Long-range Ca2+ Signaling from Growth Cone to Soma Mediates Reversal of Neuronal Migration Induced by Slit-2
Chen-bing Guan
ABSTRACT
The development of the nervous system can be divided into three steps: the regulated proliferation and differentiation of the neural stem cells; the guided migration of the neurons to their appropriate positions; the correct formation of the neural circuits. Among the development, the neuronal migration is an important step because more than 109 neurons migrate in the forebrain of the primates, and some neurons can migrate more than several centimeters. Furthermore, neurons should form different structures, such as lamination, after migrating to appropriate positions.
According to the different stages of development, the neuronal migration is known happening in two different time periods ---- the migration in embryonic stage and the migration postnatally. The former, which is involved in the lamination of the cortex and the lamination of the dorsal horn of the spinal cord, is represented by the migration of the post-mitosis neurons out of ventricular zone. The latter stage of migration includes the migration of the cerebellar granule neurons from the external granule cell layer to the inner granule cell layer and the migration of the anterior-subventricular-zone neuron to the olfactory bulb forming a rostral migratory stream. According to the relationship between the migrating neurons and the glial cells, neuronal migration also can be divided into two forms as glia-guided neuronal migration and glia-independent tangential migration.
In central nervous system, the process of the neuronal migration is composed by three steps: the commencing of the migration after the neuron’s fate was designated; the guidance of the migration to make the neuron migrate in the correct path and in the correct direction; the termination of the migration to decide the final position of the migratory neuron. No matter how the neuron is migrating, there are involving many molecular mechanisms during any of these three steps. For glia-independent migration, e.g. within the rostral migratory stream, migratory neurons are surrounded by repulsive guidance cues, such as Slits, and promoted by some other molecules, such as PS-NCAM, Ephrin, etc., by which means these neurons can migrate within a restricted pathway. For glia-guided radial migration, although it was once believed that glial cells are sufficient to guide the neuronal migration, recent findings indicate that the guidance effect can not be accomplished by the glial cells themselves, since there were neurons migrating in different directions even on the same fiber of a glial cell, which suggested the existence of guidance molecules.
Compared to the research of the guidance of neuronal migration, the research of axon guidance had already aroused most interesting. A century before after observing the stained nervous tissues, the famous neuroscientist, Ramon y Cajal, stated that the tip of the neuron axon should display a dynamic motility, and should be attracted or repelled by target tissues in the same as the leukocytes. In recent 20 years, the research of the axon guidance has fully confirmed the description from Ramon y Cajal, and a large amount of guidance cues, including attractants and repellents, and their receptors have been identified, as well as many intracellular mechanisms have also been unveiled. These guidance molecules include the classic guidance factors, e.g. Netrins, Slits, Semaphorins, Ephrins, etc., the nerve growth factors, e.g. BDNF, NGF, etc., the embryonic morphogens, e.g. Wnt, BMP, Shh, etc., and many other secreted and membrane-anchored proteins.
Interestingly, these guidance cues for axon guidance are also involved in the guidance of neuronal migration. Furthermore, the observation of the neuronal migration in brain slice or in culture indicated that the migratory neuron always bears a leading process in the front of the soma, and has a growth-cone like structure at the tip of the leading process, which we also name it as “leading growth cone”. During the migration, the motilities of the leading growth cone and the soma are well coordinated. In addition, it was also found that Netrin-1 could attract the neuronal migration perhaps through its attractive effect of the leading process. All these imply an underlying mechanism to coordinate the growth cone guidance and the directed neuronal migration. An intriguing possibility is that the guidance of neuronal migration is mainly determined by the action of guidance cues on the growth cone of the leading process, and subsequent long-range signaling from the leading growth cone to the soma coordinates the translocation of the soma. Alternatively, the soma directly senses the extracellular guidance signal in a manner that is relatively independent of the leading growth cone. In the thesis, these two possibilities were examined by using isolated cerebellar granule cells in culture, and monitoring the behavior of the growth cone and the soma in response to an extracellular gradient of Slit-2, a repulsive factor that is known to halt or to reverse the direction of neuronal migration when applied in front of the migrating neuron.
The repulsive factor, Slit, was firstly identified in 1984 by the study on the mutants of Drosophila, and its receptor, Robo, was identified in 1998 as a receptor that mediating repulsive signals. One year later, Slit was confirmed as the ligand of Robo and the Slit-Robo signaling is know involved in the repulsive effect of the commission axons crossing the mid-line, and also involved in the repulsive effect of the guidance of neuronal migration and leukocyte migration as well.
It was identified that the cerebellar granule cell in the external granule cell layer is expressing Robo2, a receptor of Slit, and the Purkinje cell is expressing Slit-2, suggesting the repulsive effect of Slit-Robo, here, prevents the immature inward migration of the cerebellar granule cells.
The mechanisms underlying axon guidance by extracellular signals have been extensively studied. Each guidance factor may activate a cascade of cytoplasmic signals, which eventually induce cytoskeleton rearrangements required for directed growth cone extension. Among various cytoplasmic second messengers, Ca2+ signal mediates both attractive and repulsive responses of growth cones induced by several guidance factors, and is also critical for neuronal migration. In the thesis, it was found by living cells monitoring and Ca2+ imaging that the leading growth cone is responsible for sensing extracellular Slit-2, and a propagating Ca2+ wave from the leading growth cone to the soma is responsible for inducing the reversal in the direction of soma translocation in response to a frontal gradient of Slit-2. Furthermore, Ca2+ wave induced by Ca2+ release from internal stores was sufficient to reverse the soma translocation. Finally, through transfecting mutated constructs and living observation, biochemical pull-down assay, and FRET-based living cell imaging, it was indicated that the reversal of migration induced by Slit-2 required RhoA activity in these neurons and correlated with an anterior-to-posterior redistribution of active RhoA in the soma. Such RhoA redistribution may lead to the cytoskeletal rearrangement underlying the reversal of soma translocation.
Our observation of single living cell migration applying micro-gradient of guidance cues and the living imaging of fluorescent proteins facilitate the research of the intracellular signaling without the interference from cell-cell interaction. Currently, the research of neuronal migration is one of the most active fields in neuroscience, and still has many puzzles untangled. These researches are showing great significance in understanding the development of the fascinating nervous system and in suggesting the method for the renaissance after the neuronal injury.
Key borad: neuronal migration, Slit-2, Ca2+ wave, RhoA
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