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

Abstract

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.

Fig. 1

Amino acid alignment of E. japonensis glutamine synthetase (EjGS) with representative GSs from various other species. The species and protein ID of the GSs listed here are as follows: Enchytraeus japonensis (BAE93509.1), sea anemone Aiptasia pallida (AAR36878.1), nematode Caenorhabditis elegans (NP_001041010.1), Pacific oyster Crassostea gigas (CAD90162.1), fruit fly Drosophila melanogaster (AAZ41780.1), sea urchin Paracentrotus lividus (AAC41562.1), zebrafish Danio rerio (AAH66735.1), clawed frog Xenopus laevis (AAH46681.1), chicken Gallus gallus (AAA48783.1), and mouse Mus musculus (AAH15086.1). Amino acid residues are indicated in blue when greater than or equal to 70% of these residues are identical in the ten representative species. The beta grasp domain, catalytic domain, and the regions that are conserved in prokaryotes and eukaryotes are underlined by broken single lines, broken double lines, and thick single lines, respectively. Ligands for Mn²⁺ ions are indicated by arrowheads. The ATP-binding sites are boxed. The protein length and overall amino acid identities with EjGS are indicated at the end of each sequence

Fig. 2

Glutamine synthetase mRNA expression during regeneration in E. japonensis. Transcripts were detected using whole-mount in situ hybridization (a–g and k–o) or in situ hybridization analysis of paraffin-embedded sections (h–j) using an antisense gs riboprobe, with the exception of the lower specimen shown in a. The anterior is to the left, and the ventral side is down in each image. Arrowheads indicate the amputated sites. a Whole-body images of regenerating fragments at about 1 day after amputation with the anterior blastema and an intact tail. The lower specimen was hybridized with a sense probe. b–f Lateral views of anterior blastemas at about 1 (b), 1.5 (c), 2 (d), 2.5 (e), and 4 days (f) after amputation. g Lateral view of an intact head. h–i Sagittal sections of anterior blastemas at about 1.5 (h) and 2 days (i) after amputation. j Sagittal section of an intact head. k–m Lateral views of posterior blastemas at about 0.5 (k), 1 (l), and 1.5 days (m) after amputation. n–o Ventral views of intact tails in an actively growing (n) and a full-grown individual (o). ba Brain anlage, br brain, bc buccal cavity, ch chloragocyte, ea esophageal appendage, ep epidermal cell, es esophagus, gl gut lumen, gz growth zone, m mouth, pc pharyngeal cavity, pp pharyngeal pad, pr prostomium, py pygidium, st stomodeum, va ventral nerve cord anlage, vn ventral nerve cord. Scale bars, 50 μm

Fig. 3

Glutamine synthetase mRNA expression in nonblastemal tissues. Transcripts were detected by in situ hybridization on paraffin-embedded sections (a–c and e) or by whole-mount in situ hybridization (d) using an antisense gs riboprobe, except for the right-hand specimen in a. The anterior is to the left, and the ventral side is down in each image. a Sagittal sections of regenerating fragments at about 0.5 days after amputation. The right-hand specimen was hybridized with a sense probe. Arrowheads indicate the amputated sites. b–c Sagittal sections of intact worms. d Whole-mount in situ hybridization analysis of an intact worm showing gs expression in the epidermal cells. e Sagittal section of a sexually mature worm. ch Chloragocyte, ep epidermal cell, ge gut epithelium, gl gut lumen, ne nephridium, oo oocyte, sc spermatocyte, st spermatid, sp sperm. Scale bars, 50 μm