An externally brooding acorn worm (Hemichordata, Enteropneusta, Torquaratoridae) from the Russian arctic.

A single specimen of a previously undescribed acorn worm in the family Torquaratoridae was trawled from a bottom depth of about 350 m in the Kara Sea (Russian Arctic). The new species is the shallowest of the exclusively deep-sea torquaratorids found to date, possibly an example of high-latitude emergence. On the basis of ribosomal DNA sequences and morphology, the worm is described here as the holotype of Coleodesmium karaensis n. gen., n. sp. It is most similar in overall body shape to the previously described enteropneust genus Allapasus, but is uniquely characterized by a tubular component of the proboscis skeleton ensheathing the collar nerve cord. Additionally, within the proboscis, the sparseness of the musculature of C. karaensis clearly distinguishes it from the much more muscular members of Allapasus. The holotype is a female bearing about a dozen embryos on the surface of her pharyngeal region, each recessed within a shallow depression in the dorsal epidermis. The embryos, ranging from late gastrula to an early stage of coelom formation, are a little more than 1 mm in diameter and surrounded by a thin membrane. Each embryo comprises an external ectoderm of monociliated cells (not arranged in obvious ciliated bands) and an internal endo-mesoderm; the blastopore is closed. In the most advanced embryos, the anterior coelom is starting to constrict off from the archenteron. Coleodesmium karaensis is the first enteropneust (and indeed the first hemichordate) found brooding embryos on the surface of the mother's body.

Biomineral ultrastructure, elemental constitution and genomic analysis of biomineralization-related proteins in hemichordates.

Here, we report the discovery and characterization of biominerals in the acorn worms Saccoglossus bromophenolosus and Ptychodera flava galapagos (Phylum: Hemichordata). Using electron microscopy, X-ray microprobe analyses and confocal Raman spectroscopy, we show that hemichordate biominerals are small CaCO(3) aragonitic elements restricted to specialized epidermal structures, and in S. bromophenolosus, are apparently secreted by sclerocytes. Investigation of urchin biomineralizing proteins in the translated genome and expressed sequence tag (EST) libraries of Saccoglossus kowalevskii indicates that three members of the urchin MSP-130 family, a carbonic anhydrase and a matrix metaloprotease are present and transcribed during the development of S. kowalevskii. The SM family of proteins is absent from the hemichordate genome. These results increase the number of phyla known to biomineralize and suggest that some of the gene-regulatory 'toolkit', if not mineralized tissue themselves, may have been present in the common ancestor to hemichordates and echinoderms.

[Electron microscopic study of the intestinal epithelium of Saccoglossus mereschkowskii (Enteropneusta, Hemichordata)].

Epithelium of the hepatic region of the intestine in Saccoglossus mereschkowskii, a representative of enteropneusts (Enteropneusta, Hemichordata) standing at the base of Chordata, has been investigated using electron microscope. The ultrastructure of ciliated and granular epithelial cells, elements of the intraepithelial nerve layer, and intercellular junctions have been characterized. The data concerning details of the organization of the ciliary apparatus and rootlets system are presented. It is justified the presence of complicated supporting construction of cilia which performs a mechanical stabilizing function and possibly also provide synchronization of ciliary movements. The presence of cilia with two centrioles is considered as an adaptation to high functional load on ciliary apparatus. Well developed bundles of myofilaments are found in the cytoplasm of the basal portions of ciliary cells that characterizes these cells as myoepithelial. The features indicating the role of ciliary cells in absorption are described. The capability of these cells to balloon-like secretion is considered. Data on the accumulation of food reserves in the form of lipid droplets and glycogen in the cell cytoplasm are presented. Ciliated cells are characterized by their function as ciliated secretory-absorptive myoepithelial cells. Based on the location of secretory granules both in the apical and basal portions of granular cells, an exocrine-endocrine function of these cells has been suggested. Typical endocrine cells in the intestinal epithelium of S. mereschkowskii are absent. Several types of granules in the nerve fibers cytoplasm are described. Junctions between the nerve fibers and basal portions of ciliary and granular epithelial cells are found. Nerve regulation of contractile and secretory functions of epithelial cells is supposed. The presence of the regulatory nerve-endocrine system that includes receptor cells of open type, secretory endocrine-like cells and nerve elements of nerve layer is supposed in the intestinal epithelium of enteropneusts.

Cilia-like structures anchor the amphioxus notochord to its sheath.

Body stiffness is important during undulatory locomotion in fish. In amphioxus, the myosepta play an important role in transmission of muscular forces to the notochord. In order to define the specific supporting role of the notochord in amphioxus during locomotion, the ultrastructure of 10 adult amphioxus specimens was analyzed using transmission electron microscopy. Numerous cilia-like structures were found on the surface of each notochordal cell at the sites of their attachment to the notochordal sheath. Ultrastructurally, these structures consisted of the characteristic arrangement of peripheral and central microtubular doublets and were anchored to the inner layer of the notochordal sheath. Immunohistochemically, a positive reaction to applied dynein and ß-tubulin antibodies characterized the area of the cilia-like structures. We propose that reduced back-and-forth movements of the cilia-like structures might contribute to the flow of the fluid content inside the notochord, thus modulating the stiffness of the amphioxus body during its undulatory locomotion.

Regeneration in the hemichordate Ptychodera flava.

When the body of P. flava is severed, the animal has the ability to regenerate its missing anterior or posterior as appropriate. We have focused on anterior regeneration when the head and branchial regions are severed from the body of the worm. After transection, the body wall contracts and heals closed in 2 to 3 days. By the third day a small blastema is evident at the point of closure. The blastema grows rapidly and begins the process of differentiating into a head with a proboscis and collar. At 5 days the blastema has increased greatly in size and differentiated into a central bulb, the forming proboscis, and two lateral crescents, the forming collar. Between 5 and 7 days a mouth opens ventral to the differentiating blastema. Over the next few days the lateral crescents extend to encircle the proboscis and mouth, making a fully formed collar. By 10 to 12 days a new head, sized to fit the worm's body, has grown attached to the severed site. At about this time the animal regains apparently normal burrowing behavior. After the head is formed, a second blastema-like area appears between the new head and the old body and a new branchial region is inserted by regeneration from this blastema over the next 2 to 3 weeks. The regenerating tissues are unpigmented and whitish such that in-situ hybridization can be used to study the expression of genes during the formation of new tissues.

Development of the enteropneust Ptychodera flava: ciliary bands and nervous system.

Ripe specimens of Ptychodera flava were collected at Paiko Peninsula, Oahu, Hawaii, USA, and the development from egg to tornaria larva was followed in the laboratory. To complete the series, large tornaria larvae were collected from the plankton off the nearby Ala Moana Beach, and followed through metamorphosis to a juvenile stage with four pairs of gill slits. Ciliary band development was examined by scanning electron microscopy, and the development of the serotonergic nervous system was followed by means of immunostaining. The development of the apical tuft and neotroch (circumoral/perioral ciliary band) and their subsequent degeneration accorded fully with previous descriptions. A perianal ciliary ring of separate cilia develops just after hatching. This later develops a midventral extension, the neurotroch, extending to the neotroch posterior to the mouth. The cilia of this ring apparently beat diaplectically, with the effective stroke in the clockwise direction when seen from behind. An additional ring of cilia develops several days later anterior to the perianal ring. This opisthotroch (called telotroch by previous authors) consists at first of separate cilia, but later they became organized as large compound cilia. The apical tuft disappears after about a week, the neotroch degenerates at the transition to the Agassiz stage, and the opisthotroch degenerates just after metamorphosis. The serotonergic nervous system of the fully grown tornaria consists of an apical ganglion with many perikarya, a paired lateral group of perikarya on the postoral ciliary band, and scattered perikarya along the opisthotroch. Serotonergic processes are found along the ciliary bands except for the ventral and perianal ciliary bands and are scattered along the epidermis. At the Spengel stage and at metamorphosis (Agassiz stage), the processes along the ciliary bands are concentrated in the three ciliated food grooves so as to form three separate nerves, and are retained on the proboscis at least until 2-3 gill slit stage. No serotonergic processes were found to extend from the proboscis to the collar region, and no serotonergic neurons were observed in the collar cord or in the ventral nerve cord. Our results therefore do not provide any clues as to the origin of the chordate neural tube relative to the dorsal-ventral orientation of the enteropneusts.

The notochordal sheath in amphioxus--an ultrastructural and histochemical study.

The notochord and notochordal sheath of 10 adult amphioxus were investigated ultrastructurally and histochemically. The notochord in amphioxus consists of parallel notochordal cells (plates) and each plate consists of parallel thicker and thinner fibrils and numerous profiles of smooth endoplasmic reticulum situated just beneath the cell membrane. Histochemical staining shows that the notochordal plates resemble neither the connective tissue notochordal sheath nor the typical muscular structure myotomes. The notochordal sheath has a complex three-layered organization with the outer, middle and inner layer The outer and middle layer are composed of collagen fibers of different thickness and course, that correspond to collagen type I and collagen type III in vertebrates, respectively, and the inner layer is amorphous, resembles basal lamina, and is closely attached to the notochord by hemidesmosome junctions. These results confirm the presence of collagen fibers and absence of elastic fibers in amphioxus.

Eggs and embryos in Xenoturbella (phylum uncertain) are not ingested prey.

Xenoturbella is an enigmatic animal that has puzzled science for almost a century. The eggs and embryos found in Xenoturbella have recently been interpreted as ingested prey. However, PCR on individual eggs as well as in situ hybridisation and in situ PCR unambiguously show that they are Xenoturbella's own. The eggs and embryos are individually enclosed within follicles with the same ultrastructure. The cortical granules in oocytes and eggs from Xenoturbella but not Nucula stained positively with an antiserum against Reissner's substance. The embryos incorporated 5-bromodeoxyuridine in vivo, i.e. they replicate their genome and are living.

Expression of AmphiCoe, an amphioxus COE/EBF gene, in the developing central nervous system and epidermal sensory neurons.

The COE/EBF gene family marks a subset of prospective neurons in the vertebrate central and peripheral nervous system, including neurons deriving from some ectodermal placodes. Since placodes are often considered unique to vertebrates, we have characterised an amphioxus COE/EBF gene with the aim of using it as a marker to examine the timing and location of peripheral neuron differentiation. A single COE/EBF family member, AmphiCoe, was isolated from the amphioxus Branchiostoma floridae. AmphiCoe lies basal to the vertebrate COE/EBF genes in molecular phylogenetic analysis, suggesting that the duplications that formed the vertebrate COE/EBF family were specific to the vertebrate lineage. AmphiCoe is expressed in the central nervous system and in a small number of scattered ectodermal cells on the flanks of neurulae stage embryos. These cells become at least largely recessed beneath the ectoderm. Scanning electron microscopy was used to examine embryos in which the ectoderm had been partially peeled away. This revealed that these cells have neuronal morphology, and we infer that they are the precursors of epidermal primary sensory neurons. These characters lead us to suggest that differentiation of some ectodermal cells into sensory neurons with a tendency to sink beneath the embryonic surface represents a primitive feature that has become incorporated into placodes during vertebrate evolution.

The dorsal compartment locomotory control system in amphioxus larvae.

Amphioxus myotomes consist of separate sets of superficial and deep muscle fibers, each with its own innervation, that are thought to be responsible for slow swimming and escape behavior, respectively. Tracings from serial EM sections of the anterior nerve cord in the larva show that the motoneurons and premotor interneurons controlling the superficial fibers (the dorsal compartment, or DC pathway) are linked by specialized junctions of a previously undescribed type, referred to here as juxta-reticular (JR) junctions for the characteristic presence of a cisterna of endoplasmic reticulum on each side. JR junctions link the DC motoneurons with each other, with the largest of the anterior paired neurons (LPN3s) and with one class of ipsilateral projection neurons (IPNs), but occur nowhere else. Because of the paucity of synaptic input to the DC system, larval behavior can only be explained if the JR junctions act as functional links between cells. An analysis of the pattern of cell contacts also suggests that the LPN3s are probably pacemakers for both slow and fast locomotion, but act through junctions in the former case and conventional synapses in the latter. The only major synaptic input to the DC system identified in somites 1 and 2 was from four neurons located in the cerebral vesicle, referred to here as Type 2 preinfundibular projection neurons (PPN2s). They have unusually large varicosities, arranged in series, that make periodic contacts with the DC motoneurons. More caudally, the DC motoneurons receive additional input via similar large varicosities from the receptor cells of the first dorsal ocellus, located in somite 5. The overall circuitry of the locomotory control system suggests that the PPN2s may be instrumental in sustaining slow swimming, whereas mechanical stimulation, especially of the rostrum, preferentially activates the fast mode.

Heads or tails? Amphioxus and the evolution of anterior-posterior patterning in deuterostomes.

In Xenopus, the canonical Wnt-signaling pathway acting through beta-catenin functions both in establishing the dorso-ventral axis and in patterning the anterior-posterior axis. This pathway also acts in patterning the animal-vegetal axis in sea urchins. However, because sea urchin development is typically indirect, and adult sea urchins have pentamerous symmetry and lack a longitudinal nerve cord, it has not been clear how the roles of the canonical Wnt-signaling pathway in axial patterning in sea urchins and vertebrates are evolutionarily related. The developmental expression patterns of Notch, brachyury, caudal, and eight Wnt genes have now been determined for the invertebrate chordate Amphioxus, which, like sea urchins, has an early embryo that gastrulates by invagination, but like vertebrates, has a later embryo with a dorsal hollow nerve cord that elongates posteriorly from a tail bud. Comparisons of Amphioxus with other deuterostomes suggest that patterning of the ancestral deuterostome embryo along its anterior-posterior axis during the late blastula and subsequent stages involved a posterior signaling center including Wnts, Notch, and transcription factors such as brachyury and caudal. In tunicate embryos, in which cell numbers are reduced and cell fates largely determined during cleavage stages, only vestiges of this signaling center are still apparent; these include localization of Wnt-5 mRNA to the posterior cytoplasm shortly after fertilization and localization of beta-catenin to vegetal nuclei during cleavage stages. Neither in tunicates nor in Amphioxus is there any evidence that the canonical Wnt-signaling pathway functions in establishment of the dorso-ventral axis. Thus, roles for Wnt-signaling in dorso-ventral patterning of embryos may be a vertebrate innovation that arose in connection with the evolution of yolky eggs and gastrulation by extensive involution.

Characterization of Amphioxus AmphiVent, an evolutionarily conserved marker for chordate ventral mesoderm.

Structure and developmental expression are described for amphioxus AmphiVent, a homolog of vertebrate Vent genes. In amphioxus, AmphiVent-expressing ventral mesoderm arises at midneurula by outgrowth from the paraxial mesoderm, but in vertebrates, Vent-expressing ventral mesoderm originates earlier, at the gastrula stage. In other embryonic tissues (nascent paraxial mesoderm, neural plate, endoderm, and tailbud), AmphiVent and its vertebrate homologs are expressed in similar spatiotemporal domains, indicating conservation of many Vent gene functions during chordate evolution. The ventral mesoderm evidently develops precociously in vertebrates because their relatively large embryos probably require an early and extensive deployment of the mesoderm-derived circulatory system. The vertebrate ventral mesoderm, in spite of its strikingly early advent, still resembles the nascent ventral mesoderm of amphioxus in expressing Vent homologs. This coincidence may indicate that Vent homologs in vertebrates and amphioxus play comparable roles in ventral mesoderm specification.

Three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) associated with the tail bud: the evolution of somitogenesis in chordates.

The amphioxus tail bud is similar to the amphibian tail bud in having an epithelial organization without a mesenchymal component. We characterize three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) and show that their early expression around the blastopore can subsequently be traced into the tail bud; in vertebrate embryos, there is a similar progression of expression domains for Wnt3, Wnt5, and Wnt6 genes from the blastopore lip (or its equivalent) to the tail bud. In amphioxus, AmphiWnt3, AmphiWnt5, and AmphiWnt6 are each expressed in a specific subregion of the tail bud, tentatively suggesting that a combinatorial code of developmental gene expression may help generate specific tissues during posterior elongation and somitogenesis. In spite of similarities within their tail buds, vertebrate and amphioxus embryos differ markedly in the relation between the tail bud and the nascent somites: vertebrates have a relatively extensive zone of unsegmented mesenchyme (i.e., presomitic mesoderm) intervening between the tail bud and the forming somites, whereas the amphioxus tail bud gives rise to new somites directly. It is likely that presomitic mesoderm is a vertebrate innovation made possible by developmental interconversions between epithelium and mesenchyme that first became prominent at the dawn of vertebrate evolution.

Sulphated polyanions in cytoplasm and nuclei of epithelial cells of Branchiostoma demonstrated by the cationic dye Cupromeronic Blue.

The intracellular occurrence and distribution of sulphated polyanions, interpreted to represent mucins, were studied in secretory epithelial cells in the primitive chordates Branchiostoma lanceolatum and B. floridae at the electron microscopical level by using Cupromeronic Blue (CMB). CMB-precipitates were mainly found within two potential types of mucin vesicles (apical and basal) and Golgi cisterns. The mucin vesicles form a distinct population of secretory granules different from another nonmucin granule population. Within the epidermal cells the staining intensity of the Golgi cisterns with CMB increased from the cis to the trans compartment. The pharyngeal mucous cells showed staining only in the trans Golgi compartment. These findings indicate, that CMB can be used for intracellular localization of mucins and that sulphation of the mucins in the investigated cells may occur within different compartments of the Golgi complex. Apparently the mucin is secreted apically but only in the epidermis it forms a dense layer covering the apical microvilli. In the Branchiostoma epidermal cells a layer of specialized basal vesicles occurred, containing unusually large and branched CMB-precipitates which possibly serve mechanical functions. In the nuclei CMB-precipitates were regularly demonstrated in the euchromatin of the cell types studied.

Electron microscopic study of the development of amphioxus, Branchiostoma belcheri tsingtauense: the neurula and larva.

The later development of Asian amphioxus was investigated by scanning and transmission electron microscopy (SEM and TEM). The age ranged from 10.5 hr (the early neurula) to 48 hr after fertilization (the larva with two gill-slits). This period was divided into 6 stages; the neurula (N) 1, 2, 3, and the larva (L) 1, 2, 3. At stage N1, the lateral edge of the flattened neural plate became stratified and the superficial layer separated from the deeper one to spread over the neural plate toward the median line where they fuse randomly with each other. The notochord and the mesoderm were formed by folding from the dorsomedian and the dorsolateral wall of the archenteron during stages N1 to N2. At stage N3, a typical triploblastic embryo was formed, consisting of the definitive ectoderm, neural tube, notochord, endoderm and mesoderm (the wall of the coelom). Ultrastructurally, paramyosin fibrils in the notochordal cells and myofibrils in the muscle cells were found at stage L1. After stage L1, the anteroposterior differentiation took place and the formation of the larval organs progressed. Self-feeding larval life was established at stage L3 when the larva acquired the mouth and two gill-slits.

Septate junctions with paired septa in the cephalochordate Branchiostoma lanceolatum.

The cells of the atrial epithelium of Branchiostoma lanceolatum are interconnected by an apical zonula adhaerens and a septate junction extending between the apical zonula adhaerens and a level corresponding to the middle of the nucleus. The spacing of the septa, which are relatively few in number (about 10), varies considerably. Within the junction paired and unpaired septa occur. The thickness of the paired septa measures 16-25 nm, the distance between the individual septa of the paired structure 6-12 nm, and the intercellular space at the site traversed by the septa 17-20 nm. At the intersection between three cells the septa (paired or unpaired) delineate a central triangular space.

The fine structure of the deep muscle lamellae and their sarcoplasmic reticulum in Branchiostoma lanceolatum.

The ultrastructure of the deep lamellae of Branchiostoma's trunk muscle was investigated electron microscopically using quantitative morphometry and extracellular markers. Earlier measurements by Flood [7] were mostly confirmed and further extended. The controversy about the arrangement of the sarcoplasmic reticulum (SR) was widely resolved. The extracellular space occupies only 4.0% of the whole muscle volume and is freely accessible to extracellular markers (ferritin, lanthanum). The SR consists of cisternae without tubular interconnections. It occupies 5 to 11% of the cell volume. A junctional gap of 11 to 15 nm width separates the cisternae from the surface membrane and is bridged by typical junctional feet, 11 nm long and 10 to 20 nm in diameter. A Markham rotation analysis revealed a well defined tetragonal pattern with a centre-to-centre feet distance of 34 to 38 nm. A transverse tubular system is absent in the main part of the sheet-like muscle cells. However in basal regions with double layers of myofibrils, extracellular channels surrounded by SR cisternae could be detected within the muscle cells. The results show that there are no principal differences in membranal contacts between the diads of Branchiostoma and vertebrate triads. From these data and physiological findings [23, 24] it seems unlikely that main differences exist in the mode of excitation-contraction coupling between these muscle cells.

[Comparative morphology of intercellular contacts]. / Sravnitel'naia morfologiia mezhkletochnykh kontaktov.

The data of the literature on ultrastructure of the intercellular contacts in main types of Metazoa (Spongia, Coelenterata, Plahelminthes, Annelides, Mollusca, Arthropods, Echinodermata and Chordata) have been analysed in view of systemic organology. Function of the intercellular contacts is considered as system-forming. Three aspects in the system-forming function of the intercellular contacts have been determined--adhesion, communication and isolation. Increase in the system-forming function of these structures has been demonstrated to be the main regularity in the evolution of the intercellular contacts in animals; this point is proved by analysing all the three aspects. A suggestion is made on the source for appearance of tight junctions.