The outgrowth and guidance of sensory axons is one of the first steps toward generating the precise patterns of neuronal connections that allow us to perceive and interpret the world around us. The growth cones at the leading edge of migrating axons follow highly stereotyped pathways and reach their target regions by recognizing molecular guidance cues in their environment. The long term objective of this proposal is to understand how these guidance molecules collaborate mechanistically to determine the behavior of growing axons and establish appropriate patterns of sensory connections. During mouse embryonic development, the maxillary nerve of the trigeminal ganglion projects toward and ultimately innervates the prospective whisker epithelium of the maxillary process. In order to understand how specific axons choose particular pathways and how a multitude of molecular guidance cues collaborate to determine these decisions, I plan to develop the trigeminal-maxillary projection into a model system for the molecular study of axon guidance. To this end, I will develop an organotypic slice culture assay for the trigeminal-maxillary projection. This will establish an easily manipulated system with the complexity to recapitulate the actual cellular environment experienced by migrating trigeminal axons. The function of relevant axon guidance molecules can thus be addressed in the context of their natural environment and multiple molecules can be manipulated either alone or in combination to investigate cooperation between molecules and address problems of functional redundancy. Development of this assay will be combined with identification and characterization of the molecules generating specificity to the guidance of the maxillary nerve. Specifically, I propose here to determine the contribution of several gene families (laminins, neurotrophins, netrins, semaphorins) that have been implicated in vitro and in vivo as signals for neuronal pathfinding and targeting. The expression of laminin along trigeminal axon pathways will be confirmed and the integrin laminin receptors on trigeminal neurons will be identified. Function blocking antibodies to these proteins will be used to investigate their contribution to trigeminal axon guidance. The neurotrophin NT-4/5 will be examined as a possible candidate for the chemoattractant activity identified for trigeminal neurons and termed maxillary factor, as my preliminary experiments have shown that -4/5 has the temporal expression patterns and biochemical activities consistent with a role as maxillary factor. To address its contribution, NT-4/5 deficient mice will be analyzed for the activity and for defects in pathfinding and targeting. I have also detected chemorepellent activities in the maxillary process. Netrin-1 and members of the semaphorin family of chemorepellents are reportedly expressed in first branchial arch tissues and thus will be analyzed for a contribution to these activities. The semaphorins expressed in the maxillary process will be identified and isolated, and along with netrin-1, will be tested for chemorepellent activity following expression in COS cells. The expression of NT-4/5, netrin-1, and the relevant integrins and semaphorins will be examined by RNA in situ hybridization and immunocytochemistry. This combination of expression and functional analysis will establish an anatomy of guidance molecules in relation to the pathway of the maxillary nerve. The development of the embryonic slice culture assay and the analysis of these guidance molecules will establish the framework and begin to generate the tools required to study how information from multiple molecules is integrated mechanistically by a migrating growth cone. Furthermore, a fundamental understanding of the strategies and mechanisms involved with normal migration processes such as axon guidance will further our understanding of the pathologic migrations (e.g. by metastatic tumor cells) wince both are functionally similar processes.
| O'Connor, R; Tessier-Lavigne, M (1999) Identification of maxillary factor, a maxillary process-derived chemoattractant for developing trigeminal sensory axons. Neuron 24:165-78 |
