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

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

Cell proliferation activity during head regeneration in <i>E. japonensis</i> and <i>E. buchholzi</i>.

<p>(A) Spontaneous fragments from the trunk region of <i>E. japonensis</i>. (B) Posterior fragments of <i>E. buchholzi</i> that were amputated at the 5th–9th segment. (C) Small head fragments of <i>E. japonensis</i> that were amputated at the 6th–7th segments (upper three specimens, with arrows indicating weakly-labeled blastemas) and a spontaneous fragment from the trunk region (lower specimen, with arrowheads indicating strongly-labeled blastemas). Fragments were incubated at 23°C, labeled with BrdU for 18 hours, fixed and immunostained for BrdU (yellow dots) and counterstained with propidium Iodide (orange). Chaetae show intense yellow autofluorescence signals. The days after amputation (including BrdU labeling time) are indicated. Broken lines mark the levels of amputation. Scale bars, 100 µm.</p

Regeneration patterns of artificially amputated individuals of <i>E. japonensis</i> and <i>E. buchholzi</i>.

<p>The upper illustrations of each panel schematically summarize the results of anterior (A, C) and posterior (B, D) regeneration of <i>E. japonensis</i> (A, B) and <i>E. buchholzi</i> (C, D) following amputation at various positions along the antero-posterior body axis. Neoblast-bearing segments and regenerated segments are indicated in gray and red, respectively. Fragmentation induced by head removal <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037319#pone.0037319-Myohara1" target="_blank">[4]</a> is omitted from the illustrations of <i>E. japonensis</i> to enable an easier comparison with <i>E. buchholzi</i>. The lower graphs of each panel show the frequency and degree of regeneration in the anterior (A, C) and posterior (B, D) direction. The bars indicate the mean numbers of regenerated segments with standard deviation (SD), with the numerical axis at the left side. The blue, red and green lines indicate the frequency of regeneration of the head, tail and undeterminable type, respectively, with the numerical axis at the right side. A total of 32, 83, 100 and 109 fragments were examined in (A), (B), (C) and (D), respectively. Bic, bicaudal; Dic, dicephalic; UD, undeterminable.</p

Representative <i>E. japonensis</i> regenerates.

<p>(A) Example of a head with four segments regenerated after amputation at the 4th–5th segment. (B) A head with seven segments regenerated after amputation in trunk region. (C) A dicephalic monster with biaxial heads formed after amputation at the 6th segment. A head with three segments was regenerated posteriorly in this case. (D) A normal worm regenerated after amputation at the 7th segment. (E) A long dicephalic monster with biaxial heads formed after amputation at the 11th segment and culture in water instead of agar medium. A complete head with seven segments was regenerated posteriorly in this case. Segments of the original fragments are numbered with Roman numerals, and regenerated segments are numbered using Arabic numerals. The broken lines mark the levels of amputation. The anterior is to the left in each image. p, prostomium; py, pygidium. Scale bars, 100 µm.</p

Comparative summary of the typical regeneration patterns of artificially amputated individuals of <i>E. japonensis</i> and <i>E. buchholzi</i> cultured in agar medium.

<p>Comparative summary of the typical regeneration patterns of artificially amputated individuals of <i>E. japonensis</i> and <i>E. buchholzi</i> cultured in agar medium.</p

Schematic illustration of the regeneration pattern during asexual reproduction in <i>E. japonensis</i>.

<p><i>E. japonensis</i> worms harbor neoblasts in all segments except for the anterior-most eight segments, i.e. the seven head-specific segments (segment I–VII) and the first trunk segment (segment VIII). Following spontaneous fragmentation, each fragment regenerates a complete head and/or tail and grows into a normal worm, irrespective of the region of the body from which the fragment was originally derived <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037319#pone.0037319-Myohara1" target="_blank">[4]</a>. Neoblast-bearing segments and regenerated segments are indicated in gray and red, respectively.</p

Representative <i>E. buchholzi</i> regenerates.

<p>(A) Example of a head with three segments regenerated after amputation at the 8th segment. (B) A head with one segment regenerated after amputation at the 12th segment. (C) Undifferentiated blastema of an undeterminable type formed after amputation at around the 20th segment. (D) A bicaudal monster with biaxial tails formed after amputation in the region close to the tail. A tail with three additional segments regenerated anteriorly. (E) A dicephalic monster with biaxial heads formed after amputation at the 11th segments. A head with two segments was regenerated posteriorly in this case. (A–E) Amputees were cultured in 0.6% plain agar for 14 days (A–C), 32 days (E), or 40 days (D), fixed, and then stained with orcein. Segments of the original fragments are numbered with Roman numerals, and regenerated segments are numbered using Arabic numerals. The broken lines mark the levels of amputation. The anterior is to the left and the ventral is down in each image. b, brain; g, gut; m, mouth; p, prostomium; py, pygidium; vnc, ventral nerve cord. Scale bars, 100 µm.</p