Identification of motor neuron pool marker genes and analysis of their roles in motor circuit assembly

In order to produce behavioral output, nervous systems critically depend on the establishment of selectively connected intricate neuronal networks. The overwhelming number and complexity of intersecting neuronal pathways complicates efforts to improve our understanding of the brain. Experiments are therefore ideally performed in well-defined neuronal networks, such as the monosynaptic stretch reflex circuits in the spinal cord (SC). The establishment of this sensory-motor feedback loop relies on the formation of highly selective synaptic contacts between group Ia proprioceptive afferents, spinal motor neurons (MNs) and their target muscles in limbs. The high degree of connective specificity exhibited by this circuit paralleled with its relative simplicity combine to a set of favorable features for experimental neurobiological research directed at the elucidation of neuronal circuit formation.
The goal of my PhD thesis was to investigate the molecular mechanisms controlling the diversification of MNs into discrete subpopulations, referred to as MN pools, which establish precise axonal trajectories to individual muscles and specific sensory connectivity patterns. Since synaptic specificity is largely controlled by genetic programs, we acquired gene expression profiles of four individual cervical motor neuron (MN) pools, supplying Cutaneous Maximus, Triceps, Pectoralis Minor and Pectoralis Major muscles, using a combination of retrograde labeling, Laser Capture Microdissection and Affymetrix gene chip technology.
Comparison of the obtained expression profiles allowed the identification of genes expressed specifically in single MN pools. Our analysis was particularly focused on the interplay between transcription factors and their cognate repertoire of cell surface molecules. MN pool expression of many such genes could be validated by in situ hybridization. We also performed a second genome-wide screen in order to determine whether identified CM marker genes were regulated by the ETS transcription factor Pea3 known for its key role in the specification of the CM MN pool.
Based on the results of our screening experiments we chose the G-protein coupled prokineticin receptor 2 (Pkr2) and the extracellular matrix protein developmental endothelial regulated locus 1 (Del1), both of which are expressed in CM MNs and positively regulated by Pea3, for further analysis. In addition, we report on the upregulation of the transcription factor Pou3f1 (Scip) in CM MNs of Pea3 mutant mice.
I addressed the functionality of Pkr2 in MNs by means of a genetic null mutant engineered in our laboratory. Based on its roles in other neuronal systems and because Pkr2 expression is dependent on Pea3, our experiments primarily explored its function with respect to potential contributions to motor circuit formation defects detected in Pea3 mutant mice. Our analysis did however not reveal any abnormalities in cell migration, sensory-motor connectivity or muscle innervation patterns or a role in axon pathfinding in DRG sensory neurons, in subpopulations of which Pkr2 is expressed as well.
As an extracellular matrix protein involved in chemotactic signaling events, the second downstream target of Pea3, Del1, also disposed of all necessary characteristics to play an essential role in neuronal circuit assembly. For this reason, the functionality of Del1 was addressed analogously to Pkr2. Although the function of Del1 in the CM MNs remains uncertain to date our experimental results are not indicative of an important role in axon pathfinding or neuronal migration.
Scip was the only gene validated as negatively regulated by Pea3 in CM MNs. We were speculating that in Pea3 mutants the transcriptional identity conferred by Scip could endow CM MNs with molecular features normally inherent to forearm projecting MN pools that express Scip in wild-type. This scenario is of interest insofar as in Pea3 mutant mice, CM motor neurons change the status and the specificity of their sensory-motor connectivity. In the future, the role of Scip could be addressed by experiments using ectopic expression and Scip loss of function mice.
In summary, I identified and functionally characterized genes expressed in distinct MN pool with the potential to contribute to the process of motor circuit assembly. To our knowledge this is the first time the expression profiles of MNs were resolved at single MN pool resolution and our findings have thus provided an entry point to a deeper understanding of the molecular events that govern the specification of MNs and the establishment of motor circuits. Future research should be directed at the functional elucidation of additional molecular factors identified by our approach and could also contribute to an increased understanding of the developmental processes underlying neuronal network formation in the brain.