The Increased Expression of an Engrailed to Sustain Shell Formation in Response to Ocean Acidification.

Yukun Zhang,Lingling Wang,Yinan Li,Linsheng Song,Yanan Zong,Zirong Han,Yan Zheng,Zhaoqun Liu

doi:10.3389/fphys.2020.530435

Abstract

Engrailed is a transcription factor required in numerous species for important developmental steps such as neurogenesis, segment formation, preblastoderm organization, and compartment formation. Recent study has proved that engrailed is also a key gene related to shell formation in marine bivalves. In the present study, the expression pattern of an engrailed gene (Cgengrailed-1) in Pacific oyster Crassostrea gigas under CO2-driven acidification was investigated to understand its possible role in the regulation of shell formation and adaptation to ocean acidification (OA). The open reading frame (ORF) of Cgengrailed-1 was obtained, which was of 690 bp encoding a polypeptide of 229 amino acids with a HOX domain. Phylogenetic analysis indicated that the deduced amino acid sequence of Cgengrailed-1 shared high homology with other engraileds from Drosophila melanogaster, Mizuhopecten yessoensi, and Crassostrea virginica. The mRNA transcripts of Cgengrailed-1 were constitutively expressed in various tissues with the highest expression levels detected in labial palp and mantle, which were 86.83-fold (p < 0.05) and 75.87-fold (p < 0.05) higher than that in hepatopancreas. The mRNA expression of Cgengrailed-1 in mantle decreased dramatically after moderate (pH 7.8) and severe (pH 7.4) acidification treatment (0.75- and 0.15-fold of that in control group, p < 0.05). The results of immunofluorescence assay demonstrated that the expression level of Cgengrailed-1 in the middle fold of mantle increased significantly upon moderate and severe acidification treatment. Moreover, after the oyster larvae received acidification treatment at trochophore stage, the mRNA expression levels of Cgengrailed-1 increased significantly in D-shape larvae stages, which was 3.11- (pH 7.8) and 4.39-fold (pH 7.4) of that in control group (p < 0.05). The whole-mount immunofluorescence assay showed that Cgengrailed-1 was mainly expressed on the margin of shell gland, and the periostracum in trochophore, early D-shape larvae and D-shape larvae in both control and acidification treatment groups, and the intensity of positive signals in early D-shape larvae and D-shape larvae increased dramatically under acidification treatment. These results collectively suggested that the expression of Cgengrailed-1 could be triggered by CO2-driven acidification treatment, which might contribute to induce the initial shell formation in oyster larvae and the formation of periostracum in adult oyster to adapt to the acidifying marine environment.

Highlights

Engrailed is a homeodomain transcription factor which is involved in plentiful species for prime developmental period, and its evolution is known to be closely related to the evolution of metazoan body plan (Loomis et al, 1996; Iwaki and Lengyel, 2002; Byrne et al, 2005)
When adult oysters received acidification treatment, positive signals were detected in the middle fold of mantle, and the signal intensity in moderate acidification treatment group was significantly stronger than that in severe acidification treatment group (Figure 6). Marine bivalves such as oysters and mussels secret matrix proteins and deposit calcium carbonate to form the calcified shells as a supporting skeleton to protect their soft bodies (Zhang et al, 2012; Hendriks et al, 2015), which is thought to be one of the key factors that trigger the expansion of bivalves at the dawn of the Cambrian times (Marie et al, 2012)
They are in great danger due to the ocean acidification (OA) caused by excessive burning of fossil fuels (Orr et al, 2005; Waldbusser et al, 2015; Wang et al, 2017a), and there is urgent need for a better understanding of the shell formation mechanism in bivalves and their stress responses and adaptation modes to CO2-driven acidification

Summary

Introduction

Engrailed is a homeodomain transcription factor which is involved in plentiful species for prime developmental period, and its evolution is known to be closely related to the evolution of metazoan body plan (Loomis et al, 1996; Iwaki and Lengyel, 2002; Byrne et al, 2005). Lots of homologues of engrailed have been identified from various animal groups including mammals, annelids, arthropods, insects, echinoderms, and chordates (Fjose et al, 1985; Joyner et al, 1985; Holland et al, 1997; Lowe and Wray, 1997; Ikuta, 2017). The sequence of engrailed contains a conserved HOX domain, which is regarded as an important DNA-binding site for transcriptional regulation in crucial developmental processes (Desplan et al, 1985; Patel et al, 1989). The HOX domain of engrailed can bind to DNA mainly relying on the helix-turn-helix structure (two α-helixes connected by a β-corner), in which one of the α-helix combines some hydrogen bonds and hydrophobic interactions to special chains and groups in the major groove of DNA (Piper et al, 1999). Engrailed is able to bind directly to the eukaryotic translation initiation factor 4E, via a sequence located in the N-terminus (Nedelec et al, 2004)

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Conclusion