Proper wiring of neuronal circuits during development is highly dependent on the establishment of precise networks of neural connectivity. Defects in the assembly of these neural networks, which include cellular processes such as axonal growth, elongation and guidance, cell body migration, dendrite arborization and proper synapse formation, lead to severe neurological deficits. Experiments described in this proposal will characterize the function of novel genes required for vertebrate motor neuron connectivity identified in a mouse genetic screen. Motor neurons mediate the control over locomotion, respiration and autonomic responses, and are profoundly affected by developmental diseases such as spinal muscle atrophy (SMA) and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Motor neurons develop in the ventral spinal cord and hindbrain and their cell bodies migrate to stereotypical positions along the mediolateral, rostrocaudal, and dorsoventral axes of the CNS while their axons simultaneously grow from specific exit points in the CNS and navigate to precise peripheral targets. Although numerous axon guidance molecules have been identified for motor neuron guidance in the limbs, our understanding of the signals that initially guide motor axons from the CNS and the mechanisms that control the precise spatiotemporal activity of guidance factors remain fragmentary. To identify novel genes that regulate motor neuron development, a transgenic mouse with GFP-labeled motor axons and td-tomato-labeled motor nuclei was generated. This reporter mouse was used in an ENU mutagenesis screen which has identified three independent mutants (Greenlight, WrongWay, and Merge), to date, that each display defects in motor axon exiting from the neural tube.
The aims proposed in this study will focus on the cloning and characterization of the Greenlight (GrL) mutation using a gene mapping strategy and in vitro guidance assays that are well established in the lab.
In Aim1, GrL will be cloned using out-crosses and SNP mapping in conjunction with state-of-the-art, high-throughput sequencing. The tissue specific expression of the gene will be examined using in situ hybridization and immunolabeling for protein, and the nature of the mutation (i.e.. null, hypomorph, gain-of-function) will be tested using mouse genetics and biochemistry.
In Aim2, functional characterization of GrL will be performed using a multi-disciplinary approach, including mouse genetics, biochemistry, in vitro guidance assays, and advanced multi-dimensional imaging techniques in order to provide significant insights into the molecular and cellular properties of GrL and elucidate its physiological role during spinal cord development. Ultimately, studying the molecular and genetic pathways that mediate neuronal pathfinding and connectivity should further contribute to our understanding of the structure of the spinal motor circuit and its effects on locomotor activity and motor behavioral response. !
Motor neurons are selectively lost in developmental diseases such as spinal motor atrophy (SMA) and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Although the mechanisms that underlie these disease pathologies are largely unknown, it is known that there is a genetic component. By identifying and characterizing genes (and their signaling pathways) that regulate motor neuron growth, guidance, and connectivity, therapies may be effectively targeted within the spinal cord, facilitating recovery of motor function that is lost in such debilitating diseases.
