Larval Apical Organ Research Articles

A feather star is born: embryonic development and nervous system organization in the crinoid Antedon mediterranea.

Crinoids belong to the Echinodermata, marine invertebrates with a highly derived adult pentaradial body plan. As the sister group to all other extant echinoderms, crinoids occupy a key phylogenetic position to explore the evolutionary history of the whole phylum. However, their development remains understudied compared with that of other echinoderms. Therefore, the aim here was to establish the Mediterranean feather star (Antedon mediterranea) as an experimental system for developmental biology. We first set up a method for culturing embryos in vitro and defined a standardized staging system for this species. We then optimized protocols to characterize the morphological and molecular development of the main structures of the feather star body plan. Focusing on the nervous system, we showed that the larval apical organ includes serotonergic, GABAergic and glutamatergic neurons, which develop within a conserved anterior molecular signature. We described the composition of the early post-metamorphic nervous system and revealed that it has an anterior signature. These results further our knowledge on crinoid development and provide new techniques to investigate feather star embryogenesis. This will pave the way for the inclusion of crinoids in comparative studies addressing the origin of the echinoderm body plan and the evolutionary diversification of deuterostomes.

The ultrastructure of the apical organ of Curini-Galletti's larva, a new polyclad larval type.

Polycladida are the only free-living flatworms with a planktonic larval stage in some species. Currently, it is not clear if a larval stage is ancestral in polyclads, and which type of larva that would be. Known polyclad larvae are Müller's larva, Kato's larva and Goette's larva, differing by body shape and the number of lobes and eyes. A valuable character for the comparison and characterisation of polyclad larval types is the ultrastructural composition of the apical organ. This organ is situated at the anterior pole of the larva and is associated with at least one ciliary tuft. The larval apical organ of Theama mediterranea features two multiciliated apical tuft sensory cells. Six unfurcated apical tuft gland cell necks are sandwiched between the apical tuft sensorycells and two anchor cells and have their cell bodies located lateral to the brain. Another type of apical gland cell necks is embedded in the anchor cells. Ventral to the apical tuft, ciliated sensory neurons are present, which are neighbouring the cell necks of two furcated apical tuft glandcells. Based on the ultrastructural organisation of the apical organ and other morphological features, like a laterally flattened wedge-shaped body and three very small lobes, we recognise the larva of T. mediterranea as a new larval type, which we name Curini-Galletti's larva after its first discoverer. The ultrastructural similarities of the apical organ in different polyclad larvae support their possible homology, that is,all polyclad larvae have likely evolved from a common larva.

Gene Regulatory Network that Shaped the Evolution of Larval Apical Organ in Cnidaria.

Among non-bilaterian animals, a larval apical sensory organ with integrated neurons is only found in cnidarians. Within cnidarians, an apical organ with a ciliary tuft is mainly found in Actiniaria. Whether this apical tuft has evolved independently in Actiniaria or alternatively originated in the common ancestor of Cnidaria and Bilateria and was lost in specific groups is uncertain. To test this hypothesis, we generated transcriptomes of the apical domain during the planula stage of four species representing three key groups of cnidarians: Aurelia aurita (Scyphozoa), Nematostella vectensis (Actiniaria), and Acropora millepora and Acropora tenuis (Scleractinia). We showed that the canonical genes implicated in patterning the apical domain of N. vectensis are largely absent in A. aurita. In contrast, the apical domain of the scleractinian planula shares gene expression pattern with N. vectensis. By comparing the larval single-cell transcriptomes, we revealed the apical organ cell type of Scleractinia and confirmed its homology to Actiniaria. However, Fgfa2, a vital regulator of the regionalization of the N. vectensis apical organ, is absent in the scleractinian genome. Likewise, we found that FoxJ1 and 245 genes associated with cilia are exclusively expressed in the N. vectensis apical domain, which is in line with the presence of ciliary apical tuft in Actiniaria and its absence in Scleractinia and Scyphozoa. Our findings suggest that the common ancestor of cnidarians lacked a ciliary apical tuft, and it could have evolved independently in the Actiniaria.

Identification of the neuropeptide precursor genes potentially involved in the larval settlement in the Echiuran worm Urechis unicinctus

BackgroundIn marine invertebrate life cycles, which often consist of planktonic larval and benthonic adult stages, settlement of the free-swimming larva to the sea floor in response to environmental cues is a key life cycle transition. Settlement is regulated by a specialized sensory–neurosecretory system, the larval apical organ. The neuroendocrine mechanisms through which the apical organ transduces environmental cues into behavioral responses during settlement are not fully understood yet.ResultsIn this study, a total of 54 neuropeptide precursors (pNPs) were identified in the Urechis unicinctus larva and adult transcriptome databases using local BLAST and NpSearch prediction, of which 10 pNPs belonging to the ancient eumetazoa, 24 pNPs belonging to the ancient bilaterian, 3 pNPs belonging to the ancient protostome, 9 pNPs exclusive in lophotrochozoa, 3 pNPs exclusive in annelid, and 5 pNPs only found in U. unicinctus. Furthermore, four pNPs (MIP, FRWamide, FxFamide and FILamide) which may be associated with the settlement and metamorphosis of U. unicinctus larvae were analysed by qRT-PCR. Whole-mount in situ hybridization results showed that all the four pNPs were expressed in the region of the apical organ of the larva, and the positive signals were also detected in the ciliary band and abdomen chaetae. We speculated that these pNPs may regulate the movement of larval cilia and chaeta by sensing external attachment signals.ConclusionsThis study represents the first comprehensive identification of neuropeptides in Echiura, and would contribute to a complete understanding on the roles of various neuropeptides in larval settlement of most marine benthonic invertebrates.

Does the frontal sensory organ in adults of the hoplonemertean Quasitetrastemma stimpsoni originate from the larval apical organ?

BackgroundThe apical organ is the most prominent neural structure in spiralian larvae. Although it has been thoroughly investigated in larvae of the class Pilidiophora in phylum Nemertea, studies on its structure in other nemertean larvae are limited. Most adult hoplonemertean worms have a frontal organ located in a position corresponding to that of the larval apical organ. The development and sensory function of the frontal organ has not been thoroughly characterized to date.ResultsThe apical organ in the early rudiment stage of Quasitetrastemma stimpsoni larvae consists of an apical plate enclosed by ducts of frontal gland cells and eight apical neurons. The apical plate is abundantly innervated by neurites of apical neurons. During the late rudiment stage, the larval apical organ has external innervation from below by two subapical-plate neurons, along with 11 apical neurons, and its plate contains serotonin-like immunoreactive (5-HT-lir) cells. In the vermicular stage (free-swimming juvenile), the number of apical neurons is reduced, and their processes are resorbed. Serotonin is detected in the apical plate with no visible connection to apical neurons. In adult worms, the frontal organ has a small apical pit with openings for the frontal gland ducts. The organ consists of 8 to 10 densely packed 5-HT-lir cells that form the roundish pit.ConclusionsAlthough the ultrastructure of the Q. stimpsoni larval apical organ closely resembles that of the apical organ of Polycladida larvae, the former differs in the presence of flask-shaped neurons typical of Spiralia. Significant differences in the structure of the apical organs of hoplonemertean and pilidia larvae point to two different paths in the evolutionary transformation of the ancestral apical organ. Ultrastructural and immunoreactive analyses of the apical organ of a hoplonemertean larva in the late rudiment and vermicular stages and the frontal organ of the adult worms identified common morphological and functional features. Thus, we hypothesize that the larval apical organ is modified during morphogenesis to form the adult frontal organ, which fulfills a sensory function in the hoplonemertean worm. This unique developmental trait distinguishes the Hoplonemertea from other nemertean groups.

Siphonariid development: Quintessential euthyneuran larva with a mantle fold innovation (Gastropoda; Panpulmonata).

Recent phylogenetic revisions of euthyneuran gastropods ("opisthobranchs" and "pulmonates") suggest that clades with a planktotrophic larva, the ancestral life history for euthyneurans, are more widely distributed along the trunk of the euthyneuran tree than previously realized. There is some indication that the planktotrophic larva of euthyneurans has distinctive features, but information to date has come mainly from traditional "opisthobranch" groups. Much less is known about planktotrophic "pulmonate" larvae. If planktotrophic larvae of "pulmonates" share unique traits with those of "opisthobranchs," then a distinctive euthyneuran larval-type has been the developmental starting template for a spectacular amount of evolved morphological and ecological disparity among adult euthyneurans. We studied development of a siphonariid by preparing sections of larval and postmetamorphic stages for histological and ultrastructural analysis, together with 3D reconstructions and data from immunolabeling of the larval apical sensory organ. We also sought a developmental explanation for the unusual arrangement of shell-attached, dorso-ventral muscles relative to the mantle cavity of adult siphonariids. Adult siphonariids ("false limpets") have a patelliform shell but their C-shaped shell muscle partially embraces a central mantle cavity, which is different from the arrangement of these components in patellogastropods ("true limpets"). It is not obvious how shell muscles extending into the foot become placed anterior to the mantle cavity during siphonariid development from a veliger larva. We found that planktotrophic larvae of Siphonaria denticulata are extremely similar to previously described, planktotrophic "opisthobranch" larvae. To emphasize this point, we update a list of distinctive characteristics of planktotrophic euthyneuran larvae, which can anchor future studies on the impressive evolvability of this larval-type. We also describe how premetamorphic and postmetamorphic morphogenesis of larval mantle fold tissue creates the unusual arrangement of shell-muscles and mantle cavity in siphonariids. This result adds to the known postmetamorphic evolutionary innovations involving mantle fold tissue among euthyneurans.

Nervous system characterization during the development of a basal echinoderm, the feather star Antedon mediterranea.

Neural development of echinoderms has always been difficult to interpret, as larval neurons degenerate at metamorphosis and a tripartite nervous system differentiates in the adult. Despite their key phylogenetic position as basal echinoderms, crinoids have been scarcely studied in developmental research. However, since they are the only extant echinoderms retaining the ancestral body plan of the group, crinoids are extremely valuable models to clarify neural evolution in deuterostomes. Antedon mediterranea is a feather star, endemic to the Mediterranean Sea. Its development includes a swimming lecithotrophic larva, the doliolaria, with basiepithelial nerve plexus, and a sessile filter-feeding juvenile, the pentacrinoid, whose nervous system has never been described in detail. Thus, we characterized the nervous system of both these developmental stages by means of immunohistochemistry and, for the first time, in situ hybridization techniques. The results confirmed previous descriptions of doliolaria morphology and revealed that the larval apical organ contains two bilateral clusters of serotonergic cells while GABAergic neurons are localized under the adhesive pit. This suggested that different larval activities (e.g., attachment and metamorphosis) are under the control of different neural populations. In pentacrinoids, the analysis showed the presence of a cholinergic entoneural system while the ectoneural plexus appeared more composite, displaying different neural populations. The expression of three neural-related microRNAs was described for the first time, suggesting that these are evolutionarily conserved also in basal echinoderms. Overall, our results set the stage for future investigations that will reveal new information on echinoderm evo-devo neurobiology.

Conserved MIP receptor–ligand pair regulates Platynereis larval settlement

Life-cycle transitions connecting larval and juvenile stages in metazoans are orchestrated by neuroendocrine signals including neuropeptides and hormones. In marine invertebrate life cycles, which often consist of planktonic larval and benthic adult stages, settlement of the free-swimming larva to the sea floor in response to environmental cues is a key life cycle transition. Settlement is regulated by a specialized sensory-neurosecretory system, the larval apical organ. The neuroendocrine mechanisms through which the apical organ transduces environmental cues into behavioral responses during settlement are not yet understood. Here we show that myoinhibitory peptide (MIP)/allatostatin-B, a pleiotropic neuropeptide widespread among protostomes, regulates larval settlement in the marine annelid Platynereis dumerilii. MIP is expressed in chemosensory-neurosecretory cells in the annelid larval apical organ and signals to its receptor, an orthologue of the Drosophila sex peptide receptor, expressed in neighboring apical organ cells. We demonstrate by morpholino-mediated knockdown that MIP signals via this receptor to trigger settlement. These results reveal a role for a conserved MIP receptor-ligand pair in regulating marine annelid settlement.

Development of the larval anterior neurogenic domains of Terebratalia transversa (Brachiopoda) provides insights into the diversification of larval apical organs and the spiralian nervous system.

BackgroundLarval features such as the apical organ, apical ciliary tuft, and ciliated bands often complicate the evaluation of hypotheses regarding the origin of the adult bilaterian nervous system. Understanding how neurogenic domains form within the bilaterian head and larval apical organ requires expression data from animals that exhibit aspects of both centralized and diffuse nervous systems at different life history stages. Here, we describe the expression of eight neural-related genes during the larval development of the brachiopod, Terebratalia transversa.ResultsRadially symmetric gastrulae broadly express Tt-Six3/6 and Tt-hbn in the animal cap ectoderm. Tt-NK2.1 and Tt-otp are restricted to a central subset of these cells, and Tt-fez and Tt-FoxQ2 expression domains are already asymmetric at this stage. As gastrulation proceeds, the spatial expression of these genes is split between two anterior ectodermal domains, a more dorsal region comprised of Tt-Six3/6, Tt-fez, Tt-FoxQ2, and Tt-otp expression domains, and an anterior ventral domain demarcated by Tt-hbn and Tt-NK2.1 expression. More posteriorly, the latter domains are bordered by Tt-FoxG expression in the region of the transverse ciliated band. Tt-synaptotagmin 1 is expressed throughout the anterior neural ectoderm. All genes are expressed late into larval development. The basiepithelial larval nervous system includes three neurogenic domains comprised of the more dorsal apical organ and a ventral cell cluster in the apical lobe as well as a mid-ventral band of neurons in the mantle lobe. Tt-otp is the only gene expressed in numerous flask-shaped cells of the apical organ and in a subset of neurons in the mantle lobe.ConclusionsOur expression data for Tt-Six3/6, Tt-FoxQ2, and Tt-otp confirm some aspects of bilaterian-wide conservation of spatial partitioning within anterior neurogenic domains and also suggest a common origin for central otp-positive cell types within the larval apical organs of spiralians. However, the field of sensory neurons within the larval apical organ of Terebratalia is broader and composed of more cells relative to those of other spiralian larvae. These cellular differences are mirrored in the broader spatial and temporal expression patterns of Tt-FoxQ2 and Tt-otp. Corresponding differences in the expression of Tt-hbn, Tt-NK2.1, and Tt-FoxG are also observed relative to their respective domains within the cerebral ganglia of spiralians. Based on these data we argue that the anterior region of the bilaterian stem species included Six3/6, NK2.1, otp, hbn, fez, and FoxQ2 expression domains that were subsequently modified within larval and adult neural tissues of protostome and deuterostome animals.

Neuromuscular development in Novocrania anomala: evidence for the presence of serotonin and a spiralian‐like apical organ in lecithotrophic brachiopod larvae

The phylogenetic position of Brachiopoda remains unsettled, and only few recent data on brachiopod organogenesis are currently available. In order to contribute data to questions concerning brachiopod ontogeny and evolution we investigated nervous and muscle system development in the craniiform (inarticulate) brachiopod Novocrania anomala. Larvae of this species are lecithotrophic and have a bilobed body with three pairs of dorsal setal bundles that emerge from the posterior lobe. Fully developed larvae exhibit a network of setae pouch muscles as well as medioventral longitudinal and transversal muscles. After settlement, the anterior and posterior adductor muscles and delicate mantle retractor muscles begin to form. Comparison of the larval muscular system of Novocrania anomala with that of rhynchonelliform (articulate) brachiopod larvae shows that the former has a much simpler muscular organization. The first signal of serotonin-like immunoreactivity appears in fully developed Novocrania anomala larvae, which have an apical organ that consists of four flask-shaped cells and two ventral neurites. These ventral neurites do not stain positively for the axonal marker alpha-tubulin in the larval stages. In the juveniles, the nervous system stained by alpha-tubulin is characterized by two ventral neurite bundles with three commissures. Our data are the first direct proof for the presence of an immunoreactive neurotransmitter in lecithotrophic brachiopod larvae and demonstrate the existence of flask-shaped serotonergic cells in the brachiopod larval apical organ, thus significantly increasing the probability that this cell type was part of the bauplan of the larvae of the last common lophotrochozoan ancestor.

Shaping the Things to Come: Ontogeny of Lophotrochozoan Neuromuscular Systems and the Tetraneuralia Concept

Despite the large variation in adult bodyplan phenotypes, a worm-shaped morphology is considered plesiomorphic for both Lophotrochozoa and Bilateria. Although almost all larval and adult lophotrochozoan worms have serially arranged ring muscles in their body wall, a comparison of their ontogeny reveals no less than six different developmental pathways that lead to this homogenous arrangement of ring muscles. However, in all taxa, with the exception of chaetodermomorph molluscs and the segmented annelids, ring muscle development starts with synchronous formation of certain pioneer myocytes, which is thus considered basal for Lophotrochozoa. Recent studies on spiralian neurogenesis revealed remnants of ancestral segmentation in echiurans and sipunculans, thus confirming molecular phylogenetic studies that propose a close relationship of these three taxa. Larval entoprocts exhibit a mosaic of larval and adult molluscan characters and, among other apomorphies, share with polyplacophoran Mollusca a complex larval apical organ and a tetraneurous nervous system, strongly suggesting a monophyletic assemblage of Entoprocta and Mollusca. The term Tetraneuralia is proposed herein for this lophotrochozoan clade. Overall, formation of the lophotrochozoan neuromuscular bodyplan appears as a highly dynamic process on both the ontogenetic and the evolutionary timescales, highlighting the importance of insights into these processes for reconstructing ancestral bodyplan features and phylogenetic relationships.

Comparative lophotrochozoan neurogenesis and larval neuroanatomy: Recent advances from previously neglected taxa

Recently, a number of neurodevelopmental studies of hitherto neglected taxa have become available, contributing to questions relating to the evolution of the nervous system of Lophotrochozoa (Spiralia + Lophophorata). As an example, neurogenesis of echiurans showed that these worm-shaped spiralians, which as adults do not exhibit any signs of segmentation, do show such traits during ontogeny, e.g. by segmentally arranged perikarya and commissures. Similarly, sipunculan worms, which have a single ventral nerve cord in the adult stage, develop this nerve cord by gradual fusion of a paired larval nerve during metamorphosis, and show transitional stages of segmentation. These findings indicate that echiurans, annelids and sipunculans stem from a segmented ancestor. By contrast, no traces of body segmentation are present during neurogenesis of basal molluscs. However, a tetraneurous condition (i.e. one pair of ventral and one pair of lateral nerve cords), as is typical for Mollusca, and a serotonergic larval apical organ that matches the complexity of polyplacophoran apical organs, were found in larval entoprocts, thus strongly supporting a mollusc-entoproct clade. Within the Lophophorata (Ectoprocta + Phoronida + Brachiopoda), data on nervous system development for any of the 3 lophophorate phyla are as of yet too scarce for profound phylogenetic inferences. Taking into account the most recent advances in molecular phylogenetics and developmental neurobiology, a scenario emerges that proposes a clade comprising Sipuncula + Annelida (including Echiura) on the one hand and a monophyletic assemblage of Entoprocta + Mollusca on the other.

The development of the serotonergic and FMRF-amidergic nervous system in Antalis entalis (Mollusca, Scaphopoda)

The morphogenesis of serotonin- and FMRF-amide-bearing neuronal elements in the scaphopod Antalis entalis was investigated by means of antibody staining and confocal laser scanning microscopy. Nervous system development starts with the establishment of two initial, flask-like, serotonergic central cells of the larval apical organ. Slightly later, the apical organ contains four serotonergic central cells which are interconnected with two lateral serotonergic cells via lateral nerve projections. At the same time the anlage of the adult FMRF-amide-positive cerebral nervous system starts at the base of the apical organ. Subsequently, the entire neuronal complex migrates behind the prototroch and the six larval serotonergic cells lose transmitter expression prior to metamorphic competence. There are no strictly larval FMRF-amide-positive neuronal structures. The development of major adult FMRF-amide-containing components such as the cerebral system, the visceral loop, and the buccal nerve cords, however, starts before the onset of metamorphosis. The anlage of the putative cerebral system is the only site of adult serotonin expression in Antalis larvae. Establishment of the adult FMRF-amidergic and serotonergic neuronal bauplan proceeds rapidly after metamorphosis. Neurogenesis reflects the general observation that the larval phase and the expression of distinct larval morphological features are less pronounced in Scaphopoda than in Gastropoda or Bivalvia. The degeneration of the entire larval apical organ before metamorphic competence argues against an involvement of this sensory system in scaphopod metamorphosis. The lack of data on the neurogenesis in the aplacophoran taxa prevent a final conclusion regarding the plesiomorphic condition in the Mollusca. Nevertheless, the results presented herein shed doubts on general theories regarding possible functions of larval "apical organs" of Lophotrochozoa or even Metazoa.

Comparative structure of the larval apical sensory organ in gastropods and hypotheses about function and developmental evolution

Summary Larval gastropods have a distinctive sensory structure located at their anterior end. This apical sensory organ typically consists of neuronal somata clustered around a medial neuropil region (the apical ganglion) and cells giving rise to two or three long tufts of cilia. The neuronal nature of the tuft cells has not been firmly established. Putative sensory neurons within the apical ganglion have a dendrite-like extension and one type has an invaginated pocket filled with cilia. Another type of putative sensory neuron shows serotonin-like immunoreactivity. Serotonin-like antigenicity may also be expressed by non-sensory neurons within the apical ganglion and is invariably expressed by neurites within the neuropil, and by peripheral neurites extending toward the peripheral, prototrochal ciliary band of the velum. I review evidence suggesting that the apical ganglion is a sensory-motor ganglion that coordinates activity of velar prototrochal cilia during the free-living larval stage and during encapsulated developmental stages. Recent evidence also implicates the apical sensory organ in the reception or integration of sensory cues that initiate metamorphosis. Finally, I map two traits of the larval sensory organ on a phylogenetic hypothesis for relationships between major gastropod clades to infer patterns of evolutionary change for this highly conserved larval structure.

Larval and Adult Characters in Animal Phytogeny

SYNOPSIS. Protostomes and deuterostomes can be characterized by two completely separate sets of characters. Protostome characters are downstream- collecting ciliary bands with compound cilia on multiciliate cells and central nervous system with apical brain and ventral paired or fused cords; spiral cleavage is only found in the protostome group Spiralia. Deuterostome characters are upstream-collecting ciliary systems with separate cilia on monociliate cells, dorsal central nervous system and loss of the larval apical organ in the adults, and archimery with prosome, mesosome and metasome. Only the ectoproct bryozoans lack all the justmentioned characters; they are referred to the protostomes on basis of their metamorphosis which resembles that of entoprocts. The two bilateral groups have probably evolved independently from a radial, gastraea-like ancestor

Metamorphosis of the marine bryozoan Membranipora membranacea: An ultrastructural study of rapid morphogenetic movements.

The colonial marine bryozoan Membranipora membranacea produces a planktotrophic cyphonautes larva that is encased by a triangular bivalved shell. Following a relatively long free-swimming phase, the cyphonautes larva settles and undergoes a dramatic metamorphosis to become the sessile progenitor of the colony, referred to as the ancestrula. This paper examines the initial morphogenetic movements of metamorphosis that transform the cyphonautes larva into an incipient ancestrula. At the onset of metamorphosis, contractions of the striated median muscles situated along the anterior and posterior margins of the larva cause a retraction of the larval apical organ and a centripetal movement of the anterior and posterior ends of the larva. Concurrently, the ciliated corona at the base of the larva is pulled within the shell by contractions of the striated lateral muscles. As the larva assumes a more spherical shape, the posterior margins of the shell are spread apart, and the internal sac is everted. Eversion of the sac is apparently achieved by contractions of the lateral muscles that cause a buckling of the shell in the apical-basal direction. The neck region of the everted sac secretes adhesive granules that attach the larva to the substratum. Subsequently, contractions of the nonstriated sac muscles fold the valves of the shell over each other and draw the larva toward the substratum. The initial events of metamorphosis that culminate in the attachment and flattening of the larva are completed in 10-15 seconds. In the subsequent few minutes, the lateral edges of the everted sac fuse with the neighboring margins of the aboral epithelium underlying the shell and thus form the fully sealed body wall of the incipient ancestrula.