ABSTRACT: 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.
