Project description:Egr3 is a zinc-finger transcription factor involved in growth and development. Egr3-deficient mice have severe sensory ataxia due to failed development of muscle spindle stretch receptors. Sensory and motor neurons that normally innervate spindles are absent in Egr3-deficient mice, presumably as a secondary consequence to the loss of trophic signals produced by spindles during development that are required for innervation and neuron survival. The molecular mechanisms involving motor neuron fate specification, target derived growth factor dependencies, and specification of target innervation have been difficult to study since select markers for functionally specific motor neurons are very poorly characterized. A more thorough understanding of the molecular mediators of motor neuron biology will be important to evaluate the efficacy of new strategies devised to thwart neuron death that occurs in a variety of human motor neuronopathies and neuropathies. To identify genes specifically expressed by spinal cord fusimotor neurons: Many motor neuron specific genes have been described over the years. However, none have been described that distinguish fusimotor neurons from skeletomotor neurons despite the fact that they have distinct muscle targets (muscle spindle stretch receptors) and comprise 25-30% of the spinal motor neuron populations. Since these motor neurons have remarkably different target innervation and function, we hypothesize that they express genes that establish their specific phenotypes during development. We hypothesize that fusimotor neurons can be distinguished in the spinal cord by characterizing fusimotor neuron specific gene expression. Once fusimotor neuron specific genes are identified, they will be used as markers to identify fusimotor neurons in complex neuroglial cell populations in vivo and in vitro. We hypothesize that by characterizing fusimotor neuron specific genes, unique marker molecules will be identified for in vivo and in vitro study of this functionally distinct and important motor neuron subtype. Moreover, we hypothesize that many of the genes that are specifically expressed by fusimotor neurons will be involved in mechanisms related to their fate specification, target innervation and growth factor dependent biology. We will use the Affymetrix microarray platform to identify genes that are specifically expressed by fusimotor neurons in mouse spinal cord. The differential expression analysis will be performed on microdissected segments of spinal cord (L3-L5) from wild type and Egr3-deficient mice. Postnatal Egr3-deficient mice lack muscle spindles and fusimotor neurons in their spinal cords. By comparing gene expression from microdissected segments of spinal cord (L3-L5) between wild type and Egr3-deficient mice, we hypothesize that fusimotor neuron selective genes can be identified. We will microdissect L3-L5 segments of spinal cord using precise anatomical landmarks to ensure that comparable spinal cord regions are anlayzed from each animal. For each microarray experiment, total RNA will be extracted from L3-L5 cords (approximately 2 mm length of spinal cord). The integrity of each RNA sample will be verified by gel electrophoresis. The intact RNA samples from mice of similar genotype will be pooled from three (3) 27-day old animals. The intact cord dissection is easier in young animals (eg: 27-day old) and the phenotype is known to exist at this developmental stage. The RNA from each animal of a similar genotype will be pooled into a single sample to minimize false positive gene calls that may represent genes related to the specific state of vigilance of a particular animal at the time of sacrifice (eg: activity dependent genes). Thus, each of the two RNA samples to be analyzed for a particular microarray experiment will represent RNA from three (3) spinal cords of each genotype. RNA amplification for probe synthesis should not be necessary since we will provide 7 ug of intact pooled total RNA for each sample. For statistical analysis, the experiment will be performed twice. Since the RNA samples are precious, they will be provided to the Array Consortium in two shipments with each of the experiments performed independently.

Project description:Egr3 is a zinc-finger transcription factor involved in growth and development. Egr3-deficient mice have severe sensory ataxia due to failed development of muscle spindle stretch receptors. Sensory and motor neurons that normally innervate spindles are absent in Egr3-deficient mice, presumably as a secondary consequence to the loss of trophic signals produced by spindles during development that are required for innervation and neuron survival. The molecular mechanisms involving motor neuron fate specification, target derived growth factor dependencies, and specification of target innervation have been difficult to study since select markers for functionally specific motor neurons are very poorly characterized. A more thorough understanding of the molecular mediators of motor neuron biology will be important to evaluate the efficacy of new strategies devised to thwart neuron death that occurs in a variety of human motor neuronopathies and neuropathies. To identify genes specifically expressed by spinal cord fusimotor neurons: Many motor neuron specific genes have been described over the years. However, none have been described that distinguish fusimotor neurons from skeletomotor neurons despite the fact that they have distinct muscle targets (muscle spindle stretch receptors) and comprise 25-30% of the spinal motor neuron populations. Since these motor neurons have remarkably different target innervation and function, we hypothesize that they express genes that establish their specific phenotypes during development. We hypothesize that fusimotor neurons can be distinguished in the spinal cord by characterizing fusimotor neuron specific gene expression. Once fusimotor neuron specific genes are identified, they will be used as markers to identify fusimotor neurons in complex neuroglial cell populations in vivo and in vitro. We hypothesize that by characterizing fusimotor neuron specific genes, unique marker molecules will be identified for in vivo and in vitro study of this functionally distinct and important motor neuron subtype. Moreover, we hypothesize that many of the genes that are specifically expressed by fusimotor neurons will be involved in mechanisms related to their fate specification, target innervation and growth factor dependent biology. We will use the Affymetrix microarray platform to identify genes that are specifically expressed by fusimotor neurons in mouse spinal cord. The differential expression analysis will be performed on microdissected segments of spinal cord (L3-L5) from wild type and Egr3-deficient mice. Postnatal Egr3-deficient mice lack muscle spindles and fusimotor neurons in their spinal cords. By comparing gene expression from microdissected segments of spinal cord (L3-L5) between wild type and Egr3-deficient mice, we hypothesize that fusimotor neuron selective genes can be identified. We will microdissect L3-L5 segments of spinal cord using precise anatomical landmarks to ensure that comparable spinal cord regions are anlayzed from each animal. For each microarray experiment, total RNA will be extracted from L3-L5 cords (approximately 2 mm length of spinal cord). The integrity of each RNA sample will be verified by gel electrophoresis. The intact RNA samples from mice of similar genotype will be pooled from three (3) 27-day old animals. The intact cord dissection is easier in young animals (eg: 27-day old) and the phenotype is known to exist at this developmental stage. The RNA from each animal of a similar genotype will be pooled into a single sample to minimize false positive gene calls that may represent genes related to the specific state of vigilance of a particular animal at the time of sacrifice (eg: activity dependent genes). Thus, each of the two RNA samples to be analyzed for a particular microarray experiment will represent RNA from three (3) spinal cords of each genotype. RNA amplification for probe synthesis should not be necessary since we will provide 7 ug of intact pooled total RNA for each sample. For statistical analysis, the experiment will be performed twice. Since the RNA samples are precious, they will be provided to the Array Consortium in two shipments with each of the experiments performed independently. Keywords: other

Dataset Information

E12.5 spinal cord scRNA-seq [e12p5-wc-neuron]

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