Transcription profiling of mouse wild type and Ctip2-/- (Bcl11b) mutant striatum at P0

Striatal medium spiny neurons (MSN) are critically involved in motor control, and their degeneration is a principal component of Huntington's disease. We find that the transcription factor Ctip2 (also known as Bcl11b) is central to MSN differentiation and striatal development. Within the striatum, it is expressed by all MSN, while it is excluded from essentially all striatal interneurons. In the absence of Ctip2, MSN do not fully differentiate, as demonstrated by dramatically reduced expression of a large number of MSN markers, including DARPP-32, FOXP1, Chrm4, Reelin, MOR1, GluR1, and Plexin-D1. Furthermore, MSN fail to aggregate into patches, resulting in severely disrupted patch-matrix organization within the striatum. Finally, heterotopic cellular aggregates invade the Ctip2-/- striatum suggesting a failure by MSN to repel these cells in the absence of Ctip2. In order to investigate the molecular mechanisms that underlie Ctip2-dependent differentiation of MSN and that underlie the patch-matrix disorganization in the mutant striatum, we directly compared gene expression between wild type and mutant striatum at P0. Because CTIP2-expressing MSN constitute 90-95% of the neurons within the striatum, we reasoned that we should be able to detect changes in medium spiny neuron gene expression in Ctip2 null mutants. We microdissected out small regions of striatum at matched locations in wild type and Ctip2-/- mutant littermates at P0 and investigated gene expression with Affymetrix microarrays. We selected the 153 most significant genes and further analyzed them to identify a smaller set of genes of potentially high biological relevance. In order to verify the microarray data and define the distribution of the identified genes in the striatum, we performed in situ hybridization or immunohistochemistry for 12 selected genes: Plexin-D1, Ngef, Nectin-3, Kcnip2, Pcp4L1, Neto1, Basonuclin 2, Fidgetin, Semaphorin 3e, Secretagogin, Unc5d, and Neurotensin. We find that all these genes are either specifically downregulated (Plexin-D1, Ngef, Nectin-3 Kcnip2, Pcp4L1, Neto1), or upregulated (Basonuclin 2, Fidgetin, Semaphorin 3e, Secretagogin, Unc5d, Neurotensin), in the Ctip2-/- striatum, confirming and extending the microarray results. Together, these data indicate that Ctip2 is a critical regulator of MSN differentiation, striatal patch development, and the establishment of the cellular architecture of the striatum. Experiment Overall Design: Matched regions of striatum from wild type and Ctip2-/- mice were obtained via 500 µm diameter punch biopsies performed in the center of the developing striatum in acutely sectioned 300 µm coronal slices of the brain at postnatal day 0 (P0). Sections were matched rostro-caudally between wild type and null mutant tissue, and fiduciary landmarks were used to assure reproducible microdissection of comparable regions. RNA was extracted using the StrataPrep Total RNA Mini Kit (Stratagene, La Jolla, CA), and RNA quality was assayed using a bioanalyzer (Agilent Technologies, Paola Alto, CA). To ensure reproducibility and biological significance, microarrays were performed with RNA samples from three independent wild type, one heterozygote, and four Ctip2-/- mice (biological replicates). Microarray data were normalized using the RMA function within Bioconductor (Irizarry et al., 2003). Statistical significance of gene expression differences between wild type and knockout was determined using Statistical Analysis of Micrarrays (SAM) (Tusher et al., 2001). Using a SAM d-score cutoff of > 2 or < -2, we selected the 153 most significant genes and further analyzed them to identify a smaller set of genes of potentially high biological relevance.纹状体中型多棘神经元（Striatal medium spiny neurons, MSN）在运动调控中发挥关键作用，其变性是亨廷顿病（Huntington's disease）的核心病理特征之一。本研究发现，转录因子Ctip2（又称Bcl11b）对MSN分化与纹状体发育至关重要。在纹状体中，Ctip2在所有MSN中均有表达，而几乎所有纹状体中间神经元均不表达该因子。在Ctip2缺失的情况下，MSN无法完成完全分化，具体表现为大量MSN标志物的表达水平显著降低，包括DARPP-32、FOXP1、Chrm4、Reelin、MOR1、GluR1及Plexin-D1。此外，MSN无法聚集成补丁状结构，导致纹状体内的补丁-基质组织结构严重紊乱。最终，异位细胞聚集体侵入Ctip2-/-小鼠的纹状体，提示在缺乏Ctip2的情况下，MSN无法有效排斥此类细胞。 为探究Ctip2依赖的MSN分化分子机制，以及突变纹状体中补丁-基质结构紊乱的成因，我们直接比较了出生后第0天（P0）野生型与突变型小鼠纹状体的基因表达谱。由于表达CTIP2的MSN占纹状体神经元总数的90%~95%，我们推测可在Ctip2全基因敲除突变体中检测到MSN基因表达的变化。我们通过显微切割获取了P0小鼠急性脑片（300 μm冠状切片）中，野生型与Ctip2-/-同窝突变体纹状体中心匹配位置的、直径为500 μm的组织样本，并采用Affymetrix基因芯片检测基因表达。我们筛选出153个差异最显著的基因，并进一步分析以筛选出一组具有潜在高生物学相关性的更小基因集。 为验证基因芯片数据并明确所鉴定基因在纹状体中的分布，我们对12个筛选出的基因开展了原位杂交或免疫组化实验，分别为Plexin-D1、Ngef、Nectin-3、Kcnip2、Pcp4L1、Neto1、Basonuclin 2、Fidgetin、Semaphorin 3e、Secretagogin、Unc5d及Neurotensin。结果显示，上述基因在Ctip2-/-纹状体中要么呈现特异性下调（Plexin-D1、Ngef、Nectin-3、Kcnip2、Pcp4L1、Neto1），要么呈现特异性上调（Basonuclin 2、Fidgetin、Semaphorin 3e、Secretagogin、Unc5d、Neurotensin），验证并拓展了基因芯片的实验结果。综上，上述数据表明Ctip2是调控MSN分化、纹状体补丁发育以及纹状体细胞架构建立的关键调节因子。 实验整体设计：我们在出生后第0天（P0）的急性脑片（300 μm冠状切片）中，于野生型与Ctip2-/-小鼠纹状体中心匹配位置，通过直径500 μm的穿刺活检获取匹配区域的纹状体组织。实验中依据吻尾轴位置匹配野生型与敲除组组织，并借助参考标志点确保可重复获取相同区域的显微切割样本。采用StrataPrep Total RNA Mini Kit（Stratagene, La Jolla, CA）提取RNA，并通过生物分析仪（Agilent Technologies, Paola Alto, CA）检测RNA质量。为保证实验可重复性与生物学意义，我们使用来自3只独立野生型小鼠、1只杂合子小鼠及4只Ctip2-/-小鼠的RNA样本开展基因芯片实验（生物学重复）。基因芯片数据采用Bioconductor中的RMA函数进行标准化处理（Irizarry等，2003）。采用微阵列统计分析工具（SAM，Tusher等，2001）确定野生型与敲除组间基因表达差异的统计学显著性。以SAM的d值阈值>2或< -2为筛选标准，我们选取了153个差异最显著的基因，并进一步分析以筛选出一组具有潜在高生物学相关性的更小基因集。