Thymocyte precursors in early-thymectomized Xenopus: migration into and differentiation in allogenic thymus grafts

In an anuran amphibian, Xenopus laevis, thymectomy of 4-day-old larvae abrogates T-cell dependent immune responsiveness. When such early-thymectomized (TX) diploid frogs were implanted with histocompatible triploid thymuses and grafted 8 weeks later with skin from third-party donors, the grafts were rejected relatively normally in 20-27 days. Microspectrophotometric determination of ploidy 3-5 months after thymus reconstitution revealed that most thymocytes were donor-derived. In contrast, when TX frogs received allogeneic triploid thymuses, they rejected skin grafts from a third-party donor relatively slowly (48-92 days) but did not reject skin from the thymus donor. Most thymocytes in such animals were of host origin. Host thymocytes were present 4 weeks after thymus implantation and became dominant population by 12 weeks. Few thymus implant-derived donor cells were detectable in the host spleen. These data suggest that existence of precursor cells in TX Xenopus that can functionally differentiate along a T-cell pathway as a result of microenvironment provided by the thymus implant.     

Immune Response to “Self” Lens in Xenopus laevis Enucleated during Larval Life

We have reinvestigated an important issue in the amphibian immunology that has not been settled for years since the pioneer work of Triplett, concerning the necessity of being exposed to organ-specific antigens early in development. It was found that syngeneic lenses were rejected by frogs, Xenopus laevis, that had been enucleated (eye removed) during early larval life. This rejection did not occur in intact frogs or in those enucleated in later larval or adult life. Whereas the splenocytes from intact frogs did not proliferate in response to a co-cultured syngeneic lens, those from frogs that had been enucleated at any of the larval stages, or even after metamorphosis, proliferated intensely. Both of these responses were shown to be thymus- dependent. In conclusion, it was demonstrated that the frog immune system rejected even syngeheic lenses by enucleation in early larval life and that it began to recognize the syngeneic lenses by lymphoid proliferation after enucleation, even in later life.     

Induction of T cell differentiation in early-thymectomized Xenopus by grafting adult thymuses from either MHC-matched or from partially or totally MHC-mismatched donors

The effect of grafting thymuses from major histocompatibility complex (MHC)-matched and mismatched (either partially or totally) adult donors on the restoration of T-cell dependent immune responses of Xenopus adults that were thymectomized (txd) during early larval life was examined. Four to 5 month-old diploid (or triploid) frogs of defined MHC haplotypes that had been txd on day 4 or 5 postfertilization were each grafted subcutaneously with a pair of thymuses from a triploid (or diploid) MHC-defined frog. Regardless of the donor-host combination, thymus grafts restored in vivo acute skin allograft rejection capacities and antibody responses to SRBC and in vitro proliferative responses of spleen cells to the T cell mitogens, PHA and Con A. Txd frogs that were grafted with MHC-mismatched thymus did not reject skin grafts with the MHC haplotype of the thymus donor. Nevertheless, their spleen cells could proliferate, in one way MLC, in response to irradiated stimulator cells with the thymus donor MHC haplotype. Ploidy analyses of mithramycin-stained thymic and splenic lymphocytes (DNA quantitation by flow cytometry) demonstrated that in certain donor-host combinations, cells from totally or partially MHC-mismatched donors as well as from MHC-matched donors persisted in the thymus grafts and/or the spleens of txd hosts. Chimerism lasted for at least 1 year after thymus grafting. In other donor-host combinations, however, (txd isogenic cloned LG15 frogs grafted with allogeneic thymus from MHC-homozygous triploid J strain frogs), no donor cells could be detected 7 months after thymus grafting. Chromosome counts of PHA-induced metaphases of spleen cells from these and other thymus-grafted frogs revealed host cells in metaphase. This suggests that thymus grafts can promote the differentiation of host precursor cells along a T cell pathway.     

Ouro proteins are not essential to tail regression during Xenopus tropicalis metamorphosis

Tail regression is one of the most prominent transformations observed during anuran metamorphosis. A tadpole tail that is twice as long as the tadpole trunk nearly disappears within 3 days in Xenopus tropicalis. Several years ago, it was proposed that this phenomenon is driven by an immunological rejection of larval‐skin‐specific antigens, Ouro proteins. We generated ouro‐knockout tadpoles using the TALEN method to reexamine this immunological rejection model. Both the ouro1‐ and ouro2‐knockout tadpoles expressed a very low level of mRNA transcribed from a targeted ouro gene, an undetectable level of Ouro protein encoded by a target gene and a scarcely detectable level of the other Ouro protein from the untargeted ouro gene in tail skin. Furthermore, congenital athymic frogs were produced by Foxn1 gene modification. Flow cytometry analysis showed that mutant frogs lacked splenic CD8⁺ T cells, which play a major role in cytotoxic reaction. Furthermore, T‐cell‐dependent skin allograft rejection was dramatically impaired in mutant frogs. None of the knockout tadpoles showed any significant delay in the process of tail shortening during the climax of metamorphosis, which shows that Ouro proteins are not essential to tail regression at least in Xenopus tropicalis and argues against the immunological rejection model.     

Expression of Class II Major Histocompatibility Complex Antigens on Adult T Cells in Xenopus is Metamorphosis- Dependent

Class II major histocompatibility complex (MHC) antigens are expressed predominantly on B lymphocytes and macrophages of tadpoles of the South African clawed frog, Xenopus laevis, as is the pattern in lymphocyte populations of most mammals. However, unlike most mammals, young postmetamorphic frogs show expression of class II MHC antigens on a high proportion of thymocytes and most peripheral T and B lymphocytes. Using the J-strain of Xenopus and the anticlass II monoclonal antibody, 14A2, we have studied, by indirect immunofluorescence, whether inhibition of metamorphosis would alter the pattern of expression of class II antigens during ontogeny. In control animals, class II antigens were virtually absent from thymic lymphocytes and peripheral T cells of normal untreated larvae, but could be found in increasing numbers in both populations after metamorphosis (10-12 weeks of age). In contrast, larvae, whose metamorphosis was inhibited by treatment with sodium perchlorate, had relatively few class II⁺ thymic lymphocytes throughout the 6-month period of study, and the proportion of class II⁺ splenic lymphocytes was approximately equal to that of IgM⁺ B lymphocytes. Thus, perchlorate-treated animals retained the larval pattern of class II epression, suggesting that emergence of class II⁺ T cells is dependent on metamorphosis.     

Expression of class II major histocompatibility complex antigens on adult T cells in Xenopus is metamorphosis-dependent

Class II major histocompatibility complex (MHC) antigens are expressed predominantly on B lymphocytes and macrophages of tadpoles of the South African clawed frog, Xenopus laevis, as is the pattern in lymphocyte populations of most mammals. However, unlike most mammals, young postmetamorphic frogs show expression of class II MHC antigens on a high proportion of thymocytes and most peripheral T and B lymphocytes. Using the J-strain of Xenopus and the anticlass II monoclonal antibody, 14A2, we have studied, by indirect immunofluorescence, whether inhibition of metamorphosis would alter the pattern of expression of class II antigens during ontogeny. In control animals, class II antigens were virtually absent from thymic lymphocytes and peripheral T cells of normal untreated larvae, but could be found in increasing numbers in both populations after metamorphosis (10-12 weeks of age). In contrast, larvae, whose metamorphosis was inhibited by treatment with sodium perchlorate, had relatively few class II+ thymic lymphocytes throughout the 6-month period of study, and the proportion of class II+ splenic lymphocytes was approximately equal to that of IgM+ B lymphocytes. Thus, perchlorate-treated animals retained the larval pattern of class II expression, suggesting that emergence of class II+ T cells is dependent on metamorphosis.     

Involvement of Thyroid Hormones in the Expression of MHC class I Antigens During Ontogeny in Xenopus

The major histocompatibility complex (MHC) is a cluster of genes encoding products central to all major functions of the vertebrate immune system. Evidence for an MHC can be found in all vertebrate groups that have been examined except the jawless fishes. Expression of MHC class I and class II antigens early in ontogeny is critically important for development of T lymphocytes capable of discriminating self from nonself. Because of this essential role in T-cell development, the ontogeny of MHC expression in the South African clawed frog, Xenopus laevis, was studied. Previous studies of MHC class I expression in Xenopus laevis suggested that class I antigens are virtually absent from tadpole tissues until climax of metamorphosis. We therefore examined the possible role of thyroid hormones (TH) in the induction of class I. By flow cytometry, a small amount of class I expression was detectable on splenocytes and erythrocytes in untreated frogs at prometamorphic stages 55-58, and the amount increased significantly at the conclusion of metamorphic climax. Thus, metamorphosis is associated with increased intensity of class I expression. Neither inhibition nor acceleration of metamorphosis altered the timing of onset of class I expression. However, inhibition of metamorphosis prevented the increase in class I expression characteristic of adult cell populations. Because expression was not accelerated in TH-treated frogs or delayed in metamorphosis-inhibited frogs, it is unlikely that TH are the direct developmental cues that induce expression, although they seem to be required for the upregulation of class I expression occurring at metamorphosis. Differences in the pattern of expression in different subpopulations of cells suggest a complex pattern of regulation of expression of class I antigens during ontogeny.     

Susceptibility of Xenopus laevis tadpoles to infection by the ranavirus Frog-Virus 3 correlates with a reduced and delayed innate immune response in comparison with adult frogs

Xenopus laevis adults mount effective immune responses to ranavirus Frog Virus 3 (FV3) infections and clear the pathogen within 2-3 weeks. In contrast, most tadpoles cannot clear FV3 and succumb to infections within a month. While larval susceptibility has been attributed to ineffective adaptive immunity, the contribution of innate immune components has not been addressed. Accordingly, we performed a comprehensive gene expression analysis on FV3-infected tadpoles and adults. In comparison to adults, leukocytes and tissues of infected tadpoles exhibited modest (10-100 time lower than adult) and delayed (3 day later than adult) increase in expression of inflammation-associated (TNF-α, IL-1β and IFN-γ) and antiviral (Mx1) genes. In contrast, these genes were readily and robustly upregulated in tadpoles upon bacterial stimulation. Furthermore, greater proportions of larval than adult PLs were infected by FV3. Our study suggests that tadpole susceptibility to FV3 infection is partially due to poor virus-elicited innate immune responses.     

Ontogeny of central serotonergic neurons in the directly developing frog, <Emphasis Type="Italic">Eleutherodactylus coqui</Emphasis>

Embryonic development of the central serotonergic neurons in the directly developing frog, Eleutherodactylus coqui, was determined by using immunocytochemistry. The majority of anuran amphibians (frogs) possess a larval stage (tadpole) that undergoes metamorphosis, a dramatic post-embryonic event, whereby the tadpole transforms into the adult phenotype. Directly developing frogs have evolved a derived life-history mode where the tadpole stage has been deleted and embryos develop directly into the adult bauplan. Embryonic development in E. coqui is classified into 15 stages (TS 1–15; 1 = oviposition / 15 = hatching). Serotonergic immunoreactivity was initially detected at TS 6 in the raphe nuclei in the developing rhombencephalon. At TS 7, immunopositive perikarya were observed in the paraventricular organ in the hypothalamus and reticular nuclei in the hindbrain. Development of the serotonergic system was steady and gradual during mid-embryogenesis. However, starting at TS 13 there was a substantial increase in the number of serotonergic neurons in the paraventricular, raphe, and reticular nuclei, a large increase in the number of varicose fibers, and a differentiation of the reticular nuclei in the hindbrain. Consequentially, E. coqui displayed a well-developed central serotonergic system prior to hatching (TS 15). In comparison, the serotonergic system in metamorphic frogs typically starts to develop earlier but the surge of development that transpires in this system occurs post-embryonically, during metamorphosis, and not in the latter stages of embryogenesis, as it does in E. coqui. Overall, the serotonergic development in E. coqui is similar to the other vertebrates.     

The metamorphic fate of supernumerary caudal vertebrae in South Asian litter frogs (Anura: Megophryidae)

Tadpoles of the Megophryidae, a South Asian family of litter frogs, are unique among anurans by virtue of their expanded caudal skeletons, which include supernumerary vertebral centra. The number of these vertebrae varies widely within the family, with tadpoles of Leptobrachella having as many as 30 and Leptolalax only five. Vertebral morphology is also quite variable, ranging from complete, perichordal centra to fragmentary ossifications. This variation in the caudal osteology of larval megophryids, however, is not manifested in the adult morphology. Post-metamorphic litter frogs have a typical anuran axial skeleton, invariably comprising eight presacral vertebrae, a single sacral vertebra and, postsacrally, the urostyle. To resolve this incongruity between life phases and to determine the precise metamorphic fate of supernumerary caudal vertebrae in megophryids, we examined metamorphic specimens from the genera Leptobrachella, Leptolalax, Ophryophryne and Megophrys. In all four, the caudal larval skeleton undergoes massive reduction, leaving only the coccyx and hypochord untouched. Caudal centra are apparently degraded by osteoclasts, which have not previously been implicated in vertebral remodelling during anuran metamorphosis. In Megophrys and Ophryophryne metamorphs, presacral centra also undergo resorption, consistent with an epichordal mode of centrum formation. The conservation of megophryid adult axial osteology in the face of extensive larval skeletal diversity reveals the role of metamorphosis in constraining anuran morphology.     

Antigen-specific CD4 T cell clonal expansion and differentiation in the aged lymphoid microenvironment. I. The primary T cell response is unaffected

Aging is associated with changes in the immune system that lead to decreased immunity in the elderly. Prior studies from humans and mice have shown that aged T cells exhibit numerous defects, including decreased proliferation following in vitro stimulation, suggesting that intrinsic defects exist within aged T cells, leading to defective T cell activation and clonal expansion. In vivo, however, cellular and soluble factors in the lymphoid microenvironment influence T cell function. To investigate the effects of the aged lymphoid microenvironment on T cell function, we monitored the immune response of CD4 T cells from DO11.10 TCR transgenic mice following adoptive transfer into young and aged hosts. After immunization with specific antigen similar rates of donor DO11.10 T cell division were observed in the two host types. However, at the peak of the response, greater numbers of DO11.10 T cells were found in the aged hosts. Regardless of the age of the host, the donor DO11.10 T cell population differentiated into functional effector cells. Despite the increased CD4 T cell growth in aged hosts, similar numbers of memory DO11.10 T cells were found in young and in aged hosts. As CD4 T cell clonal expansion and differentiation is not impaired in the aged microenvironment, our data suggest that diminished T cell immunity during aging is largely due to intrinsic T cell defects, rather than to extrinsic influences associated with the aged lymphoid microenvironment.     

Immune response to "self" lens in Xenopus laevis enucleated during larval life

We have reinvestigated an important issue in the amphibian immunology that has not been settled for years since the pioneer work of Triplett, concerning the necessity of being exposed to organ-specific antigens early in development. It was found that syngeneic Lenses were rejected by frogs, Xenopus laevis, that had been enucleated (eye removed) during early larval life. This rejection did not occur in intact frogs or in those enucleated in later larval or adult life. Whereas the splenocytes from intact frogs did not proliferate in response to a co-cultured syngeneic lens, those from frogs that had been enucleated at any of the larval stages, or even after metamorphosis, proliferated intensely. Both of these responses were shown to be thymus-dependent. In conclusion, it was demonstrated that the frog immune system rejected even syngeneic lenses by enucleation in early larval life and that it began to recognize the syngeneic lenses by lymphoid proliferation after enucleation, even in later life.     

Evidence for rhombomeric organization of multiple respiratory oscillators in the bullfrog brainstem

The anuran brainstem is segmentally organized into rhombomeres (r) and this segmental organization is uniquely preserved throughout development. We hypothesized that rhombomeres associated with cranial nerves (CN) also contain oscillators that are capable of producing rhythmic motor output (bursts) in isolation. We used in vitro brainstem preparations from pre- and post-metamorphic bullfrogs (Lithobates catesbeianus) to determine if rhombomeric organization of oscillators is present throughout development. Brainstems were transected into segments containing one or more rhombomeres and motor output was measured with suction electrodes attached to CN V, X and XII. Rhythmic motor output was observed in 85% of tadpoles and 91% of frogs in an anterior segment (r0-r5), 27% of tadpoles and 18% of frogs in the middle segment (r6-r7), and 77% of tadpoles and 55% of frogs in the caudal segment (r8). There were significant reductions in burst frequency and whole nerve amplitude following transections. These data support the hypothesis that brainstem oscillators associated with specific groups of rhombomeres are present throughout development in anurans.     

Atg7-dependent autophagy promotes neuronal health, stress tolerance, and longevity but is dispensable for metamorphosis in Drosophila

Autophagy, a cellular process of cytoplasmic degradation and recycling, is induced in Drosophila larval tissues during metamorphosis, potentially contributing to their destruction or reorganization. Unexpectedly, we find that flies lacking the core autophagy regulator Atg7 are viable, despite severe defects in autophagy. Although metamorphic cell death is perturbed in Atg7 mutants, the larval-adult midgut transition proceeds normally, with extended pupal development compensating for reduced autophagy. Atg7-/- adults are short-lived, hypersensitive to nutrient and oxidative stress, and accumulate ubiquitin-positive aggregates in degenerating neurons. Thus, normal levels of autophagy are crucial for stress survival and continuous cellular renewal, but not metamorphosis.     

Mosaic evolution of neural development in anurans: acceleration of spinal cord development in the direct developing frog<Emphasis Type="Italic"> Eleutherodactylus coqui</Emphasis>

Previous studies have shown that spinal cord development in direct developing frogs of the genus Eleutherodactylus, which have evolutionarily lost the tadpole stage, differs from that in biphasically developing anurans (with the larval and the adult stage separated by metamorphosis). The present study of spinal cord development in Eleutherodactylus coqui provides additional information about neurogenesis, neuronal differentiation and growth analyzed by immunostaining for proliferating cell nuclear antigen (PCNA), in situ hybridization for NeuroD, and morphometric measurements in various developmental stages. Furthermore, spinal cord development in the frogs Discoglossus pictus, Xenopus laevis, and Physalaemus pustulosus, which belong to different anuran families but all exhibit biphasic development, was similarly analyzed. This comparative analysis allows inference of the ancestral anuran pattern of spinal cord development and how it has been modified during the evolution of Eleutherodactylus. All biphasically developing frogs analyzed share a similar pattern of spinal cord development, suggesting that this is ancestral for anurans: after neural tube closure, levels of proliferation and neurogenesis in the spinal cord were low throughout embryogenesis until they were upregulated drastically at early larval stages followed by development of the lateral motor columns. In contrast, no such quiescent embryonic period exists in E. coqui, where rapid growth, high levels of proliferation and neurogenesis, and early formation of lateral motor columns occur shortly after neural tube closure, while other parts of the central nervous system develop more slowly. Thus, spinal cord development has been accelerated during the evolution of Eleutherodactylus relative to the development of other parts of the central nervous system, probably related to the precocious development of limbs in this lineage.     

Ontogeny of immunity in amphibians: changes in antibody repertoires and appearance of adult major histocompatibility antigens in Xenopus.

Larval Xenopus anti-2,4-dinitrophenyl antibodies of the low-molecular weight type can be analyzed by isoelectric focusing (IEF). Within a clone of genetically identical animals, tadpoles make antibodies whose IEF spectrotypes are shared by most of the individuals. Adults of the same clone also make antibodies of identical spectrotype, but the adult pattern can be very different from the larval one, although both responses are heterogeneous. The larval spectrotypes that one cannot see in a primary adult response can be found if the adult has been primed during larval life and boosted after metamorphosis. The heterogeneity of the response is somewhat lower in tadpoles (up to 12 antibody IEF bands) than in adults (up to 20 antibody IEF bands). The change in the repertoire occurs during metamorphosis at the time of the appearance of two major histocompatibility complex antigens. One, a lymphocyte antigen, appears 10-15 days before the end of metamorphosis, the other, present on red cells (and presumably also on lymphocytes), appears 1.5 month after the end of metamorphosis, as determined by immunofluorescence analysis. During the same period, the syngeneic mixed leukocyte reaction switches from a larval anti-adult to an adult anti-larval reaction.     

Hypothalamo-hypophyseal neurosecretory system inRana temporaria in early ontogenesis

The hypothalamo-hypophyseal neurosecretory system (HHNS) was investigated in tadpoles and immature frogs of the speciesRana temporaria. Heterochronia was established in the formation of the preoptic nucleus (earlier differentiation of neurosecretory cells in the dorsal than in the ventral part). Differentiation of both parts of the preoptic nucleus is preceded by increased mitotic activity in the ependyma of the corresponding areas of the lateral walls of the third ventricle and its preoptic recess.In the early stages of frog ontogenesis no neurosecretory substance (NSS) was found in the cells of the preoptic region and preoptico-hypophyseal tract or in the anlage of the median eminence; it appeared only in the posterior lobe of the hypophysis. By the beginning of metamorphosis many cells of the dorsal part of the preoptic nucleus are formed, and concentration of Gomoripositive NSS is observed in the median eminence near the blood vessels which gradually grow into this area from the meninges. In tadpoles beginning metamorphosis, NSS disappears from individual hypertrophied cells of the dorsocaudal part of the preoptic nucleus. This indicates activation of the neurosecretory cells. Absence of NSS is observed also in the median eminence, but it is present in small amounts in the posterior lobe of the hypophysis. After metamorphosis, NSS again begins to accumulate near the dilated capillaries of the median eminence and posterior lobe of the hypophysis, and all parts of the HHNS have almost completed their development.     

A non-protective T helper 1 response against the intra-macrophage protozoan Theileria annulata

Theileria annulata is a protozoan parasite which infects and transforms bovine macrophages. Infected macrophages possess augmented antigen presentation capabilities, as they are able to activate the majority of T cells from unexposed animals. In vivo, T cells in the draining lymph node (principal site of parasite development) are activated ‘non-specifically’ by the parasite. This event is followed by failure of the immune response to control the infection. Protective immune responses against intra-macrophage protozoa are usually mediated by T helper 1 (Th1) T cell responses. Here we examine the cytokine responses made by T. annulata-activated T cells. We show that the outcome of in vitro activation of T cells by parasitized macrophages is a skewing of their cytokine responses towards preferential expression of interferon-gamma (IFN-γ) mRNA. The in vitro response is mirrored during in vivo infection, as greatly elevated amounts of IFN-γ protein are found in lymph efferent from infected lymph nodes, while expression of IL-4 mRNA within the node stops. IFN-γ production does not correlate with protection against the parasite, as infected cells flourish during peak IFN-γ production, and only very small amounts of IFN-γ are produced during the effective immune response of an immunized animal. Overproduction of IFN-γ and loss of IL-4 expression are also likely to account for the failure of B cells to reach the light zone of germinal centres, a developmental step which is tightly regulated by cytokines.