The distribution of nucleoplasmin in early development and organogenesis of Xenopus laevis

Summary The fate of the germinal vesicle-derived protein, nucleoplasmin, was followed in embryos and tadpoles of Xenopus using monoclonal antibodies and indirect immunofluorescent staining. Nucleoplasmin was found in all nuclei up to feeding tadpole stages. Thereafter its level decreased in all nuclei. It was not detected in nuclei of advanced tadpoles or of adults. Contrasting with another protein, N₁, that was previously monitored in the nuclei of dividing gonia of both sexes, nucleoplasmin was only detected in the nuclei of ovarian oocytes starting at diplotene. Traces of nucleoplasmin have also been found in a rapidly-dividing fibroblastic cell-line by immunohistology and protein blotting.     

Comparative study of gill neuroepithelial cells and their innervation in teleosts and Xenopus tadpoles

Peripheral O₂ chemoreceptors initiate adaptive cardiorespiratory responses to hypoxia in vertebrates. Morphological and physiological evidence suggests that, in fish, neuroepithelial cells (NECs) of the gill perform this role. We conducted a comparative examination in three species of teleosts (zebrafish, goldfish and trout) and larvae of the amphibian Xenopus laevis, using whole-mount gill preparations and confocal immunofluorescence, to elucidate the distribution, morphology and innervation of gill NECs. Nerve fibres were immunolabelled with the neuronal marker zn-12 and were associated with serotonin-immunoreactive NECs in the gills of all species tested. With the exception of trout, innervated NECs were present on all gill arches in the filaments and respiratory lamellae in fish and on homologous structures in Xenopus (i.e. gill “tufts”, including respiratory terminal branches). Thus, the distribution and innervation of NECs of the internal gills of amphibians and teleosts are relatively well conserved, suggesting an important role for gill NECs as O₂ chemoreceptors in aquatic vertebrates. Furthermore, the size and density of gill NECs is variable among teleosts and developmental stages of Xenopus larvae and may be dependent on general gill dimensions or environmental conditions. This report constitutes the first comparative study of gill NECs in fish and amphibians and highlights the significance of gill NECs as an evolutionary model for studying O₂ sensing in vertebrates. We acknowledge the Natural Sciences and Engineering Research Council (NSERC) of Canada for funding through an operating grant to C.A.N., and the NSERC and the Ontario Graduate Scholarship (OGS) program for postgraduate scholarships to M.G.J.     

Retinofugal projections in the eel,Anguilla anguilla L. (Teleostei), visualized by the cobalt-filling technique

The retinofugal projections in the eel were studied by use of the cobalt-filling technique. The optic tract projects contralaterally to the hypothalamic optic nucleus, the anterior periventricular nucleus, the lateral geniculate nucleus, the dorsomedial optic nucleus, four pretectal recipient areas, the optic tectum, and the tegmentum. Small ipsilateral projections were demonstrated in the hypothalamic optic nucleus, the dorsomedial optic nucleus, and the optic tectum.     

Retinal projections in the chain pickerel (Esox niger Lesueur)

Summary The retinal projections inEsox niger, as determined with the aid of a modified cobalt-lysine method, are considerably more extensive in the diencephalon and pretectum than in other teleost fishes so far examined. Although most retinal axons terminate contralaterally, rare fibers can be traced to the same aggregates ipsilaterally. The retinohypothalamic projection appears larger than hitherto reported in teleosts, and the dorsomedial optic tract issues fibers to a series of cell clusters extending from the rostral thalamus to mid-torus levels. A retinal projection to a presumed ventrolateral optic nucleus (VLO) is described for the first time in a teleost. Other targets of retinal fibers include the nucleus geniculatus lateralis ipse of Meader (GLI), the pretectal nucleus (P), the cortical nucleus and a well-developed ventromedial optic nucleus (VMO). The projection to the optic tectum is principally to the stratum fibrosum et griseum superficiale (SFGS) and stratum marginale (SM), but a considerable number of axons also course through the stratum album centrale (SAC) before terminating there or piercing the stratum griseum centrale (SGC) and terminating in SFGS. Rare terminal arborizations of retinal fibers were also observed in stratum griseum centrale (SGS) and in the stratum griseum periventriculare (SGC) in restricted portions of the tectum. Because of the relatively large size of the visual structures inE. niger it is a potentially useful model for future experimental studies on the visual system.     

Presence of microtubules in isolated cortices of prophase I and metaphase II oocytes in Xenopus laevis

Summary An extensive array of microtubules has been shown to exist in the cortex of Xenopus laevis oocytes both at the prophase I and metaphase II stages. The cortical microtubules were visualized after the oocyte cortex was squashed and immunostained using anti-tubulin antibody. They are cold- and nocodazole-sensitive; their stability to both treatments decreases after meiotic maturation. Biochemical extraction of manually isolated oocyte cortices, in a microtubule-stabilizing buffer, confirms these cytological observations.     

Retinal projections in sockeye salmon smolts (Oncorhynchus nerka)

Summary The retinal projections in 2-year-old salmon smolt (Oncorhynchus nerka) are significantly different from those observed in other teleosts examined to date in that the projections are more extensive. Very noticeable are extensive projections to most of the dorsal thalamus, to all layers of the optic tectum, and into the periaqueductal gray of the torus semicircularis. The salmon smolt has bilateral retinal projections to the diencephalon and pretectum. A small retinal projection to the lateral habenular nucleus has not been described previously. Although these findings suggest striking differences in retinal projections among teleosts, this variation may relate to age differences since the previously studied teleosts were adults.     

Retinofugal and retinopetal projections in the teleost Channa micropeltes (Channiformes)

Summary Retinofugal and retinopetal projections were investigated in the teleost fish Channa micropeltes (Channiformes) by means of the cobaltous lysine and horseradish peroxidase (HRP) tracing techniques. Retinofugal fibers cross completely in the optic chiasma. A conspicious lamination is present in those parts of the optic tract that give rise to the marginal branches of the optic tract. This layering of optic fibers continues in the marginal branches to mesencephalic levels. Retinal projections to the preoptic and hypothalamic regions are sparse; they are more pronounced in the area of pretectal nuclei. The medial pretectal complex and the cortical pretectal nucleus are more fully differentiated than in other teleostean species. Further targets include the thalamus and the optic tectum. The course of major optic sub-tracts and smaller fascicles is described. Retinopetal neurons are located contralaterally in a rostral and a caudal part of the nucleus olfactoretinalis, and in a circumscribed nucleus thalamoretinalis. The present findings are compared with reports on other teleost species.     

Balbiani cytoplasm in oocytes of a primitive fish,the sturgeon <Emphasis Type="Italic">Acipenser gueldenstaedtii</Emphasis>, and its potential homology to the Balbiani body (mitochondrial cloud) of <Emphasis Type="Italic">Xenopus laevis</Emphasis> oocytes

The oocytes of many organisms, including frogs and fish, contain a distinct cytoplasmic organelle called the Balbiani body. Because of the scarcity of published information and the tremendous variability in the appearance, ultrastructure, and composition of Balbiani bodies between species, the function of the Balbiani body and its inter-species homology remain a mystery. In Xenopus laevis, the Balbiani body is known to play a role in transporting germ cell determinants and localized RNAs to the oocyte vegetal cortex. In fish, however, the molecular composition of the Balbiani body has not been studied to date, and its function remains completely unknown. We have studied the ultrastructure and molecular composition of previtellogenic oocytes of the sturgeon, Acipenser gueldenstaedtii, by using electron microscopy, in situ hybridization, and immunostaining. We have found that sturgeon oocytes contain two distinct zones of cytoplasm: homogeneous (organelle-free) and granular (organelle-rich). We have also found that the granular ooplasm, which we term the Balbiani cytoplasm, shares important homologies, in both ultrastructure and molecular composition, with Xenopus Balbiani bodies. This work was supported by funds from the research grant BW/IZ/2005 to M.Z.     

Development of Xenopus laevis skin glands producing 5-hydroxytryptamine and caerulein

Summary The granular glands in Xenopus laevis skin are known to contain large quantities of biogenic amines and bioactive peptides which closely resemble mammalian brain-gut peptides. We studied the development of glands producing 5-hydroxytryptamine (5-HT) and caerulein using immunohistochemistry, HPLC-fluorometric systems and RIA. The immunoreactivities of 5-HT and caerulein were first detected in the spherical gland rudiments in the stratum spongiosum at St. 58 (Nieuwkoop and Faber stage), or at the beginning of metamorphosis. Both immunoreactivities appeared in the same rudiment at the same time. Some of the gland rudiments have a small lumen filled with both immunoreactive materials at St. 58–59. During the rest of the metamorphic period, the glands grow in size, accumulating immunoreactive materials in the lumen. The concentrations of 5-HT and caerulein in the skin of tadpoles were below 1 ng per mg wet tissue at St. 58–59, increased as metamorphosis proceeded and reached 63 and 134 ng per mg wet tissue at St. 66, or at the end of metamorphosis, respectively. The amphibian granular glands where large quantities of biogenic amines and hormone-like peptides are rapidly synthesized may provide a useful model for the study of the development of amine- and peptide-producing cells including neurons and paraneurons.     

Neurotrophin receptors and enteric neuronal development during metamorphosis in the amphibian<Emphasis Type="Italic"> Xenopus laevis</Emphasis>

During metamorphosis, the frog intestine goes through a dramatic shortening with extensive apoptosis and regeneration in the epithelial layer and connective tissue. Our aim was to study changes in the enteric nervous system represented by one inhibitory (vasoactive intestinal polypeptide; VIP) and one excitatory (substance P, neurokinin A; SP/NKA) nerve population and concomitant changes in neurotrophin receptor occurrence during this development in the gut of Xenopus laevis adults and tadpoles at different stages of metamorphosis (NF stages 57–66). Sections were incubated with antibodies against the neurotrophin Trk receptors and p75^NTR, and the neurotransmitters VIP and SP/NKA. Trk-immunoreactive nerves increased dramatically but transiently in number during early metamorphic climax. Nerves immunoreactive for p75^NTR were present throughout the gut, decreased in number in the middle intestine during climax, and increased in the large intestine during late metamorphosis. The percentage of VIP-immunoreactive nerves did not change during metamorphosis. SP/NKA-immunoreactive nerves were first apparent at NF stages 61–62 in the middle intestine and increased in the stomach and large intestine during metamorphosis. Endocrine cells expressing SP/NKA increased in number in stomach, proximal, and middle intestine during metamorphic climax. Thus, neurotrophin receptors are expressed transiently in neurons of the enteric nervous system during metamorphosis in Xenopus laevis and SP/NKA innervation is more abundant in the intestine of the postmetamorphic frog than in the tadpole.This study was supported by grants from the Swedish Research Council to S. Holmgren     

Emigration of bilayered epidermal cell sheets from tadpole tails (Xenopus laevis)

Summary Migration of bilayered epidermal cell sheets out of explants of tadpole tails (Xenopus laevis) were investigated with time-lapse cinemicrography using reflection-contrast optics. Cell-sheet formation begins beneath the explant in a region where it is closely attached to the coverslip. A single basal cell extends a lamellipodium through the outer (surface) epidermal layer and starts moving in a direction free of attached cells. This cell remains connected to the following basal cell, which the also extends a lamellipodium onto the glass. The cell sheet develops as increasingly more adjacent basal cells start to migrate. Surface cells do not actively locomote but they remain attached to the basal cells and to adjacent surface cells. Thus, they are transported as an intact cell layer, and consequently the in situ arrangement of the tadpole epidermis is largely preserved in the cell sheet, i.e., basal cells adhere to the substratum and are covered by outer cells (surface cells) which face the culture medium. Basal cells extend lamellae beneath the rear end of the preceding cell, which is slightly fifted off the substratum. The direction of locomotion is determined by the frontal cells. Cell-sheet enlargement and locomotion cease when all the epidermal cells facing the coverslip have left the explant, and the cell sheet and epidermis covering the explant form a continuous layer.     

Interaction between SPARC and tubulin in <Emphasis Type="Italic">Xenopus</Emphasis>

Secreted protein, acidic, rich in cysteine (SPARC) is an ancient calcium-binding glycoprotein associated with the extracellular matrices of invertebrates and vertebrates. We have previously reported an intracellular association of SPARC with the 9+2 microtubule arrays of cilia on the surface ectoderm of Xenopus embryos. During early development in Xenopus, ciliated cell precursors are associated with the inner sensorial layer of the two-layered embryonic skin. The ciliated cell precursors migrate to the overlying surface ectoderm where they undergo ciliogenesis. Whole-mount immunohistochemical data indicate SPARC is associated with the ciliary tuffts until ciliated cells begin to disappear from the surface ectoderm during late tailbud development. We now report an association between SPARC and tubulin in Xenopus embryonic cell lysates by co-immunoprecipitation. Tubulin is not co-immunoprecipitated by anti-SPARC antibodies that show no cross-reactivity to Xenopus SPARC by whole-mount immunocytochemical analysis. An association of SPARC with tubulin has also been observed in pull-down assays with biotinylated SPARC as bait. These data indicate that SPARC may have intracellular and extracellular functions during development in Xenopus.     

Bromodeoxyuridine-immunohistochemistry on cellular differentiation and migration in the fundic gland of Xenopus laevis during development

Summary Cellular differentiation and migration in the fundic glands of adult and larval Xenopus laevis have been examined using bromodeoxyuridine-immunohistochemistry. In the adult fundic gland, cumulative labeling with bromodeoxyuridine revealed a proliferative cell zone between the surface mucous cells and mucous neck cells, in what is referred to as the neck portion of the gland. The labeling-index of mucous neck cells had rapidly increased by week-5. The labeling-index of oxynticopeptic cells showed a more delayed increase until week-7, coincident with the decrease in the labeling of mucous neck cells. In the immature fundic glands of larvae, the labeled proliferating cells were randomly distributed throughout the developing gastric mucosa. During metamorphosis, the labeling-index of immature epithelial cells was highest at stage 63. Following administration of bromodeoxyurdine at this, stage, there was no significant loss of labeled epithelial cells during the metamorphosing period. Furthermore, there was no significant difference in the labeling-indices among the epithelial cells, such as surface mucous cells/generative cells, mucous neck cells, and oxynticopeptic cells, 7 days after administration. Cellular differentiation and migration pathways of epithelial cells in the fundic gland of adult X. laevis and its larvae are discussed.     

Tight junction formation in early <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryos: identification and ultrastructural characterization of junctional crests and junctional vesicles

How tight junctions (TJ) form during early amphibian embryogenesis is still an open question. We used time-lapse video microscopy, scanning electron microscopy (SEM), TEM and freeze-fracture to gain new insight into TJ biogenesis in early clevages of Xenopus laevis. Video analysis suggests three phases in junction formation between blastomeres. A first "waiting" phase, where new unpigmented lateral membranes are generated. A second "mixing" phase, where the unpigmented lateral membrane is separated from the pigmented apical membrane by an area showing a limited degree of intermingling of cortical pigment. And a third "sealing" phase, characterized by the formation of cingulin-containing boundaries between membrane domains, and their rapid directional adhesion in a zipper-like fashion. By SEM, we characterized these boundaries ("junctional crests", JC) as arrays of villiform protrusions at the border between old and new membranes. In the 2-cell embryo, JC are deeply located, and thus not visible at the surface, but they become increasingly more superficial as cleavages progress. After adjacent blastomeres have adhered to each other, fractured JC display linear arrays of junctional vesicles (JV) of 1-3 mum diameter. TEM analysis shows that JV are symmetrically located near the apposed membranes of adjacent blastomeres, and that the membranes near the JV display focal sites of intimate contact, typical of TJ. Freeze-fracture analysis confirms that intramembrane fibrils, typical of TJ, are present at adhesion sites. We conclude that TJ are formed following the sealing of JC, through the recruitment, sorting and assembly of membrane and cytoplasmic proteins at or near JV.     

Localization of xenopsin and xenopsin precursor fragment immunoreactivities in the skin and gastrointestinal tract of Xenopus laevis

Summary Xenopsin (Xp) and xenopsin precursor fragment (XPF) are bioactive peptides derived from a single precursor molecule; both were isolated previously from extracts of Xenopus laevis skin. The present immunohistochemical study was undertaken to determine the specific cellular localization of these two peptides in the skin and also in the gastrointestinal tract of adult Xenopus. We report here that Xp-like and XPF-like immuno-reactivities co-exist in the granular glands of the skin and specific granular cells in the lower esophagus and stomach. However, only Xp-like immunoreactivity, not XPF-like immunoreactivity, was detected in tall, thin cells of the duodenum and in club-shaped cells of the large intestine. The immunochemical co-localization of the two peptides in specific cells of the skin, lower esophagus and stomach suggests that the same gene is expressed in each of these cells, and that the precursor molecule undergoes similar post-translational processing. In contrast, the observation that certain cells of the duodenum and large intestine display only one peptide immunoreactivity suggests an alternative phenomenon, possibly involving selective peptide accumulation or expression of a different gene.     

Ultrastructural Identification of gastrin-like immunoreactive nerve fibres in the brain of Xenopus laevis by means of colloidal gold or ferritin immunocytochemical methods

Summary By use of an anti-gastrin serum and colloidal gold- or ferritin-labelled sheep anti-rabbit -globulins, nerve fibres and nerve terminals containing a gastrin-like substance were characterized at the ultrastructural level in the median eminence of Xenopus laevis. These immunoreactive fibres contain neurosecretory granules displaying medium to high electron density and a mean diameter of 75 nm. Labelling intensity varies from granule to granule. This is the first demonstration at the ultrastructural level of the precise location of a gastrin-like hormone in the median eminence of a vertebrate.Supported by the D.G.R.S.T., Contrat no 80.7.0242     

Immunohistochemical demonstration of TSR-, LH- and ACTH-cells in the hypophysis of tadpoles of Xenopus laevis D.

Summary By use of the immunofluorescence technique TSH-, LH- and ACTH-cells were localized in the hypophysis of tadpoles of Xenopus laevis. The first signs of the activity of these cells were observed in early stages of the development, i.e., stage 39 for ACTH, and stage 42 for TSH and LH.     

Prey-capture in the African clawed toad (Xenopus laevis): comparison of turning to visual and lateral line stimuli

Separately delivered visual and lateral line stimuli elicit similar but not identical orientation and approach by intact, sighted Xenopus. Response frequencies for visual stimuli declined sharply for distant or caudal stimuli while those for lateral line stimuli changed little. Turn angles correlated highly with stimulus angles but were smaller on average, so regression slopes were less than one. Regression slopes were smaller for visual than for lateral line stimuli, but this apparent difference was due to different distributions of stimulus distance interacting with the toad’s rotation center. Errors in final headings, most often under-rotations, did not differ by modality. Frequencies of lunges and arm capture movements were higher for visual stimuli both overall and especially for rostral proximal stimuli. The results demonstrate accurate orientation by sighted Xenopus to visual and lateral line stimuli; they are consistent with expectations based on in-register tectal maps. Orientation to lateral line stimuli is similar to previous results with blinded animals, revealing no heightened acuity in the latter. Modality differences indicate that the lateral line system is better for omnidirectional orientation and approach to distant stimuli whereas the visual system is more attuned to nearby rostral stimuli and more apt to mediate strikes.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Distribution of calbindinlike immunoreactive structures in the optic tectum of normal and eye-enucleated cyprinid fish

Summary Using the ABC immunohistochemical method, we investigated the distribution of calbindinlike immunoreactive structures in the optic tectum of normal fish, Tinca tinca, and from normal and unilaterally eye-enucleated fish, Cyprinus carpio. In nonoperated individuals of both species the optic tectum contained numerous immunoreactive neurons with strongly positive somata located in the stratum periventriculare and a thick immunolabeled dendritic shaft ascending radially toward the stratum fibrosum et griseum superficiale. The retinorecipient layers contained many fibrous immunoreactive structures. Some varicose fibers, isolated or in small bundles, were localized to the stratum album centrale, especially in the dorsal tectal half. Unilateral eye removal produced the disappearance of the immunoreactive fibrous structures located in the retinorecipient layers of the tectum contralateral to the enucleation. The present work shows that calbindinlike immunoreactive substances are localized in specific neural circuits of the fish optic tectum and suggests that the calbindin-like immunoreactive fibers in the retinorecipient strata are of retinal origin.     

Dopaminergic neurons in the retina of Xenopus laevis: amacrine vs. interplexiform subtypes and relation to bipolar cells

Presumed dopaminergic neurons were visualized in the retina of the clawed frog, Xenopus laevis, by anti-tyrosine hydroxylase (TH) immunoreactivity. The studied cells constitute a uniform population with perikarya at the junction of inner nuclear (INL) and inner plexiform (IPL) layers. Each cell body gives rise to 4–6 relatively stout processes (0.5–2.0 m in diameter) which run for up to 1.2 mm in strata 4–5 of the IPL. These processes have a very asymmetric distribution in the horizontal plane of the retina. A dense plexus of TH fine fibers is distributed uniformly in stratum 1 of the IPL. TH cells are distributed evenly but sparsely (16–20 cells/mm²) across the retina. About 20% of the TH neurons emit 1–3 distally directed fine processes, the majority of which extend < 20 m, which barely suffices to reach the outer plexiform layer (OPL). Other longer processes are typically unbranched; some reach the OPL, others run tangentially in the INL. The axon terminals of Golgi-impregnated bipolar cells are characterized according to the strata of the IPL in which they arborize. About 80% are confined either to strata 1–2 or 3–5, conforming to the off and on zones defined by Famiglietti and Kolb (1976). The remainder appear to end in both zones, some extending across the entire width of the IPL. EM examination showed that TH processes receive bipolar synaptic input in both distal and proximal portions of the IPL.