Hypothalamic transcriptome plasticity in two rodent species reveals divergent differential gene expression but conserved pathways. Hypothalamic transcriptome plasticity in two rodent species reveals divergent differential gene expression but conserved pathways

We have addressed the question of how different rodent species cope with the life-threatening homeostatic challenge of dehydration at the level of transcriptome modulation in the supraoptic nucleus (SON), a specialised hypothalamic neurosecretory apparatus responsible for the production of the antidiuretic peptide hormone arginine vasopressin (AVP). AVP maintains water balance by promoting water conservation at the level of the kidney. Dehydration evokes a massive increase in the regulated release of AVP from SON axon terminals located in the posterior pituitary, and this is accompanied by a plethora of changes in the morphology, electrophysiological properties, biosynthetic and secretory activity of this structure. Microarray analysis was used to generate a definitive catalogue of the genes expressed in the mouse SON, and to describe how the gene expression profile changes in response to dehydration. Comparison of the genes differentially expressed in the mouse SON as a consequence of dehydration with those of the rat has revealed many similarities, pointing to common processes underlying the function-related plasticity in this nucleus. In addition we have identified many genes that are differentially expressed in a species-specific manner. However, in many cases, we have found that the hyperosmotic cue can induce species-specific alterations in the expression of different genes in the same pathway. The same functional end can be served by different means, via differential modulation, in different species, of different molecules in the same pathway. We suggest that pathways, rather than specific genes, should be the focus of integrative physiological studies based on transcriptome data. Overall design: Animals. Adult male C57BL/6 mice (Harlan Sera-Lab, Loughborough, UK) were group housed (4 per cage) under controlled temperature (21+ 2ºC) and diurnal light conditions (14-h light, 10-h dark, lights on at 05.00). Food and water were available ad libitum until the experiment commenced. Complete fluid deprivation was imposed for 48 hours starting at 11.00 a.m. Control animals maintained free access to drinking water, and both groups had access to standard laboratory rodent chow. Experiments on adult male rats described previously (27). All procedures were conducted in strict accordance with the Animal Scientific Procedures Act (1986), UK, and were approved by the local University of Bristol Ethical Review Process. Tissue collection. Mice were killed using cervical dislocation and the brain was carefully removed from the cranium and snap frozen using powdered dry ice and stored at -80oC for no more than 14 days. Sections of brain (14μm) were cut using an RNase free cryostat and mounted onto RNase free membrane coated glass slides (P.A.L.M. Membrane slides; P.A.L.M. Microlaser Technologies). Immediately after sectioning, frozen sections were thawed and fixed (30s; in 95% [v/v] EtOH), rehydrated (30s in each of 75% [v/v] and 50% [v/v] EtOH) before being stained (60s 1% [v/v] cresyl violet). Sections were then dehydrated in a graded EtOH series (30s in each of 50% [v/v], 75% [v/v] and 95% [v/v]. then 2x 30s in 100% [v/v]). Laser microdissection was performed using a P.A.L.M. MicrolaserSystem (P.A.L.M. Microlaser Technologies). The SON was identified with reference to Franklin and Paxinos (28) and the tissue from each animal was independently pooled into collection vials containing RNAlater® (Ambion, Huntingdon, UK). A single operative carried out all dissections. Total RNA was isolated without delay (within 24h) according to standard procedures that accompany the Ambion RNAqueous MicroKit (Ambion). Microarray analysis. Separate microarrays (n=4) were probed using independently generated target. For each completely independent replicate, tissue from 1 mouse was used for RNA extraction.

本研究探讨了不同啮齿类物种如何通过视上核（supraoptic nucleus, SON）的转录组调控，应对脱水这一危及生命的稳态挑战。视上核是下丘脑特化的神经分泌结构，负责合成抗利尿肽类激素精氨酸加压素（arginine vasopressin, AVP）。AVP通过促进肾脏的水重吸收以维持机体水平衡。脱水会触发位于垂体后叶的SON轴突末梢大量释放AVP，同时伴随该结构在形态、电生理特性、生物合成与分泌活性等多方面的广泛改变。
本研究采用微阵列（microarray）分析技术，构建了小鼠SON中基因表达的完整目录，并描述了脱水状态下小鼠SON的基因表达谱变化。将脱水诱导的小鼠SON差异表达基因与大鼠的同类数据进行比对后，发现二者存在诸多共性，提示该核团功能相关可塑性存在共同的分子机制。此外，本研究还鉴定出诸多以物种特异性方式差异表达的基因。然而在多数情况下，高渗刺激可诱导同一通路内不同基因在不同物种中出现特异性表达改变。不同物种可通过对同一通路内不同分子的差异化调控，以不同途径实现相同的功能终点。据此我们提出，基于转录组数据的整合生理学研究应将通路而非单一基因作为核心研究靶点。
总体实验设计：
1. 实验动物
选用成年雄性C57BL/6小鼠（购自英国拉夫堡的Harlan Sera-Lab公司），每笼4只群居饲养，环境温度控制为21±2℃，光照周期为14小时光照/10小时黑暗（每日05:00开灯）。实验开始前，小鼠可自由采食标准啮齿类饲料并自由饮水。自当日上午11:00起，实验组小鼠完全禁水48小时；对照组小鼠可自由饮水，两组小鼠均持续提供标准啮齿类饲料。关于成年雄性大鼠的实验细节参见既往研究（27）。所有实验操作均严格遵循英国《1986年动物科学实验法案》，并经英国布里斯托大学当地伦理审查委员会批准。
2. 组织采集
采用颈椎脱臼法处死小鼠，小心取出脑组织，使用干冰粉末快速冷冻后于-80℃保存，保存时长不超过14天。使用无RNase冰冻切片机将脑组织切成14μm厚的切片，粘贴于无RNase的覆膜载玻片（P.A.L.M. Membrane slides；P.A.L.M. Microlaser Technologies公司）上。切片完成后，立即将冰冻切片解冻固定（95%[v/v]乙醇中固定30秒），依次经75%[v/v]乙醇、50%[v/v]乙醇各浸泡30秒进行复水，随后用1%[v/v]甲酚紫染色60秒。之后再依次经50%[v/v]乙醇、75%[v/v]乙醇、95%[v/v]乙醇各浸泡30秒，最后用100%[v/v]乙醇浸泡2次，每次30秒进行脱水。使用P.A.L.M.激光显微切割系统（P.A.L.M. Microlaser Technologies公司）进行激光显微切割。参照Franklin与Paxinos的脑图谱（28）定位视上核，将每只小鼠的目标组织分别收集至含有RNA later®（Ambion公司，英国亨廷顿）的收集管中。由同一名实验人员完成所有解剖切割操作。按照Ambion RNAqueous MicroKit（Ambion公司）配套的标准操作流程，在24小时内完成总RNA的提取。
3. 微阵列分析
采用独立制备的靶标探针分别检测4张独立的微阵列芯片（n=4）。每一次独立重复实验均使用1只小鼠的组织进行RNA提取。