Gene profiling of identified neurons to dissect molecular mechanisms involved in spinal reflex assembly

The central question during my PhD studies was to understand the molecular mechanisms and genetic cascades controlling the sequential specification of distinct classes of dorsal root ganglia (DRG) sensory neurons, with a particular focus on genes involved in controlling connectivity between Ia proprioceptive afferents and motor neurons in the spinal cord. The underlying genetic mechanisms controlling the formation of specific synaptic connections between Ia proprioceptive afferents and motor neurons in the lumbar spinal cord are currently only poorly understood. The main reason for the difficulty of isolating genes responsible for controlling aspects of connectivity was due to the fact that an enormous number of distinct subpopulations exist in the nervous system. In the spinal monosynaptic reflex circuit, proprioceptive afferents in the dorsal root ganglion (DRG) represent only 10-20% of all neurons. Moreover, cell bodies of given sensory neuron subpopulations in the DRG are highly dispersed. Therefore, initial technical difficulties were faced when performing gene expression analysis experiments of individual neuronal subtypes. In our study, we have used mouse genetics to selectively label distinct neuronal subpopulations. These tools allowed purifying defined populations of DRG sensory neurons (Klein et al., 1994) by Fluorescent Activated Cell Sorting (FACS) and subsequent gene expression profiling analysis using Affymetrix GeneChip technology. The aim of the first part of my PhD was the identification of genes involved in the specification and differentiation of DRG SN subtypes. The second major part of this project was the verification of candidate genes isolated from the Affymetrix chip screen experiments and to perform functional experiments to address their role in controlling connectivity between Ia proprioceptive afferents and motor neurons in the spinal cord. First, selected putative regulators were analyzed for their expression profile using in situ hybridization experiments on wild-type embryos and TrkC-/- and Er81-/- mutant backgrounds. We focused in particular on genes that were expressed in subpopulations of DRG neurons in wild-type embryos, but are not expressed in either TrkC-/- or Er81-/- mutant mice. Such genes are selectively expressed in proprioceptive DRG neurons or regulated by the transcription factor Er81 and they therefore represented the most interesting population of genes to assay for function (Arber et al., 2000; Klein et al., 1994).
Our initial gene expression profiling analysis was extended to also isolate novel proprioceptive afferent markers, the expression of which is potentially restricted to distinct sensory neuron pools. We pushed the technical limitations further and used methods to profile proprioceptive afferents from different spinal levels.
Some of the genes identified in our screen were also analyzed functionally. One of these genes is the orphan nuclear receptor estrogen-related receptor gamma (Err3). We analyzed its function in proprioceptive afferent neuron specification and connectivity in greater detail in the third part of my PhD thesis. Analysis of Err3 expression revealed expression specifically in gamma motor neurons, a motor neuron subpopulation to which no marker gene has been correlated to date. We used various mutant animals to show that muscle spindles are required for gamma motor neuron survival.
Moreover, chapter IV of this thesis addresses a potential role of Err3 in a neurodegenerative disease model for amyotrophic lateral sclerosis (ALS).