Alex L. Kolodkin
Molecular Mechanisms of Growth Cone Guidance
A central issue in neurobiology is determining how neurons find their targets. Neuronal growth cones are guided to their targets by both short and long range cues. Though the major emphasis to date has been on attractive cues, recently it has become clear that repulsion plays an equally important role in enabling growth cones to find their appropriate targets. In our laboratory, we utilize both innvertebrate and vertebrate model systems to understand the cellular and molecular basis of repulsive guidance mechanisms. The insect nervous system allows us to study the molecular basis of these guidance cues, exploiting genetics and molecular biology in the fruit fly Drosophila and cellular manipulations and molecular biology in the larger grasshopper embryo. The similarity in guidance mechanisms and the molecules that mediate them in invertebrates and vertebrates allows us to apply what is learned from our insect studies to our vertebrate neuronal culture systems, in particular rat embryonic primary culture and mouse genetic models. The semaphorins are a family of growth cone guidance molecules, conserved from insects to mammals, which includes proteins strongly implicated in mediating repulsive guidance during neurodevelopment and neuronal regeneration. The present focus of our efforts is an elucidation how semaphorins function as growth cone guidance cues during neurodevelopment.
Semaphorins, which include both transmembrane and secreted proteins, were discovered in studies aimed at identifying cell surface molecules capable of functioning as pathway labels for embryonic growth cones . The first member of this gene family to be identified, Grasshopper semaphorin I (G-sema I), functions in vivo to stall and steer a pair of well studied growth cones in the developing limb bud as they encounter a stripe of G-sema I expressing epithelial cells. Other members of this gene family were identified in Drosophila (D-sema I, D-sema II) and in mammals (H-sema III, M-sema III). Included in this gene family is the molecule chick collapsin, which in vitro is capable of causing the collapse of dorsal root ganglion (DRG) growth cones. Our studies show that a rodent homologue of H-Sema III/collapsin, M-Sema III, is expressed in the ventral embryonic spinal cord. Sema III can function as a selective chemorepellent for specific populations of DRG sensory afferents during their innervation of the embryonic spinal cord. Recently, a very large number of semaphorins have been identified in several vertebrate and invertebrate species. These semaphorins have been shown to be specifically expressed in both neuronal and non-neuronal tissues during development and in the adult, underscoring the potential functions these proteins might perform in a variety of developmental and regenerative events.
Sema III is a secreted protein that in vitro causes neuronal growth cone collapse and chemorepulsion of neurites, and in vivo is required for correct sensory afferent innervation and other aspects of development. The mechanism of Sema III function, however, is unknown. To begin to understand how Sema III imparts changes in the growth cone cytoskeleton that in turn result in repulsive steering events, we searched for a Sema III receptor. We have recently shown that neuropilin, a type I transmembrane protein implicated in aspects of neurodevelopment, is a Sema III receptor or a component of a receptor complex. We have also identified neuropilin-2, a related neuropilin family member, and have shown that neuropilin and neuropilin-2 are expressed in overlapping yet distinct populations of neurons in the rat embryonic nervous system (see Figure below). These studies raise the exciting possibility that the neuropilins contribute to a family of semaphorin receptor or receptor complexes that differentially interact with semaphorins and provide for a diversity of semaphorin-mediated neuronal responses. Currently, we are using biochemical and molecular strategies to identify proteins that might form receptor complexes with neuropilins, and also to find the downstream signal transduction components that transmit semaphorin cues into intracellular events.
In addition, we are also very interested in the cellular basis of transmembrane semaphorin function. To this end, we have initiated a genetic analysis in Drosophila to determine the role of the transmembrane semaphorin Drosophila semaphorin I (D-sema I) in well characterized growth cone guidance decisions. Our analysis thus far shows that D-sema I is essential for peripheral and central axon guidance events, and suggests that this transmembrane semaphorin, like secreted semaphorins, can also function as a repulsive guidance cue. Our work has also identified several paradigms suitable for identifying gene products that interact with D-sema I, using powerful suppressor and enhancer genetic mutagenesis approaches. These include screens for visible adult and embryonic phenotypes that are dependent upon D-sema I. In addition, we are undertaking several complementary molecular and biochemical approaches to identify important components of the D-sema I signaling cascade.
These studies will further our understanding of how the Semaphorins function in growth cone steering decisions during neurodevelopment, and of the role that repulsive growth cone guidance plays in this process. Our future goals include an extension of our studies to address how semaphorins and their receptors function during adult neuronal regeneration. Our preliminary results in several systems strongly suggest that this will be an informative direction, and that semaphorins may contribute to the repulsive characteristics known to prevent regeneration in the adult mammalian CNS.