Pet-1 Switches Transcriptional Targets Postnatally to Regulate Maturation of Serotonin Neuron Excitability.

Newborn neurons enter an extended maturation stage, during which they acquire excitability characteristics crucial for development of presynaptic and postsynaptic connectivity. In contrast to earlier specification programs, little is known aboutthe regulatory mechanisms that control neuronal maturation. The Pet-1 ETS (E26 transformation-specific) factor is continuously expressed in serotonin (5-HT) neurons and initially acts in postmitotic precursors to control acquisition of 5-HT transmitter identity. Using a combination of RNA sequencing, electrophysiology, and conditional targeting approaches, we determined gene expression patterns in maturing flow-sorted 5-HT neurons and the temporal requirements for Pet-1 in shaping these patterns for functional maturation of mouse 5-HT neurons. We report a profound disruption of postmitotic expression trajectories in Pet-1 / neurons, which prevented postnatal maturation of 5-HT neuron passive and active intrinsic membrane properties, G-protein signaling, and synaptic responses to glutamatergic, lysophosphatidic, and adrenergic agonists. Unexpectedly, conditional targeting revealed a postnatal stage-specific switch in Pet-1 targets from 5-HT synthesis genes to transmitter receptor genes required for afferent modulation of 5-HT neuron excitability. 5-HT1a autoreceptor expression depended transiently on Pet-1, thus revealing an early postnatal sensitive period for control of 5-HT excitability genes. Chromatin immunoprecipitation followed by sequencing revealed that Pet-1 regulates 5-HT neuron maturation through direct gene activation and repression. Moreover, Pet-1 directly regulates the 5-HT neuron maturation factor Engrailed 1, which suggests Pet-1 orchestrates maturationthrough secondary postmitotic regulatoryfactors. The early postnatal switch in Pet-1targets uncovers a distinct neonatal stage-specific function for Pet-1, during which it promotes maturation of 5-HT neuron excitability. 5-HT neuron mRNA profiles of E11.5, E15.5, and postnatal (P1-P3) wild type (WT) and Pet-1-/- mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. Myc-tagged Pet-1 ChIP-seq was performed on E12.5 to E14.5 hindbrains and sequencing using NextSeq 500.新生神经元会进入一段延长的成熟阶段，在此期间它们会获得与突触前和突触后连接发育至关重要的兴奋性特征。与早期的神经元特化程序不同，目前对控制神经元成熟的调控机制知之甚少。Pet-1 ETS（E26转化特异性，E26 transformation-specific）因子在血清素（5-HT，serotonin）能神经元中持续表达，并最初在有丝分裂后前体细胞中发挥作用，以调控5-HT递质表型的获得。本研究结合RNA测序（RNA sequencing）、电生理学（electrophysiology）与条件性靶向技术，对成熟的流式分选5-HT能神经元的基因表达模式，以及Pet-1在塑造小鼠5-HT能神经元功能成熟所需的这些表达模式中的时序需求进行了解析。我们发现，Pet-1基因敲除（Pet-1-/-）的神经元中，有丝分裂后表达轨迹发生了显著紊乱，这阻断了5-HT能神经元的出生后成熟过程，包括其被动和主动内在膜特性、G蛋白信号通路，以及对谷氨酸能、溶血磷脂酸和肾上腺素能激动剂的突触反应。出乎意料的是，条件性靶向实验揭示了Pet-1靶点在出生后发生了阶段特异性转换：从5-HT合成基因转换为调控5-HT能神经元兴奋性传入调节所需的递质受体基因。5-HT1a自身受体的表达短暂依赖于Pet-1，这揭示了调控5-HT兴奋性基因的出生后早期敏感窗口期。染色质免疫共沉淀测序（ChIP-seq，chromatin immunoprecipitation followed by sequencing）结果显示，Pet-1通过直接激活和抑制基因来调控5-HT能神经元的成熟。此外，Pet-1可直接调控5-HT能神经元成熟因子Engrailed 1，这表明Pet-1可通过次级有丝分裂后调控因子协同调控成熟过程。Pet-1靶点的出生后早期转换，揭示了Pet-1具有独特的新生儿阶段特异性功能，在此期间它可促进5-HT能神经元兴奋性的成熟。本研究通过Illumina HiSeq 2500平台进行三次重复深度测序，获取了E11.5、E15.5以及出生后（P1-P3）野生型（WT，wild type）和Pet-1基因敲除（Pet-1-/-）小鼠的5-HT能神经元mRNA表达谱。对E12.5至E14.5的后脑进行了Myc标签标记的Pet-1染色质免疫共沉淀测序，并使用NextSeq 500平台完成测序。