop in a trans- common ancestor of Deuterostomia Andreas Altenburger for sharing bra- verse orientation – and only reorient might have already lost (or radically chiopod data and insightful comments. later to become longitudinal [112] modified) the transverse muscles, as (Fig. 5B). This reorientation has been they are absent from all living repre- proposed to underlie, at least in part, sentatives. By vacuolization, the ven- the “somite rotation” affecting the tromedian muscle gave rise to the References Xenopus myotome [113–115]. The Platy- chordate notochord, which came to lie 1. Erwin D, Valentine J. 2013. The nereis ventral oblique muscles undergo dorsally after dorsoventral inversion. A Explosion: The Construction of Animal Bio- a more limited 45˚ rotation, from an rigorous ancestral state reconstruction diversity., United States: Roberts and Com- initially transverse orientation to the based on morphological data supports pany Publishers. “oblique” direction that gave them their the presence of a ventromedian muscle 2. Gee H. 1996. Before the Backbone: Views on the Origin of the Vertebrates. United name (Fig. 5A). In vertebrates, the axial in bilaterian ancestors [14]. These con- States: Springer. complex composed of notochord and verging and mutually supportive evi- 3. Satoh N, Tagawa K, Takahashi H. 2012. attached longitudinal muscles has been dence from comparative How was the notochord born? Evol Dev 14: 56–75. complemented by the evolution of a (covering many phyla) and molecular 4. Bateson W. 1884. Note on the Later Stages new structure: the rigid backbone (see developmental biology (from verte- in the Development of Balanoglossus Kowa- Box 1). brates and annelids) make the axochord levskii (Agassiz), and on the Affinities of the Enteropneusta. United : Royal hypothesis a plausible and stimulating Society of London. explanation for the origin of the chor- 5. Bateson W. 1886. Memoirs: the Ancestry Conclusions and outlook date notochord. However, alternative of the Chordata. Q J Microsc Sci s2-26: hypotheses cannot yet be ruled out: for 535–72. 6. Gislen TRE. 1930. Affinities between the Emerging comparative molecular and example, the axochord and the noto- echinodermata, enteropneusta, and chor- morphological data make it possible, for chord could have evolved independ- donia. Zool Bidr Upps 12: 199–304. the first time, to propose a groundplan ently from mesenchymal cells, which 7. Gaskell WH. 2009. The Origin of Verte- brates. United States: Cornell University for the ventral musculature of the last would have acquired contractility sep- Library. common bilaterian ancestor – including arately in the protostome and deuter- 8. Goodrich ES. 1930. Studies on the Struc- a ventromedian muscle, transverse ostome lineages. Cell type-level ture & Development of Vertebrates. United muscles attached to it, and paired comparisons between a broader range Kingdom: Macmillan and co., limited. 9. Engelbrecht D, van Z. 1969. The annelid ventrolateral muscles. Morphological of bilaterian phyla will help elucidating ancestry of the chordates and the origin of data suggest that a circular muscle this issue. In the future, better resolu- the chordate central nervous system and the layer around the body could be tion of the bilaterian tree and extension notochord. J. Zool Syst Evol Res 7: 18–30. 10. Remane J. 1983. The concept of homology added [55]. A very similar plan can still of the molecular and developmental in phylogenetic research — its meaning be recognized in some groups, such as studies to more groups will be key to and possible applications. Palaontol€ Z 57: (like Platynereis), kino- further assessing these hypotheses. 267–9. 11. Wagner GP. 1989. The biological homology rhynchs, and larval molluscs and bra- concept. Annu Rev Ecol Syst 20: 51–69. chiopods (all of which, however, lost 12. Laubichler MD. 2000. Homology in devel- the circular musculature). We hypothe- Acknowledgements opment and the development of the homol- size that this groundplan was still We thank the whole Arendt lab, Sabrina ogy concept. Am Zool 40: 777–88. 13. De Pinna MCC. 1991. Concepts and tests present, in a largely unmodified fash- Kaul-Strehlow, Chris Lowe, Georg of homology in the cladistic paradigm. ion, in the last common ancestors of Mayer, Martin V. Sørensen, and Katrine Cladistics 7: 367–94.

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