Gene expression profile of retina and pineal gland of NeuroD1 conditional knockout mice

NeuroD1 encodes a basic helix-loop-helix transcription factor involved in the development of neural and endocrine structures. NeuroD1 mRNA is highly abundant in the adult mammalian pineal gland and exhibits a developmental expression pattern similar to the retina. This is consistent with the common evolutionary origin of pinealocytes and retinal photoreceptors. Pinealocytes and retinal photoreceptors express a shared set of phototransduction genes and submammalian pinealocytes are photosensitive. In contrast to the retina, the pineal gland is a relatively homogeneous structure, composed 95% of pinealocytes. This makes the pineal gland a particularly useful model for understanding photoreceptor cell biology. The loss of NeuroD1 in the retina results in progressive photoreceptor degeneration and the molecular mechanisms underlying this retinal degeneration phenotype remain unknown. Similarly, the role that NeuroD1 plays in the pineal gland is unknown. To determine the function of NeuroD1 in the pineal gland and retina, a Cre/loxP recombination strategy was used to selectively target a NeuroD1 floxed allele and generate NeuroD1 conditional knockout (cKO) mice. Tissue specificity was conferred using Cre recombinase expressed under the control of the promoter of Crx, a transcription factor selectively expressed in the pineal gland and retina. Pineal and retinal tissues from two-month-old NeuroD1 cKO and control animals were used in microarray studies to identify candidate genes responsible for the photoreceptor degeneration phenotype. The Cre/loxP recombination strategy was used to target a NeuroD1 floxed allele and generate NeuroD1 conditional knockout mice (NeuroD1floxed::Crx-Cre+/-). NeuroD1 floxed mice (NeuroD1floxed::Crx-Cre-/-) served as the controls. Pineal glands and retinas from two-month-old control and conditional knockout mice were collected at ZT6 and ZT20. 3 pools of 6 pineal glands per genotype and respective time of day were collected for each sample. Similarly, 3 pools of 6 retinas each were also collected. RNA from each pool was extracted and hybridized on the Affymetrix Mouse 430 2.0 array.神经源性分化因子1（NeuroD1）编码一种碱性螺旋-环-螺旋转录因子，参与神经与内分泌结构的发育过程。NeuroD1 mRNA在成年哺乳动物的松果体内含量极高，其发育表达模式与视网膜高度相似，这与松果体细胞与视网膜感光细胞的共同进化起源相符。松果体细胞与视网膜感光细胞共享一套光转导基因，且亚哺乳动物的松果体细胞具备感光性。与视网膜不同，松果体属于相对均质的结构，其中95%的细胞为松果体细胞，这使其成为研究感光细胞生物学的理想模型。视网膜中NeuroD1的缺失会引发进行性感光细胞变性，但该视网膜变性表型背后的分子机制尚未明确；同样，NeuroD1在松果体内的功能也尚未阐明。为探明NeuroD1在松果体与视网膜中的功能，研究人员采用Cre/loxP重组策略，特异性靶向NeuroD1的floxed等位基因，构建得到NeuroD1条件性敲除（conditional knockout, cKO）小鼠。研究利用受Crx启动子调控表达的Cre重组酶实现组织特异性敲除——Crx是一种仅在松果体与视网膜中特异性表达的转录因子。研究人员收集了2月龄NeuroD1 cKO小鼠与对照小鼠的松果体与视网膜组织，通过微阵列分析筛选与感光细胞变性表型相关的候选基因。本研究采用Cre/loxP重组策略靶向NeuroD1的floxed等位基因，构建得到NeuroD1条件性敲除小鼠（NeuroD1floxed::Crx-Cre+/-）；以NeuroD1 floxed小鼠（NeuroD1floxed::Crx-Cre-/-）作为对照。研究人员分别于授时时间（Zeitgeber Time, ZT）6与ZT20时刻，收集2月龄对照与条件性敲除小鼠的松果体与视网膜组织。针对每个基因型与对应时间点，每类样本均收集3个混合池，每个混合池包含6个松果体；同理，每类样本也收集3个混合池，每个混合池包含6个视网膜。提取各混合池的总RNA后，将其在Affymetrix Mouse 430 2.0芯片上进行杂交实验。