The overall goal of the proposed experiments is to investigate three aspects of the of sensory innervation patterns--(l) potential guidance cues for cutaneous and muscle afferents, (2) the development of cutaneous projections to dorsal horn and (3) development of sensory neuron phenotype-using the chick embryo as a model preparation. (1) To test whether outgrowing sensory neurons receive guidance cues from their respective targets, cutaneous afferents from the dorsomedial trigeminal ganglion (DM-TG) and proprioceptive afferents from the trigeminal mesencephalic nucleus (TMN) will be co-cultured with skin or myotubes, and the outgrowth of neurites in the vicinity of the targets followed with timelapse videomicroscopy. To determine whether sensory axons receive guidance cues from outgrowing motor axons interactions between DM-TG and TMN neurons and motor axons in vitro will be similarly recorded. (2) The projections of selected cutaneous nerves to the superficial dorsal horn will be labeled with fluorescent dyes in paraformaldehyde-fixed embryos o different ages to determine whether central projections are established correctly from the outset. To learn how and when dorsal horn laminae develop, the birthdates of neurons in laminae 11 and III will be determined with 3H-thymidine autoradiography. In addition, replication-deficient retrovirus bearing the lacZ gene will be introduced into the neural tube of young embryos, and the clones derived from infected cells analyzed to determine the lineage relationships among neurons in different laminae. (3) To investigate the development phenotypic heterogeneity among sensory neurons, the expression of a glycoconjugate that distinguishes a subset of neurons will be studied. Extracts of skin, muscle or spinal cord will be applied to DRG neurons in vitro to determine whether the target turns on glycoconjugate expression. In addition, to determine the neurotrophic requirements of different subpopulations of DRG neurons, neurons will be grown in vitro in either NGF or brain-derived neurotrophic factor and the surviving neurons stained with markers known to label subsets of sensory neurons. In some cases neurons will be retrogradely labeled from skin or muscle prior to culture to determine the trophic requirements of neurons with different targets. Together the results of these experiments should advance our understanding of the development of sensory neurons, giving insight into how the neurons acquire their central and peripheral connection's and phenotypic identity, which are essential for normal sensory function.
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