N-acetyltransferase (nat) Is a Critical Conjunct of Photoperiodism between the Circadian System and Endocrine Axis in Antheraea pernyi

Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16∶8 (LD) and LD12∶12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4°C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNANAT caused dysfunction of photoperiodism. dsRNAPER upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNANAT decreased melatonin while dsRNAPER increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNANAT, to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism.自1923年被发现以来，光周期现象（photoperiodism）的生物学机制在诸多方面仍未被完全阐明。本研究以柞蚕（Antheraea pernyi）为实验材料，旨在探寻连接昼夜节律系统（circadian system）与内分泌开关（endocrine switch）的关键通路。研究人员在原脑（protocerebrum）的两对背外侧神经元中同时检测到PER、CLK和CYC的免疫反应阳性（immunoreactive，下文简称ir），提示此类神经元即为同时表达褪黑素（melatonin）、N-乙酰基转移酶（N-acetyltransferase, NAT）和羟基吲哚O-甲基转移酶（hydroxyindole O-methyltransferase, HIOMT）免疫反应阳性的昼夜节律神经元，结果表明昼夜节律神经元中存在褪黑素信号通路。在紧邻节律神经元的促前胸腺激素（prothoracicotropic hormone, PTTH）免疫反应阳性神经元中检测到褪黑素受体（melatonin receptor, MT2或MEL-1B-R）免疫反应阳性，提示褪黑素可调控PTTH的释放。放射免疫测定（radioimmunoassay, RIA）结果显示，在长日照16小时光照/8小时黑暗（LD16∶8，简称LD组）和12小时光照/12小时黑暗（LD12∶12，简称SD组）培养条件下，成虫脑组织中的褪黑素均呈现节律性波动，且均在关灯后4小时达到峰值；同时LD组的褪黑素峰值与基础水平均高于SD组，表明光周期对其具有显著调控作用。当处于滞育（diapause）状态的蛹接受10个周期的LD光照处理，或在恒暗条件下4℃储存4个月后，在PTTH释放蜕皮激素（ecdysone）时，可观察到NAT活性显著升高。nat基因上游的DNA序列包含E盒（E-box），可被CYC/CLK复合物结合，且靶向clk或cyc的双链RNA（double-stranded RNA, dsRNA）可抑制nat基因的转录。靶向NAT的双链RNA（dsRNANAT）会导致光周期现象功能失常。基于黑腹果蝇（Drosophila melanogaster）昼夜节律系统的研究结果，预期靶向PER的双链RNA（dsRNAPER）会上调nat基因的转录，实验结果也证实了这一推测。nat、cyc和clk的转录均在昼夜节律时间（Zeitgeber Time, ZT）12时达到峰值。RIA检测结果显示，dsRNANAT可降低褪黑素水平，而dsRNAPER则可升高褪黑素水平。综上，作为受节律时钟调控的基因，nat是连接昼夜节律时钟与内分泌开关的关键因子。褪黑素与受体结合可能会触发PTTH释放，从而终止滞育过程。本研究在柞蚕的光周期调控通路中，对从节律时钟的基本功能单元——作为nat基因mRNA积累体的光周期计数器，到介导光周期现象的内分泌开关进行了全面解析，表明该调控系统无需额外的光周期特异性装置即可自主完成完整功能。