Genome-wide expression analysis of the mouse pars tuberalis (PT) under chronic short-day and long-day conditions

Living organisms detect seasonal changes in day length (photoperiod), and alter their physiological functions accordingly, to fit seasonal environmental changes. This photoperiodic system is implicated in seasonal affective disorders and the season-associated symptoms observed in bipolar disease and schizophrenia. Thyroid-stimulating hormone beta subunit (Tshb), induced in the pars tuberalis (PT), plays a key role in the pathway that regulates animal photoperiodism. However, the upstream inducers of Tshb expression remain unknown. Here we show that late-night light stimulation acutely triggers the Eya3-Six1 pathway, which directly induces Tshb expression. Using melatonin-proficient CBA/N mice, which preserve the photoperiodic Tshb-expression response, we performed a genome-wide expression analysis of the PT under chronic short-day and long-day conditions. These data comprehensively identified long-day and short-day genes, and indicated that late-night light stimulation induces long-day genes. We verified this by advancing and extending the light period by 8 hours, which acutely induced Tshb expression, within one day. In a genome-wide expression analysis under this condition, we searched for candidate upstream genes by looking for expression that preceded Tshb’s, and identified Eya3 gene. These results elucidate the comprehensive transcriptional photoperiodic response in the PT, revealing the complex regulation of Tshb expression and unexpectedly rapid response to light changes in the mammalian photoperiodic system. Mice were separated into 2 groups. One group was maintained under the short-day conditions (light: dark = 8 h:16 h, ZT0 = lights on, ZT8 = lights off, 400 lux) and the other was housed under long-day conditions (light:dark = 16 h:8 h, ZT0 = lights on, ZT16 = lights off, 400 lux) for 2 weeks. The PTs of both groups were retrieved every 4 h for 1 day (6 time points for each group), starting at ZT0. For the experiments performed during the first day of the long-day conditions, we applied two different conditions, following 3 weeks under short-day conditions. In one, the light-onset was advanced by 8 hours (advance condition), and in the other, the dark period was delayed by 8 hours (delay condition). PTs from both groups were obtained every 4 h for 1 day, starting at the lights-on time. (Lights on for the advance condition was ZT16 as defined by the short-day condition. Lights on for the delay condition was ZT0). We sampled 25 mice at each time point. This whole procedure was repeated twice (n = 2) to obtain experimental replicates.

生物体可感知日照时长（光周期，photoperiod）的季节性变化，并相应调整自身生理功能，以适应季节性环境波动。该光周期系统与季节性情感障碍、双相情感障碍及精神分裂症中观察到的季节相关症状密切相关。在垂体结节部（pars tuberalis, PT）中诱导产生的促甲状腺激素β亚基（Thyroid-stimulating hormone beta subunit, Tshb），在调控动物光周期现象的通路中发挥关键作用。然而，目前尚不明确Tshb表达的上游诱导因子。本研究证实，深夜光照刺激可快速激活Eya3-Six1通路，进而直接诱导Tshb的表达。本研究利用保留了光周期依赖性Tshb表达响应的褪黑素正常型CBA/N小鼠，对长期短日照和长日照条件下的垂体结节部进行了全基因组表达谱分析。该分析全面鉴定了长日照相关基因与短日照相关基因，并提示深夜光照刺激可诱导长日照相关基因的表达。本研究通过将光照起始时刻提前并将光照时长延长8小时，在1天内快速诱导了Tshb的表达，从而验证了上述结论。在此条件下开展的全基因组表达谱分析中，本研究通过筛选早于Tshb表达的基因，找到了潜在的上游调控因子，并鉴定出Eya3基因。上述结果阐明了垂体结节部中全面的转录水平光周期响应机制，揭示了Tshb表达的复杂调控模式，以及哺乳动物光周期系统对光照变化出乎意料的快速响应能力。

将小鼠分为两组：一组在短日照条件下饲养（光照：黑暗=8小时：16小时，ZT0为开灯时刻，ZT8为关灯时刻，光照强度400勒克斯），另一组在长日照条件下饲养（光照：黑暗=16小时：8小时，ZT0为开灯时刻，ZT16为关灯时刻，光照强度400勒克斯），饲养周期均为2周。两组小鼠均从ZT0时刻开始，每4小时采集一次垂体结节部样本，持续1天（每组共6个时间点）。针对长日照条件首日的实验，我们先将小鼠在短日照条件下饲养3周，随后设置两种不同的光照干预方案：其一为光照起始时刻提前8小时（提前组），其二为黑暗周期延迟8小时（延迟组）。两组小鼠均从开灯时刻开始，每4小时采集一次垂体结节部样本，持续1天。（提前组的开灯时刻对应短日照条件下的ZT16；延迟组的开灯时刻为ZT0。）每个时间点采集25只小鼠的样本。整个实验流程重复两次（n=2）以设置生物学重复。