Laevis Larvae Research Articles

The development of primary afferents to the lumbar spinal cord in Xenopus laevis

The development of dorsal root ganglia (DRG) and the primary afferent system of the hindlimb was studied during metamorphosis in Xenopus laevis larvae. The first DRG cells appeared at stage 40 and at stage 48 the first primary afferent fibers entered the lumbar spinal cord where they bifurcated into ascending and descending branches. Primary afferent fibers and the dendrites of the secondary motoneurons contacted first in a lateral (state 56) and later (stage 58) in a dorsomedial terminal field. Reflexogenic hindlimb behaviour at stage 56 concurred with the presence of the lateral terminal field and many unipolar dorsal root ganglion cells.

Ultrastructural identification of the primitive muscle spindle in the Xenopus laevis larvae.

The initial formation of muscle spindles was studied with electron microscopy using the toe muscle of Xenopus laevis. At the larval stage 57 (Nieuwkoop and Faber 1967), muscle spindles were first identified primarily by the presence of sensory endings associated with a thin bundle of myotubes, e.g. intrafusal (IF) myotubes which were partly invested by a single cellular layer. The number of IF myotubes per spindle was 5 to 6; the adult complement. IF- and extrafusal (EF) myotubes were almost identical in their size and structure. A few thinner IF myotubes with scarce myofibrils were also present. The reticular zone had been undeveloped. Sensory endings were smaller in size and in number per spindle than those in the adult, forming irregular beaded chains with occasional tubular expansions. The endings and IF myotubes were rarely in direct contact, being frequently interposed by a satellite cell and its process. Incipient fusimotor endings were widely distributed from the juxta-equatorial to the polar region. Large cored vesicles resembling the neurosecretory vesicles occurred in sensory and motor endings as well as in intramuscular nerve fibers. The vesicles may be involved in the neuronal influence upon the spindle differentiation. The results were compared with the formative process of mammalian spindles.

The development of the Merkel cells in the tentacles of Xenopus laevis larvae.

The Merkel cells in the larval tentacles of Xenopus laevis were examined by TEM. Different forms of Merkel cells were found, depending on the age of the larvae or the location in the tentacles. These forms have the appearance of intermediate states between Merkel cells and superficial epidermal cells; thus an epidermal origin for the Merkel cells seems more likely than an immigration from the neural crest. The forms differ in (1) their location in the epidermis, (2) their shape, (3) the number and extension of their desmosomes, (4) the content and distribution of dense-core granules, and (5) the outgrowth of their finger-like processes. Also the relation to a nerve ending is different. By marking Merkel cells with quinacrine, fluorescence spots were observed between the superficial and basal epidermal cells or, in the very tip, within the superficial epidermal cells. These latter spots represent immature Merkel cells, as confirmed by TEM. This indicates a development of Merkel cells from superficial epidermal cells and migration towards the basal layer. Dermal Merkel cells were never observed.

Transdifferentiation of eye tissues in anuran amphibians: Analysis of the transdifferentiation capacity of the iris of Xenopus laevis larvae

Lensectomized Xenopus laevis larvae are capable of regenerating a lens from the cells of the outer cornea. Unlike the outer cornea, the iris of larval Xenopus exhibits a high degree of phenotypic stability, even when it has been damaged to various degrees in order to stimulate its latent transdifferentiative competence. However, when isolated from its surrounding tissues and implanted in an appropriate site, the dorsal iris of larval Xenopus is capable of following a differentiative pathway different to that normally followed in situ. Our results show that, when such an implant is placed in the vitreous chamber of a lensectomized eye, the pigmented epithelial cells of the iris transdifferentiate into neural retina regardless of whether the iris stroma is present or not. Unlike the vitreous chamber, the environment of the anterior chamber of a lensectomized eye does not promote the transdifferentiative process of the iris. We suggest the existence of eye factors that promote retina-forming transformation of the iris and that are distributed in a gradient in lensectomized eyes.

Survey of the vestibulum, and behavior of xenopus laevis larvae developed during a 7-days space flight

Aquatic animals have almost no body weight related proprioception for spatial orientation. Xenopus larvae, like fish, maintain their attitude in water by continuous correction with their fin(s). For these reasons a special performance of the equilibrium system compared to terrestrial animals is necessary. Evidently fish therefore have more compact (dense) otoliths; Xenopus larvae have less dense otolith (membranes) similar to land vertebrates; but their sacculus-otoliths are vertically positioned, which also may lead to a higher g-sensitivity. For plausibility reasons gravity should influence the embryonic development of gravity receptors. Yet, evaluations of photographs taken from the surface of cut deep-frozen objects by incident light show no aberration of the shape of the whole vestibulum and of the shape, density, size and position of the otolith membrane in larvae developed under near-zero g (NEXPA-BW-STATEX in D1-Mission). The further evaluation of the “weightless-larvae” revealed a probably not yet described statolith-like formation in the dorsal wall of the vestibulum. In the weightless larvae this formation outnumbers, also qualitatively, strongly the 1-g controls. An extra result is the lack of striking effects of cosmic radiation on the embryonic development of the flown Xenopus eggs. The swimming behavior of the larvae which was observed about one hour after landing of the Space Shuttle showed a typical anomaly (loop swimming), which is known from larvae developed on the clinostat or from fish flown aboard Apollo capsules.

The development of serotonergic raphespinal projections in Xenopus laevis

The development of serotonin-immunoreactive neurons in the central nervous system of Xenopus laevis larvae has been studied with special emphasis on the development of the raphe nuclei and raphespinal projections. The first serotonergic neurons were observed in the rostral part of the brain stem at stage 25, only 28 hr after fertilization. By stage 28 some 20 serotonin-immunoreactive neurons were found in the rostral part of the brain stem, bearing small protrusions on the ventromedial side of the soma. These initial axonal outgrowths reach the rostral part of the spinal cord at stage 32. By stage 35/36 the growth cones of the descending serotonergic axons in the spinal cord have reached the level of the anus (10th to 15th myotome). Up to stage 45 the majority of the descending serotonergic axons was found in the dorsolateral part of the marginal zone of the spinal cord. After stage 45 some serotonergic axons were also found scattered over other parts of the spinal marginal zone. Collateral oranches were first observed in the caudal part of the brain stem at stage 35/36. Later they occurred also in the rostral (stage 43) and caudal (stage 50) spinal cord, usually on fibers in the ventral half of the spinal cord. The number of serotonergic neurons in the central nervous system (brain stem and hypothalamus) increased steadily throughout development until stage 45. After that the total number of serotonergic neurons in the central nervous system increased about two times faster than the number of serotonergic neurons in the raphe nuclei, due to a massive increase of serotonergic neurons in the hypothalamus.The present study shows that young, just differentiated raphe neurons already contain serotonin. The generation of these neurons appears to take place in the ventricular zone (matrix) of the brain stem between the caudal border of the mesencephalon and the entrance of the nervus octavus. From here these neurons seem to migrate to their final destination. The distribution of serotonin-immunoreactive neurons in the brain stem suggests that a superior (not described so far in Anura) and an inferior raphe nucleus can be distinguished in Xenopus. A rostrocaudal gradient seems to be present in the production of serotonergic neurons which project to the spinal cord. Spinal projections from the raphe nuclei are particularly extensive from the nucleus raphes inferior and gradually decrease rostralwards. In the rostral part of the nucleus raphes superior almost no neurons projecting to the spinal cord are found.

Growth and death of cells of the mesencephalic fifth nucleus in Xenopus laevis larvae.

Positions, numbers, and cell and nuclear sizes of mesencephalic fifth nucleus (M-V) cells were determined in Xenopus laevis larvae in stages 47 through the end of metamorphosis at stage 66. M-V cells may be found at every tectal level, from the rostralmost section almost to the caudal pole, and in the anterior medullary velum. A large majority of the cells lie between 15 and 65% caudad of the rostral tip of the tectum. At anterior and middle tectal levels the cells lie lateral to but mainly above the optic ventricle. At posterior levels, to which the ventricle does not extend, a few cells may be seen at middle and lower tectal levels, as if in transition to the anterior medullary velum. At stage 47 fewer than ten cells are seen in each animal. The numbers rise to 20-40 by stage 50, and are uniformly above 100 after stage 51. Initially many M-V cells were small, i.e., 6-7 microns in diameter, but grew to a mean diameter of about 19 microns at stage 59, with a maximum value of 29 microns. A single individual at stage 57 had 581 cells. The peak of mean cell numbers, 387, occurred at stage 59, which was also the stage with the highest mean values for nuclear and cell sizes. Pyknotic M-V cells at low frequency were seen at stages 55 and 57, and at all stages thereafter. Cell death frequency peaked at stage 62, but continued through stage 66. By stage 66 mean cell numbers had been reduced to about 240, indicating survival of about 60% of cells present at stage 59.

Use of Xenopus laevis larvae in 96-hour, flow-through toxicity tests with naphthalene.

Use of Xenopus laevis larvae in 96-hour, flow-through toxicity tests with naphthalene.

B-lymphocyte differentiation in Xenopus laevis larvae

Direct immunofluorescence was used to detect the appearance of pre-B cells in different tissues of Xenopus laevis embryos. Cells with intracellular heavy chains belonging to high-molecular-weight immunoglobulin, but devoid of any detectable surface immunoglobulin, were first observed in the liver at stage 46. These cells were also observed in the developing spleen at stage 49 and they were present in these organs up to the beginning of metamorphosis. At stage 58 (climax of metamorphosis) their number dropped to zero. Results of organ culture experiments suggest that the stem cells enter the liver just before stage 39, where they differentiate to become pre-B cells at stage 46.

Xenopus laevis larvae (Amphibia, Anura) as model suspension feeders

Larvae of the South African clawed frog, Xenopus laevis (Daudin), are efficient, obligate suspension feeders. We examine the relationship between the ambient particle concentration offered these larvae as food and their filtering, ingestion, and buccal pumping rates. We demonstrate that: (i) the larvae can sense and respond to a broad range of particle concentrations, down to 0.2 mg 1−1 (dry weight); (ii) their metabolic needs theoretically can be met by particle concentrations as low as 5 mg 1−1; and (iii) their patterns of regulation of filtering and ingestion fit predictions from certain models used to describe zooplankton feeding dynamics. Two such models are discussed: the modified Monod (Michaelis-Menten) model, with a lower threshold below which the tadpoles do not feed, and an energy optimization model. Both the models and the observed behavior of the tadpoles allow for stability of populations of food organisms. Tadpole feeding dynamics apparently are compatible with both the predictions and assumptions of these models, suggesting similar regulation of feeding by tadpoles and zooplankton. However, the size, morphology, and behavior of X. laevis larvae make their feeding regulation uniquely accessible to direct observation.

Replication of Ribosomal DNA in Xenopus laevis

The study of the localization of the replication origins of rDNA in Xenopus laevis has been approached by two different methods. 1. The DNA of X. laevis larvae was fractionated by CsCl gradient centrifugation in bulk and ribosomal DNA and examined in the electron microscope. In bulk DNA, clusters of microbubbles, which are related with the origins of replication, appear to be spaced along the DNA molecules at intervals comparable with the size of the 'average' replicon of X. laevis. In ribosomal DNA, the distance between adjacent clusters is much shorter and corresponds to the size of the rDNA repeating unit. When ribosomal DNA was submitted to digestion with restriction enzymes (Eco RI and HindIII) the microbubbles are observed in the non-transcribed spacer-containing fragment. 2. Cultured cells of X. laevis were synchronized by mitotic selection and incubated with 5-fluoro-2-deoxyuridine for a time longer than the G1 phase. This treatment synchronizes the replicons and allows them to start replicating very slowly. It was thus possible to obtain a preferential labelling of the regions containing the origins. The analysis by gel electrophoresis of the Eco Ri-digested rDNA showed that the radioactivity was preferentially incorporated in the fragments which contain the non-transcribed spacer. The results of these two approaches indicate that the rRNA gene cluster consists of multiple units of replication, possibly one per gene unit. Furthermore they show that the origins of replication are localized into the non-transcribed spacer.

An analysis of school structure for tadpoles (Anura: Amphibia)

The spatial relationships of Bufo woodhousei larvae were examined in the laboratory and field and compared with laboratory data for Xenopus laevis larvae. 1. Tadpoles of both species avoid positions directly in front of or behind neighbors. Bufo larvae demonstrate particularly strong preference for being directly lateral to conspecifics. Bearing relationships are different in light than in dark for Xenopus, but not for Bufo. 2. In the absence of other cues, Bufo and Xenopus tadpoles orient parallel to nearest neighbors under a variety of conditions of density, size, and in both the light and dark, although more strongly in the light. Bufo larvae show little tendency to orient antiparallel in contrast to Xenopus. Bufo typically parallelorient over greater distances than Xenopus, whether interactive distance is measured in number of nearest neighbors, centimeters, or tadpole body lengths. 3. Schooling tadpoles, in contrast to fish, tend to be relatively stationary and at random distances from their nearest neighbors.

Patterns of protein synthesis in livers of Xenopus laevis during metamorphosis: Effects of estrogen in normal and thyrostatic animals

Two-dimensional gel electrophoresis has been used to analyse protein synthesis in the livers of Xenopus laevis larvae during metamorphosis. The patterns found at different developmental stages have been characterised and compared to those found in developmentally static tadpoles and estrogen-treated tadpoles. The results suggest that the majority of proteins synthesized by the larval liver during metamorphosis can be divided equally into three main categories: those which are synthesized continuously, those whose synthesis is lost, and those whose synthesis is gained during development. The synthesis of proteins tends to be lost earlier in metamorphosis than it is gained. The pattern of liver protein synthesis in thyrostatic animals is not characteristic of any single stage of normal development, and displays features characteristic of many different stages. About half the changes in protein synthesis which occur during normal metamorphosis are dependent upon it. All the stages examined are responsive to estrogen, and each has a characteristic response. Half of the estrogen-induced changes in protein synthesis are independent of metamorphosis, while the other half require metamorphosis.

Influence of the olfactory placode on the development of the brain in Xenopus laevis (Daudin) : I. Axonal growth and connections of the transplanted olfactory placode

The olfactory placode of Xenopus laevis larvae at different stages of development has been removed and transplanted onto the head of host larvae to determine the effect of the donor/host age on the viability of the transplant. Viability of the transplant (defined as penetration of the nerve of the transplanted placode in the host's CNS) has been found to be maximal when both donor and host were at about stage29/30. Using stage 29/30 larvae, three distinct zones have been mapped on the host's head from which the transplanted olfactory placode consistently sent its nerve to three different regions of the brain, namely to the telencephalon, the diencephalon and to the myelencephalon, respectively. The additional olfactory input induced a hypertrophy where it penetrated the telencephalon. When the olfactory input penetrated the diencephalon a marked hyperplasia of the neuronal elements and formation of glomeruli was observed in the dorsal thalamus. There was no overall change in the organization of the myelencephalon when the input reached this region. Donors up to and including stage 41 remade the placode. Removal of the placode after this stage resulted in permanent loss of the organ and hypoplasia of the olfactory bulb and telencephalon. The results illustrate the unique property of the olfactory sensory neurons to induce hyperplasia in anomalous regions of the host's CNS and to determine, in these regions, the characteristic terminal structures which are commonly described as glomeruli.

Lens formation from cornea in the presence of the old lens in larval Xenopus laevis.

Three different types of experiments were carried out to investigate the role of the lens in lens-forming transformations of the outer cornea of Xenopus laevis tadpoles (at stage 51, as defined by Nieuwkoop and Faber, '56): 1) simple lentectomy; 2) incision of outer and inner cornea in the region opposite the dorsal iris without lens removal; and 3) incision of outer and inner cornea and perforation of dorsal iris without lens removal. The results indicate that the outer cornea of Xenopus laevis larvae has lens-forming competence, even in the presence of the old lens, only when direct communication has been set up between the outer cornea and the environment of the vitreous chamber. These data indicate that the inhibition exerted by the lens on lens-forming transformations of the outer cornea is mechanical in nature.

The role of thyroxine in the transition of vitellogenin synthesis from noninducibility to inducibility during metamorphosis in Xenopus laevis

Vitellogenin synthesis can be induced in vivo by oestrogen in the livers of Xenopus laevis larvae during metamorphic climax. The timing of the appearance of vitellogenin inducibility has been precisely defined in relation to changing morphological characteristics, and occurs during normal development at stage 62. This suggests that thyroid hormones may be involved in the transition from the uninducible to the inducible state of the vitellogenin gene. Chemical inhibition of the larval thyroid by propyl thiouracil or thiourea was used to produce developmentally static larvae. Tests on these animals for response to oestrogen showed that the transition depends on metamorphic stage, not on chronological age. Immersion of normal tadpoles or developmentally static tadpoles in high concentrations of thyroxine induces rapid metamorphosis, but does not alter the developmental stage at which vitellogenin inducibility is acquired in either case. Induction of vitellogenin synthesis in animals before stage 62 was only achieved by permitting thyrostatic animals to continue a delayed metamorphosis. It is apparent that thyroxine does not directly confer inducibility on the vitellogenin gene.

Lens formation from pericorneal epidermis in the presence of the old lens in larval Xenopus laevis

AbstractThe lens‐forming capacity of the pericorneal epidermis of Xenopus laevis larvae at stage 51 has been investigated. The results obtained show that, whether or not the lens is present, the pericorneal epidermis can form a lens when it is in direct contact with the vitreous chamber of the eye. It is concluded that the posterior pole of the lens either does not produce any inhibiting substances or else is unable effectively to remove the factor(s) contained in the vitreous chamber which are responsible for the lens‐forming transformations of the pericorneal epidermis.

Trochlear nerve regeneration in Xenopus laevis larvae.

The trochlear nerve of Xenopus laevis larvae was sectioned in the orbit, and the nerve distal to the section was removed, so that it could not serve to guide the return of the regenerating nerve to the superior oblique muscle (SOM). In a second series, in addition to removing the distal nerve segment, the SOM was removed or damaged. The regeneration pattern of the trochlear nerve fibers was recorded in situ at 1 to 5-day intervals with the aid of methylene blue staining. The early growth or regenerating fibers was multidirectional; only some fibers encountered the SOM by day 6 or 7; others reached nerves, inappropriate muscle, and other tissues. Ultimately, the fibers that reached the SOM persisted and were reinforced, while those that reached other locations were withdrawn. By 20 days or so, the regenerated trochlear nerve usually had an appearance that was close to that of an uncut nerve. In the second series, in which the SOM was removed or damaged, the multidirectional character of the growth persisted through 20 days after section. these data suggest that: 1) Trochlear nerve regeneration is not guided by cues that closely control fiber growth along a direct path to the SOM, and 2) The SOM acts as a trap for regenerating trochlear nerve fibers, promoting the maintenance and the fasciculation of the trochlear nerve fibers that reach it, and the degeneration of the fibers that do not.

A comparative study of the buccal pumping mechanism of tadpoles

To understand the ecological significance of diversity in the buccal pump morphology of anuran larvae we have developed a descriptive geometric model of the tadpole buccal pump. The model allows comparison of the buccal puinping mechanism of different species. The reliability of the model was established by comparing buccal volumes predicted by the model with buccal volumes reported in the literature for Rana sylvatica and Rana catesbeiana as well as those obtained from our own feeding experiments with Xenopus laevis larvae. The model was used to examine the following features for the larvae of 40 species from 11 anuran families: (1) the amount of deflection in the buccal floor with each stroke of the buccal pump (angle of rotation of the ceratohyal); (2) the mechanical advantage of the pump (the relative length of the lever arm on the ceratohyal); and (3) buccal volume. We identify patterns in diese three features that correlate with the feeding ecology of a variety of tadpoles. Macrophagous larvae tend to have a long lateral lever arm on the ceratohyal (high mechanical advantage), and produce a large buccal volume by deflecting a large buccal floor area through a shallow arc (i.e. a large bore, short stroke system). Midwater, microphagous larvae tend to have poor mechanical advantage, but nevertheless achieve a large buccal volume; in these forms the large buccal volume is obtained by deflecting a smaller buccal floor area through a larger arc (i.e. a small bore, long stroke system). Benthic larvae, such as stream tbrms with suctorial mouths, tend to have high mechanical advantage, but only modest buccal volume. Larvae can regulate their feeding rate by varying either the p umping rate (frequency modulation) or the buccal floor displacement (amplitude modulation). The anatomy of the buccal pump may determine not only the feeding ecology of a larva, but the predominant behavioral pattern it uses to regulate its feeding rate. Buccal volume has a negative allometric relationship to body length both intraspecifically and interspecihcally.

Thyroid hormone induces competence for oestrogen-dependent vitellogenin synthesis in developing Xenopus laevis liver.

OESTROGEN-DEPENDENT synthesis of vitellogenin, the precursor of the major yolk proteins, in the liver of egg-laying vertebrates is an attractive model for investigating regulatory mechanisms of gene expression. Induction of vitellogenin synthesis by oestrogen occurs not only in the liver of females but also in males which normally do not produce this protein, thus showing that responsiveness to oestrogen is not linked to sex (for review see ref. 1). However, Follet and Redshaw2 reported that liver of Xenopus laevis larvae is refractory to oestrogen, becoming responsive shortly after completion of metamorphosis. As amphibian metamorphosis is known to be obligatorily dependent on thyroid hormones (for review see ref. 3), the question arises as to whether acquisition of competence to respond to oestrogen by the Xenopus liver cells is part of the hormonally regulated metamorphic changes or merely reflects age-dependent maturation. Here we present evidence that during normal development of Xenopus laevis the liver cells become responsive to oestrogen during metamorphosis and before vitellogenin synthesis has been initiated by endogeneous oestrogens. Moreover, we demonstrate that responsiveness to oestrogen fails to appear when metamorphosis is suppressed, but readily appears after induction of metamorphosis by thyroxine, suggesting that acquisition of competence to respond to oestrogen by the Xenopus liver cells is controlled by thyroid hormones.