The goal of this proposal is to determine the function of a new Netrin gene family member, Netrin5, in the development of motor and sensory neurons in the mammalian nervous system. The development of motor and sensory neurons in the spinal cord and cranial nerves is well studied;however, major questions remain, including how motor axons identify the ventral motor exit point, how sensory axons identify the dorsal root entry zone, and how the boundary between the central and peripheral nervous system is established. The nature of this boundary is unclear. No physical barrier, such as a basal lamina, has been observed, and the signals that underlie these important functions are poorly defined. We recently identified a new member of the Netrin family of genes that may be such a signal. Netrins are well known to direct axon guidance and cell migration in the developing nervous system, and we identified Netrin5 in mice in the course of our studies on Netrin signaling in motor axon guidance. The gene is conserved in the human genome and in mice, Netrin5 is expressed in a population of cells found at the motor exit points and dorsal root entry zones in the mouse embryonic spinal cord, and is associated with the sensory trigeminal nerve and possibly other cranial nerves. These cells appear to be boundary cap cells, a transient neural crest-derived cell population that functions to restrict motor neuron cell bodies to the ventral horn and to contribute to dorsal root Schwann cell and dorsal root ganglia nociceptive neuron populations. The expression pattern of Netrin5 suggests a role for this developmental signal in motor and sensory neuron development and peripheral axon guidance into and out of the spinal cord, and possibly similar functions in cranial nerves. We have generated expression constructs for in vitro studies, and knockout mice lacking Netrin5 expression for an in vivo assessment of its function. In our first Aim, we will examine motor and sensory neuron development in the absence of Netrin5. These studies will be informed by the known function of boundary cap cells, such as restricting motor neuron cell bodies to the ventral horn or differentiating into nociceptive neurons found in the dorsal root ganglia. We will also examine known Netrin-dependent processes for which the specific Netrin gene responsible has not been determined, such as trochlear nerve guidance. In addition to anatomical studies, we will assess motor and sensory behaviors in the mice to determine the functional consequences of the loss of Netrin5. In our second Aim, we will determine the effect of NETRIN5 protein on motor and sensory axon outgrowth and guidance in vitro, and determine the Netrin receptors required. These studies will be informed by the expression patterns of netrin receptors, and in vitro results will be confirmed genetically with neurons cultured from Netrin receptor knockout mice and by an in vivo assessment of possible phenocopy in the relevant receptor knockout strains. Together, these experiments will determine the role of Netrin5 in mammalian neurodevelopment and address important, unanswered questions about motor and sensory neuron development.
The development of motor and sensory neurons is essential for normal function in adults, and motor and sensory deficits lead to a number of debilitating conditions, including ulcerations and movement disorders. Netrins are a family of genes known to be critically important for normal neurodevelopment, and here we describe a new, previously unstudied member of this family that circumstantially has a role in the development of motor and sensory neurons based on where the gene is expressed. Mutations in other genes in the Netrin pathway result in congenital mirror movement disorder and are candidate causes of Rett syndrome and autism spectrum disorder, demonstrating their importance in human neurodevelopment. Furthermore, emerging stem cell-based regenerative medicine approaches will require a thorough understanding of such developmental signaling pathways to optimize outcomes. Understanding the function of Netrin5 as a new member of this important family of neurodevelopmental genes is central to these efforts.