Nervous system dynamics during fragmentation and regeneration in Enchytraeus japonensis (Oligochaeta, Annelida).

Enchytraeus japonensis is a small terrestrial oligochaete which primarily reproduces asexually by fragmentation and regeneration. In order to introduce a molecular approach to the study of regeneration we developed a whole-mount immunostaining procedure for the worm. Using an antibody directed against acetylated tubulin in conjunction with confocal laser-scanning microscopy, we succeeded in clarifying the three- dimensional structure of the entire nervous system in the full-grown worm and its dynamics during the fragmentation and regeneration process. In addition, we examined the expression of neurotransmitters and neuropeptides in the worm using a fluorescently-labeled antagonist and various antibodies. In particular, we found two circumferential structures in the body wall muscle of each segment that react strongly with alpha-bungarotoxin, an antagonist of nicotinic acetylcholine receptors, and detected nerve fibers just underneath these structures. During the fragmentation process, the circular body wall muscles contract near one of these circumferential structures in the middle of the segment, which causes constriction and results in fission of the body. This alpha-bungarotoxin-positive structure was designated the neuromuscular junction of the circular muscle. During the regeneration process nerve fibers grow from the remaining ventral nerve cord and gradually form networks in both the anterior and posterior regeneration buds. The growing fibers extend to the prostomium (a sensory organ) at the anterior end prior to connecting to the presumptive brain rudiment. A neural network appears around the pygidium, and this is followed by growth of the body at the posterior end. The nervous system appears to play an important role in both anterior and posterior regeneration.

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.

Exon-intron organization of Xenopus MHC class II beta chain genes.

The amphibian Xenopus laevis is the most primitive vertebrate in which the major histocompatibility complex (MHC) has been defined at the biochemical, functional, and molecular genetic levels. We previously described the isolation and characterization of cDNA clones encoding X. laevis MHC class II beta chains. In the present study, genomic clones encoding class II beta chains were isolated from X. laevis homozygous for the MHC f haplotype. Three class II beta chain genes, designated Xela-DAB, Xela-DBB, and Xela-DCB, were identified. Sequence analysis of these genes showed that Xela-DBB and Xela-DCB correspond to the previously characterized cDNA clones F3 and F8, respectively, whereas Xela-DAB encodes a third, hitherto unidentified class II beta chain of the MHC f haplotype. As a representative of X. laevis class II beta chain genes, the Xela-DAB gene underwent detailed structural analysis. In addition, the nucleotide sequence of Xela-DABf cDNA clones was determined. The Xela-DAB gene is made up of at least six exons, with an exon-intron organization similar to that of a typical mammalian class II beta chain gene. The 5'-flanking region of the Xela-DAB gene contains transcriptional control elements known as X1, X2, and Y, but lacks typical TATA or CCAAT boxes. A notable feature of the X. laevis class II beta chain genes is that the sizes of the introns are larger than those of their mammalian counterparts. As assessed by northern blot analysis, the three class II beta chain genes had similar expression patterns, with the highest level of transcription detected in the intestine. Identification of the Xela-DAB, -DBB, and -DCB genes is consistent with our previous observations, which suggested that the MHC of the tetraploid frog X. laevis is diploidized at the genomic level and contains three class II beta chain genes per haplotype that cross-hybridize to one another under reduced stringency conditions.