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

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

JOURNAL=Frontiers in Physiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2020.530435

DOI=10.3389/fphys.2020.530435

ISSN=1664-042X

ABSTRACT=<p>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 (<italic>Cg</italic>engrailed-1) in Pacific oyster <italic>Crassostrea gigas</italic> under CO<sub>2</sub>-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 <italic>Cg</italic>engrailed-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 <italic>Cg</italic>engrailed-1 shared high homology with other engraileds from <italic>Drosophila melanogaster</italic>, <italic>Mizuhopecten yessoensi</italic>, and <italic>Crassostrea virginica</italic>. The mRNA transcripts of <italic>Cg</italic>engrailed-1 were constitutively expressed in various tissues with the highest expression levels detected in labial palp and mantle, which were 86.83-fold (<italic>p</italic> &lt; 0.05) and 75.87-fold (<italic>p</italic> &lt; 0.05) higher than that in hepatopancreas. The mRNA expression of <italic>Cg</italic>engrailed-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, <italic>p</italic> &lt; 0.05). The results of immunofluorescence assay demonstrated that the expression level of <italic>Cg</italic>engrailed-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 <italic>Cg</italic>engrailed-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 (<italic>p</italic> &lt; 0.05). The whole-mount immunofluorescence assay showed that <italic>Cg</italic>engrailed-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 <italic>Cg</italic>engrailed-1 could be triggered by CO<sub>2</sub>-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.</p>