Transcription factor Arx null brains (fulp-affy-mouse-364520)

Arx is a paired-box homeodomain transcription factor and the vertebrate ortholog to the Drosophila aristaless (al) gene. Mutations in Arx are associated with a variety of human diseases, including X-linked infantile spasm syndrome (OMIM: 308350), X-linked myoclonic epilepsy with mental retardation and spasticity (OMIM: 300432), X-linked lissencephaly with ambiguous genitalia (OMIM: 300215), X-linked mental retardation 54 (OMIM: 300419), and agenesis of the corpus callosum with abnormal genitalia (OMIM: 300004). Arx-deficient mice exhibit a complex, pleiotrophic phenotype, including decreased proliferation of neuroepithelial cells of the cortex, dysgenesis of the thalamus and olfactory bulbs, and abnormal nonradial migration of GABAergic interneurons. It has been suggested that deficits in interneuron specification, migration, or function lead to loss of inhibitory neurotransmission, which then fails to control excitatory activity and leads to epilepsy or spasticities. Given that Arx mutations are associated with developmental disorders in which epilepsy and spasticity predominate and that Arx-deficient mice exhibit deficits in interneuron migration, understanding the function of Arx in interneuron migration will prove crucial to understanding the pathology underlying interneuronopathies. Yet, downstream transcriptional targets of Arx, to date, remain unidentified. The aim of this project is to identify bona fide transcriptional targets for the Arx, a transcription factor required for normal migration of interneurons from the ganglionic eminences to the cortex, and to investigate the functions of these genes in the Arx-dependent pathway regulating nonradial neuronal migration. We hypothesize that the genes regulated by the Arx transcription factor will play a critical role in the nonradial migration of interneurons and that the results of this study will provide novel insights into the molecular mechanisms of nonradial neuronal migration, in particular, and possibly the molecular and biochemical pathogenesis underlying epilepsy, mental retardation, infantile spasm syndromes, and other so-called interneuronopathies; We have recently generated a transgenic mouse with a floxed Arx allele (Arxflox). We have generated conditional knockouts in which Arx is removed specifically from the brain by mating Arxflox mice with transgenic mice expressing Cre behind the neural tube-specific transcriptional regulatory elements of the POU domain, class 3, transcription factor 4 promoter. Preliminary analyses of these mice suggest that conditional knockout mice recapitulate the nonradial migration defects associated with conventional knockout mice. We will compare the gene expression profiles of ganglion eminences (GEs; the anatomical source for nonradially migrating interneurons) from male Arxflox mice that express Pou3f-Cre to those from male mice without Arxflox allele. Animals will be prepared and sacrificed following our institutional protocol. Tissue will be rapidly dissected from E14.5 (the temporal peak of interneuron migration) GEs (MGE and LGE from both left and right hemispheres). Preliminary experiments suggest that the amounts of RNA that can be isolated from a pair of GEs is in the range of 2000-3500 ng, which should be sufficient for microarray analysis following linear amplification of RNA. The GEs from each animal will be combined, snap frozen in liquid nitrogen, and stored at -80 C until RNA is extracted. Total RNA will be extracted using Trizol followed by RNA purification with the RNeasy cleanup kit. We will be providing total RNA samples from four wildtype and four transgenic animals (true biological replicates) from three separate litters to mitigate any expression differences resulting from mouse to mouse or litter to litter variation.

Arx是一种配对盒同源域转录因子（paired-box homeodomain transcription factor），亦是果蝇aristaless（al）基因的脊椎动物同源物。Arx突变与多种人类疾病密切相关，包括X连锁婴儿痉挛综合征（OMIM: 308350）、伴智力障碍与痉挛的X连锁肌阵挛性癫痫（OMIM: 300432）、伴生殖器歧义的X连锁无脑回畸形（OMIM: 300215）、X连锁智力障碍54型（OMIM: 300419），以及伴生殖器异常的胼胝体发育不全（OMIM: 300004）。
Arx缺陷小鼠表现出复杂的多效性表型，包括大脑皮层神经上皮细胞增殖水平降低、丘脑与嗅球发育异常，以及γ-氨基丁酸能中间神经元（GABAergic interneurons）的异常非径向迁移。已有研究表明，中间神经元的特化、迁移或功能缺陷会导致抑制性神经递质传递丧失，进而无法有效调控兴奋性活动，最终引发癫痫或痉挛症状。
鉴于Arx突变与以癫痫和痉挛为主要特征的发育障碍相关，且Arx缺陷小鼠存在中间神经元迁移缺陷，阐明Arx在中间神经元迁移中的功能，对于解析中间神经元病的病理机制至关重要。然而截至目前，Arx的下游转录靶标仍未被明确鉴定。本项目的研究目标为：鉴定Arx——一种调控中间神经元从神经节隆起向皮层正常迁移的转录因子——的真实转录靶标，并探究这些基因在Arx依赖的非径向神经元迁移调控通路中的功能。我们提出如下假说：受Arx转录因子调控的基因在中间神经元的非径向迁移过程中发挥关键作用；本研究的结果将为非径向神经元迁移的分子机制，尤其是癫痫、智力障碍、婴儿痉挛综合征及其他所谓中间神经元病的分子与生化发病机制，提供全新的认知。
我们近期构建了携带loxP位点修饰的Arx等位基因（Arxflox）的转基因小鼠。通过将Arxflox小鼠与在POU结构域3类转录因子4（POU domain, class 3, transcription factor 4, Pou3f4）启动子的神经管特异性转录调控元件后表达Cre重组酶的转基因小鼠交配，我们获得了特异性在脑中敲除Arx的条件性基因敲除小鼠。对这些小鼠的初步分析显示，条件性敲除小鼠能够重现传统Arx敲除小鼠的非径向迁移缺陷。我们将比较表达Pou3f4-Cre的雄性Arxflox小鼠与不携带Arxflox等位基因的雄性小鼠的神经节隆起（ganglionic eminences, GEs；非径向迁移中间神经元的解剖学来源）的基因表达谱。实验动物将按照我们机构的实验规程进行饲养与安乐死。在胚胎发育14.5天（E14.5，中间神经元迁移的时间峰值）时，快速摘取双侧大脑半球的内侧神经节隆起（medial ganglionic eminence, MGE）与外侧神经节隆起（lateral ganglionic eminence, LGE）组织。预实验结果显示，一对神经节隆起可提取到2000~3500 ng的RNA，该产量足以在RNA线性扩增后用于芯片分析（microarray analysis）。将每只小鼠的神经节隆起组织合并，经液氮快速冷冻后保存于-80℃，直至RNA提取。总RNA将采用Trizol试剂提取，随后通过RNeasy纯化试剂盒进行RNA纯化。我们将从3窝独立的仔鼠中获取4只野生型小鼠与4只转基因小鼠的总RNA样本（真正的生物学重复），以抵消小鼠个体间或窝别间的表达差异带来的影响。