Marcia G. Honig - US grants

During development, neurons establish synaptic connections in a precise and specific manner. In many systems, the correct connections are made because growing axons have the ability to choose correctly among several available pathways and thus arrive at the correct target sites. The long-range goal of this research is to further our understanding of specificity and axonal guidance by elucidating how the axons of sensory neurons choose among pathways in the chick hindlimb during development. The experiments proposed here will employ a tissue culture system to examine mechanisms at a more cellular level than has been possible in the intact embryo. The neurons to be studied will be labeled prior to culturing with fluorescent carbocyanine dyes, so that it is possible to unambiguously identify the different types of neurons after they are placed into culture. The three major aims are: 1). To elucidate how sensory neuron axons may use other axons as cues, and more specifically, how motoneurons axons may guide sensory neuron axons along pathways in the limb, as in vivo studies suggest they do, time-lapse videotaping will be used to examine the behavior of sensory neuron growth cones as they encounter the processes of motoneurons. 2). To elucidate why motoneuron axons never grow to the skin whereas sensory neurons axons normally grow to skin and to muscle, a series of experiments will examine if motoneuron and sensory neuron axons are attracted by the appropriate type of target tissue. 3) To determine whether muscle sensory neurons and cutaneous sensory neurons are intrinsically specified, a series of experiments will assess if the two types of sensory neurons differ either in terms of the responses of their growth cones to motoneuron axons or to other sensory neuron axons or with respect to their growth to the two different types of target tissues. If differences are found, sensory neurons from younger embryos will be examined to ascertain if the observed differences are intrinsic or if they are a consequence of the neurons having become specified by contact with their peripheral target tissues. Together these studies will increase our understanding of the differences between different types of neurons and of the cellular interactions that may play a role in pathway selection during development. Understanding these processes is important because abnormalities in these processes during development would result in defects in the organization, and consequently the function, of the nervous system.

The mechanisms involved in the growth of axons along specific, pathways will be studied in the chick hindlimb. Select sets of neurons will be labeled in ovo with different fluorescent dyes and then removed from the embryo and studied in culture. Two types of sensory neurons, those that innervate skin and muscle, will be studied to determine if these differ in their responses to contact with processes of motoneurons, expression of various cell adhesion molecules and growth toward specific target tissues. More generally, these studies promise to elucidate mechanisms involved in the development of neuronal specificity and regeneration.

DESCRIPTION (provided by applicant): The goal of my research is to understand how axons grow along the correct pathways during development. To address this issue, we study sensory axons projecting into the chick hindlimb. In contrast to motoneuron axons, which do not require the presence of muscle per se to form muscle nerves, the skin is essential for the normal development of cutaneous projections. Our surgical manipulations have shown that the initial formation of a major cutaneous nerve in the embryonic chick limb, the lateral femoral cutaneous (LFCt) nerve requires the presence of the target ectoderm during a critical time period, when those axons are about to diverge from the hindlimb plexus. To elucidate the underlying mechanisms, we characterized the effects of ectoderm removal on a variety of molecules expressed in the limb. Importantly, Bmp4 expression was downregulated and BMP4-soaked beads placed on the denuded limb surface rescued LFCt nerve formation. In the proposed studies, we will continue our ongoing efforts to elucidate the mechanisms responsible for cutaneous nerve formation, taking advantage of the accessibility of chick embryos for carrying out surgical and molecular manipulations. 1 major goal will be to determine whether BMP4 exerts its effects by directly affecting the outgrowth of cutaneous axons or by signaling the limb mesenchyme in a way that it in turn elicits LFCt nerve formation. To distinguish between these possibilities and elucidate the molecular mechanisms underlying BMP4 action, the proposed experiments will characterize the expression of BMP receptors in the DRGs and in the limb;perturb BMP4 signaling, using various loss-of-function approaches, targeted by means of electroporation, to either the limb bud or to the neural crest (to transfect DRG neurons);and use complementary tissue culture assays to examine the cellular mechanisms by which BMP4 rescues LFCt nerve formation. Other studies will determine how cues from the skin contribute to LFCt nerve formation. These will focus on NGF and NTS, characterizing their expression in the limb, the expression of the relevant receptors on the first cutaneous axons to project to the skin, and examining whether exogenous NGF and/or NT3 rescues LFCt nerve formation after ectoderm removal. The proposed studies will help us understand how discrete regions of the skin bring about the formation of the cutaneous nerve projecting to it, and thereby also provide insight into ways to improve regeneration following peripheral nerve injury.