Hox, homology, and parsimony: An organismal perspective.

Hox genes are important regulators in animal development. They often show a mosaic of conserved (e.g., longitudinal axis patterning) and lineage-specific novel functions (e.g., development of skeletal, sensory, or locomotory systems). Despite extensive research over the past decades, it remains controversial at which node in the animal tree of life the Hox cluster evolved. Its presence already in the last common metazoan ancestor has been proposed, although the genomes of both putative earliest extant metazoan offshoots, the ctenophores and the poriferans, are devoid of Hox sequences. The lack of Hox genes in the supposedly "simple"-built poriferans and their low number in cnidarians and the basally branching bilaterians, the xenacoelomorphs, seems to support the classical notion that the number of Hox genes is correlated with the degree of animal complexity. However, the 4-fold increase of the Hox cluster in xiphosurans, a basally branching chelicerate clade, as well as the situation in some teleost fishes that show a multitude of Hox genes compared to, e.g., human, demonstrates, that there is no per se direct correlation between organismal complexity and Hox number. Traditional approaches have tried to base homology on the morphological level on shared expression profiles of individual genes, but recent data have shown that, in particular with respect to Hox and other regulatory genes, complex gene-gene interactions rather than expression signatures of individual genes alone are responsible for shaping morphological traits during ontogeny. Accordingly, for sound homology assessments and reconstructions of character evolution on organ system level, additional independent datasets (e.g., morphological, developmental) need to be included in any such analyses. If supported by solid data, proposed structural homology should be regarded as valid and not be rejected solely on the grounds of non-parsimonious distribution of the character over a given phylogenetic topology.

A mosaic of conserved and novel modes of gene expression and morphogenesis in mesoderm and muscle formation of a larval bivalve.

The mesoderm gives rise to several key morphological features of bilaterian animals including endoskeletal elements and the musculature. A number of regulatory genes involved in mesoderm and/or muscle formation (e.g., Brachyury (Bra), even-skipped (eve), Mox, myosin II heavy chain (mhc)) have been identified chiefly from chordates and the ecdysozoans Drosophila and Caenorhabditis elegans, but data for non-model protostomes, especially those belonging to the ecdysozoan sister clade, Lophotrochozoa (e.g., flatworms, annelids, mollusks), are only beginning to emerge. Within the lophotrochozoans, Mollusca constitutes the most speciose and diverse phylum. Interestingly, however, information on the morphological and molecular underpinnings of key ontogenetic processes such as mesoderm formation and myogenesis remains scarce even for prominent molluscan sublineages such as the bivalves. Here, we investigated myogenesis and developmental expression of Bra, eve, Mox, and mhc in the quagga mussel Dreissena rostriformis, an invasive freshwater bivalve and an emerging model in invertebrate evodevo. We found that all four genes are expressed during mesoderm formation, but some show additional, individual sites of expression during ontogeny. While Mox and mhc are involved in early myogenesis, eve is also expressed in the embryonic shell field and Bra is additionally present in the foregut. Comparative analysis suggests that Mox has an ancestral role in mesoderm and possibly muscle formation in bilaterians, while Bra and eve are conserved regulators of mesoderm development of nephrozoans (protostomes and deuterostomes). The fully developed Dreissena veliger larva shows a highly complex muscular architecture, supporting a muscular ground pattern of autobranch bivalve larvae that includes at least a velum muscle ring, three or four pairs of velum retractors, one or two pairs of larval retractors, two pairs of foot retractors, a pedal plexus, possibly two pairs of mantle retractors, and the muscles of the pallial line, as well as an anterior and a posterior adductor. As is typical for their molluscan kin, remodelling and loss of prominent larval features such as the velum musculature and various retractor systems appear to be also common in bivalves. Supplementary information: The online version contains supplementary material available at 10.1007/s13127-022-00569-5.

Morphology of the bryozoan Cinctipora elegans (Cyclostomata, Cinctiporidae) with first data on its sexual reproduction and the cyclostome neuro-muscular system.

BACKGROUND: Cyclostome bryozoans are an ancient group of marine colonial suspension-feeders comprising approximately 700 extant species. Previous morphological studies are mainly restricted to skeletal characters whereas data on soft tissues obtained by state-of-the-art methods are still lacking. In order to contribute to issues related to cyclostome ground pattern reconstruction, we analyzed the morphology of the neuromuscular system Cinctipora elegans by means of immunocytochemical staining, confocal laser scanning microscopy, histological sections and microCT imaging. RESULTS: Polypides of C. elegans are located in elongated tubular skeletal cystids. Distally, the orifice leads into a prominent vestibulum which is lined by an epithelium that joins an almost complete perimetrical attachment organ, both containing radially arranged neurite bundles and muscles. Centrally, the prominent atrial sphincter separates the vestibulum from the atrium. The latter is enclosed by the tentacle sheath which contains few longitudinal muscle fibers and two principal neurite bundles. These emerge from the cerebral ganglion, which is located at the lophophoral base. Lateral ganglia are located next to the cerebral ganglion from which the visceral neurite bundles emerge that extend proximally towards the foregut. There are four tentacle neurite bundles that emerge from the ganglia and the circum-oral nerve ring, which encompasses the pharynx. The tentacles possess two striated longitudinal muscles. Short buccal dilatators are situated at the lophophoral base and short muscular sets are present at the abfrontal and frontal side of the tentacle base. The pharynx is myoepithelial and triradiate in cross-section. Oocytes are found inside the pharyngeal myoepithelium. The digestive tract contains dense circular musculature and few longitudinal muscles. The membranous sac contains regular, thin, circular and diagonal muscles and neurites in its epithelial lining. CONCLUSIONS: The general structure of the neuro-muscular system is more reminiscent of the condition found in Gymnolaemata rather than Phylactolaemata, which supports a close relationship between Cyclostomata and Gymnolaemata. Several characters of C. elegans such as the lateral ganglia or loss of the cardia are probably apomorphic for this species. For the first time, oocytes that surprisingly develop in the pharyngeal wall are reported for this species.

Expression of six3 and otx in Solenogastres (Mollusca) supports an ancestral role in bilaterian anterior-posterior axis patterning.

The homeodomain transcription factors six3 and otx are involved in patterning the anterior body and parts of the central nervous system (CNS) in bilaterians. Their similar expression patterns have been used as an argument for homology of heads, brains, segmentation, and ciliated larvae. We investigated the developmental expression of six3 and otx in the aplacophoran mollusk Wirenia argentea. Six3 is expressed in subepithelial cells delimiting the apical organ of the solenogaster pericalymma larva. Otx is expressed in cells of the prototroch and adjacent regions as well as in posterior extensions of the prototrochal expression domain. Advanced larvae also show pretrochal otx expression in the developing CNS. Comparative analysis of six3 and otx expression in bilaterians argues for an ancestral function in anterior-posterior body axis patterning but, due to its presence in animals lacking a head and/or a brain, not necessarily for the presence of these morphological structures in the last common ancestor (LCA) of bilaterians. Likewise, the hypothesis that the posterior border of otx expression corresponds to the border between the unsegmented head and the segmented trunk of the LCA of protostomes is not supported, since otx is extensively expressed in the trunk in W. argentea and numerous other protostomes.

Staggered Hox expression is more widespread among molluscs than previously appreciated.

Hox genes are expressed along the anterior-posterior body axis in a colinear fashion in the majority of bilaterians. Contrary to polyplacophorans, a group of aculiferan molluscs with conserved ancestral molluscan features, gastropods and cephalopods deviate from this pattern by expressing Hox genes in distinct morphological structures and not in a staggered fashion. Among conchiferans, scaphopods exhibit many similarities with gastropods, cephalopods and bivalves, however, the molecular developmental underpinnings of these similar traits remain unknown. We investigated Hox gene expression in developmental stages of the scaphopod Antalis entalis to elucidate whether these genes are involved in patterning morphological traits shared by their kin conchiferans. Scaphopod Hox genes are predominantly expressed in the foot and mantle but also in the central nervous system. Surprisingly, the scaphopod mid-stage trochophore exhibits a near-to staggered expression of all nine Hox genes identified. Temporal colinearity was not found and early-stage and late-stage trochophores, as well as postmetamorphic individuals, do not show any apparent traces of staggered expression. In these stages, Hox genes are expressed in distinct morphological structures such as the cerebral and pedal ganglia and in the shell field of early-stage trochophores. Interestingly, a re-evaluation of previously published data on early-stage cephalopod embryos and of the gastropod pre-torsional veliger shows that these developmental stages exhibit traces of staggered Hox expression. Considering our results and all gene expression and genomic data available for molluscs as well as other bilaterians, we suggest a last common molluscan ancestor with colinear Hox expression in predominantly ectodermal tissues along the anterior-posterior axis. Subsequently, certain Hox genes have been co-opted into the patterning process of distinct structures (apical organ or prototroch) in conchiferans.

Unity in diversity: a survey of muscular systems of ctenostome Gymnolaemata (Lophotrochozoa, Bryozoa).

BACKGROUND: Myoanatomical studies of adult bryozoans employing fluorescent staining and confocal laser scanning microscopy (CLSM) have been chiefly conducted on freshwater bryozoans. The diversity of muscular systems in the marine bryozoans is currently not well known with only two species being studied in more detail. The aim of this study is to unravel the diversity of muscle systems of 15 ctenostome bryozoans by phalloidin-coupled fluorescence stainings combined with CLSM. RESULTS: In general, the myoanatomy of the selected ctenostomes shows significant similarities and consists of 1) muscles associated with the body wall, 2) apertural muscles, 3) lophophoral muscles, 4) tentacle sheath muscles, 5) digestive tract muscles and 6) the prominent retractor muscles. Differences are present in the arrangement of the apertural muscles from generally three muscles sets of four bundles, which in some species can be partially reduced or modified into a bilateral arrangement. The cardiac region of the digestive tract shows a distinct sphincter in four of the six studied clades. In some cases the cardiac region forms a prominent proventriculus or gizzard. Tentacle sheath muscles in victorelloideans and walkerioideans are arranged diagonally and differ from the simple longitudinal muscle arrangements common to all other taxa. Lophophoral base muscles consist of four sets that vary in the size of the sets and in the shape of the inner lophophoral ring, which either forms a complete ring or separate, intertentacular muscle bundles. The stolon-forming walkeridiodean ctenostomes show prominent transverse muscles in their stolons. These are always present in the shorter side stolons, but their occurrence in the main stolon seems to depend on the colony form, being present in creeping but absent in erect colony forms. CONCLUSIONS: This study represents the first broad survey of muscular systems in adult ctenostome bryozoans and shows a certain degree of conservation in a series of diverse colony forms belonging to five major clades. However, several myoanatomical features such as the cardiac sphincter, basal (possibly transitory) cystid muscles, tentacle sheath muscles or apertural muscle arrangement vary across taxa and thus show a high potential for the assessment of character evolution within ctenostomes. As such, this study represents an essential contribution towards determining and reconstructing the character states of the bryozoan ground pattern once a reliable phylogenetic tree of the whole phylum becomes available.

Towards a ground pattern reconstruction of bivalve nervous systems: neurogenesis in the zebra mussel Dreissena polymorpha.

Bivalvia is a taxon of aquatic mollusks that includes clams, oysters, mussels, and scallops. Within heterodont bivalves, Dreissena polymorpha is a small, mytiliform, freshwater mussel that develops indirectly via a planktotrophic veliger larva. Currently, only a few studies on bivalve neurogenesis are available, impeding the reconstruction of a ground pattern in Bivalvia. In order to inject novel data into this discussion, we describe herein the development of the serotonin-like and α-tubulin-like immunoreactive (lir) neuronal components of D. polymorpha from the early trochophore to the late veliger stage. Neurogenesis starts in the early trochophore stage at the apical pole with the appearance of one flask-shaped serotonin-lir cell. When larvae reach the veliger stage, four flask-shaped serotonin-lir cells are present in the apical organ. At the same time, the anlagen of the cerebral ganglia start to form at the base of the apical organ. From the apical organ, one pair of cerebro-visceral connectives projects posteriorly and connects to a posterior larval sensory organ that contains serotonin- and α-tubulin-like flask-shaped cells. Additional, paired serotonin-lir neurites originate from the apical organ and project into the velum. One unpaired stomatogastric serotonin-lir cell develops ventrally to the stomach at the veliger stage. The low number of serotonin-lir cells in the apical organ of bivalve veligers is shared with larvae of basally branching gastropods and scaphopods and is thus considered a feature of the last common ancestor of Conchifera, while the overall simplicity of the larval neural architecture appears to be a specific trait of Bivalvia.

Reconstructing the muscular ground pattern of phylactolaemate bryozoans: first data from gelatinous representatives.

BACKGROUND: Phylactolaemata is commonly regarded the earliest branch within Bryozoa and thus the sister group to the other bryozoan taxa, Cyclostomata and Gymnolaemata. Therefore, the taxon is important for the reconstruction of the bryozoan morphological ground pattern. In this study the myoanatomy of Pectinatella magnifica, Cristatella mucedo and Hyalinella punctata was analysed by means of histology, f-actin staining and confocal laser-scanning microscopy in order to fill gaps in knowledge concerning the myoanatomy of Phylactolaemata. RESULTS: The retractor muscles and muscles of the aperture, gut, body wall, tentacle sheath, lophophore constitute the most prominent muscular subsets in these species. The lophophore shows longitudinal muscle bands in the tentacles, lophophoral arm muscles, epistome musculature and hitherto undescribed muscles of the ring canal. In general the muscular system of the three species is very similar with differences mainly in the body wall, tentacle sheath and epistome. The body wall contains an orthogonal grid of musculature. The epistome exhibits either a muscular meshwork in the epistomal wall or muscle fibers traversing the epistomal cavity. The whole tentacle sheath possesses a regular mesh of muscles in Pectinatella and Cristatella, whereas circular muscles are limited to the tentacle sheath base in Hyalinella. CONCLUSION: This study is the first to describe muscles of the ring canal and contributes to reconstructing muscular features for the last common ancestor of all bryozoans. The data available suggest that two longitudinal muscle bands in the tentacles, as well as retractor muscles and longitudinal and circular muscles in the tentacle sheath, were present in the last common bryozoan ancestor. Comparisons among bryozoans shows that several apomorphies are present in the myoanatomy of each class- level taxon such as the epistomal musculature and musculature of the lophophoral arms in phylactolaemates, annular muscles in cyclostomes and parietal muscles in gymnolaemates.

Ancestral and novel roles of Pax family genes in mollusks.

BACKGROUND: Pax genes are transcription factors with significant roles in cell fate specification and tissue differentiation during animal ontogeny. Most information on their tempo-spatial mode of expression is available from well-studied model organisms where the Pax-subfamilies Pax2/5/8, Pax6, and Paxα/ß are mainly involved in the development of the central nervous system (CNS), the eyes, and other sensory organs. In certain taxa, Pax2/5/8 seems to be additionally involved in the development of excretion organs. Data on expression patterns in lophotrochozoans, and in particular in mollusks, are very scarce for all the above-mentioned Pax-subfamilies, which hampers reconstruction of their putative ancestral roles in bilaterian animals. Thus, we studied the developmental expression of Pax2/5/8, Pax6, and the lophotrochozoan-specific Paxß in the worm-shaped mollusk Wirenia argentea, a member of Aplacophora that together with Polyplacophora forms the Aculifera, the proposed sister taxon to all primarily single-shelled mollusks (Conchifera). RESULTS: All investigated Pax genes are expressed in the developing cerebral ganglia and in the ventral nerve cords, but not in the lateral nerve cords of the tetraneural nervous system. Additionally, Pax2/5/8 is expressed in epidermal spicule-secreting or associated cells of the larval trunk and in the region of the developing protonephridia. We found no indication for an involvement of the investigated Pax genes in the development of larval or adult sensory organs of Wirenia argentea. CONCLUSIONS: Pax2/5/8 seems to have a conserved role in the development of the CNS, whereas expression in the spicule-secreting tissues of aplacophorans and polyplacophorans suggests co-option in aculiferan skeletogenesis. The Pax6 expression pattern in Aculifera largely resembles the common bilaterian expression during CNS development. All data available on Paxß expression argue for a common role in lophotrochozoan neurogenesis.

Hox and ParaHox gene expression in early body plan patterning of polyplacophoran mollusks.

Molecular developmental studies of various bilaterians have shown that the identity of the anteroposterior body axis is controlled by Hox and ParaHox genes. Detailed Hox and ParaHox gene expression data are available for conchiferan mollusks, such as gastropods (snails and slugs) and cephalopods (squids and octopuses), whereas information on the putative conchiferan sister group, Aculifera, is still scarce (but see Fritsch et al., 2015 on Hox gene expression in the polyplacophoran Acanthochitona crinita). In contrast to gastropods and cephalopods, the Hox genes in polyplacophorans are expressed in an anteroposterior sequence similar to the condition in annelids and other bilaterians. Here, we present the expression patterns of the Hox genes Lox5, Lox4, and Lox2, together with the ParaHox gene caudal (Cdx) in the polyplacophoran A. crinita. To localize Hox and ParaHox gene transcription products, we also investigated the expression patterns of the genes FMRF and Elav, and the development of the nervous system. Similar to the other Hox genes, all three Acr-Lox genes are expressed in an anteroposterior sequence. Transcripts of Acr-Cdx are seemingly present in the forming hindgut at the posterior end. The expression patterns of both the central class Acr-Lox genes and the Acr-Cdx gene are strikingly similar to those in annelids and nemerteans. In Polyplacophora, the expression patterns of the Hox and ParaHox genes seem to be evolutionarily highly conserved, while in conchiferan mollusks these genes are co-opted into novel functions that might have led to evolutionary novelties, at least in gastropods and cephalopods.

Cell Proliferation Pattern and Twist Expression in an Aplacophoran Mollusk Argue Against Segmented Ancestry of Mollusca.

The study of aplacophoran mollusks (i.e., Solenogastres or Neomeniomorpha and Caudofoveata or Chaetodermomorpha) has traditionally been regarded as crucial for reconstructing the morphology of the last common ancestor of the Mollusca. Since their proposed close relatives, the Polyplacophora, show a distinct seriality in certain organ systems, the aplacophorans are also in the focus of attention with regard to the question of a potential segmented ancestry of mollusks. To contribute to this question, we investigated cell proliferation patterns and the expression of the twist ortholog during larval development in solenogasters. In advanced to late larvae, during the outgrowth of the trunk, a pair of longitudinal bands of proliferating cells is found subepithelially in a lateral to ventrolateral position. These bands elongate during subsequent development as the trunk grows longer. Likewise, expression of twist occurs in two laterally positioned, subepithelial longitudinal stripes in advanced larvae. Both, the pattern of proliferating cells and the expression domain of twist demonstrate the existence of extensive and long-lived mesodermal bands in a worm-shaped aculiferan, a situation which is similar to annelids but in stark contrast to conchiferans, where the mesodermal bands are usually rudimentary and ephemeral. Yet, in contrast to annelids, neither the bands of proliferating cells nor the twist expression domain show a separation into distinct serial subunits, which clearly argues against a segmented ancestry of mollusks. Furthermore, the lack of twist expression during the development of the ventromedian muscle argues against homology of a ventromedian longitudinal muscle in protostomes with the notochord of chordates.

Neural architecture of Galathowenia oculata Zach, 1923 (Oweniidae, Annelida).

BACKGROUND: Oweniids are marine tubeworms burrowing in muddy sediments that in current phylogenies form an early branching lineage within Annelida. Little is known about their general morphology, in particular the nervous system. Here we provide an immunocytochemical investigation of the nervous system of Galathowenia oculata in order to discuss putative ancestral neuronal features in Oweniidae. RESULTS: Adult Galathowenia oculata have neither a supraesophageal ganglion nor ganglia associated with the ventral nerve cord. Instead, there is a dorsal brain commissure in the head collar that is engulfed by a cellular cortex. Accordingly, we herein term this neural structure "medullary brain commissure". The anterior margin of the head collar exhibits numerous neurites that emerge from the brain commissure. The dorsolateral folds are innervated by the ventrolateral neurite bundles extending from the circumesophageal connectives. In the anterior uniramous and biramous segments immunoreactive somata are distributed evenly along the ventral nerve cord and arranged metamerically in the posterior-most short segments. One dorsal and two pairs of lateral neurite bundles extend longitudinally along the body. Numerous serially arranged circular neurite bundles were labeled in anteriormost long segments. Metameric arrangement of the circular neurite bundles stained against FMRFamide and acetylated α-tubulin is revealed in posterior short segments. For the first time immunoreactive somata arranged in clusters are reported within the pygidium in oweniids. CONCLUSIONS: Due to the lack of head appendages and a sedentary mode of life, G. oculata exhibits a single dorsal commissure (versus a brain with four commissures in most annelids). A "medullary brain commissure" is known so far only in Oweniidae and Echiura. Lack of ganglia and metamery in the ventral nerve cord of the anteriormost segments might be the result of the elongation of these segments. In the short posterior segments the metamery of immunoreactive somata and circular neurite bundles is conserved. We hypothesize that the unpaired ventral nerve cord in adult oweniids might be a result of an initially paired ventral nerve cord that fuses during development, a condition not uncommon within Annelida.

A putative species complex in the Sea of Japan revealed by DNA sequence data: a study on Lottia cf. kogamogai (Gastropoda: Patellogastropoda).

A putative new limpet species (Patellogastropoda) from the Sea of Japan is revealed by molecular genetic analyses using the mitochondrial markers 16S rRNA and cytochrome c oxidase subunit I (CO1), as well as the DNA marker 18S rRNA. Our data indicate that the limpet, collected in the Peter the Great Bay (Russian Federation), is not, as its morphology suggests, the Japanese species Lottia kogamogai Sasaki and Okutani, 1994, and might also hint towards another putative species complex in the Sea of Japan. The different currents between the Far East Asian mainland (cold, subpolar jet running southwards) and the Japanese archipelago (warm, subtropical jet running northwards) are likely to act as a barrier that has a substantial influence on species distribution in these waters. Accordingly, our results indicate that it is about time for a revision of patellogastropod species with a reported distribution in Japanese and Far Eastern Russian waters by an integrative approach using molecular genetic and morphological characters. The species investigated herein is referred to as Lottia cf. kogamogai until it is morphologically re-examined and compared with primary type specimens of known species.

Neuromuscular development in Patellogastropoda (Mollusca: Gastropoda) and its importance for reconstructing ancestral gastropod bodyplan features.

Within Gastropoda, limpets (Patellogastropoda) are considered the most basal branching taxon and its representatives are thus crucial for research into evolutionary questions. Here, we describe the development of the neuromuscular system in Lottia cf. kogamogai. In trochophore larvae, first serotonin-like immunoreactivity (lir) appears in the apical organ and in the prototroch nerve ring. The arrangement and number of serotonin-lir cells in the apical organ (three flask-shaped, two round cells) are strikingly similar to those in putatively derived gastropods. First, FMRFamide-lir appears in veliger larvae in the Anlagen of the future adult nervous system including the cerebral and pedal ganglia. As in other gastropods, the larvae of this limpet show one main and one accessory retractor as well as a pedal retractor and a prototroch muscle ring. Of these, only the pedal retractor persists until after metamorphosis and is part of the adult shell musculature. We found a hitherto undescribed, paired muscle that inserts at the base of the foot and runs towards the base of the tentacles. An apical organ with flask-shaped cells, one main and one accessory retractor muscle is commonly found among gastropod larvae and thus might have been part of the last common ancestor.

The serotonin-lir nervous system of the Bryozoa (Lophotrochozoa): a general pattern in the Gymnolaemata and implications for lophophore evolution of the phylum.

BACKGROUND: Serotonin represents an evolutionary ancient neurotransmitter that is ubiquitously found among animals including the lophotrochozoan phylum Bryozoa, a group of colonial filter-feeders. Comparatively little is known on their nervous system, and data on their serotonin-lir nervous system currently are mostly limited to the basal phylactolaemates. Previous investigations indicated a common ground-pattern of the serotonin-lir nervous system in these animals, but in order to assess this on a larger scale, 21 gymnolaemate species from 21 genera were comparatively analysed herein. METHODS: Twenty-one species from 21 gymnolaemate genera were analysed by immunocytochemical stainings and confocal laser scanning microscopy. RESULTS: In all species the serotonin-lir signal is concentrated in the cerebral ganglion from where a nerve tract emanates laterally and traverses orally to engulf the foregut. Serotonin-lir perikarya are situated at the base of the tentacles that almost always correspond to the number of tentacles minus two. The oral side in almost all species shows three serotonin-lir perikarya followed by a 'serotonergic gap' that to our knowledge is not reflected in the morphology of the nervous system. Some species show additional serotonin-lir signal in tentacle nerves, visceral innervation and pore complexes. Paludicella articulata is exceptional as it shows signal in the latero-visceral nerves with serotonin-lir perikarya in the esophagus, parts of the tentacle sheath nerves as well as the frontal body wall around the parietal muscle bundles. CONCLUSIONS: In general, the serotonin-lir nervous system in the Bryozoa shows a consistent pattern among its different clades with few deviations. Preliminary data on phylactolaemates suggest the presence of a 'serotonergic gap' similar to gymnolaemates. Both show a subset of oral tentacles and the remaining tentacles in gymnolaemates which correspond to the lateral tentacles of phylactolaemates. The lophophoral concavity lacks serotonin-lir perikarya indicating that due to their larger sizes and increased tentacle number, the horse-shoe shaped arrangement could represent an apomorphy of phylactolaemates.

Muscular anatomy of an entoproct creeping-type larva reveals extraordinary high complexity and potential shared characters with mollusks.

BACKGROUND: Entoprocta (Kamptozoa) is an enigmatic, acoelomate, tentacle-bearing phylum with indirect development, either via a swimming- or a creeping-type larva and still debated phylogenetic position within Lophotrochozoa. Recent morphological and neuro-anatomical studies on the creeping-type larva support a close relationship of Entoprocta and Mollusca, with a number of shared apomorphies including a tetraneurous nervous system and a complex serotonin-expressing apical organ. However, many morphological traits of entoproct larvae, in particular of the putative basal creeping-type larva, remain elusive. RESULTS: Applying fluorescent markers and 3D modeling, we found that this larval type has the most complex musculature hitherto described for any lophotrochozoan larva. The muscle systems identified include numerous novel and most likely creeping-type larva-specific structures such as frontal organ retractors, several other muscle fibers originating from the frontal organ, and longitudinal prototroch muscles. Interestingly, we found distinct muscle sets that are also present in several mollusks. These include paired sets of dorso-ventral muscles that intercross ventrally above the foot sole and a paired enrolling muscle that is distinct from the musculature of the body wall. CONCLUSION: Our data add further morphological support for an entoproct-mollusk relationship (Tetraneuralia) and strongly argue for the presence of an enrolling musculature as well as seriality (but not segmentation) in the last common tetraneuralian ancestor. The evolutionary driving forces that have led to the emergence of the extraordinarily complex muscular architecture in this short-lived, non-feeding entoproct larval type remain unknown, as are the processes that give rise to the highly different and much simpler muscular bodyplan of the adult entoproct during metamorphosis.

From complex to simple: myogenesis in an aplacophoran mollusk reveals key traits in aculiferan evolution.

BACKGROUND: Recent studies suggest a bifurcation at the base of Mollusca, resulting in the primarily single-shelled Conchifera (Bivalvia, Gastropoda, Scaphopoda, Monoplacophora, Cephalopoda) and the spicule-bearing Aculifera (Polyplacophora, Neomeniomorpha, Chaetodermomorpha). A recent study revealed a complex larval musculature exclusively shared by Neomeniomorpha and Polyplacophora, supporting a close relationship of both taxa. However, the ontogenetic transition from the complex larval to the simple adult neomeniomorph musculature, which mainly consists of a three-layered body-wall musculature and serially iterated dorsoventral muscles, remains unknown. To close this gap in knowledge, we studied remodeling of the larval musculature during metamorphosis in the neomeniomorph Wirenia argentea. A comparative analysis with a novel data set of a polyplacophoran, Leptochiton asellus, allows us to infer the morphology of the last common ancestor of Aculifera and the evolution of its subclades therefrom. RESULTS: The complex larval musculature of Wirenia argentea persists through metamorphosis and becomes modified to form two of the three muscle layers of the adult body wall. The innermost longitudinal layer of the three-layered body wall musculature is generated by transformation and expansion of distinct larval longitudinal muscle bundles. The larval ventrolateral muscle strands are remodeled and eventually become the most ventral part of the adult longitudinal layer of the body wall musculature. The paired larval enrolling muscle forms the lateral parts and the former rectus muscle is destined to become the most dorsal part of the longitudinal layer of the body wall musculature. The transient ventromedian muscle is lost during postmetamorphic development. CONCLUSIONS: Postmetamorphic remodeling in W. argentea supports the hypothesis of a complex myoanatomy rather than a three-layered body wall musculature at the base of Aculifera, and thus argues against homology of the body wall musculature of adult Neomeniomorpha and other potential molluscan sister groups. Our data show that the neomeniomorph body wall musculature is a derived condition and not an aculiferan or molluscan plesiomorphy.

Ancestral role of Pax2/5/8 in molluscan brain and multimodal sensory system development.

BACKGROUND: Mollusks represent the largest lophotrochozoan phylum and exhibit highly diverse body plans. Previous studies have demonstrated that transcription factors such as Pax genes play important roles during their development. Accordingly, in ecdysozoan and vertebrate model organisms, orthologs of Pax2/5/8 are among others involved in the formation of the midbrain/hindbrain boundary, the auditory/geosensory organ systems, and the excretory system. METHODS: Pax2/5/8 expression was investigated by in situ hybridization during the development of representatives of the two major molluscan subclades, Aculifera and Conchifera. RESULTS: Compared to the investigated polyplacophoran and bivalve species that lack larval statocysts as geosensory organs and elaborate central nervous systems (CNS), cephalopods possess highly centralized brains and statocysts. Pax2/5/8 is expressed in regions where sensory cells develop subsequently during ontogenesis. Expression domains include esthetes and the ampullary system in polyplacophorans as well as the eyes of cephalopods. No Pax2/5/8 expression was observed in the less centralized CNS of bivalve, polyplacophoran, and gastropod embryos, thus arguing for a loss of Pax2/5/8 involvement in CNS development in these lineages. In contrast, Pax2/5/8 is expressed among others in brain lobes along the trajectory of the esophagus that divides the cephalopod brain. CONCLUSIONS: Our results, along with those on Otx- and Hox-gene expression, demonstrate that the cephalopod condition is similar to that in mouse and fruit fly, with Otx being expressed in the anterior-most brain region (except for the vertical lobe) and a Pax2/5/8 expression domain separating the Otx-domain from a Hox-gene expressing posterior brain region. Thus, Pax2/5/8 appears to have been recruited independently into regionalization of non-homologous complex brains of organisms as different as squid, fruit fly, and mouse. In addition, Pax2/5/8 is expressed in multimodal sensory systems in mollusks such as the esthetes and the ampullary system of polyplacophorans as well as the eyes of cephalopods. Pax2/5/8-expressing cells are present in regions where the future sensory cells such as the polyplacophoran esthetes are situated and hence Pax2/5/8 expression probably predates sensory cell development during ontogeny. In mollusks, Pax2/5/8 is only expressed in derivatives of the ectoderm and hence an ancestral role in molluscan ectoderm differentiation is inferred.

Mantle margin morphogenesis in Nodipecten nodosus (Mollusca: Bivalvia): new insights into the development and the roles of bivalve pallial folds.

BACKGROUND: Despite extensive knowledge on bivalve anatomy and development, the formation and differentiation of the mantle margin and its associated organs remain largely unclear. Bivalves from the family Pectinidae (scallops) are particularly promising to cast some light on these issues, because they exhibit a complex mantle margin and their developmental stages are easily obtained from scallop farms. We investigated the mantle margin of the scallop Nodipecten nodosus (L. 1758) during larval and postmetamorphic development. METHODS: A thorough analysis of the mantle margin development in Nodipecten nodosus, from veliger larvae to mature adults, was conducted by means of integrative microscopy techniques, i.e., light, electron, and confocal microscopy. RESULTS: Initially unfolded, the pallial margin is divided into distal and proximal regions by the periostracum-forming zone. The emergence of the pallial musculature and its neural innervation are crucial steps during bivalve larval development. By the late pediveliger stage, the margin becomes folded, resulting in a bilobed condition (i.e., outer and inner folds), a periostracal groove, and the development of different types of cilia. After metamorphosis, a second outgrowth process is responsible for emergence of the middle mantle fold from the outer surface of the inner fold. Once the three-folded condition is established, the general adult features are rapidly formed. CONCLUSIONS: Our data show that the middle mantle fold forms from the outer surface of the inner fold after metamorphosis and that the initial unfolded mantle margin may represent a common condition among bivalves. The first outgrowth process, which gives rise to the outer and inner folds, and the emergence of the pallial musculature and innervation occur during larval stages, highlighting the importance of the larval period for mantle margin morphogenesis in Bivalvia.