Transcription profiling of mouse spinal cord (L3-L5) between wild type and Egr3-deficient animals to identify spinal cord fusimotor neuron-specific genes

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.Egr3是一种参与生长与发育进程的锌指转录因子(zinc-finger transcription factor)。Egr3缺陷小鼠可因肌梭牵张受体(muscle spindle stretch receptors)发育障碍，出现严重的感觉性共济失调。正常情况下支配肌梭的感觉神经元与运动神经元在Egr3缺陷小鼠体内缺失，推测该表型为发育过程中肌梭产生的营养信号缺失所导致的次级效应——此类营养信号是神经元支配与存活所必需的。 由于功能特异性运动神经元的选择性标记特征至今仍未得到充分阐明，涉及运动神经元命运特化、靶源性生长因子依赖性以及靶组织支配特化的分子机制一直难以开展研究。深入解析运动神经元生物学的分子介导机制，对于评估针对多种人类运动神经元病(motor neuronopathies)及神经病变(neuropathies)中神经元死亡的新型干预策略的有效性，具有重要意义。 为鉴定脊髓梭内肌运动神经元(fusimotor neurons)特异性表达的基因：多年来已有诸多运动神经元特异性基因被报道，但目前尚无能够区分梭内肌运动神经元与骨骼运动神经元(skeletomotor neurons)的特异性标记基因——尽管二者拥有截然不同的肌肉靶组织（肌梭牵张受体），且在脊髓运动神经元群体中占比达25%~30%。 鉴于这类运动神经元具有显著差异的靶组织支配模式与功能，我们提出假说：其在发育过程中会表达特异性基因以确立自身的表型特征。我们进一步提出假说：可通过解析梭内肌运动神经元的特异性基因表达谱，在脊髓中区分出该类神经元。 一旦鉴定出梭内肌运动神经元特异性基因，便可将其作为标记物，在体内(in vivo)与体外(in vitro)的复杂神经胶质细胞群体中识别梭内肌运动神经元。我们提出假说：通过解析梭内肌运动神经元的特异性基因表达特征，可鉴定出该功能独特且具有重要意义的运动神经元亚型的专属标记分子，用于体内外研究。此外，我们还提出假说：梭内肌运动神经元特异性表达的诸多基因，将参与其命运特化、靶组织支配以及生长因子依赖性生物学过程相关的分子机制。 本研究将使用Affymetrix微阵列(microarray)平台，鉴定小鼠脊髓中梭内肌运动神经元特异性表达的基因。差异表达分析将基于野生型与Egr3缺陷小鼠的脊髓显微切割节段（L3~L5节段）进行。 出生后Egr3缺陷小鼠的脊髓中缺乏肌梭与梭内肌运动神经元。通过比较野生型与Egr3缺陷小鼠脊髓显微切割节段（L3~L5节段）的基因表达谱，我们提出假说：可鉴定出梭内肌运动神经元选择性表达的基因。 我们将借助精确的解剖学标志显微切割L3~L5节段脊髓，以确保每只动物所取的脊髓区域具有可比性。对于每一次微阵列实验，我们将从L3~L5节段脊髓（约2mm长度）中提取总RNA(total RNA)。通过凝胶电泳(gel electrophoresis)验证每份RNA样品的完整性。将相同基因型、27日龄的3只小鼠的完整RNA样品进行混合，以降低假阳性基因检出率。幼年动物（例如27日龄）的脊髓解剖操作更为简便，且该发育阶段已表现出明确的表型特征。 将相同基因型的每只小鼠的RNA混合为单一样品，可减少假阳性基因的检出——这类假阳性可能源于实验动物处死时的特定警觉状态相关的基因（如活动依赖性基因）。因此，每一次微阵列实验所需的两份RNA样品，将分别对应两种基因型各3只小鼠的脊髓RNA。 由于我们将为每份样品提供7μg完整混合总RNA，因此无需进行RNA扩增以制备探针。 对于统计分析，本实验将重复开展两次。鉴于RNA样品较为珍贵，我们将分两批将样品提交至Array Consortium，分两次独立开展实验。