View Project fulp-affy-mouse-364520

Project Summary
Status:	Public
Publications:	1 Published
Project Detail		Data Detail
Platform:	Affymetrix	MIAME Areas	Compliance
Species:	Mouse	Array Design Detail	true
Organ/Tissue Type:	telencephalon	Experiment Detail	true
Organ Region:		Sample Detail	true
Cell Type:	neural cells	Hybridization Detail	true
Study Type:	subclassification	Measurement Detail	false
Disease/Condition:	neuronal migration defect
Replicates:	4
Expected Samples:	8

Available Actions

Investigator Contact Detail
Name	Mr. Carl T Fulp
Institution:	Children's Hospital of Philadelphia
Street Address:	Abramson Research Center
	34th and Civic Center Blvd.
City, State/Province:	Philadelphia , PA
Zip/Postal Code:	19104
Country:	United States
Work Phone:	(215) 590-5672
Fax:
E-mail:	fulp@mail.med.upenn.edu

Proposal Detail

Grant:

5R01NS046616-02

Status:

Public

Service Type:

Start to Finish Profiling

IACUC:

2005-12622

IACUC date:

2005-01-26

Study Relevance:

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.

Hypothesis:

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;

Specific Aim:

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.

Experimental Procedure and Design:

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.

Experimental Factors:

Conditions that are tested in the experiment. At least one is required. Experimental factors are the independent variables in the experiment.

Factor Name	Description	Factor Category
knockout vs. wild-type	Arx-/- GE vs. wild-type littermate co...	organism_status

Project Samples

Samples associated with this project.

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Name	Description	Bio-Source	Extracts
Arx-/Y #1	Total RNA (Trizol + RNeasy ...	Litter 3/21 Mouse #8	1
Arx-/Y #2	Total RNA (Trizol + RNeasy ...	Litter 4/18 Mouse #7	1
Arx-/Y #3	Total RNA (Trizol + RNeasy ...	Litter 4/18 Mouse #11	1
Arx-/Y #4	Total RNA (Trizol + RNeasy ...	Litter 5/10B Mouse #7	1
Arx+/Y #1	Total RNA (Trizol + RNeasy ...	Litter 3/21 Mouse #5	1
Arx+/Y #2	Total RNA (Trizol + RNeasy ...	Litter 3/21 Mouse #7	1
Arx+/Y #3	Total RNA (Trizol + RNeasy ...	Litter 5/10A Mouse#1	1
Arx+/Y #4	Total RNA (Trizol + RNeasy ...	Litter 5/10B Mouse #3	1

Project Hybridizations

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Name	Array	Labeled Extract	Hybridization Protocol
Hybridization8	Mouse Genome 430 2.0 Array_7	Arx+/Y #3_le1	Affymetrix
Hybridization9	Mouse Genome 430 2.0 Array_1	Arx-/Y#1_le1	Affymetrix
Hybridization10	Mouse Genome 430 2.0 Array_8	Arx+/Y #4_le1	Affymetrix
Hybridization11	Mouse Genome 430 2.0 Array_3	Arx-/Y #3_le1	Affymetrix
Hybridization12	Mouse Genome 430 2.0 Array_5	Arx+/Y #1_le1	Affymetrix
Hybridization13	Mouse Genome 430 2.0 Array_4	Arx-/Y #4_le1	Affymetrix
Hybridization14	Mouse Genome 430 2.0 Array_2	Arx-/Y #2_le1	Affymetrix
Hybridization15	Mouse Genome 430 2.0 Array_6	Arx+/Y #2_le1	Affymetrix

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