Gene chips, or microarrays, have proven to be immensely important in measuring the activity of thousands of genes at once in such cells as cancer cells or immune cells. The use of these chips has given scientists snapshots of gene activity that lead to better understanding of the genetic machinery of the cells. This understanding has led to new ways to kill cancers or to manipulate the immune system, for example.
Gene chips consist of vast arrays of thousands of specific genetic segments spotted onto tiny chips. When gene extracts of cells are applied to the chips, labeled with fluorescent indicators, genes from the cell extracts attach to their complementary counterparts on the chips. Measurements of the fluorescence of each spot give scientists an indication of the activity of particular genes.
As vital as they are to studies of individual types of cells, gene chips have proven to be less useful in efforts to understand the genetic signatures of specific brain cells, because a myriad of subtly different subtypes of brain cells are intertwined in brain tissue.
Now, however, researchers led by Jeffrey Macklis, Bradley Molyneaux, and Paola Arlotta of the MGH-HMS Center for Nervous System Repair at Harvard Medical School and Massachusetts General Hospital and Harvard Stem Cell Institute have developed a way to distinguish particular brain cell subtypes in tissue and to separate them for genetic analysis with microarrays. Their technique will prove enormously helpful to neuroscientists studying the development and function of the brain. For example, it will enable researchers to genetically tag, manipulate, and even knock out the function of specific subtypes of neurons to study their function. Also, by comparing genetic profiles of cells in normal and diseased brains, researchers can gain invaluable clues to the origins of neurological disorders.
In their technique, the scientists first labeled a specific brain cell in living brain tissue using fluorescent microspheres. They then used microdissection, biochemical methods, and fluorescence-activated cell sorting to separate out the particular brain cell subtype for genetic analysis using DNA microarrays. Such cell sorting isolates those cells that have absorbed the fluorescent microspheres.
In their paper, the scientists report using their new technique to unravel the genes that are active in corticospinal motor neurons (CSMN), which connect the cortex and spinal cord and carry the signals that operate muscles. These neurons are important because their degeneration contributes critically to amyotrophic lateral sclerosis (Lou Gehrig's disease) and to the loss of muscle function in spinal cord injury. Better understanding of the genes that control the development of these neurons could aid in the development of treatments for these disorders.
In their experiments, the scientists isolated the neurons and analyzed the genes that were active in CSMNs during stages of embryonic development in mice. They compared these active genes with those of two other closely related subtypes of such cortical neurons to discover specific genes that are likely critical to CSMN development.
To demonstrate that their technique had, indeed, identified functionally important genes, they knocked out one of the genes, called Ctip2, in mice. The resulting animal had defects in the connections between the cortex and spinal cord that showed that the gene was critical for CSMN development.
"The data here support the idea that a precise molecular classification of distinct classes of projection neurons is possible and provide a foundation for increasingly sophisticated analysis of stage-specific genes controlling corticospinal motor neuron development," concluded the scientists.
Paola Arlotta, Bradley J. Molyneaux, Jinhui Chen, Jun Inoue, Ryo Kominami, and Jeffrey D. Macklis: "Neuronal Subtype-Specific Genes that Control Corticospinal Motor Neuron Development In Vivo"
The other members of the research team included Jinhui Chen of the MGH-HMS Center for Nervous System Repair at Harvard Medical School and Massachusetts General Hospital and the Harvard Stem Cell Institute [presently at the Spinal Cord and Brain Injury Research Center of University of Kentucky]; and Jun Inoue and Ryo Kominami of the Graduate School of Medical and Dental Sciences at Niigata University. This work was partially supported by grants from the NIH, Christopher Reeve Paralysis Foundation, and ALS Association (to J.D.M.). P.A. was supported by a Wills Foundation Postdoctoral Fellowship. B.J.M. was supported by the Harvard M.S.T.P..
The context and implications of this work are discussed in a Preview by Joseph D. Dougherty and Daniel H. Geschwind.
Publishing in Neuron, Volume 45, Number 2, January 20, 2005, pages 207–221.
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