The Xenopus retinal ganglion cell as a model neuron to study the establishment of neuronal connectivity

Neurons receive inputs through their multiple branched dendrites and pass this information on to the next neuron via long axons, which branch within the target. The shape the neuron acquires is thus the key to its proper functioning in the neural circuit in which it participates. Both axons and dendrites grow in a directed fashion to their target partner neurons by responding to a large number of molecular cues in the milieu through which they extend. They then go through the process of synaptogenesis, first choosing a neuron on which to synapse, and then the appropriate subcellular location. How a neuron acquires its unique shape, establishes and modifies appropriate synaptic connectivity, and the molecular signals involved, are key questions in developmental neurobiology. Such questions of nervous system wiring are being pursued actively with a variety of different animal models and neuron types, each with its own unique advantages. Among these, the developing retinal ganglion cell (RGC) of the South African clawed frog, Xenopus laevis, has proven particularly fruitful for revealing the secrets of how axons and dendrites acquire their final morphology and connectivity. In this review, we describe how this system can be used to understand the multiple molecular events that instruct the incorporation of RGCs into the neural circuit that controls vision.     

Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology

Neurons establish diverse dendritic morphologies during development, and a major challenge is to understand how these distinct developmental programs might relate to, and influence, neuronal function. Drosophila dendritic arborization (da) sensory neurons display class-specific dendritic morphology with extensive coverage of the body wall. To begin to build a basis for linking dendrite structure and function in this genetic system, we analyzed da neuron axon projections in embryonic and larval stages. We found that multiple parameters of axon morphology, including dorsoventral position, midline crossing and collateral branching, correlate with dendritic morphological class. We have identified a class-specific medial-lateral layering of axons in the central nervous system formed during embryonic development, which could allow different classes of da neurons to develop differential connectivity to second-order neurons. We have examined the effect of Robo family members on class-specific axon lamination, and have also taken a forward genetic approach to identify new genes involved in axon and dendrite development. For the latter, we screened the third chromosome at high resolution in vivo for mutations that affect class IV da neuron morphology. Several known loci, as well as putative novel mutations, were identified that contribute to sensory dendrite and/or axon patterning. This collection of mutants, together with anatomical data on dendrites and axons, should begin to permit studies of dendrite diversity in a combined developmental and functional context, and also provide a foundation for understanding shared and distinct mechanisms that control axon and dendrite morphology.     

Extracellular signal-regulated kinase 1/2 mediates survival, but not axon regeneration, of adult injured central nervous system neurons in vivo

Neurotrophins play important roles in the response of adult neurons to injury. The intracellular signaling mechanisms used by neurotrophins to regulate survival and axon growth in the mature CNS in vivo are not well understood. The goal of this study was to define the role of the extracellular signal-regulated kinases 1/2 (Erk1/2) pathway in the survival and axon regeneration of adult rat retinal ganglion cells (RGCs), a prototypical central neuron population. We used recombinant adeno-associated virus (AAV) to selectively transduce RGCs with genes encoding constitutively active or wild-type mitogen-activated protein kinase kinase 1 (MEK1), the upstream activator of Erk1/2. In combination with anterograde and retrograde tracing techniques, we monitored neuronal survival and axon regeneration in vivo. MEK1 gene delivery led to robust and selective transgene expression in multiple RGC compartments including cell bodies, dendrites, axons and targets in the brain. Furthermore, MEK1 activation induced in vivo phosphorylation of Erk1/2 in RGC bodies and axons. Quantitative analysis of cell survival demonstrated that Erk1/2 activation promoted robust RGC neuroprotection after optic nerve injury. In contrast, stimulation of the Erk1/2 pathway was not sufficient to induce RGC axon growth beyond the lesion site. We conclude that the Erk1/2 pathway plays a key role in the survival of axotomized mammalian RGCs in vivo, and that activation of other signaling components is required for axon regeneration in the growth inhibitory CNS environment.     

Rho proteins: their function in neurons

Neurons possess a polarized morphology. In general, each neuron has several dendrites but only one axon. Such morphology is the basis for directionalized rapid signaling, information flowing from the short dendrites to the long axon. The mechanisms involved in the establishment of the neuronal polarity remain largely unknown. However, recently, members of Rho family proteins have been implicated in the regulation of neuronal morphology especially development of neuronal polarity, axon outgrowth and guidance, dendritic tree elaboration and synapse formation. Moreover, the Rho GTPases have been reported to be directly or indirectly involved in some neurological conditions such as X-linked mental retardation as well as Alzheimer's and Parkinson's diseases. These findings demonstrate the importance of Rho GTPases in the development, maintenance and function of the nervous system.     

Rac proteins and the control of axon development

Rac GTPases and their effectors control cellular morphogenesis in a wide range of developmental contexts by regulating the structure and dynamics of the actin cytoskeleton. Although much is known about the biochemistry of Racs and Rac regulators, less is known about how Racs control cellular morphogenesis, including axon development, in vivo. Recent loss-of-function genetic studies using model organisms have shown that Racs and their effectors are required for multiple aspects of axon development, including axon outgrowth, axon guidance and axon branching. Interestingly, these studies have also revealed that Rac activity is required to prune spurious axons and branches. Analyses of Racs and their upstream and downstream effectors suggest that Rac signaling is complex. Different neurons utilize distinct combinations of upstream Rac regulators during axon development, possibly reflecting responses to different axon path-finding signals, and Racs use distinct downstream effectors to mediate different aspects of axon development, possibly reflecting differential regulation of the lamellipodial and filopodial growth-cone actin-cytoskeleton domains underlying axon developmental events.     

Kidins220/ARMS Modulates the Activity of Microtubule-regulating Proteins and Controls Neuronal Polarity and Development

In order for neurons to perform their function, they must establish a highly polarized morphology characterized, in most of the cases, by a single axon and multiple dendrites. Herein we find that the evolutionarily conserved protein Kidins220 (kinase D-interacting substrate of 220-kDa), also known as ARMS (ankyrin repeat-rich membrane spanning), a downstream effector of protein kinase D and neurotrophin and ephrin receptors, regulates the establishment of neuronal polarity and development of dendrites. Kidins220/ARMS gain and loss of function experiments render severe phenotypic changes in the processes extended by hippocampal neurons in culture. Although Kidins220/ARMS early overexpression hinders neuronal development, its down-regulation by RNA interference results in the appearance of multiple longer axon-like extensions as well as aberrant dendritic arbors. We also find that Kidins220/ARMS interacts with tubulin and microtubule-regulating molecules whose role in neuronal morphogenesis is well established (microtubule-associated proteins 1b, 1a, and 2 and two members of the stathmin family). Importantly, neurons where Kidins220/ARMS has been knocked down register changes in the phosphorylation activity of MAP1b and stathmins. Altogether, our results indicate that Kidins220/ARMS is a key modulator of the activity of microtubule-regulating proteins known to actively regulate neuronal morphogenesis and suggest a mechanism by which it contributes to control neuronal development.     

Developmental mechanisms that regulate retinal ganglion cell dendritic morphology

One of the fundamental features of retinal ganglion cells (RGCs) is that dendrites of individual RGCs are confined to one or a few narrow strata within the inner plexiform layer (IPL), and each RGC synapses only with a small group of presynaptic bipolar and amacrine cells with axons/dendrites ramified in the same strata to process distinct visual features. The underlying mechanisms which control the development of this laminar-restricted distribution pattern of RGC dendrites have been extensively studied, and it is still an open question whether the dendritic pattern of RGCs is determined by molecular cues or by activity-dependent refinement. Accumulating evidence suggests that both molecular cues and activity-dependent refinement might regulate RGC dendrites in a cell subtype-specific manner. However, identification of morphological subtypes of RGCs before they have achieved their mature dendritic pattern is a major challenge in the study of RGC dendritic development. This problem is now being circumvented through the use of molecular markers in genetically engineered mouse lines to identify RGC subsets early during development. Another unanswered fundamental question in the study of activity-dependent refinement of RGC dendrites is how changes in synaptic activity lead to the changes in dendritic morphology. Recent studies have started to shed light on the molecular basis of activity-dependent dendritic refinement of RGCs by showing that some molecular cascades control the cytoskeleton reorganization of RGCs.     

BDNF increases synapse density in dendrites of developing tectal neurons in vivo

Neuronal connections are established through a series of developmental events that involve close communication between pre- and postsynaptic neurons. In the visual system, BDNF modulates the development of neuronal connectivity by influencing presynaptic retinal ganglion cell (RGC) axons. Increasing BDNF levels in the optic tectum of Xenopus tadpoles significantly increases both axon arborization and synapse density per axon terminal within a few hours of treatment. Here, we have further explored the mechanisms by which BDNF shapes synaptic connectivity by imaging tectal neurons, the postsynaptic partners of RGCs. Individual neurons were co-labeled with DsRed2 and a GFP-tagged postsynaptic density protein (PSD95-GFP) to visualize dendritic morphology and postsynaptic specializations simultaneously in vivo. Immunoelectron microscopy confirmed that PSD95-GFP predominantly localized to ultrastructurally identified synapses. Time-lapse confocal microscopy of individual, double-labeled neurons revealed a coincident, activity-dependent mechanism of synaptogenesis and axon and dendritic arbor growth, which is differentially modulated by BDNF. Microinjection of BDNF into the optic tectum significantly increased synapse number in tectal neuron dendritic arbors within 24 hours, without significantly influencing arbor morphology. BDNF function-blocking antibodies had opposite effects. The BDNF-elicited increase in synapse number complements the previously observed increase in presynaptic sites on RGC axons. These results, together with the timescale of the response by tectal neurons, suggest that the effects of BDNF on dendritic synaptic connectivity are secondary to its effects on presynaptic RGCs. Thus, BDNF influences synaptic connectivity in multiple ways: it enhances axon arbor complexity expanding the synaptic territory of the axon, while simultaneously coordinating synapse formation and stabilization with individual postsynaptic cells.     

The Rac activator DOCK7 regulates neuronal polarity through local phosphorylation of stathmin/Op18

The polarization of a neuron generally results in the formation of one axon and multiple dendrites, allowing for the establishment of neuronal circuitry. The molecular mechanisms involved in priming one neurite to become the axon, particularly those regulating the microtubule network, remain elusive. Here we report the identification of DOCK7, a member of the DOCK180-related protein superfamily, as a Rac GTPase activator that is asymmetrically distributed in unpolarized hippocampal neurons and selectively expressed in the axon. Knockdown of DOCK7 expression prevents axon formation, whereas overexpression induces formation of multiple axons. We further demonstrate that DOCK7 and Rac activation lead to phosphorylation and inactivation of the microtubule destabilizing protein stathmin/Op18 in the nascent axon and that this event is important for axon development. Our findings unveil a pathway linking the Rac activator DOCK7 to a microtubule regulatory protein and highlight the contribution of microtubule network regulation to axon development.     

BDNF instructs the kinase LKB1 to grow an axon

Determining which neurite of a differentiating neuron is to become the axon is a crucial step in neuronal morphogenesis. Two groups (Barnes et al., 2007; Shelly et al., 2007) now report that axon specification in vivo is mediated by extracellular signals acting through the serine/threonine kinase LKB1.     

Both the establishment and the maintenance of neuronal polarity require active mechanisms: critical roles of GSK-3beta and its upstream regulators

Axon-dendrite polarity is a cardinal feature of neuronal morphology essential for information flow. Here we report a differential distribution of GSK-3beta activity in the axon versus the dendrites. A constitutively active GSK-3beta mutant inhibited axon formation, whereas multiple axons formed from a single neuron when GSK-3beta activity was reduced by pharmacological inhibitors, a peptide inhibitor, or siRNAs. An active mechanism for maintaining neuronal polarity was revealed by the conversion of preexisting dendrites into axons upon GSK-3 inhibition. Biochemical and functional data show that the Akt kinase and the PTEN phosphatase are upstream of GSK-3beta in determining neuronal polarity. Our results demonstrate that there are active mechanisms for maintaining as well as establishing neuronal polarity, indicate that GSK-3beta relays signaling from Akt and PTEN to play critical roles in neuronal polarity, and suggest that application of GSK-3beta inhibitors can be a novel approach to promote generation of new axons after neural injuries.     

Ganglion cell axon pathfinding in the retina and optic nerve

The eye is a highly specialized structure that gathers and converts light information into neuronal signals. These signals are relayed along axons of retinal ganglion cells (RGCs) to visual centers in the brain for processing. In this review, we discuss the pathfinding tasks RGC axons face during development and the molecular mechanisms known to be involved. The data at hand support the presence of multiple axon guidance mechanisms concentrically organized around the optic nerve head, each of which appears to involve both growth-promoting and growth-inhibitory guidance molecules. Together, these strategies ensure proper optic nerve formation and establish the anatomical pathway for faithful transmission of information between the retina and the brain.     

Tissue-specific neuro-glia interactions determine neurite differentiation in ganglion cells

Guided formation and extension of axons versus dendrites is considered crucial for structuring the nervous system. In the chick visual system, retinal ganglion cells (RGCs) extend their axons into the tectum opticum, but not into glial somata containing retina layers. We addressed the question whether the different glia of retina and tectum opticum differentially affect axon growth. Glial cells were purified from retina and tectum opticum by complement-mediated cytolysis of non-glial cells. RGCs were purified by enzymatic delayering from flat mounted retina. RGCs were seeded onto retinal versus tectal glia monolayers. Subsequent neuritic differentiation was analysed by immunofluorescence microscopy and scanning electron microscopy. Qualitative and quantitative evaluation revealed that retinal glia somata inhibited axons. Time-lapse video recording indicated that axonal inhibition was based on the collapse of lamellipodia- and filopodia-rich growth cones of axons. In contrast to retinal glia, tectal glia supported axonal extension. Notably, retinal glia were not inhibitory for neurons in general, because in control experiments axon extension of dorsal root ganglia was not hampered. Therefore, the axon inhibition by retinal glia was neuron type-specific. In summary, the data demonstrate that homotopic (retinal) glia somata inhibit axonal outgrowth of RGCs, whereas heterotopic (tectal) glia of the synaptic target area support RGC axon extension. The data underscore the pivotal role of glia in structuring the developing nervous system.     

Fibroblast growth factors redirect retinal axons in vitro and in vivo

Growth factors have been shown previously to participate in the process of axon target recognition. We showed that fibroblast growth factor receptor (FGFR) signaling is required for Xenopus laevis retinal ganglion cell (RGC) axons to recognize their major midbrain target, the optic tectum [neuron 17 (1996), 245]. Therefore, we have hypothesized that a change in expression of a fibroblast growth factor (FGF) at the entrance of the optic tectum, the border between the diencephalon and mesencephalon, may serve as a signal to RGC axons that they have reached their target. To determine whether RGC axons can sense changes in FGF levels, we asked whether they altered their behavior upon encountering an ectopic source of FGF. We found that in vivo RGC growth cones avoided FGF-misexpressing cells along their path, and that FGF-2 directly repelled RGC growth cones in an in vitro growth cone turning assay. These data support the idea that RGC axons can sense changes in FGF levels, and as such provide a mechanism by which FGFR signaling is involved in RGC axon target recognition.     

Differentiated neurons retain the capacity to generate axons from dendrites

Cutting the axon of a morphologically polarized neuron (stage 3) close to the cell body causes another neurite to grow as an axon [1-3]. Stage 3 neurons still lack molecular segregation of axonal and dendritic proteins, however. Axonal and dendritic compartments acquire their distinct composition at stage 4 (4-5days in culture), when proteins such as the microtubule-associated protein 2 (MAP-2) and the glutamate receptor subunit GluR1 localize to the dendrites and disappear from the axon [4,5]. We investigated whether cultured hippocampal neurons retained axon/dendrite plasticity after axons and dendrites have created their distinct cytoskeletal architecture and acquired their specific membrane composition. We found that axotomy of stage 4 neurons transformed a dendrite into an axon. Using axonal and dendritic markers, we tested whether cytoskeletal changes could cause similar transformations, and found that actin depolymerization induced multiple axons in unpolarized neurons. Moreover, depletion of actin filaments from both morphologically and molecularly polarized cells also resulted in the growth of multiple axons from pre-existing dendrites. These results imply that dendrites retain the potential to become axons even after molecular segregation has occurred and that the dendritic fate depends on the integrity of the actin cytoskeleton.