Effects of light on pigment development in the echiuran worm, Bonellia viridis

In rhe echiuran Bonellia viridis the green pigment first develops in the gastrula, before the formation of the trochal rings, and the trochophores acquire a deep green pigmentation before hatching. Eggs that were cultured under continuous illumination (2000–4000 lx) did not develop any green pigment, and although the trochophores hatched normally, they lacked pigment entirely. The unpigmented trochophores that were kept under continuous illumination developed into unpigmented males and females. The far-blue (380–400 nm) and red (620–640 nm) regions were more effective in preventing pigment development than the blue, green and yellow regions of the spectrum. Illumination of the eggs before the gastrula stage did not affect the development of pigment when these were subsequently returned to the dark. When unpigmented trochophores, that had been cultured in the light, were placed in the dark they acquired a slight pigmentation, but this was much less intense than that of trochophores that had been cultured in the dark throughout. Pigmented trochophores were more vulnerable to high light intensities than unpigmented ones.     

Larval development of Myzostoma cirriferum (Myzostomida)

The larval development of Myzostoma cirriferum is described by means of SEM, TEM, and cLSM. It is similar to that of other myzostomids and includes three stages: the protrochophore, the trochophore, and the metatrochophore. The protrochophore is a ball-shaped larva present in culture from 18-48 h after egg laying. It has no internal organs and its body is made of three cell types: covering cells and ciliated cells that are external and surrounded by a cuticle, and resting cells that fill the blastocoel. The trochophore is a pear-shaped larva that develops 20-72 h after egg laying; the body includes the same three cell types as the previous stage. The metatrochophore is a pear-shaped larva that develops between 40 h and 14 days and is characterized by the presence of two bundles of four chaetae. When fully developed, the metatrochophore has a digestive system (made of a pharynx, an esophagus, and a blind digestive pouch), two pairs of protonephridia, and a nervous system composed of a supraesophageal ganglion, circumesophageal connectives, and dorsal and ventral nerves. Metamorphosis generally occurs 7 days after egg laying. At that time, the metatrochophore loses its chaetae and becomes pleated ventrally. This ultrastructural analysis suggests that chaetae and the five ventral longitudinal nerve cords of M. cirriferum metatrochophores are homologous structures to those observed in some polychaete trochophores. Coupled with recent phylogenetic analyses, where the Myzostomida are placed outside the Annelida, homologies between myzostomid and polychaete larvae support the view that a trochophore appeared early during the spiralian evolution.     

Ultrastructure of the eyespot in three polychaete trochophore larvae (Annelida)

Summary The eyespot is structurally similar in trochophore larvae of Harmothoe imbricata, Serpula vermicularis and Spirobranchus giganteus. In the receptor cell parallel lamellae lie below a tuft of microvilli which extends into a hollow in one side of the pigment cell. In 1-eyed trochophores this space connects with the outside via a small pore. In eyes preserved during the day there is evidence of a membrane breakdown in both lamellae and microvilli as well as indications of a membrane-fragment disposal system involving the receptor cell, the pigment cell and the eyespot pore. The orientation of the eyespot of S. giganteus is the reverse of that found in S. vermicularis, a situation that may be associated with ecologically significant differences in behaviour.     

Behavior of pigment cells in gastrula-stage embryos of Hemicentrotus pulcherrimus and Scaphechinus mirabilis

The behavior of pigment cells in sea urchin embryos, especially at the gastrula stage, is not well understood, due to the lack of an appropriate method to detect pigment cells. We found that pigment cells emanated autofluorescence when they were fixed with formalin and irradiated with ultraviolet or green light. In Hemicentrotus pulcherrimus, fluorescent pigment cells became visible at the archenteron tip at the mid-gastrula stage. The cells detached from the archenteron slightly before the initiation of secondary invagination and migrated toward the apical plate. Most pigment cells entered the apical plate. This entry site seemed to be restricted, because pigment cells could not enter the ectoderm and remained in the blastocoele at the vegetal pole side when elongation of archenteron was blocked. Pigment cells that had entered the apical plate soon began to migrate in the aboral ectoderm toward the vegetal pole. In contrast, pigment cells of Scaphechinus mirabilis embryos were first detected in the vegetal plate before the onset of gastrulation. Without entering the blastocoele, these cells began to migrate preferentially in the aboral ectoderm toward the animal pole. When the archenteron tip reached the apical plate, pigment cells had already distributed throughout the aboral ectoderm. Thus, the behavior of pigment cells was quite different between H. pulcherrimus and S. mirabilis.     

The golden age of comparative morphology: laser scanning microscopy and neurogenesis of trochophore animals

Immunochemical labeling of neuronal elements and laser confocal microscopy have considerably expanded the capacity of comparative morphology and allowed us to monitor the neurogenesis of various trochophore animals at the level of individual identified neurons and their projections. It has been demonstrated that many generally accepted concepts of the larval nervous system and the phylogenetic theories constructed on this basis are incorrect. Comparative analysis has demonstrated that the orthogonal brain is absent at all developmental stages in the representative Lophotrochozoa members. Fundamental differences in the structure and development of the nervous system have been found in the trochophores belonging to different taxonomic groups within Lophotrochozoa; these differences demonstrate that the trochophore larva in these groups are not homologous, while their similarity is most likely a result of convergence. Our results challenge the concept of trochophore as the ancestral form common for all trochophore animals. It is necessary to exclude from phylogenetic discussions the orthogon as a basic plan for the structure of the nervous system and the trochophore as an ancestral form for all Lophtrochozoa.     

Inductive effect of the eye tissues of adult clawed toads on the gastrula ectoderm

The inducing influence of adult eye tissues on the early gastrula ectoderm was studied in vitro. Both retina and pigment epithelium induced in the early gastrula ectoderm similar spectra of cell types, including nervous tissue, retina, pigment epithelium, lentoids, ectomesenchyme, and melanophores. It is suggested that the correspondence of these cell types with those arising at a spontaneous transdifferentiation of the isolated retina and pigment epithelium cells in vitro or at the induction of the early gastrula ectoderma by archencephalic endomesoderm during the normal development can be accounted for by that in these eye cells molecular determinants appeared as a result of induction and maintaina the stability of their differentiation and their potencies to transdifferentiation in vitro being reproduced during the lifetime of these cells.     

The golden age of comparative morphology: Laser scanning microscopy and neurogenesis in trochophore animals

Immunochemical labelling of neuronal elements and laser confocal microscopy have considerably expanded the capacity of comparative morphology, and allowed us to study neurogenesis in various trochophore animals at the level of identified neurons. The data obtained have impeached some popular phylogenetic theories. Thus, the comparative study has shown that in the representative Trochozoan species, the orthogonal brain is absent at all developmental stages. Fundamental differences in neurogenesis and neuroarchitecture between trochophores from different taxa suggest that trochophore-like larvae are not homologous, and their likeness is most probably the result of convergence. Our data support the “intercalation hypothesis” of the origin of indirect development with a trochophore-like larva. It seems reasonable to exclude from phylogenetic discussions the orthogon and the trochophore as an ancestral form common for Lophotrochozoa.     

The role of MAPK signaling in patterning and establishing axial symmetry in the gastropod Haliotis asinina

Gastropods are members of the Spiralia, a diverse group of invertebrates that share a common early developmental program, which includes spiral cleavage and a larval trochophore stage. The spiral cleavage program results in the division of the embryo into four quadrants. Specification of the dorsal (D) quadrant is intimately linked with body plan organization and in equally cleaving gastropods occurs when one of the vegetal macromeres makes contact with overlying micromeres and receives an inductive signal that activates a MAPK signaling cascade. Following the induction of the 3D macromere, the embryo begins to gastrulate and assumes a bilateral cleavage pattern. Here we inhibit MAPK activation in 3D with U0126 and examine its effect on the formation and patterning of the trochophore, using a suite of territory-specific markers. The head (pretrochal) region appears to maintain quadri-radial symmetry in U0126-treated embryos, supporting a role for MAPK signaling in 3D in establishing dorsoventral polarity in this region. Posterior (posttrochal) structures - larval musculature, shell and foot - fail to develop in MAPK inhibited trochophores. Inhibition of 3D specification by an alternative method - monensin treatment - yields similar abnormal trochophores. However, genes that are normally expressed in the ectodermal structures (shell and foot) are detected in U0126- and monensin-perturbed larvae in patterns that suggest that this region has latent dorsoventral polarity that is manifested even in the absence of D quadrant specification.     

Änderungen der Ultrastrukturen in den Grünalgen Ankistrodesmus braunii und Chlorella fusca var. rubescens bei Stickstoffmangel

Summary Ultrastructural changes as well as changes in pigment and lipid content induced by nitrogen deficiency in the green algae Ankistrodesmus braunii and Chlorella fusca var. rubescens have been studied. Electron micrographs (freezeetching technique) and analyses show that the content of chlorophylls and the number of thylakoids decrease under these conditions synchronously with the increase of secondary carotenoids (astaxanthin-like type) and lipid vacuoles. However, there is no change, as compared with the green control, in the number and structure of the plastoglobuli localized in the chloroplasts. After addition of nitrogen, synthesis of the thylakoids and chlorophylls begins. The lipid vacuoles and secondary carotenoids disappear gradually. These extraplastidic lipid vacuoles do not correspond with the intraplastidic plastoglobuli (lipid globuli) found in higher plants and in Acetabularia.     

Cardiac differentiation induced by dopamine in undifferentiated cells of early chick embryo

Evidence suggests that neurotransmitters can act as possible chemical signals involved in cell division and morphogenetic movements long before neurons appear in the embryo. However, whether they are playing a role in differentiation is now unknown. It was recently observed (M. Sarasa and S. Climent, 1987, J. Exp. Zool. 241, 181-190) that the neurotransmitter dopamine exerted a stimulating effect on cardiac differentiation in the chick in ovo. We show here that dopamine acts as a specific inducer of heart muscle differentiation in vitro. When cells of the gastrula of embryos treated with dopamine were dissociated and reaggregated, the aggregates obtained almost entirely underwent cardiac muscle differentiation. Also, when small postnodal pieces obtained from the most posterior region of the gastrula were cultivated in the presence of dopamine, they differentiated into myocardic tissue instead of following their fate map. Therefore, dopamine can trigger a process that both causes undifferentiated cells to differentiate into heart muscle and compels cells already determined to another way of differentiation to become myocardic tissue.     

Morphogenesis and phenotypic divergence in two developmental morphs of Streblospio benedicti (Annelida,Spionidae)

Abstract. The morphology of marine invertebrate larvae is strongly correlated with egg size and larval feeding mode. Planktotrophic larvae typically have suites of morphological traits that support a planktonic, feeding life style, while lecithotrophic larvae often have larger, yolkier bodies, and in some cases, a reduced expression of larval traits. Poecilogonous species provide interesting cases for the analysis of early morphogenesis, as two morphs of larvae are produced by a single species. We compared morphogenesis in planktotrophic and lecithotrophic morphs of the poecilogonous annelid Streblospio benedicti from the trochophore stage through metamorphosis, using observations of individuals that were observed alive, with scanning electron microscopy, or in serial sections. Offspring of alternate developmental morphs of this species are well known to have divergent morphologies in terms of size, yolk content, and the presence of larval bristles. We found that some phenotypic differences between morphs occur as traits that are present in only one morph (e.g., larval bristles, bacillary cells on the prostomium and pygidium), but that much of the phenotypic divergence is based on heterochronic changes in the differentiation of shared traits (e.g., gut and coelom). Tissue and organ development are compared in both morphs in terms of their structure and ontogenetic change throughout early development and metamorphosis.     

Comparative morphology of photoreceptors in free-living plathelminths — a survey

In free-living Plathelminthes, the best-known photoreceptors are pigment-cup ocelli, eyes formed of one or several pigmented supportive cells into whose cup-shaped cavity project the light-sensitive elements of one or several sensory cells. Besides these, so-called Sehkolben, photoreceptors lacking pigment granules, are found in some species. Sensory cells in plathelminth photoreceptors most commonly use microvilli as the light-sensitive organelles, but some use cilia and combinations of microvilli and cilia. Lamellate ciliary bodies with cilia whose membranes are strongly flattened and rolled and pericerebral ciliary aggregations with interwoven cilia protruding into an intracellular cavity are likely photoreceptors in that they show amplification of membrane likely to bear photoreceptive pigments. Cells with ballooned cilia and tubular vacuoles are other differentiations to which light-sensitivity has been attributed. A variety of structures serve as lenses, all usually formed from parts of the pigment cell.     

From larval bodies to adult body plans: patterning the development of the presumptive adult ectoderm in the sea urchin larva

Echinoderms are unique among bilaterians for their derived, nonbilateral adult body plan. Their radial symmetry emerges from the bilateral larval body plan by the establishment of a new axis, the adult oral–aboral axis, involving local mesoderm–ectoderm interactions. We examine the mechanisms underlying this transition in the direct-developing sea urchin Heliocidaris erythrogramma. Adult ectoderm arises from vestibular ectoderm in the left vegetal quadrant. Inductive signals from the left coelom are required for adult ectodermal development but not for initial vestibule formation. We surgically removed gastrula archenteron, making whole-ectoderm explants, left-, right-, and animal-half ectoderm explants, and recombinants of these explants with left coelom. Vestibule formation was analyzed morphologically and with radioactive in situ hybridization with HeET-1, an ectodermal marker. Whole ectodermal explants in the absence of coelom developed vestibules on the left side or ventrally but not on the right side, indicating that left–right polarity is ectoderm autonomous by the gastrula stage. However, right-half ectodermal explants robustly formed vestibules that went on to form adult structures when recombined with the left coelom, indicating that the right side retains vestibule-forming potential that is normally suppressed by signals from the left-side ectoderm. Animal-half explants formed vestibules only about half the time, demonstrating that animal–vegetal axis determination occurs earlier. However, when combined with the left coelom, animal-half ectoderm always formed a vestibule, indicating that the left coelom can induce vestibule formation. This suggests that although coelomic signals are not required for vestibule formation, they may play a role in coordinating the coelom–vestibule interaction that establishes the adult oral–aboral axis.     

LOCALIZATION OF ACID PHOSPHATASE IN LIPOFUSCIN GRANULES AND POSSIBLE AUTOPHAGIC VACUOLES IN INTERSTITIAL CELLS OF THE GUINEA PIG TESTIS

The intracellular localization of acid phosphatase in guinea pig testicular interstitial cells was investigated by incubating nonfrozen thick sections of glutaraldehyde-perfused testis in a modified Gomori medium and preparing the tissue for electron microscopy. Lipofuscin pigment granules in these cells contain dense pigment, granular matrix, and often a lipid droplet. Reaction product is seen in the matrix of the pigment granules, and they may therefore be called residual bodies. At least some of the dense pigment appears to be derived from myelin figures and membrane whorls, since suitable intermediates can be seen. Lipid droplets found free in the cytoplasm are another possible source of pigment. In both cases the chemical mechanism is presumed to be autoxidation of unsaturated lipid. Acid phosphatase is present in the inner cisterna of Golgi elements. Enzyme activity also appears in possible autophagic vacuoles bounded by double membranes; the reaction product lies between the membranes. Consideration of the enzyme as a tracer suggests that the autophagic vacuoles are derived from the Golgi complex. Possible stages in the formation of these vacuoles by the inner Golgi cisternae are observed.     

Cryopreservation of oyster (Crassostrea gigas) embryos

Several critical variables associated with successful cryopreservation of oyster embryos (Crassostrea gigas) were examined. These were 1) embryo developmental stage, 2) kind and concentration of cryoprotectant, 3) equilibration time, and 4) freezing rate. The percentage of survival was scored as the number of recovered embryos that swam actively 12 h after thawing and had developed into veliger stage. The oyster embryos became increasingly susceptible to the cryoprotectants as the concentration was increased and the equilibration time was lengthened. The stage of development appears to be a critical factor for survival of oyster embryos, with trochophore stage embryos more resistant than morula and gastrula stages embryos to cryoprotectant exposure and having better surviving after freezing. The optimum cryoprotectant concentration for the trochophore embryos differed markedly from the morula stage. Cryopreservation of fertilized eggs (2 to 8 cells) was unsuccessful. Varying degrees of success were achieved using gastrula- and trochophore-stage embryos. Maximum survival was obtained when trochophore embryos incubated in 10% propylene glycerol-artificial sea water were cooled at -2.5 degrees C/min to -30 degrees C and were then directly placed into liquid nitrogen. The results showed a clear effect of the stage of development on survival.     

Defence function of pigmentocysts in the karyorelictid ciliate Loxodes striatus

The karyorelictid ciliate Loxodes striatus has pigment granules which are similar in size, structure and distribution to the pigmentocysts in the heterotrich ciliates, Blepharisma japonicum and Stentor coeruleus, which are known to be extrusomes for chemical defence against predators. We examined whether the pigment granules of L. striatus are also defensive organelles. We showed that: (1) pigment granules of L. striatus are extrusive organelles; (2) bleached cells of L. striatus produced by inducing a massive discharge of pigment granules are more vulnerable than normally pigmented cells to the raptorial ciliate Dileptus margaritifer and the turbellarian Stenostomum sphagnetorum, while they are indistinguishable from intact cells in external morphology and the capacity to grow; (3) the cell-free fluid (CFF) which contains the pigment discharged from pigment granules of L. striatus induced in D. margaritifer behavioural and pathological reactions which are essentially the same as those observed in the interaction with L. striatus, and this effect of the CFF disappeared when the pigment was bleached by light. We conclude that pigment granules of L. striatus are extrusomes for chemical defence against predators, and that the defence is based on the toxic pigment contained in these organelles.     

The BMP/CHORDIN antagonism controls sensory pigment cell specification and differentiation in the ascidian embryo

We have investigated the role of the bone morphogenetic protein (BMP) pathway during neural tissue formation in the ascidian embryo. The orthologue of the BMP antagonist, chordin, was isolated from the ascidian Halocynthia roretzi. While both the expression pattern and the phenotype observed by overexpressing chordin or BMPb (the dpp-subclass BMP) do not suggest a role for these factors in neural induction, BMP/CHORDIN antagonism was found to affect neural patterning. Overexpression of BMPb induced ectopic sensory pigment cells in the brain lineages that do not normally form pigment cells and suppressed pressure organ formation within the brain. Reciprocally, overexpressing chordin suppressed pigment cell formation and induced ectopic pressure organ. We show that pigment cell formation occurs in three steps. (1) During cleavage stages ectodermal cells are neuralized by a vegetal signal that can be substituted by bFGF. (2) At the early gastrula stage, BMPb secreted from the lateral nerve cord blastomeres induces those neuralized blastomeres in close proximity to adopt a pigment cell fate. (3) At the tailbud stage, among these pigment cell precursors, BMPb induces the differentiation of specifically the anterior type of pigment cell, the otolith; while posteriorly, CHORDIN suppresses BMP activity and allows ocellus differentiation.     

Development and differentiation of the eye in Platynereis dumerilii (Annelida,Polychaeta)

The nereid polychaete, Platynereis dumerilii, possess two pairs of post-trochophoral eyes with one vitreous body each. The development of these eyes has first been observed in 2-day-old larvae. Whether the eye anlagen arise from stem cells or from undifferentiated ectodermal tissue was not determined. At first, the anlagen of the anterior and the posterior eyes adjoin each other. They separate in late 3-day-old larvae. The first separated eye complexes consist each of two supporting and two sensory cells. The supporting cells synthesize two different kinds of granules, the pigment granules of the pigment cup and the prospective tubules of the vitreous body. These tubules accumulate in the distal process of the supporting cell. The vitreous body is formed by compartments of the supporting cells filled with the osmiophilic vitreous body tubules. The short, bulbar photosensory processes bear microvilli that emerge into the ocular cavity. At the apex of each sensory cell process, a single cilium (or occasionally two) arises. The sensory cells contain a different kind of pigment granule within their necks at the level of the pigment cup. The rate of eye development and differentiation varies. New supporting cells are added to the rim of the eye cup. They contribute to the periphery of the vitreous body like onion skins, and sensory cells move between supporting cells. The older the individual compartments of the vitreous body are, the more densely packed is their content of vitreous body tubules. Elongation of the sensory and supporting cell processes of the older cells increases the volume of the eye. The eyespots of the trochophore are briefly described as of the two-celled rhabdomeric type with a single basal body with ciliary rootlet.     

Development of the coelomic cavities in larvae of the living isocrinid sea lily Metacrinus rotundus

Coelomogenesis in the isocrinid sea lily, Metacrinus rotundus, is described through the swimming larval stages. After the late gastrula stage, the archenteron separates from the ectoderm to form an archenteral sac, which develops into a dumbbell shape consisting of anterior and posterior lobes, and a middle part connecting both lobes. The anterior and posterior lobes, and the middle part, become separated into an axo-hydrocoel, the left and right somatocoels and an enteric sac, respectively. The hydrocoel forms from the left lower edge of the axo-hydrocoel and becomes separated from the axocoel by the late dipleurula stage, when chambered organs and coelom X bud off from the anterior tip of the right and left somatocoels, respectively. Coelom X does not occur in comatulid crinoids (feather stars), and its fate is unclear. The pore canal extends from the axocoel. The hydrocoel differentiates into a crescent shape at the overtime semidoliolaria stage, a few days after the semidoliolaria becomes competent to settle. Coelomogenesis in M. rotundus is much simpler than in the comatulids and probably represents the ancestral mode of the crinoids. As each portion of the dumbbell sac differentiates almost in situ into each coelom, presumptive fates in the sac are easily followed in M. rotundus.