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.     

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.     

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.     

Xenopus laevis tadpole limb regeneration in vivo and in vitro: thyroxine directly promotes blastemal cell proliferation and morphogenesis

Regeneration in hindlimbs of Xenopus laevis larvae which were amputated at stage 53 and 55 through the tarsalia region is promoted by thyroxine (T4), while propyl-thiouracil (PTU) inhibits regeneration when compared to controls. In this paper, by in vivo and in vitro experiments, we demonstrate that the promoting effect of T4 on the regenerative processes of larval X. laevis hindlimbs is a direct effect of this hormone on the blastemal cells. By contrast, the inhibitory effect of PTU on the regenerative process is not due to a direct effect on blastemal cells or to a general toxic effect on the treated larvae, but is related to hypothyroidism induced by the drug. We find that: (i) an increase in blastemal cell proliferation is observed not only in blastemata of T4-treated larvae, but also in blastemata cultured in vitro in a medium supplemented with T4; (ii) the renegerative process is accelerated not only in larvae reared in T4 but also in larvae submitted to a combined treatment of T4 and PTU; (iii) inhibition of cell proliferation is observed in blastemata of PTU-reared larvae but not in blastemata cultured in vitro in a medium supplemented with PTU. Experiments on thyroidless larvae (which were submitted to transplantation of hindlimbs from larvae at stages 53 and 55 followed by amputation of their own right hindlimb and the transplanted limbs) have shown that without thyroid hormone the regenerative process is arrested at cone stage and the promoting effect of T4 treatment is dependent on limb stage and amputation level.     

The morphology of cultured melanophores from tadpoles of Xenopus laevis: Scanning electron microscopical observations

Summary Tail-fin melanophores of tadpoles of Xenopus laevis (Daudin) in primary culture were examined scanning electron microscopically in the aggregated and in the dispersed state. After isolation, the melanophores are spherical, but within 24 h they develop thin filopodia for attachment to the substratum. Subsequently, cylinder-like as well as flat sheet-like processes are formed, which adhere to the substratum with terminal pseudopodia and filopodia. The processes of adjacent melanophores contact each other, thus forming an interconnecting network between the melanophores.In the aggregated state the central part of the melanophore is spherical and voluminous. Both the central part and the processes bear microvilli. In melanophores with dispersed melanosomes the central part is much flatter; the distal parts have a thickness that equals a monolayer of melanosomes. The surface of the cell bears only scarce microvilli.These features indicate that melanophores do not have a fixed shape and that pigment migration is accompanied by reciprocal volume transformation between the cell body and its processes.     

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.     

Engineered telomeres in transgenic Xenopus laevis

The expanding roles of telomeres in epigenetic gene regulation, nuclear organization, and human disease have necessitated the establishment of model organisms in which to study telomere function under normal developmental conditions. We present an efficient system for generating numerous vertebrate animals containing engineered telomeres using a Xenopus laevis transgenesis technique. Our results indicate Xenopus zygotes efficiently recognize telomeric repeats at chromosome break points and form telomeric complexes thus generating a new telomere. The resulting transgenic animals progress through normal development and successfully metamorphose into froglets despite the chromosome breakage. Overall, this presents an efficient mechanism for generating engineered telomeres in a vertebrate system and provides an opportunity to investigate epigenetic aspects of telomere function during normal vertebrate development.     

Ultrastructural observations of the tunica muscularis in the small intestine of Xenopus laevis, with special reference to the interstitial cells of Cajal

The distribution and ultrastructure of the interstitial cells of Cajal (ICC) has been examined in the small intestine of the frog Xenopus laevis, as the physiological significance of these cells remains obscure in amphibians and other lower vertebrates. The present study has revealed the existence of a special type of interstitial cell in the tunica muscularis of the small intestine of Xenopus; this cell is characterized by the presence of numerous caveolae, many small mitochondria, and the formation of intercellular connections with the same type of cell. Since these ultrastructural features are shared with mammalian ICC, the cells in the small intestine of Xenopus probably correspond to ICC. These cells also form close contacts with neighboring smooth muscle cells and with nerve varicosities containing accumulations of synaptic vesicles. These cellular networks are likely to be involved in the transmission of nerve impulses to muscle cells, as has been suggested for mammalian tissues. However, true gap junctions have not been detected; they occur neither between the same type of cells nor between the putative ICC and smooth muscle cells. The widespread distribution of ICC or equivalent cells in different groups of vertebrates, together with the conservation of their ultrastructural features, suggests that they differentiated early in vertebrate evolution to play key regulatory roles in gastrointestinal movement.     

Intracellular signals trigger ultrastructural events characteristic of meiotic maturation in oocytes of Xenopus laevis

Summary Oocytes of Xenopus laevis were treated with agents which induce individual intracellular signals normally evoked during the process of meiotic maturation. Ultrastructural analysis of these oocytes allowed identification of specific second messengers that individually trigger single ultrastructural changes characteristic of the meiotic maturation process: Manipulation of intracellular cAMP levels induced changes in cortical granule position. Cytoplasmic alkalinization triggered a disruption of the annulate lamellae, a specialized organelle in the periphery of oocytes. Activation of protein kinase C caused rapid formation of a cortical endoplasmic reticulum and subsequent disruption of cortical granules. Manipulation of transmembrane calcium flux had varied results dependent upon the agent employed. Two of the treatments, Verapamil and zero external calcium, induced a reorganization in the oocyte periphery. The results indicate that these ultrastructural events are under the control of specific intracellular signals known to be elicited during meiotic maturation.     

Ultrastruktur der spinalen Liquorkontaktneurone beim Krallenfrosch (Xenopus laevis)

Zusammenfassung Zwischen und unter den Ependymzellen des Zentralkanals des Rückenmarkes von Xenopus laevis kommen Nervenzellen vor. Die intraependymalen Neurone sind rundlich und stehen mit dem Liquor cerebrospinalis durch eine breite Oberfläche in Berührung, von der sich längere und kürzere Fortsätze und ein Cilium (Typ 9+2) in das Lumen erheben. Die hypendymalen Neurone sind bipolar; ihr Dendrit verzweigt sich im Liquor ebenfalls in fingerförmige Fortsätze. Die Liquorkontaktfortsätze beider Zelltypen sind von feinen Filamenten ausgefüllt. Der Reissnersche Faden lagert sich manchen Fortsätzen an.In den intra- und hypendymalen Perikaryen findet man neben endoplasmatischem Retikulum, Golgi-Arealen und Mitochondrien kleine dense-core Vesikel (Durchmesser 600–900 Å). Der distale Fortsatz beider Neurontypen hat Neuritennatur. Axone, die synaptische und granulierte (Durchmesser 800–1200 Å) Vesikel enthalten, bilden relativ wenige Synapsen mit den Liquorkontaktneuronen. Im hypendymalen Neuropil findet man multipolare Nervenzellen, die 1000–1200 Å große granulierte Vesikel enthalten. Aufgrund des morphologischen Bildes wird die mögliche Rolle der Liquorkontaktfortsätze und des Ciliums bei der Funktion der Liquorkontaktneurone diskutiert.

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Ultrastructure of the spinal liquor contacting neurons in the clawed toad (Xenopus laevis)

Summary Nerve cells are situated between and below the ependymal cells of the central canal of the spinal cord of Xenopus laevis. The intraependymal neurons are round-shaped; they contact the cerebrospinal fluid by a large surface from which longer and shorter processes and a cilium (type 9+2) arise into the lumen. The hypendymal neurons are bipolar; their dendrite ramifies also into finger-like processes in the cerebrospinal fluid. The liquor contacting processes of both cell types contain fine filaments. The Reissner's fibre contacts some of the processes.In the intra- and hypendymal perikarya, small dense-core vesicles (diameter 600–900 Å) are found besides of endoplasmic reticulum, Golgi-areas and mitochondria. The distal process of both neuron types has neurite character. Axons containing synaptic and granulated (diameter 800–1200 Å) vesicles, form relatively few synapses with the liquor contacting neurons. In the hypendymal neuropile, multipolar nerve cells occur that contain granulated vesicles with a diameter of about 1000–1200 Å. On the basis of the morphological picture, the possible role of the liquor contacting processes and of the cilium in the function of the liquor contacting neurons are discussed.

     

Morphological and physiological aspects of melanophores in primary culture from tadpoles of Xenopus laevis

Summary Melanophores from tadpoles of Xenopus laevis (Daudin) were isolated by digestion of tail fins with acetyltrypsin and collagenase and maintained in primary culture for 6 weeks up to 3 months. Within 36 to 72 h the melanophores develop one to eight dendritic processes per cell; secondary and tertiary branchings of the processes were frequently observed. The melanophores in primary culture disperse under the influence of -MSH or cyclic AMP; upon rinsing out these substances the cells aggregate. In darkness, about 40 % of the cells disperse their pigment, whereas under illumination the pigment of the melanophores aggregates. To date, attempts to initiate cell division in melanophores have not been successful.     

Structure of the main ganglioside from the brain of Xenopus laevis

The main component of the ganglioside¹ mixture from the brain of the adult amphibian Xenopus laevis accounts for 35% of the total, as lipid bound sialic acid. This ganglioside has been purified and characterized by thin layer chromatography, partial and exhaustive enzymatic hydrolysis with sialidase, TLC-overlay procedures with anti-Gg₄Cer and anti-Neu5Ac6GalNAc specific monoclonal antibodies and mass spectrometry. All together the results suggest the following structure:Neu5Ac8Neu5Ac3Gal3(Neu5Ac8Neu5Ac6)GalNAc4Gal4Glc1Ceror, IV³--Neu5Ac₂,III⁶--Neu5Ac₂-Gg₄Cer.     

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.     

Lens formation from cornea implanted into amputated hindlimbs of Xenopus laevis larvae requires innervation or proliferating cell populations in the stump

The capacity of amputated early and late limbs of larval Xenopus laevis to promote lens-forming transformations of corneal implants in the absence of a limb regeneration blastema has been tested by implanting outer cornea fragments from donor larvae at stage 48 (according to Nieuwkoop and Faber 1956), into limb stumps of larvae at stage 52 and 57. Blastema formation has been prevented either by covering the amputation surface with the skin or by reconnecting the amputated part to the limb stump. Results show that stage 52 non-regenerating limbs could promote lens formation from corneal implants not only when innervated but also when denervated. A similar result was observed in stage 57 limbs where blastema formation was prevented by reconnecting the amputated part to the stump. In this case, relevant tissue dedifferentiation was observed in the boundary region between the stump and the autografted part of the limb. However, stage 57 limbs, where blastema formation was prevented by covering the amputation surface with skin, could promote lens formation from the outer cornea only when innervated. In this case, no relevant dedifferentiation of the stump tissues was observed. These results indicate that blastema formation is not a prerequisite for lens-forming transformations of corneal fragments implanted into amputated hindlimbs of larval X. laevis and that lens formation can be promoted by factors delivered by the nerve fibres or produced by populations of undifferentiated or dedifferentiated limb cells.     

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.     

Neural-inducing activity of nuclei and nuclear fractions from Xenopus laevis embryos

Summary From embryos (Xenopus laevis) of different developmental stages nuclei were isolated which exert neural inducing activity in the biological test. The active material could partly be extracted from the nuclei. Experiments for the isolation of nuclear ribonucleoprotein (RNP) particles have shown that the activity is localized at least in part in these particles. On the other hand, some neural inducer is not detached from chromatin and the nuclear matrix even with ionic detergents. Inducing activity was found in germinal vesicles and to a higher degree in the cytoplasm of oocytes, but in a masked, biologically inactive state.     

Changing complexity of endogenous lectin activities during juvenile development of Xenopus laevis

Galactoside-binding lectin has been isolated from whole Xenopus laevis embryos and tadpoles at four development stages: st. 24–26, 32, 41 and 47. The main lectin activity at st. 24–26 is -galactoside specific, producing a 34/35.5K doublet on SDS-PAGE. Later in development, lectin activities specific for a wide range of other sugars appear concommitant with the detection of a number of new protein bands on SDS-PAGE gels. The greatest variety of new lectin activities exists at st. 32 when lectins specific for all of the main sugar families found in nature are detected. After this stage and up to st. 47 (the beginning of metamorphosis), fewer different lectin activities are again detected. The results suggest that a complex, developmentally regulated battery of different lectins are present during early Xenopus development, perhaps with stage-specific roles to play in the control of tissue morphogenesis.