Maroko Myohara

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.

Induction of ecdysterone-stimulated chromosomal puffs in permeabilized Drosophila salivary glands: A new method for assaying the gene-regulating activity of cytoplasm☆

Abstract A simple assay system for gene regulation using chromosomal puffing as an index of gene activity was established. Salivary glands of Drosophila melanogaster treated with a mild detergent, digitonin, were permeable to high molecular substances, including β-galactosidase (MW 465,000). The permeabilized salivary glands retained the ability to form puffs at the ecdysterone-stimulated loci (74EF and 75B) in response to the hormone. Incubation of the permeabilized salivary glands at puff stage 1 (PS1) for 2 hr in a medium containing both ecdysterone and a homogenate of intact salivary glands at puff stage 8–9 (PS8–9) induced a puff at 78C, where puffing occurs only at puff stages 6–11 in vivo. The puff at 78C was not induced when the permeabilized PS1 glands were incubated with the combination of ecdysterone and a homogenate of the PS1 salivary glands. Likewise, the 78C puff was not induced in intact PS1 salivary glands by a 2-hr incubation with ecdysterone and PS8–9 gland homogenate. These results indicate that a factor(s) required for 78C puff formation is present in PS8–9 but not in PS1 salivary glands and that factor(s) can permeate digitonin-treated salivary glands but not intact glands. The effectiveness of the permeabilized salivary glands as an assay system for gene-regulating factors is discussed.

Possible neural control of asexually reproductive fragmentation in Enchytraeus japonensis (Oligochaeta, Enchytraeidae)

Summary The enchytraeid oligochaete Enchytraeus japonensis reproduces asexually by fragmentation under laboratory conditions. In the fragmentation process, fully-grown worms break into several fragments. Each fragment regenerates into a small but complete worm in 4 days, grows rapidly and divides again in another 10 days. We found that this fragmentation can be induced artificially by amputating the head if the worms are at least 5–6 mm in length, but not in shorter worms. The fragmentation is inducible by removing the most anterior two segments, if the worms are large enough. When a worm is cut into two, fragmentation occurs more readily in the posterior section. Moreover, even a small incision made in the ventral side of the trunk causes fragmentation in the body posterior to the incision. Immersion of the worms in water is found to inhibit fragmentation even in decapitated worms. When a worm is placed in water immediately after decapitation, the ability to fragment is gradually lost as a new head regenerates. From these results it is postulated that the ability to fragment acquired early in the growth phase is suppressed by head-derived signal(s) until spontaneous fragmentation occurs. The signals seem to be constantly transmitted through the ventral nerve cord until the head matures and lifts its blockade of fragmentation, allowing this process to proceed. At present, however, we cannot exclude the possibility that the mature head of the worm produces fragmentation-stimulating signals.

Activation of Heat-Shock Genes by Digitonin is Selectively Repressed in Preheated Drosophila Salivary Glands

Treatment of Drosophila salivary glands with a mild detergent, digitonin, activates puffing at 35 chromosome loci. These digitonin‐activated puffs include all of the nine heat‐shock puffs known in D. melanogaster. Here we show that the activation of heat‐shock genes, but not of other digitoninstimulated puffs, is repressed in salivary glands which have been subjected to and have recovered from heat shock before being treated with digitonin. The findings indicate that, (a) the activation of heat‐shock genes by digitonin, as that by temperature elevation, is self‐regulated by the heat‐shock proteins (HSPs). (b) the gene repressive activity of HSPs is heat‐shock‐gene specific, and (c) the repression mechanism of heat‐shock genes by HSPs is resistant to digitonin, in contrast to that the suppression of heat‐shock genes is prevented by the detergent in non‐heat‐shocked salivary glands. The selective repression of heat‐shock genes in preheated salivary glands suggests that the heat‐shock genes and other digitonin‐activated genes may be controlled by a different mechanism(s).

Digitonin Activates Different Sets of Puff Loci Depending on Developmental Stages in Drosophila melanogaster Salivary Glands

We showed previously that treatment of Drosophila melanogaster salivary glands with a mild detergent, digitonin, induces heat shock puffs and many developmentally regulated puffs. To find if the mechanism underlying the puff induction by digitonin is related to the temporal control of gene expression in salivary glands, we examined effects of digitonin on salivary glands at various puff stages from late third instar larva to white prepupa. The results indicate that (a) all the heat shock puffs are induced by digitonin irrespective of the developmental stage of the treated glands, (b) intermolt and early puff loci are always irresponsive to digitonin, and (c) late puff loci respond to digitonin to form puffs only before the stage of their developmentally programmed puffing. Based on the stage at which the locus becomes digitonin responsive, the digitonin‐responsive late puff loci were divided into two groups: group A loci, responsive to digitonin continuously from PS1 until programmed puffing begins, and group B loci, responsive to digitonin only in a short period of time immediately before the programmed puffing. The results suggest that a digitonin‐sensitive suppression mechanism(s) is involved in the temporal control of gene expression in Drosophila salivary glands.

Two-step regulation of ecdysone-inducible late puffs in salivary glands of Drosophila melanogaster

Our previous study showed that some ecdysone‐inducible late puffs could also be induced by a mild detergent (digitonin) in Drosophila salivary glands. However, they could only be induced at the stage immediately prior to when developmentally programmed puffing occurred, suggesting that these late puff loci were under two‐step regulation. Using an in vitro culture of salivary glands, we have examined whether ecdysone or the protein products of early puff genes participate in either of the two steps of late puff regulation. This study has revealed that (i) the acquisition of digitonin‐responsiveness (the first step) could be induced in vitro by incubating salivary glands with ecdysone; (ii) the first step could also be induced by protein synthesis inhibition even in the absence of ecdysone; (iii) the second step required both ecdysone and protein synthesis unless treated with digitonin; and (iv) the first step, rather than the second step, determines the timing of normal puff formation in the loci. These results suggest that, during normal development, ecdysone controls both steps by activating two types of early genes; the first type, whose function can be mimicked by cycloheximide, renders the loci responsive to digitonin and the second type, whose function can be mimicked by digitonin, activates the loci to form puffs.