The effect of adult thymectomy upon helper function in Xenopus laevis, the South African clawed toad.

Carrier-primed helper activity enhancing anti-hapten (TNP-) responses in young adult Xenopus laevis reached peak levels 2-4 days after a low dose challenge (0.005% RBC) of unconjugated carrier. It fell quickly to negligible levels by 10 days. Adult thymectomized (ATx) animals were consistently unable to generate substantial levels of hapten-specific antigen-binding spleen cell (ABC) activity. Since ATx does not severely reduce splenic ABC responses to the high dose (10% RBC) of carrier used for hapten challenge, the results suggested a role for Xenopus adult thymus in carrier-specific enhancement of an anti-hapten response.     

Genetic control of T helper cell function in the clawed toad Xenopus laevis

The genetic control of the collaboration between Xenopus T and B cells has been analyzed in vitro using cells from five strains of major histocompatibility complex-defined Xenopus. When carrier (fowl gamma-globulin)-primed T cells and hapten (dinitrophenylated keyhole limpet hemocyanin)-primed B cells differed by minor histocompatiblity antigens or by only one haplotype of the major histocompatibility complex, the collaboration was efficient in the sense that large numbers of plaques, low-molecular weight antibodies and high-affinity IgM antibodies could be recorded in the cultures challenged with dinitrophenylated fowl gamma-globulin. However, when T and B cells differed at both alleles of the major histocompatibility complex, lower numbers of plaques were obtained, no low-molecular weight anti-hapten antibodies could be detected, and the IgM antibodies that were sometimes synthesized were of low affinity. This suggests that the major histocompatibility complex, or a gene linked with it, affects the collaboration between Xenopus T and B cells in a way perhaps similar to that described in mammals.     

Fine structure of the forelimb regenerate of the African clawed toad, Xenopus laevis

Forelimb regenerates from postmetamorphic Xenopus froglets were examined at various stages postamputation by light microscopy and scanning and transmission electron microscopy. The outside surface of the wound epithelium was found to exhibit progressive changes following amputation with a distinct difference in appearance between stump epidermis and wound epithelium at all stages examined. The internal structure of the wound epithelium is characterized by loosely arranged cells with numerous cell junctions and abundant intracellular filaments. The wound epithelium is separated at an early stage from the underlying cells by a thick band of extracellular matrix. Cells accumulating beneath the wound epithelium were morphologically similar to blastemal cells from completely regenerating limbs in other species but no evidence of myogenesis or abortive myogenesis was seen. Blastemal cells from the central portion of the regenerate were observed at varius stages of chrondrogenesis with those immediately beneath the wound epithelium least advanced in this respect. Those located more laterally appear not to be directly involved in chondrogenesis. Although the usual explanation for lack of complete regeneration in this species is inadequate innervation of the regenerate, the fine structure of the regenerating spikes noted here is markedly different than that of denervated, amputated newt limbs.     

Thymic involvement in memory responses after primary challenge with TNP-Ficoll in Xenopus laevis, the South African clawed toad

Memory to TNP-Ficoll in Xenopus laevis is demonstrable as a more rapid secondary response rather than an augmented response. While adult thymectomy abrogates ability to respond to both primary and secondary challenge with soluble TNP-Ficoll, memory to this antigen can be revealed in thymectomized animals by challenge with TNP-Ficoll on bentonite beads. Memory to TNP-Ficoll cannot be revealed by secondary challenge with TNP-LPS or TNP-RBC, but memory to TNP-LPS is revealed by TNP-Ficoll challenge. These results are consistent with the hypothesis that, whereas B1 cells (capable of responding to TI-2 antigens) can develop from a pool of B1 and B2 memory cells (generated in response to TD or TI-1 antigens), initial challenge with TI-2 antigen does not produce a pool containing both B-cell subsets.     

Phosphatidylserine expression on apoptotic lymphocytes of Xenopus laevis, the South African clawed toad, as a signal for macrophage recognition

Inflammation is avoided in apoptosis by early removal of dying cells by macrophages (MOs). In mammalian cells, an early aspect of apoptosis is the translocation of phosphatidylserine (PS) from the inner leaflet of the cell membrane to the surface. PS recognition can serve as a signal for triggering removal of dying cells. PS expression on splenocytes and thymocytes of Xenopus laevis was quantified using FITC-Annexin and flow cytometry following exposure in vitro to several known apoptogens for this species. All apoptogens used induced PS expression. Dose dependency and the kinetics of PS expression following exposure to the calcium ionophore, A23187, were also examined. Peritoneal exudate cells (PEC's) were cultured with A23187-treated thymocytes to test MO capacity for recognition of PS. MO binding to apoptotic thymocytes was reduced following exposure of PEC's to a water soluble analogue of PS, phospho-L-serine. The presence of a phagocytic PS-dependent recognition system in amphibia is supportive of the evolutionary conservation of this function in mammals that is crucial in limiting inflammation induced by dying cells.     

Lectin effects on thymic suppression of hemagglutinin production in vitro by spleen fragments from Xenopus laevis, the South African clawed toad

Suppression of hemagglutinin (HA) production was studied in co-cultures of thymus and spleen fragments from the South African clawed toad, Xenopus laevis. Concanavalin (Con)A and peanut (PNA) and wheatgerm (WGA)agglutinins were tested in conjunction with, or as a substitute for antigenic induction of thymus suppression. While thymuses from animals injected with PNA or WGA and antigen expressed suppressor function, those treated with Con A and antigen did not. Spleen fragments from animals challenged with PNA or WGA and antigen were resistant to suppression by normal thymus in reciprocal co-cultures; those receiving Con A and antigen alone were equally suppressible. All three lectins can substitute for antigen in the induction of thymus suppression. However, only PNA and WGA induce suppressor function in Xenopus spleen. Since WGA can induce splenic suppression in adult thymectomized animals, it is now possible to distinguish thymic and peripheral aspects of suppression of HA in this primitive vertebrate.     

Multisensory interaction in the torus semicircularis of the clawed toad Xenopus laevis

Interactions between the lateral-line, general somatosensory and auditory system were studied using field potential analysis and single unit recordings in the torus semicircularis of the clawed toad Xenopus laevis. As a response to paired stimuli (electric shocks applied to the peripheral nerves or acoustic clicks), in all systems a reduction of the second evoked potential occurred for intervals of up to 5 s. Following consecutive stimulation of two different systems, the amplitude of the second evoked potential was also reduced, indicating mutual interaction of different systems. Single unit recordings revealed the existence of both inhibitory and excitatory interaction between different modalities.     

Observations on the development of ascending spinal pathways in the clawed toad, Xenopus laevis

Summary The development of ascending spinal pathways has been studied in the clawed toad, Xenopus laevis. From stage 35 (hatching) on, HRP was applied at the spinomedullary border or to the area of the developing dorsal column nucleus, to analyze the development of ascending spinal pathways to the brain stem, and the onset and development of spinal projections to the dorsal column nucleus, respectively. Several populations of spinal neurons with ascending projections at least as far as the spinomedullary border were successively labeled. In early stages ascending spinal projections arise from Rohon-Beard cells and ascending interneuron populations located at the margin of the gray and white matter, i.e., marginal neurons. The ascending interneuron populations could be characterized as dorsolateral commissural and commissural interneurons projecting contralaterally, and as ipsilaterally projecting ascending interneurons and distinguished by Roberts and co-workers. Such a subdivision could be made until about stage 57. Then these ascending and commissural interneuron populations become intermingled with other populations of ascending tract neurons. Rohon-Beard cells could be labeled, more or less shrunken, until stage 55. Around stage 48 (at the time of the appearance of the limb buds) spinal ganglion cells could be labeled from the spinomedullary border and the developing dorsal column nucleus. At stage 48 such ascending primary spinal afferents were found to arise only from non-limbbud-innervating dorsal root ganglia. Gradually also the limb-bud-innervating ganglia give rise to ascending collaterals, so that by stage 53 all spinal ganglia send ascending collaterals to the brain stem. The number of cells of origin of secondary spinal afferents to the brain stem increases during development, and their distribution becomes more extensive. Particularly impressive is a large population of neurons in the dorsal horn projecting ipsilaterally to the dorsal column nucleus. Part of the latter population represents non-primary spinal afferents to the dorsal column nucleus.     

Comparative study of ultrastructures of the lateral-line organs and the palatal taste organs in the African clawed toad,Xenopus laevis

The fine structure of the lateral-line organ and the palatal taste organ in the African clawed toad, Xenopus laevis, was examined by means of electron microscopy. The lateral-line organ consisted of hair and accessory cells. The apical surface of a hair cell was studded with one kinocilium and 20 to 40 stereocilia. Synaptic bodies and subsynaptic cisternae were found in the cytoplasm of a hair cell adjacent to the synaptic contacts with the afferent and the efferent nerve endings, respectively. Crystalline bodies were observed in both the nucleoplasm and the cytoplasm of almost all hair cells. The palatal taste organ consisted of three types of cells: the taste, sustentacular, and the Merkel cells. The taste cells contained numerous dense-cored vesicles which accumulated in close association with both the afferent synapses and the basal plasma membrane. The possibility was raised from the ultrastructural results that these vesicles had dual functions as both neurotransmitter and hormone. The existence of Merkel cells in the palatal taste organ suggested that this organ might function not only as a chemoreceptor but also as a mechanoreceptor. In spite of possible chemosensory function of the lateral-line organ in Xenopus, its ultrastructure was significantly different from that of the typical gustatory organ, the palatal taste organ, in this animal.     

Differential cyclophosphamide sensitivity of suppressor function in Xenopus, the clawed toad

Thymocyte and Splenocyte cultures from in vivo immunised Xenopus were assayed to test their suppressive capacity. Immunisation with TNP-Polyvinylpyrrolidone induced suppression. Suppression induced by the haptenated antigens, TNP-Red blood cells, TNP-Lipopolysaccharide, and TNP-Ficoll affected the anti-TNP antibody response of splenocytes from TNP-PVP immunised animals. Pretreatment with cyclophosphamide revealed both a sensitive and insensitive suppression capacity in Xenopus laevis.     

Basolateral uptake and tubular metabolism of L-citrulline in the isolated-perfused non-filtering kidney of the African clawed toad (Xenopus laevis)

The kidney forms arginine (Arg) by using citrulline (Cit) as precursor, and is the main source of Arg for systemic protein synthesis. Even if the filtered and reabsorbed load (in rats) is sufficient for normal Arg synthesis, the following questions remain. (a) Can Cit be taken up across the contraluminal membrane of the tubule cells also? If so, (b) by what kind of mechanism? And (c) is this Cit, entering the cell from the peritubular side, metabolized to Arg and ornithine (Orn)? Although these questions are raised mainly in connection with mammals, we used the amphibian kidney, which is especially suitable because of its double blood supply, for an initial approach to the problem. After the toad was decapitated, the portal vein, the caval vein and the ureters were catheterized, and the kidneys were perfused through the portal vein (Ringer solution +l- or d-Cit+inulin+p-aminohippurate +l-aspartate). Exclusive peritubular perfusion was assured by showing that inulin perfused into the portal vein did not appear in the urine. During perfusion of the portal vein with l-Cit in a physiological concentration (65 μmol/l), an initial peritubular net uptake of l-Cit of 170±27 (n=10) nmol·h^?1·g kidney^?1 (wet weight) was observed, whereas the value for d-Cit (65 μmol/l) was only 18±7 (n=6) nmol·h^?1·g^?1. After perfusion for 50 min, the uptake of l-Cit reached a steady state with an uptake rate of 108±5 nmol·h^?1·g^?1. Adding l-phenylalanine (l-Phe; 20 mmol/l) to the solution or substituting mannitol for NaCl in the perfusate, decreased this l-Cit uptake to values similar to those for d-Cit. During perfusion with 65 μmol/l l-Cit, the Arg delivery into the venous blood was 40±4.8nmol·h^?1·g^?1, corresponding to 36% of the peritubular Cit uptake in the steady state, and the Orn delivery was 49.6±3.2nmol·h^?1·g^?1, corresponding to 46% of peritubular Cit uptake in the steady state. The venous Arg delivery increased to 51 nmol·h^?1·g^?1 while the kidney was perfused with 1 mmol/l l-Cit. At higher l-Cit concentrations no further increase of venous Arg delivery could be observed. During perfusion with d-Cit (65 μmol/l) or when adding l-Phe (20 mmol/l) no Arg or Orn could be detected in the venous outflow. In conclusion, the amphibian kidney perfused in situ is a suitable model for studying peritubular amino acid uptake and metabolism in the kidney. In the toad kidney, a peritubular uptake mechanism for l-Cit exists, which is stereospecific, saturable, NaCl-dependent and can be inhibited by l-Phe. In the tubule cells, l-Cit is transformed into Arg. Part of the Arg is metabolized further to Orn and urea, and Arg and Orn are released into the venous outflow.     

Spontaneous thyroid-containing teratoma associated with impaired development in the African clawed frog, Xenopus laevis

Teratomas are rare in amphibians and the neoplasm described here, which had a significant thyroid carcinoma component, is the first tumour of this type to be reported in Xenopus laevis. The thyroid component contained moderately to well-differentiated acinar glands showing much hyperplasia, dysplasia, and reduced and distorted colloid reservoirs. Cartilaginous, neural, muscular, mesenchymal and gut-like epithelial components were also observed in this ventral mediastinal neoplasm, indicating aberrant proliferation from all three germ layers. This teratoma was only one abnormality in a complex of developmental changes, followed for 28 months, which appeared in a single generation of sibling 2-week-old Xenopus larvae. Two hundred larvae produced by an apparently normal adult pair initially showed ocular defects, including microphthalmia, anophthalmia and tumours projecting near the eyes. During further development up to 28 months, mediastinal tumours developed in nine frogs; these tumours were associated with reduced growth, the frogs reaching only 13-20% of normal weight, and greatly enhanced ventral pigmentation.     

Synaptic organization of dorsal root projections to lumbar motoneurons in the clawed toad (Xenopus laevis)

Summary Synaptic connexions between dorsal root primary afferents and lumbar motoneurons have been investigated in the isolated spinal cord of the clawed toad. The study of monosynaptic actions evoked in motoneurons by 9th or 10th dorsal root stimulation or by impulses in single primary afferents provided evidence for both electrical and chemical junctional transmission at the sensory-motor synapses. The anterograde filling of the 9th and 10th dorsal roots with horseradish peroxidase (HRP) shows that afferents do project to the motoneuron field of the segments IX and X. Some of the fibres not only reach the dorsally located motoneurons, but also cross the lateral motor column (LMC) and terminate in the marginal zone of ventral horn gray matter. The projections of the 9th and 10th dorsal root fibres are most numerous in the caudal part of segment X. Simultaneous HRP labeling of single motoneurons and the whole 10th dorsal root has revealed that afferent fibres make contacts not only on the distal dendrites of the motor cells, but also on the proximal ones. This latter finding is in a good agreement with the electrophysiological data.Dr. Shiriaev died September 11, 1984     

In vitro evidence for T-B lymphocyte collaboration in the clawed toad, Xenopus

Requirements for an in vitro secondary antibody response to the soluble antigens dinitrophenylated keyhole limpet hemocyanin and dinitrophenylated flow gamma-globulin are described for the clawed toad, Xenopus. Priming of both hapten and carrier-specific cells is required in order to obtain good responses to the hapten. The carrier-reactive ("helper") cells do not adhere to nylon wool, are X ray-resistant and surface Ig-negative. The cell adherence and X ray sensitivity characteristics of these Xenopus "T" cells and their ability to generate a mixed lymphocyte response, allogeneic cytotoxic and helper activities, are identical to those of thymus-derived (T) cells in higher vertebrates. The hapten-reactive, antibody-secreting cell precursors are nylon-adherent, X ray-sensitive and surface Ig-positive. In the absence of other distinguishing serological markers for T cells in frogs, and considering the thymus dependency of the helper activity described here, the evidence presented is interpreted as demonstrating the requirement for antigen-specific T-B cell cooperation in response to soluble protein antigens in vitro for Xenopus.