Zhang Yukun, Liu Zhaoqun, Zong Yanan, Zheng Yan, Li Yinan, Han Zirong, Wang Lingling, Song Linsheng
Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, China.
Liaoning Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian, China.
Front Physiol. 2020 Dec 1;11:530435. doi: 10.3389/fphys.2020.530435. eCollection 2020.
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 (engrailed-1) in Pacific oyster under CO-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 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 engrailed-1 shared high homology with other engraileds from , , and . The mRNA transcripts of engrailed-1 were constitutively expressed in various tissues with the highest expression levels detected in labial palp and mantle, which were 86.83-fold ( < 0.05) and 75.87-fold ( < 0.05) higher than that in hepatopancreas. The mRNA expression of 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, < 0.05). The results of immunofluorescence assay demonstrated that the expression level of 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 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 ( < 0.05). The whole-mount immunofluorescence assay showed that 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 engrailed-1 could be triggered by CO-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.
Engrailed是众多物种在神经发生、体节形成、胚盘前组织形成和区域形成等重要发育过程中所需的一种转录因子。最近的研究证明，Engrailed也是与海洋双壳贝类贝壳形成相关的关键基因。在本研究中，对太平洋牡蛎中一种Engrailed基因（Engrailed-1）在CO₂驱动的酸化条件下的表达模式进行了研究，以了解其在调节贝壳形成和适应海洋酸化（OA）中的可能作用。获得了Engrailed-1的开放阅读框（ORF），其长度为690 bp，编码一个含有HOX结构域的229个氨基酸的多肽。系统发育分析表明，Engrailed-1推导的氨基酸序列与来自[具体物种1]、[具体物种2]和[具体物种3]的其他Engrailed具有高度同源性。Engrailed-1的mRNA转录本在各组织中组成性表达，在唇瓣和外套膜中检测到的表达水平最高，分别比肝胰腺中的表达水平高86.83倍（P<0.05）和75.87倍（P<0.05）。在中度（pH 7.8）和重度（pH 7.4）酸化处理后，外套膜中Engrailed-1的mRNA表达显著下降（分别为对照组的0.75倍和0.15倍，P<0.05）。免疫荧光分析结果表明，在中度和重度酸化处理后，外套膜中间褶皱中Engrailed-1的表达水平显著增加。此外，牡蛎幼虫在担轮幼虫阶段接受酸化处理后，D形幼虫阶段Engrailed-1的mRNA表达水平显著增加，分别为对照组的3.11倍（pH 7.8）和4.39倍（pH 7.4）（P<0.05）。整体免疫荧光分析表明，在对照组和酸化处理组的担轮幼虫、早期D形幼虫和D形幼虫中，Engrailed-1主要在壳腺边缘和壳皮上表达，在酸化处理下，早期D形幼虫和D形幼虫中的阳性信号强度显著增加。这些结果共同表明，CO₂驱动的酸化处理可触发Engrailed-1的表达，这可能有助于诱导牡蛎幼虫的初始贝壳形成和成年牡蛎的壳皮形成，以适应酸化的海洋环境。
