The long-term goal of this research is to determine how different types of nerve cells (neurons) are made in a developing vertebrate embryo. This proposal specifically focuses on five distinct types of neurons that are located in the ventral spinal cord and regulate locomotion. Despite the importance of these neurons, very little is known about how they develop their functional characteristics. This is a critical gap in our knowledge as it dramatically impedes our ability to treat spinal cord injuries and neuronal diseases that affect locomotion. This proposal has two specific aims: (1) to identify all of the regulatory genes that are turned on (expressed) in these different neurons and (2) to identify genes that are strong candidates for specifying particular neuronal characteristics (this proposal focuses on axon trajectories and neurotransmitter phenotypes). This project will concentrate on regulatory genes that encode for transcription factors (TFs;proteins that bind DNA and regulate the expression of other genes). All of the data so far, suggests that most functional characteristics of spinal cord neurons are determined by the TFs that these cells express as they stop dividing and start to differentiate. However, most of the details of this process are still unclear. A critical barrier to progress in this field is the fact that the full complement of TFs present in any of these cell types is still unknown. This project will use zebrafish embryos as a model system. Zebrafish embryos have several advantages for this area of research. For example, the embryos develop outside the mother and are optically transparent. This enables neuron morphology to be observed in intact, live embryos and it makes it relatively easy to isolate large numbers of fluorescently labeled neurons. In addition, it is much faster and cheaper to test the functions of different genes in zebrafish embryos than in mammals. This research will use transgenic lines of zebrafish, in which particular neurons fluoresce red and/or green to isolate each of the five distinct types of ventral spinal cord neurons. Each cell type will be expression profiled using novel microarrays, which contain probes for all zebrafish TF genes. These experiments will produce a comprehensive description of all of the TFs that are expressed by each of these different neurons (Specific Aim 1). All of the analyses that have been published so far, suggest that these ventral spinal cord neurons are highly conserved between zebrafish and mammals, in terms of the genes that they express, their functional properties and their roles in controlling locomotion. Therefore the TFs identified by this research should also be present in equivalent neurons in humans. The knowledge produced by this research will have a huge impact on the fields of developmental neurobiology, neural stem cell biology and spinal cord injury and repair. It will enable us to identify TFs with key roles in regulating neuronal development (Specific Aim 2 and future work). It will also be invaluable for researchers trying to generate neurons with particular properties from stem cells for therapeutic purposes.
This proposal investigates how different types of nerve cells are made in a growing vertebrate embryo. It will identify important regulatory genes that determine the functional characteristics of distinct classes of nerve cells that normally regulate locomotion. This knowledge should dramatically improve our ability to treat spinal cord injuries and neuronal diseases that affect locomotion.