Project description:The Dlx homeodomain transcription factors are implicated in regulating the function of inhibitory GABAergic interneurons; therefore understanding their functions will provide insights into disorders such as epilepsy, mental retardation and autism. Identifying genes that are downstream of Dlx1/2 function and are relevant for the differentiation and survival of GABAergic interneurons. During embryonic development, cortical GABAergic interneurons are generated in the proliferative zone of the medial ganglionic eminence (MGE), from where they migrate to reach their final positions in the cortex. The differentiation of these interneuron precursors is dependent on Dlx genes, as shown by Dlx1/Dlx2 double mutants, which have a block in GABAergic cell differentiation and in cell migration. When interneuron progenitors are isolated from the mutant MGE and growth in culture, they are able to proceed along their differentiation program. However, mutant cells growth in vitro show defects in cell morphogenesis and increased cell apoptosis. We hypothesize that Dlx transcription factors regulate important aspects of GABAergic neuron differentiation such as the formation and growth of axon and dendrites, and the formation of inhibitory synapses. We generated E15.5 mouse embryos that are Dlx1/2 -/- or Dlx1/2 +/?. Genotype was confirmed by PCR. A total of 8 litters were used. For each experiment, we pooled tissue from at least 6 different embryos of the same genotype. We dissected the ventricular and subventricular zones of the MGE (rostral part). This area contains ~1 million of progenitor cells per embryo. We isolated total RNA using the Stratagene RNA Miniprep kit (these samples are called MGE+/ and MGE-/- in our proposal). In addition, we used the same area (ventricular and subventricular zones of the rostral MGE) to perform primary neuronal cultures. Cells were maintained 3 days in vitro. After that, we isolated total RNA using the Stratagene RNA Miniprep kit (samples called primary cells+/ and primary cells-/- in our proposal). We would like to perform gene expression comparison between: 1) MGE+/ and MGE-/-, and 2) primary cells+/ and primary cells-/-. Keywords: Dlx mutants, medial ganglionic eminence, neuronal cultures

Project description:The Dlx homeodomain transcription factors are implicated in regulating the function of inhibitory GABAergic interneurons; therefore understanding their functions will provide insights into disorders such as epilepsy, mental retardation and autism. Identifying genes that are downstream of Dlx1/2 function and are relevant for the differentiation and survival of GABAergic interneurons. During embryonic development, cortical GABAergic interneurons are generated in the proliferative zone of the medial ganglionic eminence (MGE), from where they migrate to reach their final positions in the cortex. The differentiation of these interneuron precursors is dependent on Dlx genes, as shown by Dlx1/Dlx2 double mutants, which have a block in GABAergic cell differentiation and in cell migration. When interneuron progenitors are isolated from the mutant MGE and growth in culture, they are able to proceed along their differentiation program. However, mutant cells growth in vitro show defects in cell morphogenesis and increased cell apoptosis. We hypothesize that Dlx transcription factors regulate important aspects of GABAergic neuron differentiation such as the formation and growth of axon and dendrites, and the formation of inhibitory synapses. We generated E15.5 mouse embryos that are Dlx1/2 -/- or Dlx1/2 +/?. Genotype was confirmed by PCR. A total of 8 litters were used. For each experiment, we pooled tissue from at least 6 different embryos of the same genotype. We dissected the ventricular and subventricular zones of the MGE (rostral part). This area contains ~1 million of progenitor cells per embryo. We isolated total RNA using the Stratagene RNA Miniprep kit (these samples are called MGE+/ and MGE-/- in our proposal). In addition, we used the same area (ventricular and subventricular zones of the rostral MGE) to perform primary neuronal cultures. Cells were maintained 3 days in vitro. After that, we isolated total RNA using the Stratagene RNA Miniprep kit (samples called primary cells+/ and primary cells-/- in our proposal). We would like to perform gene expression comparison between: 1) MGE+/ and MGE-/-, and 2) primary cells+/ and primary cells-/-.

Project description:The Dlx homeobox genes have central roles in controlling patterning and differentiation of the brain and craniofacial primordia. In the brain, loss of Dlx function results in defects in the production, migration and differentiation of GABAergic neurons, that can lead to epilepsy. In the branchial arches, loss of Dlx function leads to craniofacial malformations that include trigeminal axon pathfinding defects. To determine how these genes function, we wish to identify the transcriptional circuitry that lies downstream of these transcription factors by comparing gene expression in wild type with Dlx mutant CNS and craniofacial tissues. 1) Compare gene expression in the maxillay branch of the first branchial arch (BA) of E10.5 wild type and Dlx2 -/- mutants. 2) Compare gene expression in the maxillary branch of the first BA of E10.5 wild type and Dlx1/2 -/- mutants. 3) Compare gene expression in wild type maxillary and mandibular branchial arches. 4) Compare gene expressionin mandibular branch of Dlx5/6 -/- mutants with wild type mandibular branch. The Dlx transcription factors are essential for controlling patterning of the brain and craniofacial primordia. In the brain, they control differentiation of GABAergic neurons of the basal ganglia. In the branchial arches, they control regional patterning. I hypothesize that there will be some conserved and some divergent mechanisms that the Dlx genes use in controlling brain and craniofacial development. We have already performed array analyses on Dlx function in the developing basal ganglia (with TGEN) by comparing expressed genes in wild type and Dlx1/2 mutants. Here we will compare gene expression in the brachial arches of wild type and Dlx mutant mice. 1) Generate E10.5 mouse embryos that are either wild type, Dlx2-/-, Dlx1/2 -/- or Dlx5/6 -/-. 2) Determine genotype by PCR. 3) Dissect branchial arches from the different genotypes. 4) Separate maxillary and mandibular branch of each branchial arch. 5) Prepare total RNA from the specimens. Obtain sufficient tissue to obtain 10 ug of total RNA - based on previous experience we anticipate that this will require ~ 10 branchial arches. We will pool the tissue from different embryos of the same genotype. 6) Send total RNA to TGEN for probe preparation, hybridization and array result analysis.

Project description:The Dlx homeobox genes have central roles in controlling patterning and differentiation of the brain and craniofacial primordia. In the brain, loss of Dlx function results in defects in the production, migration and differentiation of GABAergic neurons, that can lead to epilepsy. In the branchial arches, loss of Dlx function leads to craniofacial malformations that include trigeminal axon pathfinding defects. To determine how these genes function, we wish to identify the transcriptional circuitry that lies downstream of these transcription factors by comparing gene expression in wild type with Dlx mutant CNS and craniofacial tissues. 1) Compare gene expression in the maxillay branch of the first branchial arch (BA) of E10.5 wild type and Dlx2 -/- mutants. 2) Compare gene expression in the maxillary branch of the first BA of E10.5 wild type and Dlx1/2 -/- mutants. 3) Compare gene expression in wild type maxillary and mandibular branchial arches. 4) Compare gene expressionin mandibular branch of Dlx5/6 -/- mutants with wild type mandibular branch. The Dlx transcription factors are essential for controlling patterning of the brain and craniofacial primordia. In the brain, they control differentiation of GABAergic neurons of the basal ganglia. In the branchial arches, they control regional patterning. I hypothesize that there will be some conserved and some divergent mechanisms that the Dlx genes use in controlling brain and craniofacial development. We have already performed array analyses on Dlx function in the developing basal ganglia (with TGEN) by comparing expressed genes in wild type and Dlx1/2 mutants. Here we will compare gene expression in the brachial arches of wild type and Dlx mutant mice. 1) Generate E10.5 mouse embryos that are either wild type, Dlx2-/-, Dlx1/2 -/- or Dlx5/6 -/-. 2) Determine genotype by PCR. 3) Dissect branchial arches from the different genotypes. 4) Separate maxillary and mandibular branch of each branchial arch. 5) Prepare total RNA from the specimens. Obtain sufficient tissue to obtain 10 ug of total RNA - based on previous experience we anticipate that this will require ~ 10 branchial arches. We will pool the tissue from different embryos of the same genotype. 6) Send total RNA to TGEN for probe preparation, hybridization and array result analysis. Keywords: other

Project description:GABAergic interneurons are lost in conditions including epilepsy and CNS injury, but there are few culture models available to study their function. Towards the goal of obtaining renewable sources of GABAergic neurons, we used the molecular profile of a functionally-incomplete GABAergic precursor clone to screen 17 new clones isolated from GFP+ rat E14.5 cortex and ganglionic eminence (GE) that were generated by viral introduction of v-myc. The clones grow as neurospheres in medium with FGF2, and after withdrawal of FGF2 they exhibit varying patterns of differentiation. Transcriptional profiling and qPCR indicated that one clone (GE6) expresses high levels of mRNAs encoding Dlx1, 2, 5 and 6, glutamate decarboxylases, and presynaptic proteins including neuropeptide Y and somatostatin. Protein expression confirmed that GE6 is a progenitor with restricted differentiation giving rise mostly to neurons with GABAergic markers. In co-cultures with hippocampal neurons, GE6 neurons became electrically excitable and received both inhibitory and excitatory synapses. After withdrawal of FGF2 in cultures of GE6 alone, neurons matured to express BetaIII-tubulin, and staining for synaptophysin and vesicular GABA transporter (VGAT) were robust after 1-2 weeks of differentiation. GE6 neurons also became electrically excitable and displayed synaptic activity, but synaptic currents were carried by chloride and were blocked by bicuculline. The results suggest that the GE6 clone, which is ventrally derived from the GE, resembles GABAergic interneuron progenitors that migrate into the developing forebrain. This is the first report of a relatively stable fetal clone that can be differentiated into GABAergic interneurons with functional synapses. The purpose was to compare differentiation patterns of several different immortalized rat neural progenitor clones to identify early stages in differentiation. The cell clones studies were: GE6 (GABAergic neuronal precursor), GE2 (non-neuronal precursor, CTX8 (multipotential precursor), L2.2 (interneuronal precursor), and L2.3 (multipotential precursor). Five rat neural precursor cell clones were compared at three different time points following FGF2 withdrawal, which triggers differentiation. Three sister culture replicates were performed for each cell clone and time point, yielding 45 samples. One microarray failed so we have 44 microarray results in the dataset.

Project description:GABAergic interneurons are lost in conditions including epilepsy and CNS injury, but there are few culture models available to study their function. Towards the goal of obtaining renewable sources of GABAergic neurons, we used the molecular profile of a functionally-incomplete GABAergic precursor clone to screen 17 new clones isolated from GFP+ rat E14.5 cortex and ganglionic eminence (GE) that were generated by viral introduction of v-myc. The clones grow as neurospheres in medium with FGF2, and after withdrawal of FGF2 they exhibit varying patterns of differentiation. Transcriptional profiling and qPCR indicated that one clone (GE6) expresses high levels of mRNAs encoding Dlx1, 2, 5 and 6, glutamate decarboxylases, and presynaptic proteins including neuropeptide Y and somatostatin. Protein expression confirmed that GE6 is a progenitor with restricted differentiation giving rise mostly to neurons with GABAergic markers. In co-cultures with hippocampal neurons, GE6 neurons became electrically excitable and received both inhibitory and excitatory synapses. After withdrawal of FGF2 in cultures of GE6 alone, neurons matured to express BetaIII-tubulin, and staining for synaptophysin and vesicular GABA transporter (VGAT) were robust after 1-2 weeks of differentiation. GE6 neurons also became electrically excitable and displayed synaptic activity, but synaptic currents were carried by chloride and were blocked by bicuculline. The results suggest that the GE6 clone, which is ventrally derived from the GE, resembles GABAergic interneuron progenitors that migrate into the developing forebrain. This is the first report of a relatively stable fetal clone that can be differentiated into GABAergic interneurons with functional synapses. The purpose was to compare differentiation patterns of several different immortalized rat neural progenitor clones to identify early stages in differentiation. The cell clones studies were: GE6 (GABAergic neuronal precursor), GE2 (non-neuronal precursor, CTX8 (multipotential precursor), L2.2 (interneuronal precursor), and L2.3 (multipotential precursor).

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Genomic Resolution of DLX-orchestrated Transcriptional Circuits Driving Development of Forebrain GABAergic Neurons

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