Project description:Malformations of cortical development are the underlying eitiology of many cases of Mental Retardation and Epilepsy. Subtle, below the resolution of current MRI, cortical dysplasias are probably involved in many cases of MR, Epilepsy and Autism for which no diagnosis can currently be made. Therefore, understanding the process of cortical development will be vital in diagnosing and eventual treatment of many patients with these conditions. More specifically, the cortex forms from two major populations of neuroblasts which reach their final destination in the cortex by differerent mechanisms. One is radial migration from ventricular neuroblasts to the cortical plate. These cells are excititory projection neurons and glia. The second pathway is from the ventral ganglionic eminences and tangential migration of the interneuronal population of primarily inhibitory neurons. Much less is known about the control of the latter process, and many of these currently undiagnosed subtle malformations may stem from abnormalities of this tangential migration. This project focuses on the understanding the control of the tangentially migrating inhibitory interneurons. We aim to uncover the transcriptional differences between two distinct neuronal populations, GFP positive cells in the ganglionic emience and GFP positive cells within the cortical plate, in order to understand the genetic control of tangential migration. We will acomplish this aim by targeting two different labeled transcripts againts the affymetrix mouse genome arrays. We hypothesis that there will be distinct differences in mRNA transcription profiles between the ganglionic eminence and cortical plate within developing interneurons. The differences in transcript profiles will be genes that regulate the migration and differentiation of these developing neurons. Transgenic mice have been generated which express green flourscent protien (GFP) based on the activity of the Dlx 5-6 promotor. Dlx 5-6 are highly regulated genes, and are expressed only in developing interneurons with in the embryonic brain. The expression of this gene begins at around E8 within the medial and lateral ganglionic eminence (GE) and then remain present as cells born within these regions migrate out into the developing cortex. Therefore, we can take advantage of these mice by harvesting embryos at a midpoint in their development (E13.5-14.5) and dissect out GFP positive cells from the ganglionic eminence and from the cortical plate. In order to obtain enough cells to extract sufficent mRNA, we have choosen to perform microdisction of the entire GE and entire cortical plate then break up the tissue into single cell suspension and Florescent activated cell sort (FACs) the populations of GFP + and GFP - cells. We then will extract the total RNA from the GFP postive cell populations using a Trizol based method and purify the RNA with a Quiagen column purification method. The purified RNA will then be amplified using the Affymetrix 1 or 2 round T7 based amplification procedure in order to generate the microgram quantities of labelled cRNA. The labelled biotin cRNA will be sent to the microarray consortium for hybridization againt the Affy mouse genome chip. Three embryos will be pooled in order to obtain sufficent RNA and 6 GFP positive GE and cortical plate samples will be used. The six replicates will be obtained from at least 3 different pregnant mice.

Project description:Malformations of cortical development are the underlying eitiology of many cases of Mental Retardation and Epilepsy. Subtle, below the resolution of current MRI, cortical dysplasias are probably involved in many cases of MR, Epilepsy and Autism for which no diagnosis can currently be made. Therefore, understanding the process of cortical development will be vital in diagnosing and eventual treatment of many patients with these conditions. More specifically, the cortex forms from two major populations of neuroblasts which reach their final destination in the cortex by differerent mechanisms. One is radial migration from ventricular neuroblasts to the cortical plate. These cells are excititory projection neurons and glia. The second pathway is from the ventral ganglionic eminences and tangential migration of the interneuronal population of primarily inhibitory neurons. Much less is known about the control of the latter process, and many of these currently undiagnosed subtle malformations may stem from abnormalities of this tangential migration. This project focuses on the understanding the control of the tangentially migrating inhibitory interneurons. We aim to uncover the transcriptional differences between two distinct neuronal populations, GFP positive cells in the ganglionic emience and GFP positive cells within the cortical plate, in order to understand the genetic control of tangential migration. We will acomplish this aim by targeting two different labeled transcripts againts the affymetrix mouse genome arrays. We hypothesis that there will be distinct differences in mRNA transcription profiles between the ganglionic eminence and cortical plate within developing interneurons. The differences in transcript profiles will be genes that regulate the migration and differentiation of these developing neurons. Transgenic mice have been generated which express green flourscent protien (GFP) based on the activity of the Dlx 5-6 promotor. Dlx 5-6 are highly regulated genes, and are expressed only in developing interneurons with in the embryonic brain. The expression of this gene begins at around E8 within the medial and lateral ganglionic eminence (GE) and then remain present as cells born within these regions migrate out into the developing cortex. Therefore, we can take advantage of these mice by harvesting embryos at a midpoint in their development (E13.5-14.5) and dissect out GFP positive cells from the ganglionic eminence and from the cortical plate. In order to obtain enough cells to extract sufficent mRNA, we have choosen to perform microdisction of the entire GE and entire cortical plate then break up the tissue into single cell suspension and Florescent activated cell sort (FACs) the populations of GFP + and GFP - cells. We then will extract the total RNA from the GFP postive cell populations using a Trizol based method and purify the RNA with a Quiagen column purification method. The purified RNA will then be amplified using the Affymetrix 1 or 2 round T7 based amplification procedure in order to generate the microgram quantities of labelled cRNA. The labelled biotin cRNA will be sent to the microarray consortium for hybridization againt the Affy mouse genome chip. Three embryos will be pooled in order to obtain sufficent RNA and 6 GFP positive GE and cortical plate samples will be used. The six replicates will be obtained from at least 3 different pregnant mice. Keywords: other

Project description:The proper balance of excitatory and inhibitory neurons is crucial to normal processing of somatosensory information in the dorsal spinal cord. Two neural basic helix-loop-helix transcription factors, Ascl1 and Ptf1a, are essential for generating the correct number and sub-type of neurons in multiple regions of the nervous system. M-BM- In the dorsal spinal cord, Ascl1 and Ptf1a have contrasting functions in specifying inhibitory versus excitatory neurons. To understand how Ascl1 and Ptf1a function in these processes, we identified their direct transcriptional targets genome-wide in the embryonic mouse neural tube using ChIP-Seq and RNA-Seq. We show that Ascl1 and Ptf1a regulate the specification of excitatory and inhibitory neurons in the dorsal spinal cord through direct regulation of distinct homeodomain transcription factors known for their function in neuronal sub-type specification. Besides their roles in regulating these homeodomain factors, Ascl1 and Ptf1a each function differently during neuronal development with Ascl1 directly regulating genes with roles in several steps of the neurogenic program including, Notch signaling, neuronal differentiation, axon guidance, and synapse formation. In contrast, Ptf1a directly regulates genes encoding components of the neurotransmitter machinery in inhibitory neurons, and other later aspects of neural development distinct from those regulated by Ascl1. Moreover, Ptf1a represses the excitatory neuronal fate by directly repressing several targets of Ascl1. Examination of the Ascl1 and Ptf1a bound sequences shows they are enriched for a common E-Box with a GC core and with additional motifs used by Sox, Rfx, Pou, and Homeodomain factors. Ptf1a bound sequences are uniquely enriched in an E-Box with a GA/TC core and in the binding motif for its co-factor Rbpj, providing two keys to specificity of Ptf1a binding. The direct transcriptional targets identified for Ascl1 and Ptf1a provide a molecular understanding for how they function in neuronal development, particularly as key regulators of homeodomain transcription factors required for neuronal sub-type specification. Examination of gene expression in Ascl1 and Ptf1a lineage cells in the developing neural tube.

Project description:The proper balance of excitatory and inhibitory neurons is crucial to normal processing of somatosensory information in the dorsal spinal cord. Two neural basic helix-loop-helix transcription factors, Ascl1 and Ptf1a, are essential for generating the correct number and sub-type of neurons in multiple regions of the nervous system. M-BM- In the dorsal spinal cord, Ascl1 and Ptf1a have contrasting functions in specifying inhibitory versus excitatory neurons. To understand how Ascl1 and Ptf1a function in these processes, we identified their direct transcriptional targets genome-wide in the embryonic mouse neural tube using ChIP-Seq and RNA-Seq. We show that Ascl1 and Ptf1a regulate the specification of excitatory and inhibitory neurons in the dorsal spinal cord through direct regulation of distinct homeodomain transcription factors known for their function in neuronal sub-type specification. Besides their roles in regulating these homeodomain factors, Ascl1 and Ptf1a each function differently during neuronal development with Ascl1 directly regulating genes with roles in several steps of the neurogenic program including, Notch signaling, neuronal differentiation, axon guidance, and synapse formation. In contrast, Ptf1a directly regulates genes encoding components of the neurotransmitter machinery in inhibitory neurons, and other later aspects of neural development distinct from those regulated by Ascl1. Moreover, Ptf1a represses the excitatory neuronal fate by directly repressing several targets of Ascl1. Examination of the Ascl1 and Ptf1a bound sequences shows they are enriched for a common E-Box with a GC core and with additional motifs used by Sox, Rfx, Pou, and Homeodomain factors. Ptf1a bound sequences are uniquely enriched in an E-Box with a GA/TC core and in the binding motif for its co-factor Rbpj, providing two keys to specificity of Ptf1a binding. The direct transcriptional targets identified for Ascl1 and Ptf1a provide a molecular understanding for how they function in neuronal development, particularly as key regulators of homeodomain transcription factors required for neuronal sub-type specification. Examination of Ascl1 and Ptf1a genome-wide binding in developing neural tube.

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Transcriptomic disruption and functional hypoactivity in DYT-SGCE MGE-patterned inhibitory neurons

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