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）的季节变化，并据此调整自身生理功能以适应季节性环境变迁。该光周期系统与季节性情感障碍，以及双相情感障碍和精神分裂症中观察到的季节相关症状密切相关。在结节部（pars tuberalis，PT）中诱导产生的促甲状腺激素β亚基（Tshb），在调控动物光周期现象的信号通路中发挥关键作用。然而，目前尚不清楚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，该通路可直接诱导Tshb表达。本研究使用褪黑素功能完备的CBA/N小鼠（该小鼠保留了光周期依赖性Tshb表达响应），在慢性短日照和长日照条件下对其结节部组织开展全基因组表达分析。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。本研究通过将光照周期提前并延长8小时，在一天内快速诱导了Tshb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分析中，本研究通过筛选表达时序早于Tshb的基因，找到了潜在上游候选因子，并最终鉴定出Eya3基因。本研究结果阐明了结节部组织中全面的转录水平光周期响应机制，揭示了哺乳动物光周期系统中Tshb表达的复杂调控模式，以及其对光照变化出人意料的快速响应能力。实验将小鼠分为两组：一组饲养于短日照条件下（光照:黑暗=8小时:16小时，授时因子时间0（ZT0，灯光点亮时刻），ZT8为灯光熄灭时刻，光照强度400勒克斯），另一组饲养于长日照条件下（光照:黑暗=16小时:8小时，ZT0为灯光点亮时刻，ZT16为灯光熄灭时刻，光照强度400勒克斯），持续2周。从ZT0时刻开始，两组小鼠的结节部组织每4小时采集一次，持续1天（每组各设6个时间点）。针对长日照条件首日开展的实验，本研究在短日照条件饲养3周后设置了两种不同处理条件：其中一组将光照起始时间提前8小时（提前组），另一组将黑暗时段延后8小时（延后组）。两组小鼠的结节部组织均从灯光点亮时刻开始，每4小时采集一次，持续1天。（提前组的灯光点亮时刻对应短日照条件下的ZT16；延后组的灯光点亮时刻为ZT0。）每个时间点采集25只小鼠的组织样本。整个实验流程重复两次（n=2）以获得生物学重复样本。