Environmental fluctuation during embryonic and fetal development can permanently alter an organism’s morphology, physiology, and behaviour. This phenomenon, known as developmental plasticity, is particularly relevant to reptiles that develop in subterranean nests with variable oxygen tensions. Previous work has shown hypoxia permanently alters the cardiovascular system of snapping turtles and may improve cardiac anoxia tolerance later in life. The mechanisms driving this process are unknown but may involve epigenetic regulation of gene expression via DNA methylation. To test this hypothesis, we assessed in situ cardiac performance during 2 h of acute anoxia in juvenile turtles previously exposed to normoxia (21% oxygen) or hypoxia (10% oxygen) during embryogenesis. Next, we analysed DNA methylation and gene expression patterns in turtles from the same cohorts using whole genome bisulfite sequencing, which represents the first high-resolution investigation of DNA methylation patterns in any reptilian species. Genome-wide correlations between CpG and CpG island methylation and gene expression patterns in the snapping turtle were consistent with patterns observed in mammals. As hypothesized, developmental hypoxia increased juvenile turtle cardiac anoxia tolerance and programmed DNA methylation and gene expression patterns. Programmed differences in expression of genes such as SCN5A may account for differences in heart rate, while genes such as TNNT2 and TPM3 may underlie differences in calcium sensitivity and contractility of cardiomyocytes and cardiac inotropy. Finally, we identified putative transcription factor-binding sites in promoters and in differentially methylated CpG islands that suggest a model linking programming of DNA methylation during embryogenesis to differential gene expression and cardiovascular physiology later in life. Binding sites for hypoxia inducible factors (HIF1A, ARNT, and EPAS1) and key transcription factors activated by MAPK and BMP signaling (RREB1 and SMAD4) are implicated. Our data strongly suggests that DNA methylation plays a conserved role in the regulation of gene expression in reptiles. We also show that embryonic hypoxia programs DNA methylation and gene expression patterns and that these changes are associated with enhanced cardiac anoxia tolerance later in life. Programming of cardiac anoxia tolerance has major ecological implications for snapping turtles, because these animals regularly exploit anoxic environments throughout their lifespan.

中文翻译：

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DNA甲基化和基因表达模式的发育编程与普通鳄龟对缺氧的极端心血管耐受性有关

胚胎和胎儿发育过程中的环境波动会永久性地改变生物体的形态、生理和行为。这种被称为发育可塑性的现象与在具有可变氧张力的地下巢穴中发育的爬行动物特别相关。以前的研究表明，缺氧会永久性地改变鳄龟的心血管系统，并可能在以后的生活中提高心脏缺氧耐受性。驱动这一过程的机制尚不清楚，但可能涉及通过 DNA 甲基化对基因表达的表观遗传调控。为了验证这一假设，我们评估了在胚胎发育过程中先前暴露于常氧（21% 氧气）或缺氧（10% 氧气）的幼龟在急性缺氧 2 小时内的原位心脏表现。下一个，我们使用全基因组亚硫酸氢盐测序分析了来自同一群体的海龟的 DNA 甲基化和基因表达模式，这是对任何爬行动物物种中 DNA 甲基化模式的第一次高分辨率研究。鳄龟中 CpG 和 CpG 岛甲基化与基因表达模式之间的全基因组相关性与在哺乳动物中观察到的模式一致。正如假设的那样，发育性缺氧增加了幼龟心脏缺氧耐受性和程序化的 DNA 甲基化和基因表达模式。SCN5A 等基因表达的程序性差异可能是心率差异的原因，而 TNNT2 和 TPM3 等基因可能是心肌细胞钙敏感性和收缩性以及心肌收缩力差异的基础。最后，我们在启动子和差异甲基化的 CpG 岛中鉴定了推​​定的转录因子结合位点，这表明了将胚胎发生过程中 DNA 甲基化编程与生命后期差异基因表达和心血管生理学联系起来的模型。涉及缺氧诱导因子（HIF1A、ARNT 和 EPAS1）和由 MAPK 和 BMP 信号通路激活的关键转录因子（RREB1 和 SMAD4）的结合位点。我们的数据强烈表明 DNA 甲基化在爬行动物基因表达的调节中起着保守的作用。我们还表明，胚胎缺氧会影响 DNA 甲基化和基因表达模式，并且这些变化与生命后期心脏缺氧耐受性的增强有关。心脏缺氧耐受性编程对鳄龟具有重要的生态意义，