Comprehensive screens were made for genes that change their expression during a brief critical period in development when neonatal mammalian central nervous system (CNS) loses its capacity to regenerate. In newly born opossums older than... more
Comprehensive screens were made for genes that change their expression during a brief critical period in development when neonatal mammalian central nervous system (CNS) loses its capacity to regenerate. In newly born opossums older than 12 days regeneration ceases to occur in the cervical spinal cord. It continues for 5 more days in lumbar regions. The mRNA’s expressed in cords that do and do not regenerate were analyzed by polymerase chain reaction-based subtractive hybridization. The mRNAs extracted from cervical cords of animals aged 9 and 12 days were subtracted reciprocally, old from young and young from old. Additional subtractions were made between lumbar regions of 12 day-old cords (which can regenerate) and cervical regions (which cannot). Mini libraries of approximately 2000 opossum cDNA clones resulted from each subtraction. Many sequences were novel. Others that were expressed differentially were related to cell growth, proliferation, differentiation, motility, adhesion, cytoskeleton and extracellular matrix. A major task was to narrow the search and to eliminate genes that were not associated with regeneration. Clones from different subtractions were cross-hybridized. After those common to regenerating and nonregenerating cords were rejected, approximately 284 sequences of interest remained. Our results revealed novel sequences, as well as genes involved in transcription, cell signaling, myelin formation, growth cone motility, liver regeneration, and nucleic acid and protein management as the candidates important for neuroregeneration. For selected genes of potential interest for regeneration (for example cadherin, catenin, myelin basic protein), their temporal and spatial distributions and levels of expression in the CNS were measured by Northern blots, semiquantitative and real-time RT-PCR, and in situ hybridization. Our experiments set the stage for testing the efficacy of candidate genes in turning on or off the capacity for spinal cord regeneration. Opossum spinal cords in vitro provide a reliable and rapid assay for axon outgrowth and synapse formation.
Research Interests: Gene expression, In Situ Hybridization, Humans, Mice, Cellular and Molecular Neurobiology, and 13 moreAnimals, Spinal Cord, Nucleic acid hybridization, Central Nervous System, Nerve Regeneration, Monodelphis, Critical Period, Genetic variation, Spinal Cord Injuries, Neurosciences, Biochemistry and cell biology, Gene expression profiling, and Differential expression
Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson's disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons. In particular, transplantation of... more
Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson's disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons. In particular, transplantation of embryonic-stem-cell-derived dopaminergic neurons has been shown to be efficient in restoring motor symptoms in conditions of dopamine deficiency. However, the use of pluripotent-derived cells might lead to the development of tumours if not properly controlled. Here we identified a minimal set of three transcription factors--Mash1 (also known as Ascl1), Nurr1 (also known as Nr4a2) and Lmx1a--that are able to generate directly functional dopaminergic neurons from mouse and human fibroblasts without reverting to a progenitor cell stage. Induced dopaminergic (iDA) cells release dopamine and show spontaneous electrical activity organized in regular spikes consistent with the pacemaker activity featured by brain dopaminergic neurons. The three factors were able to elicit dopaminergic neuronal conversion in prenatal and adult fibroblasts from healthy donors and Parkinson's disease patients. Direct generation of iDA cells from somatic cells might have significant implications for understanding critical processes for neuronal development, in vitro disease modelling and cell replacement therapies.
Research Interests: Cellular Reprogramming, Transcription Factors, Regenerative Medicine, Multidisciplinary, Nature, and 17 morePatch-clamp and imaging techniques, Dopamine, Cell Differentiation, Humans, Mice, Animals, Embryonic Stem Cell, Skin, Neuronal Development, Neurons, Transcription Factor, Degeneration, Human Fibroblasts, Action Potentials, Parkinson Disease, Gene expression profiling, and Somatic Cells
Comprehensive screens were made for genes that change their expression during a brief critical period in development when neonatal mammalian central nervous system (CNS) loses its capacity to regenerate. In newly born opossums older than... more
Comprehensive screens were made for genes that change their expression during a brief critical period in development when neonatal mammalian central nervous system (CNS) loses its capacity to regenerate. In newly born opossums older than 12 days regeneration ceases to occur in the cervical spinal cord. It continues for 5 more days in lumbar regions. The mRNA’s expressed in cords that do and do not regenerate were analyzed by polymerase chain reaction-based subtractive hybridization. The mRNAs extracted from cervical cords of animals aged 9 and 12 days were subtracted reciprocally, old from young and young from old. Additional subtractions were made between lumbar regions of 12 day-old cords (which can regenerate) and cervical regions (which cannot). Mini libraries of approximately 2000 opossum cDNA clones resulted from each subtraction. Many sequences were novel. Others that were expressed differentially were related to cell growth, proliferation, differentiation, motility, adhesion, cytoskeleton and extracellular matrix. A major task was to narrow the search and to eliminate genes that were not associated with regeneration. Clones from different subtractions were cross-hybridized. After those common to regenerating and nonregenerating cords were rejected, approximately 284 sequences of interest remained. Our results revealed novel sequences, as well as genes involved in transcription, cell signaling, myelin formation, growth cone motility, liver regeneration, and nucleic acid and protein management as the candidates important for neuroregeneration. For selected genes of potential interest for regeneration (for example cadherin, catenin, myelin basic protein), their temporal and spatial distributions and levels of expression in the CNS were measured by Northern blots, semiquantitative and real-time RT-PCR, and in situ hybridization. Our experiments set the stage for testing the efficacy of candidate genes in turning on or off the capacity for spinal cord regeneration. Opossum spinal cords in vitro provide a reliable and rapid assay for axon outgrowth and synapse formation.
Research Interests: Gene expression, In Situ Hybridization, Humans, Mice, Cellular and Molecular Neurobiology, and 13 moreAnimals, Spinal Cord, Nucleic acid hybridization, Central Nervous System, Nerve Regeneration, Monodelphis, Critical Period, Genetic variation, Spinal Cord Injuries, Neurosciences, Biochemistry and cell biology, Gene expression profiling, and Differential expression
Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson's disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons. In particular, transplantation of... more
Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson's disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons. In particular, transplantation of embryonic-stem-cell-derived dopaminergic neurons has been shown to be efficient in restoring motor symptoms in conditions of dopamine deficiency. However, the use of pluripotent-derived cells might lead to the development of tumours if not properly controlled. Here we identified a minimal set of three transcription factors--Mash1 (also known as Ascl1), Nurr1 (also known as Nr4a2) and Lmx1a--that are able to generate directly functional dopaminergic neurons from mouse and human fibroblasts without reverting to a progenitor cell stage. Induced dopaminergic (iDA) cells release dopamine and show spontaneous electrical activity organized in regular spikes consistent with the pacemaker activity featured by brain dopaminergic neurons. The three factors were able to elicit dopaminergic neuronal conversion in prenatal and adult fibroblasts from healthy donors and Parkinson's disease patients. Direct generation of iDA cells from somatic cells might have significant implications for understanding critical processes for neuronal development, in vitro disease modelling and cell replacement therapies.
