Shin Tochinai

Differential participation of ventral and dorsolateral mesoderms in the hemopoiesis of Xenopus, as revealed in diploid-triploid or interspecific chimeras☆

The thymocytes in the early larvae of Xenopus laevis have been shown to be derived from precursor cells immigrating interstitially through the mesenchyme into the organ rudiments at 3-4 days of age (Nieuwkoop and Faber stages 42-45). Orthotopic grafting of diploid tissues onto triploid stage 22 embryos followed by ploidy analyses of their hemopoietic cells revealed that both thymocytes and erythrocytes in early larvae are derived from the ventral blood islands (VBI), whereas those in late larvae and adults come mainly from the dorsolateral plate (DLP). To study how the VBI cells of embryos at stage 22 participate in hemopoiesis, a number of interspecific chimeras were produced in X. laevis and X. borealis embryos. Sections of the chimeras at various developmental stages were examined by employing the unique stainability of X. borealis nuclei to quinacrine as a marker; the results show that the VBI-derived cells enter into the circulation around stage 35/36, and that some of them leave the blood vessels to migrate interstitially through the mesenchyme toward the thymic rudiment during stages 43-45. A minor population of the VBI-derived cells was also found extravascularly in the mesonephric primordia. In contrast to the VBI, the DLP-derived cells contributed to the hemopoietic cell population not in early larvae, but in late ones as a major constituent in the mesonephros, thymus, liver, and peripheral blood.

COMPLETE ABROGATION OF IMMUNE RESPONSE TO SKIN ALLOGRAFTS AND RABBIT ERYTHROCYTES IN THE EARLY THYMECTOMIZED XENOPUS

Larvae of the clawed toad, Xenopus laevis, were thymectomized at the Nieuwkoop‐Faber stages 45–47 (4‐ to 7‐day‐old) and 51 (18‐day‐old), and raised beyond metamorphosis to study their immune reactivities. The toads thymectomized at stage 51 and the unoperated and sham‐operated controls rejected allografted skins in 16–37 days. In contrast, the toads thymectomized at stages 45–47 did not reject the grafts at all, and there was no lymphoid invasion into the grafted area. The toads thymectomized at stage 51 produced antibodies against rabbit red blood cells (RRBC), as did the controls. However, those thymectomized at stages 45–47 did not produce specific antibodies. Electrophoresis on cellulose acetate membrane and immunoelectrophoretic analyses proved that in spite of the total lack of observed immune responses, the thymectomized toads do possess a certain level of immunoglobulins. It is concluded that the thymus exerts its decisive role in the establishment of immune competence when it is still in the initial state of differentiation. In relation to the immunoglobulins found in the early‐thymectomized toads, a discussion was also offered of the presence of a thymus‐independent humoral immune system.

Phylogenetic and expression analysis of amphibian Xenopus Toll-like receptors

An anuran amphibian, South African clawed frog (Xenopuslaevis), is used to study the immune system, as it possesses a set of acquired immune system represented by T and B lymphocytes and the immunoglobulins. The acquired immune system is impaired throughout the larva and the metamorphosis stage in the amphibians. On the other hand, the role of innate immune system in the tadpole remains unclear. Recently, insect Toll protein homologues, namely, Toll-like receptors (TLRs), have been identified as sensors recognizing microbe-pattern molecules in vertebrates. Whole-genome analysis of Xenopus tropicalis supported the existence of the tlr genes in the frog. In this study, we annotated 20 frog tlr gene nucleotide sequences from the latest genome assembly version 4.1 on the basis of homology and identified cDNAs of the predicted frog TLR proteins. Phylogenetic analysis showed that the repertoire of the frog TLRs consisted of both fish- and mammalian-type TLRs. We showed that the frog TLRs are constitutively expressed in the tadpole as well as in the adult frog. Our results suggest that tadpoles are protected from microbes by the innate system that includes TLRs, despite impaired acquired immune system in tadpoles. This is the first report on the properties of TLRs in the most primitive terrestrial animals like amphibia.

Fragmenting oligochaete Enchytraeus japonensis: A new material for regeneration study

Enchytraeus japonensis, a recently described terrestrial oligochaete, reproduces asexually by fragmentation and subsequent regeneration. Taking notice of its high potential as a new material for regeneration study, detailed studies were undertaken on the regeneration and reproduction of E. japonensis. The full‐grown body divided into 6–13 fragments that regenerated into complete individuals in 4 days, grew to full length in 10 days, and then fragmented again. Regeneration of the head and tail was epimorphic, involving blastema formation, while old segments in the regenerating fragment morphallactically transformed into the appropriate segments to retain the proper body proportions, which could be visualized by histochemistry for alkaline phosphatase. Artificially cut fragments regenerated either normally or into dicephalic monsters with biaxial heads depending on the conditions. Fragmentation could be induced by decapitation, and sexual reproduction was also found inducible in the laboratory. These findings, together with its simple metameric morphology and ease of culture and handling, suggest that E. japonensis is an excellent material for studying animal regeneration.

Brain regeneration in anuran amphibians.

Urodele amphibians are highly regenerative animals. After partial removal of the brain in urodeles, ependymal cells around the wound surface proliferate, differentiate into neurons and glias and finally regenerate the lost tissue. In contrast to urodeles, this type of brain regeneration is restricted only to the larval stages in anuran amphibians (frogs). In adult frogs, whereas ependymal cells proliferate in response to brain injury, they cannot migrate and close the wound surface, resulting in the failure of regeneration. Therefore frogs, in particular Xenopus, provide us with at least two modes to study brain regeneration. One is to study normal regeneration by using regenerative larvae. In this type of study, the requirement of reconnection between a regenerating brain and sensory neurons was demonstrated. Functional restoration of a regenerated telencephalon was also easily evaluated because Xenopus shows simple responses to the stimulus of a food odor. The other mode is to compare regenerative larvae and non‐regenerative adults. By using this mode, it is suggested that there are regeneration‐competent cells even in the non‐regenerative adult brain, and that immobility of those cells might cause the failure of regeneration. Here we review studies that have led to these conclusions.

Surprisingly complex T‐box gene complement in diploblastic metazoans

SUMMARY Ctenophores and cnidarians are two metazoan groups that evolved at least 600 Ma, predating the Cambrian explosion. Although both groups are commonly categorized as diploblastic animals without derivatives of the mesodermal germ layer, ctenophores possess definitive contractile “muscle” cells. T‐box family transcription factors are an evolutionarily ancient gene family, arising in the common ancestor of metazoans, and have been divided into eight groups in five distinct subfamilies, many of which are involved in the specification of mesodermal as well as ectodermally and endodermally derived structures. Here, we report the cloning and expression of five T‐box genes from a ctenophore, Mnemiopsis leidyi. Phylogenetic analyses demonstrated that ctenophores possess members of at least three of the five T‐box subfamilies, and expression studies suggested distinct roles of each T‐box genes during gastrulation and early organogenesis. Moreover, genome searches of the sea anemone, Nematostella vectensis (anthozoan cnidarian), showed at least 13 T‐box genes in Nematostella, which are divided into at least six distinct groups in the same three subfamilies found in ctenophores. Our results from two diploblastic animals indicate that the common ancestor of eumetazoans had a complex set of T‐box genes and that two distinct subfamilies might have appeared during triploblastic evolution.

Early Segregation of Germ and Somatic Lineages during Gonadal Regeneration in the Annelid Enchytraeus japonensis

Although regeneration studies are useful for understanding how organs renew, little information is available about regeneration of reproductive organs and germ cells. We here describe the behavior of germ-cell precursors during regeneration of the oligochaete annelid worm Enchytraeus japonensis, which has the remarkable feature of undergoing asexual (by fission) and sexual reproduction . We first found that the gonad can regenerate from any body fragment yielded by fission during asexual reproduction. We then examined behavior of germ-cell lineage during this regenerative process, by using a homolog of the Piwi gene (Ej-piwi) as a marker. We found that in asexually growing animals, specialized cells expressing Ej-piwi are distributed widely in the body as single cells. These cells seem to serve as a reservoir of germ-cell precursors because during asexual propagation these cells migrate into the regenerating tissue, where they ultimately settle in the prospective gonads, and give rise to germ cells upon sexualization. These cells are distinct from the neoblasts, thought to be stem cells in other animals. This is the first report to directly show that the germ and somatic lineages are segregated in asexually growing animals and behave differently during regeneration.

Stem cell system in asexual and sexual reproduction of Enchytraeus japonensis (Oligochaeta, Annelida)

Enchytraeus japonensis is a small oligochaete species that proliferates asexually via fragmentation and regeneration. As sexual reproduction can also be induced, it is a good model system for the study of both regenerative and germline stem cells. It has been shown by histological study that putative mesodermal stem cells called neoblasts, and dedifferentiated epidermal and endodermal cells are involved in blastema formation. Recently, we isolated three region‐specific marker genes expressed in the digestive tract and showed by in situ hybridization that morphallactic as well as epimorphic regulation of the body patterning occurs during regeneration. We also cloned two vasa‐related genes and analyzed their expression during development and in mature worms that undergo sexual reproduction. The results arising form these studies suggest that the origin and development of germline stem cells and neoblasts may be independent. Furthermore, we carried out functional analysis using RNA interference (RNAi) and showed that a novel gene termed grimp is required for mesodermal cell proliferation at the initial stages of regeneration. These findings indicate that the stem cell system in E. japonensis is regulated by both internal and external environmental factors.

Abortive meiosis in the oogenesis of parthenogenetic Daphnia pulex

Most daphnid species adopt parthenogenesis and sexual reproduction differentially in response to varied environmental cues, resulting in the production of diploid progenies in both cases. Previous studies have reportedly suggested that daphnids produce their parthenogenetic eggs via apomixis; the nuclear division of mature oocytes should be an equational division similar to somatic mitosis. However, it seems premature to conclude that this has been unequivocally established in any daphnids. Therefore, the objective of our research was to precisely reveal the process and mechanism of parthenogenetic oogenesis and maintenance of diploidy in Daphnia pulex through histology, karyology, and immunohistochemistry. We found that, when a parthenogenetic egg entered the first meiosis, division was arrested in the early first anaphase. Then, two half-bivalents, which were dismembered from each bivalent, moved back to the equatorial plate and assembled to form a diploid equatorial plate. Finally, the sister chromatids were separated and moved to opposite poles in the same manner as the second meiotic division followed by the extrusion of one extremely small daughter cell (resembling a polar body). These results suggest that parthenogenetic D. pulex do not adopt typical apomixis. We hypothesize that D. pulex switches reproductive mode depending on whether the egg is fertilized or not.

Thymocyte stem cell inflow in Xenopus laevis, after grafting diploid thymic rudiments into triploid tadpoles

Diploid thymus rudiments from 3–11 day (stage 42–49) Xenopus laevis larvae were grafted into histocompatible, triploid 40 day (stage 56–58) tadpoles. Lymphocyte ploidy in differentiated thymuses determined by microspectrophotometry of nuclear DNA revealed a sharp increase of graft-type lymphocytes in thymus grafts derived from 3 day (mean 43 %) and 4 day (mean 63 %) rudiments. Thus, in grafts derived from 5–11 day old larvae most lymphocytes (mean 82 %) were of graft origin. These migration patterns strongly support the view that in Xenopus, lymphoid precursor cells of the thymus are of extrinsic origin, immigrating into rudiments 3–4 days after fertilization.