Regeneration of the acorn worm pygochord with the implication for its convergent evolution with the notochord.

The origin of the notochord is a central issue in chordate evolution. This study examined the development of the acorn worm pygochord, a putative homologue of the notochord. Because the pygochord differentiates only after metamorphosis, the developmental was followed process by inducing regeneration after artificial amputation in Ptychodera flava. It was found that although the regeneration of the posterior part of the body did not proceed via formation of an obvious regeneration bud, pygochord regeneration was observed within a few weeks, possibly via trans-differentiation of endoderm cells. The expression of the fibrillary collagen gene (Fcol) and elav in the pygochord during regeneration was detected. This indicates that pygochord cells are not part of gut epithelial cells, but that they differentiated as a distinct cell type. Our gene expression analyses do not provide supporting evidence for the homology between the pygochord and notochord, but rather favored the convergent evolution between them.

Evolution of vertebrates as viewed from the crest.

The origin of vertebrates was accompanied by the advent of a novel cell type: the neural crest. Emerging from the central nervous system, these cells migrate to diverse locations and differentiate into numerous derivatives. By coupling morphological and gene regulatory information from vertebrates and other chordates, we describe how addition of the neural-crest-specification program may have enabled cells at the neural plate border to acquire multipotency and migratory ability. Analysis of the topology of the neural crest gene regulatory network can serve as a useful template for understanding vertebrate evolution, including elaboration of neural crest derivatives.

Dissecting the determinants of light sensitivity in amphioxus microvillar photoreceptors: possible evolutionary implications for melanopsin signaling.

Melanopsin, a photopigment related to the rhodopsin of microvillar photoreceptors of invertebrates, evolved in vertebrates to subserve nonvisual light-sensing functions, such as the pupillary reflex and entrainment of circadian rhythms. However, vertebrate circadian receptors display no hint of a microvillar specialization and show an extremely low light sensitivity and sluggish kinetics. Recently in amphioxus, the most basal chordate, melanopsin-expressing photoreceptors were characterized; these cells share salient properties with both rhabdomeric photoreceptors of invertebrates and circadian receptors of vertebrates. We used electrophysiology to dissect the gain of the light-transduction process in amphioxus and examine key features that help outline the evolutionary transition toward a sensor optimized to report mean ambient illumination rather than mediating spatial vision. By comparing the size of current fluctuations attributable to single photon melanopsin isomerizations with the size of single-channels activated by light, we concluded that the gain of the transduction cascade is lower than in rhabdomeric receptors. In contrast, the expression level of melanopsin (gauged by measuring charge displacements during photo-induced melanopsin isomerization) is comparable with that of canonical visual receptors. A modest amplification in melanopsin-using receptors is therefore apparent in early chordates; the decrease in photopigment expression-and loss of the anatomical correlates-observed in vertebrates subsequently enabled them to attain the low photosensitivity tailored to the role of circadian receptors.

The light-sensitive conductance of melanopsin-expressing Joseph and Hesse cells in amphioxus.

Two types of microvillar photoreceptors in the neural tube of amphioxus, an early chordate, sense light via melanopsin, the same photopigment as in "circadian" light detectors of higher vertebrates. Because in amphioxus melanopsin activates a G(q)/phospholipase C cascade, like phototransduction in arthropods and mollusks, possible commonalities in the photoconductance were investigated. Unlike other microvillar photoreceptors, reversal of the photocurrent can only be attained upon replacement of extracellular Na(+). In addition to Na(+), Ca(2+) is also permeant, as indicated by the fact that (a) in normal ionic conditions the photocurrent remains inward at V(m) > E(Na); (b) in Na-free solution a small residual inward photocurrent persists at V(m) near resting level, provided that Ca is present; and (c) V(rev) exhibits a modest shift with [Ca](o) manipulations. The unusual reversal is accounted for by an uncommonly low permeability of the light-dependent channels to K(+), as [K](o) only marginally affects the photocurrent amplitude and its reversal. Lanthanum and ruthenium red (RuR), two TRP channel antagonists, reversibly suppress the response to photostimulation of moderate intensity; therefore, the melanopsin-initiated cascade may recruit ion channels of the same family as those of rhabdomeric photoreceptors. With brighter lights, blockage declines, so that both La(3+) and RuR induce a right shift in the sensitivity curve without a reduction of its asymptote. Nonetheless, an effect on the transduction cascade, rather than the channels, was ruled out on the basis of the voltage dependency of the blockade and the lack of effects of intracellular application of the same substances. The mechanisms of action of these antagonists thus entail a state-dependent blockade, with a higher affinity for the channel in the closed conformation. Collectively, the results indicate a kinship of the light-sensitive channels of amphioxus with those of invertebrate rhabdomeric visual cells and support the representation of this lineage of photoreceptors among chordates.

Morphological characterization of the asexual reproduction in the acorn worm Balanoglossus simodensis.

The acorn worm Balanoglossus simodensis reproduces asexually by fragmentation and subsequent regeneration from the body fragments. We examined the morphogenesis of its asexual reproduction. At first, we collected asexually reproducing specimens and observed their morphogenesis. Then, we succeeded in inducing the asexual reproduction artificially by cutting the worm at the end of the genital region. The process of morphogenesis is completely the same between naturally collected and artificially induced specimens. The stages during morphogenesis were established on the basis of the external features of the asexually reproducing fragments. The internal features of the fragments were also examined at each stage. In a separate phase of the study, the capacity for regeneration of some body parts was also examined by dividing intact worms into about 10 fragments. Although the capacity for regeneration varied among the different body parts, some fragments regenerated into complete individuals in 1 month. The process of regeneration was the same as that in the asexually produced fragments.

Ontogeny of the collar cord: neurulation in the hemichordate Saccoglossus kowalevskii.

The chordate body plan is characterized by a central notochord, a pharynx perforated by gill pores, and a dorsal central nervous system. Despite progress in recent years, the evolutionary origin of each of theses characters remains controversial. In the case of the nervous system, two contradictory hypotheses exist. In the first, the chordate nervous system is derived directly from a diffuse nerve net; whereas, the second proposes that a centralized nervous system is found in hemichordates and, therefore, predates chordate evolution. Here, we document the ontogeny of the collar cord of the enteropneust Saccoglossus kowalevskii using transmission electron microscopy and 3D-reconstruction based on completely serially sectioned stages. We demonstrate that the collar cord develops from a middorsal neural plate that is closed in a posterior to anterior direction. Transversely oriented ependymal cells possessing myofilaments mediate this morphogenetic process and surround the remnants of the neural canal in juveniles. A mid-dorsal glandular complex is present in the collar. The collar cord in juveniles is clearly separated into a dorsal saddle-like region of somata and a ventral neuropil. We characterize two cell types in the somata region, giant neurons and ependymal cells. Giant neurons connect via a peculiar cell junction that seems to function in intercellular communication. Synaptic junctions containing different vesicle types are present in the neuropil. These findings support the hypotheses that the collar cord constitutes a centralized element of the nervous system and that the morphogenetic process in the ontogeny of the collar cord is homologous to neurulation in chordates. Moreover, we suggest that these similarities are indicative of a close phylogenetic relationship between enteropneusts and chordates.

Anterior regeneration in the hemichordate Ptychodera flava.

Ptychodera flava is a hemichordate whose anterior structures regenerate reproducibly from posterior trunk pieces when amputated. We characterized the cellular processes of anterior regeneration with respect to programmed cell death and cell proliferation, after wound healing. We found scattered proliferating cells at day 2 of regeneration using a proliferating cell nuclear antigen antibody. On day 4, most proliferating cells were associated with the nerve tract under the epidermis, and on day 6, a small proboscis derived from proliferated cells was regenerated, and a mouth had broken though the epidermis. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling) detected elevated levels of apoptosis in the endoderm that began furthest away from the region of wound healing, then moved anteriorly over 8 days. Posterior to anterior apoptosis is likely to remove digestive endoderm for later differentiation of pharyngeal endoderm. We hypothesize that P. flava regeneration is nerve dependent and that remodeling in the gut endoderm plays an important role in regeneration.

The expression of AmphiTCTP, a TCTP orthologous gene in amphioxus related to the development of notochord and somites.

The translationally controlled tumor protein (TCTP) is highly conserved and has been widely found in eukaryotic organisms. Here, we report the phylogenetic analysis and developmental expression of AmphiTCTP, a TCTP homologous gene in cephalochordate amphioxus. Phylogenetic analysis indicates that the putative protein of AmphiTCTP is close to its vertebrate orthologs. The mRNA of AmphiTCTP is found in fertilized eggs, early cleavage embryo and most of the early developmental stages by in situ hybridization and RT-PCR, but its expression is not detectable from late cleavage stage to mid-gastrula. The expression of AmphiTCTP in zygotes and early cleavage stages shows that AmphiTCTP may be a maternal gene. From the early neurula stage onward, AmphiTCTP transcript is localized in the presumptive notochord, presomitic mesoderm, and nascent somites. However, its expression is gradually down-regulated after the notochord and somites have been formed. The expression pattern of AmphiTCTP thus coincides with the differentiation of the notochord and somites, this suggests that AmphiTCTP may not be a housekeeping gene and may play an important role in mesoderm development.

The identification of lymphocyte-like cells and lymphoid-related genes in amphioxus indicates the twilight for the emergence of adaptive immune system.

To seek evidence of a primitive adaptive immune system (AIS) before vertebrate, we examined whether lymphocytes or lymphocyte-like cells and the related molecules participating in the lymphocyte function existed in amphioxus. Anatomical analysis by electron microscopy revealed the presence of lymphocyte-like cells in gills, and these cells underwent morphological changes in response to microbial pathogens that are reminiscent of those of mammalian lymphocytes executing immune response to microbial challenge. In addition, a systematic comparative analysis of our cDNA database of amphioxus identified a large number of genes whose vertebrate counterparts are involved in lymphocyte function. Among these genes, several genes were found to be expressed in the vicinity of the lymphocyte-like cells by in situ hybridization and up-regulated after exposure to microbial pathogens. Our findings in the amphioxus indicate the twilight for the emergence of AIS before the invertebrate-vertebrate transition during evolution.

Stage- and tissue-specific patterns of cell division in embryonic and larval tissues of amphioxus during normal development.

The distribution of dividing cells is described for embryos and larvae of amphioxus (Branchiostoma floridae) pulse labeled with bromodeoxyuridine. Because cell division is assessed for all of the developing tissues, this is the first comprehensive study of developmental cell proliferation for an animal lacking a stereotyped cell lineage. In amphioxus, cell divisions are virtually synchronous during cleavage, but become asynchronous at the blastula stage. Starting at the neurula stage, after the origin of the mesoderm, the proportion of dividing cells progressively declines in the somitic mesoderm and notochord. Other tissues, however, deviate from this pattern. For example, in the mid-neurula, there is a brief, intense burst of mitosis at the anterior end of the neural plate. Also, from the neurula through the early larval stage, all of the ectoderm cells cease dividing and develop cilia that propel the animal through the water; subsequently, in the epidermis of later larvae, mitosis resumes and the proportion of ciliated cells declines as muscular undulation gradually replaces ciliation for swimming. Finally, in the early larvae, there is a terminal arrest of cell division in three cell types that differentiate early to participate in feeding as soon as the mouth opens-namely the ciliated pharyngeal cells that produce the feeding current and the secretory cells of the club-shaped gland and endostyle that export food-trapping mucus into the pharynx. In sum, these stage- and tissue-specific changes in cell proliferation intensity illustrate how the requirements of embryonic and larval natural history can shape developmental programs.

Acrosome reaction in spermatozoa from the amphioxus Branchiostoma belcheri (Cephalochordata, Chordata).

The formation of an acrosomal process at acrosomal exocytosis in spermatozoa of the amphioxus was described in the present report for the first time. A non-reacted acrosome was located in front of the nucleus, where a cup-shaped acrosomal vesicle covered a conical accumulation of subacrosomal material. When naturally spawned spermatozoa were treated with a calcium ionophore, ionomycin, the acrosomal vesicle opened at the apex and an acrosomal process was projected. The process exhibited a filamentous structure. The reaction followed the mode typically seen in marine invertebrates. These observations suggest that the features and function of the acrosome of amphioxus, whose position is on the border between invertebrates and vertebrates, reflect their ecological adaptation and phylogenic position.

The chordate amphioxus: an emerging model organism for developmental biology.

The cephalochordate amphioxus is the closest living invertebrate relative of the vertebrates. It is vertebrate-like in having a dorsal, hollow nerve cord, notochord, segmental muscles, pharyngeal gill slits and a post-anal tail that develops from a tail bud. However, amphioxus is less complex than vertebrates, lacking neural crest and having little or no mesenchyme. The genetic programs patterning the amphioxus embryo are also similar to those patterning vertebrate embryos, although the amphioxus genome lacks the extensive gene duplications characteristic of vertebrates. This relative structural and genomic simplicity in a vertebrate-like organism makes amphioxus ideal as a model organism for understanding mechanisms of vertebrate development.

Cytoarchitecture, topography, and descending supraspinal projections in the anterior central nervous system of Branchiostoma lanceolatum.

The central nervous system (CNS) of the chordate amphioxus (Branchiostoma lanceolatum) is divisible into a spinal cord and an anterior portion in some ways equivalent to the brain of craniates. The present study reports on this anterior portion, with respect to general topography, cytoarchitecture, and cells that give rise to descending supraspinal projections. The anterior portion of the CNS is located adjacent to the first four myomeres and rostral to the first giant cell of Rohde-it can be divided into several regions that differ with respect to their cytoarchitecture. The tip of the neural tube is formed by a small anterior vesicle; caudally, there is a much larger region that is intercalated between the anterior vesicle and the first cell of Rohde. This intercalated region, in turn, consists of three subdivisions: an anterior subdivision adjacent to myomere 1, an intermediate subdivision adjacent to myomere 2, and a posterior one adjacent to myomeres 3 and 4. After injections of tracers into the spinal cord a large number of cells were labeled in the intercalated region. The spinally projecting cells were not evenly distributed: their number was decreased in the center of the intermediate subdivision. These subdivisions, which have previously not been noted, may be aligned with the expression domains of regulatory genes (e.g., AmphiOtx, AmphiHox) in larval lancelets. In particular, the center of the intermediate subdivision may correspond to a "nonHox/nonOtx" domain in the CNS of the larva. A similar embryonic domain occurs in the brain of craniates in which it develops into the isthmus cerebri that separates mid- and hindbrain. A close structural and topographical inspection of the corresponding region of adult lancelets reveals, however, that this region is not the homolog of an isthmus, but a uniquely derived, autapomorphic feature of lancelets.

Chordate evolution in a new light.

Gene expression studies in embryos often provide insights into evolutionary relationships across phyla. In this issue of Cell, Lowe et al. examine the patterning of the bilaterian nervous system by studying gene expression in a hemichordate, the acorn worm. Although these animals have an unstructured nervous system, they show surprisingly conserved gene expression domains, shedding new light on the evolution of the central nervous system and the phylogenetic placement of chordates.

Anteroposterior patterning in hemichordates and the origins of the chordate nervous system.

The chordate central nervous system has been hypothesized to originate from either a dorsal centralized, or a ventral centralized, or a noncentralized nervous system of a deuterostome ancestor. In an effort to resolve these issues, we examined the hemichordate Saccoglossus kowalevskii and studied the expression of orthologs of genes that are involved in patterning the chordate central nervous system. All 22 orthologs studied are expressed in the ectoderm in an anteroposterior arrangement nearly identical to that found in chordates. Domain topography is conserved between hemichordates and chordates despite the fact that hemichordates have a diffuse nerve net, whereas chordates have a centralized system. We propose that the deuterostome ancestor may have had a diffuse nervous system, which was later centralized during the evolution of the chordate lineage.

Isolation of an early neural maker gene abundantly expressed in the nervous system of the ascidian, Halocynthia roretzi.

Ascidian tadpole larvae possess a primitive nervous system, which is a prospective prototype of the chordate nervous system. It is composed of relatively few cells but sufficient for complex larval behavior. Here we report on HrETR-1, a gene zygotically expressed in a large proportion of the developing neural cells of the ascidian, Halocynthia roretzi. HrETR-1 is an early neural marker which can be used for analyzing neural differentiation. HrETR-1 expression intensified in most neural cells of genes isolated to date, in both central and peripheral nervous systems including palps as early as the 110-cell stage. Using this gene as a probe, we characterized neural cells in the nervous system as well as confirming their origins. Also, we recognized three types of peripheral epidermal neurons which presumably correlate to the larval neurons previously reported for another ascidian. Among these, five bilateral neurons located in the anterior region of the trunk appeared to be derived from a8.26 blastomeres.

Cell morphology in amphioxus nerve cord may reflect the time course of cell differentiation.

Amphioxus embryos elongate following neurulation, and this lengthens the developing nerve cord. Most neurons and support cells remain attached at their apices to the neuroepithelium, and the apices themselves become correspondingly longer. In consequence, apex length can be used in some instances as a measure of whether a given cell last divided before elongation or after, and approximately when. The data indicate that most floorplate, ependymoglial and infundibular cells are generated comparatively early, before most neurons. Among the neurons, the segmentally arranged DC (dorsal compartment) motoneurons appear to be among the first to develop, which accords with molecular data on the time course of neural development, using neurogenin and islet as markers.

Primordial germ cells and oocytes of Branchiostoma virginiae (Cephalochordata, Acrania) are flagellated epithelial cells: relationship between epithelial and primary egg polarity.

Primordial germ cells (PGCs) are described from the gonad of c. 2 cm juvenile Branchiostoma virginiae; early oocytes (c. 10 microm) and enlarging, previtellogenic oocytes (c. 35 microm) are described from the ovary of c. 5 cm adults. The germinal epithelium of the juvenile gonad and adult ovary is composed of both germinal and somatic cells. In the juvenile, somatic cells retain contact with the basal lamina of the germinal epithelium though their perikarya may be displaced towards the lumen; the germinal epithelium is, therefore, a simple but pseudostratified epithelium. In the adult ovary, somatic cells may lose contact with the basal lamina and the epithelium appears to become stratified. PGCs and oocytes are identified as germ cells by the presence of nuage. PGCs and oocytes are polarised epithelial cells. They rest on a basal lamina, extend apically towards a lumen, form adhering junctions with neighbouring cells, and exhibit apical-basal polarity. PGCs and early oocytes have an apical flagellum with an associated basal body, accessory centriole, and one or more striated rootlet fibres. The flagellum is surrounded by a collar of microvilli. Once oocytes begin to enlarge and bulge basally into the connective tissue layer, the flagellum is lost, but the basal bodies and ciliary rootlets are present at the apex of 35 microm oocytes. Similarities of the oogenic pattern in cephalochordates and echinoderms indicate that the establishment of egg polarity in deuterostomes is influenced by the polarity of the germinal epithelium.

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.