What role do annelid neoblasts play? A comparison of the regeneration patterns in a neoblast-bearing and a neoblast-lacking enchytraeid oligochaete.

The term 'neoblast' was originally coined for a particular type of cell that had been observed during annelid regeneration, but is now used to describe the pluripotent/totipotent stem cells that are indispensable for planarian regeneration. Despite having the same name, however, planarian and annelid neoblasts are morphologically and functionally distinct, and many annelid species that lack neoblasts can nonetheless substantially regenerate. To further elucidate the functions of the annelid neoblasts, a comparison was made between the regeneration patterns of two enchytraeid oligochaetes, Enchytraeus japonensis and Enchytraeus buchholzi, which possess and lack neoblasts, respectively. In E. japonensis, which can reproduce asexually by fragmentation and subsequent regeneration, neoblasts are present in all segments except for the eight anterior-most segments including the seven head-specific segments, and all body fragments containing neoblasts can regenerate a complete head and a complete tail, irrespective of the region of the body from which they were originally derived. In E. japonensis, therefore, no antero-posterior gradient of regeneration ability exists in the trunk region. However, when amputation was carried out within the head region, where neoblasts are absent, the number of regenerated segments was found to be dependent on the level of amputation along the body axis. In E. buchholzi, which reproduces only sexually and lacks neoblasts in all segments, complete heads were never regenerated and incomplete (hypomeric) heads could be regenerated only from the anterior region of the body. Such an antero-posterior gradient of regeneration ability was observed for both the anterior and posterior regeneration in the whole body of E. buchholzi. These results indicate that the presence of neoblasts correlates with the absence of an antero-posterior gradient of regeneration ability along the body axis, and suggest that the annelid neoblasts are more essential for efficient asexual reproduction than for the regeneration of missing body parts.

Glutamine synthetase gene expression during the regeneration of the annelid Enchytraeus japonensis.

Enchytraeus japonensis is a highly regenerative oligochaete annelid that can regenerate a complete individual from a small body fragment in 4-5 days. In our previous study, we performed complementary deoxyribonucleic acid subtraction cloning to isolate genes that are upregulated during E. japonensis regeneration and identified glutamine synthetase (gs) as one of the most abundantly expressed genes during this process. In the present study, we show that the full-length sequence of E. japonensis glutamine synthetase (EjGS), which is the first reported annelid glutamine synthetase, is highly similar to other known class II glutamine synthetases. EjGS shows a 61-71% overall amino acid sequence identity with its counterparts in various other animal species, including Drosophila and mouse. We performed detailed expression analysis by in situ hybridization and reveal that strong gs expression occurs in the blastemal regions of regenerating E. japonensis soon after amputation. gs expression was detectable at the cell layer covering the wound and was found to persist in the epidermal cells during the formation and elongation of the blastema. Furthermore, in the elongated blastema, gs expression was detectable also in the presumptive regions of the brain, ventral nerve cord, and stomodeum. In the fully formed intact head, gs expression was also evident in the prostomium, brain, the anterior end of the ventral nerve cord, the epithelium of buccal and pharyngeal cavities, the pharyngeal pad, and in the esophageal appendages. In intact E. japonensis tails, gs expression was found in the growth zone in actively growing worms but not in full-grown individuals. In the nonblastemal regions of regenerating fragments and in intact worms, gs expression was also detected in the nephridia, chloragocytes, gut epithelium, epidermis, spermatids, and oocytes. These results suggest that EjGS may play roles in regeneration, nerve function, cell proliferation, nitrogenous waste excretion, macromolecule synthesis, and gametogenesis.

Molecular approach to annelid regeneration: cDNA subtraction cloning reveals various novel genes that are upregulated during the large-scale regeneration of the oligochaete, Enchytraeus japonensis.

To identify genes specifically activated during annelid regeneration, suppression subtractive hybridization was performed with cDNAs from regenerating and intact Enchytraeus japonensis, a terrestrial oligochaete that can regenerate a complete organism from small body fragments within 4-5 days. Filter array screening subsequently revealed that about 38% of the forward-subtracted cDNA clones contained genes that were upregulated during regeneration. Two hundred seventy-nine of these clones were sequenced and found to contain 165 different sequences (79 known and 86 unknown). Nine clones were fully sequenced and four of these sequences were matched to known genes for glutamine synthetase, glucosidase 1, retinal protein 4, and phosphoribosylaminoimidazole carboxylase, respectively. The remaining five clones encoded an unknown open-reading frame. The expression levels of these genes were highest during blastema formation. Our present results, therefore, demonstrate the great potential of annelids as a new experimental subject for the exploration of unknown genes that play critical roles in animal regeneration.

Differential tissue development during embryogenesis and regeneration in an Annelid.

The fragmenting potworm Enchytraeus japonensis (Oligochaeta, Annelida) reproduces asexually by dividing the body into several fragments that then regenerate to complete individuals in 4-5 days. Such large-scale regeneration, however, occurs only in some invertebrates. To better our understanding of why regeneration is so limited in many animals, despite their ability to undergo embryonic development from the single cell of a fertilized egg, comparisons were made between regeneration and embryonic development of E. japonensis by using two methods: histochemistry for alkaline phosphatase (ALP) and immunohistochemistry with an antibody against acetylated tubulin that visualizes nervous system development. The analyses revealed that both ALP expression patterns and central nervous system development differ between embryogenesis and the regeneration, suggesting that regeneration is not a simple reiteration of embryogenesis but involves different regulatory mechanisms. The study provides a basis for the elucidation of mechanisms that are unique and crucial to regeneration.

Real-time observation of autophagic programmed cell death of Drosophila salivary glands in vitro.

Autophagy, a form of programmed cell death (PCD) that is morphologically distinguished from apoptosis, is thought to be as prevalent as apoptosis, at least during development. In insect metamorphosis, the steroid hormone 20-hydroxyecdysone (ecdysone) activates autophagic PCD to eliminate larval structures that are no longer needed. However, in comparison with apoptosis, there are not many studies on the regulation mechanisms of autophagy. To provide a useful model for studying autophagic PCD, I established an in vitro culture system that enables real-time observation of the autophagic cell destruction of Drosophila salivary glands. The new system revealed that de novo gene expression was still required for the destruction of salivary glands dissected from phanerocephalic pupae. This indicates the usefulness of the system for exploring genes that participate in the last processes of autophagic PCD.

Fate mapping of the larval silk glands of Bombyx mori by UV laser irradiation of the egg at fertilisation.

Localised UV laser irradiation of Bombyx eggs at the fertilisation stage causes localised cuticle defects in the integument of the resultant larvae. Based on the correlation between the site of irradiation and the site of defects, I previously established a fate map of the larval integument of Bombyx mori. Although most of the internal organs of the irradiated specimens were eliminated in the acidic fixative used for cuticle preparation, silk proteins which had accumulated in the gland lumen were resistant to the acid treatment and silk gland morphology was thus preserved. Taking advantage of this phenomenon, I continued fate mapping, now of the larval silk glands of Bombyx mori, utilising the same specimens that were prepared and analysed in the previous study. Of 710 irradiated specimens analysed, 452 showed two normal glands, 27 showed one normal and one deformed gland, 21 showed one normal gland only, and 210 showed no silk gland. The sites of irradiation that caused the deletion of a silk gland were located within a limited area that entirely overlapped the ventral half of the area where irradiation caused defects in the labium. These results are consistent with previous histological descriptions stating that the silk glands develop from the paired invaginations in the labial segment. Based on the data, the presumptive region for the larval silk glands was located on the previously established Bombyx fate map.

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.

Responses to localized UV-laser irradiation are different between Bombyx and Drosophila eggs.

It has been found previously that in the silkworm, Bombyx mori, localized ultra-violet (UV) irradiation of eggs at as early as the fertilization stage could induce cuticle defects in resultant larvae. To compare the responses to the UV irradiation between Bombyx and Drosophila, embryos at various stages were irradiated with a scanning UV-laser microbeam (355 nm) over an area of 1.4-1.6% of the total egg surface. The results revealed that: (i) in Bombyx, embryonic mortality after irradiation was always low and independent of the stage of embryos at irradiation, while in Drosophila, the mortality was high at the cleavage stage and decreased as the development proceeded; (ii) in Bombyx, frequency of defective larvae after the irradiation was always high and independent of the stage of embryos at irradiation, whereas in Drosophila, high defect frequencies could be obtained only when irradiations were performed at or after the syncytial blastoderm stage; (iii) in Bombyx, the hypoderm surrounding the site of defect was always fused together to produce a continuous cuticle, while in Drosophila, the surrounding hypoderm was rarely fused together thus producing a hole in the cuticle; (iv) in Bombyx, the extent of the defects was constant from the fertilization through to the cellularization stage, whereas in Drosophila, the extent of the defects was largest at the cleavage stage and decreased as the development proceeded. These results suggest that there may be differences between the two insects in the mechanisms and/or the timing of embryonic pattern specification.

Digitonin Activates Different Sets of Puff Loci Depending on Developmental Stages in Drosophila melanogaster Salivary Glands: (salivary gland chromosomes/detergent/gene regulation/ecdysone-inducible puffs/Drosophila melanogaster).

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.

Activation of Heat-Shock Genes by Digitonin is Selectively Repressed in Preheated Drosophila Salivary Glands: (Drosophila/heat-shock puff/digitonin/gene expression).

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).