Rif-mDia1 interaction is involved in filopodium formation independent of Cdc42 and Rac effectors

Filopodia are cellular protrusions important for axon guidance, embryonic development, and wound healing. The Rho GTPase Cdc42 is the best studied inducer of filopodium formation, and several of its effectors and their interacting partners have been linked to the process. These include IRSp53, N-WASP, Mena, and Eps8. The Rho GTPase, Rif, also drives filopodium formation. The signaling pathway by which Rif induces filopodia is poorly understood, with mDia2 being the only protein implicated to date. It is thus not clear how distinct the Rif-driven pathway for filopodium formation is from the one mediated by Cdc42. In this study, we characterize the dynamics of Rif-induced filopodia by time lapse imaging of live neuronal cells and show that Rif drives filopodium formation via an independent pathway that does not involve the Cdc42 effectors N-WASP and IRSp53, the IRSp53 binding partner Mena, or the Rac effectors WAVE1 and WAVE2. Rif formed filopodia in the absence of N-WASP or Mena and when IRSp53, WAVE1, or WAVE2 was knocked down by RNAi. Rif-mediated filopodial protrusion was instead reduced by silencing mDia1 expression or overexpressing a dominant negative mutant of mDia1. mDia1 on its own was able to form filopodia. Data from acceptor photobleaching FRET studies of protein-protein interaction demonstrate that Rif interacts directly with mDia1 in filopodia but not with mDia2. Taken together, these results suggest a novel pathway for filopodia formation via Rif and mDia1.     

Stimulation-induced changes in filopodial dynamics determine the action radius of growth cones in the snail Helisoma trivolvis

Filopodia on neuronal growth cones constantly extend and retract, thereby functioning as both sensory probes and structural devices during neuronal pathfinding. To better understand filopodial dynamics and their regulation by encounters with molecules in the environment, we investigated filopodial dynamics of identified B5 neurons from the buccal ganglion of the snail Helisoma trivolvis before and after treatment with nitric oxide (NO). We have previously demonstrated that treatment with several NO-donors caused a transient, cGMP-mediated elevation in [Ca(2+)](i), which was causally related to an increase in filopodial length and a reduction in the number of filopodia on growth cones. We demonstrate here that these effects were the result of distinct changes in filopodial dynamics. The NO-donor SIN-1 induced a general increase in filopodial motility. Filopodial elongation after treatment with SIN-1 resulted from a significant increase in the rate at which filopodia extended, as well as a significant increase in the time filopodia spent elongating. The reduction in filopodial number was caused by a significant decrease in the frequency with which new filopodia were inserted into the growth cone. With the exception of the back where filopodia appeared less motile, filopodial dynamics appeared to be mostly independent of the location on the growth cone. These results suggest that NO can regulate filopodial dynamics on migrating growth cones and might function as a messenger to adjust the action radius of a growth cone during pathfinding.     

Synaptotagmin‐1 promotes the formation of axonal filopodia and branches along the developing axons of forebrain neurons

Synaptotagmin‐1 (syt1) is a Ca²⁺‐binding protein that functions in regulation of synaptic vesicle exocytosis at the synapse. Syt1 is expressed in many types of neurons well before synaptogenesis begins both in vivo and in vitro. To determine if expression of syt1 has a functional role in neuronal development before synapse formation, we examined the effects of syt1 overexpression and knockdown on the growth and branching of the axons of cultured primary embryonic day 8 chicken forebrain neurons. In vivo these neurons express syt1, and most have not yet extended axons. We present evidence that syt1 plays a role in regulating axon branching, while not regulating overall axon length. To study the effects of overexpression of syt1, we used adenovirus‐mediated infection to introduce a syt1‐YFP construct, or control GFP construct, into neurons. Syt1 levels were reduced using RNA interference. Overexpression of syt1 increased the formation of axonal filopodia and branches. Conversely, knockdown of syt1 decreased the number of axonal filopodia and branches. Time‐lapse analysis of filopodial dynamics in syt1‐overexpressing cells demonstrated that elevation of syt1 levels increased both the frequency of filopodial initiation and their lifespan. Taken together these data indicate that syt1 regulates the formation of axonal filopodia and branches before engaging in its conventional functions at the synapse. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

The Rho family GTPase Rif induces filopodia through mDia2

Eukaryotic cells produce a variety of specialized actin-rich surface protrusions. These include filopodia-thin, highly dynamic projections that help cells to sense their external environment. Filopodia consist of parallel filaments of actin, bundled by actin crosslinking proteins. The filaments are oriented with their rapidly growing "barbed" ends at the protruding tip and their slowly growing "pointed" ends at the base. Extension occurs by polymerization at the tip and is controlled by regulation of filament capping. The Rho GTPase Cdc42 is a key mediator of filopodia formation, which it regulates through binding CRIB domain-containing effectors. Cdc42 binds and activates the WASP proteins, which in turn activate the actin-nucleating complex Arp2/3. It also binds and activates IRSp53, which recruits the Ena/WASP family protein Mena to the filopodial tip and protects elongating actin filaments from capping. Previously, we identified another Rho family GTPase, Rif, as a potent stimulator of filopodial protrusion through a mechanism that does not require Cdc42. Here we characterize the differences between filopodia induced by these two small GTPases and show that the Rif effector in this pathway is the Diaphanous-related formin mDia2. Thus, Rif and Cdc42 represent two distinct routes to the induction of filopodia-producing structures with both shared and unique properties.     

Polarized targeting of peripheral membrane proteins in neurons

Differential targeting of neuronal proteins to axons and dendrites is essential for directional information flow within the brain, however, little is known about this protein-sorting process. Here, we investigate polarized targeting of lipid-anchored peripheral membrane proteins, postsynaptic density-95 (PSD-95) and growth-associated protein-43 (GAP-43). Whereas the N-terminal palmitoylated motif of PSD-95 is necessary but not sufficient for sorting to dendrites, the palmitoylation motif of GAP-43 is sufficient for axonal targeting and can redirect a PSD-95 chimera to axons. Systematic mutagenesis of the GAP-43 and PSD-95 palmitoylation motifs indicates that the spacing of the palmitoylated cysteines and the presence of nearby basic amino acids determine polarized targeting by these two motifs. Similarly, the axonal protein paralemmin contains a C-terminal palmitoylated domain, which resembles that of GAP-43 and also mediates axonal targeting. These axonally targeted palmitoylation motifs also mediate targeting to detergent-insoluble glycolipid-enriched complexes in heterologous cells, suggesting a possible role for specialized lipid domains in axonal sorting of peripheral membrane proteins.     

Dynamin1 Is a Novel Target for IRSp53 Protein and Works with Mammalian Enabled (Mena) Protein and Eps8 to Regulate Filopodial Dynamics

Filopodia are dynamic actin-based structures that play roles in processes such as cell migration, wound healing, and axonal guidance. Cdc42 induces filopodial formation through IRSp53, an Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain protein. Previous work from a number of laboratories has shown that IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics through its Src homology 3 domain binding partners. Here, we show that dynamin1 (Dyn1), the large guanosine triphosphatase, is an interacting partner of IRSp53 through pulldown and Förster resonance energy transfer analysis, and we explore its role in filopodial formation. In neuroblastoma cells, Dyn1 localizes to filopodia, associated tip complexes, and the leading edge just behind the anti-capping protein mammalian enabled (Mena). Dyn1 knockdown reduces filopodial formation, which can be rescued by overexpressing wild-type Dyn1 but not the GTPase mutant Dyn1-K44A and the loss-of-function actin binding domain mutant Dyn1-K/E. Interestingly, dynasore, an inhibitor of Dyn GTPase, also reduced filopodial number and increased their lifetime. Using rapid time-lapse total internal reflection fluorescence microscopy, we show that Dyn1 and Mena localize to filopodia only during initiation and assembly. Dyn1 actin binding domain mutant inhibits filopodial formation, suggesting a role in actin elongation. In contrast, Eps8, an actin capping protein, is seen most strongly at filopodial tips during disassembly. Taken together, the results suggest IRSp53 partners with Dyn1, Mena, and Eps8 to regulate filopodial dynamics.     

Short Lives with Long-Lasting Effects: Filopodia Protrusions in Neuronal Branching Morphogenesis

The branching behaviors of both dendrites and axons are part of a neuronal maturation process initiated by the generation of small and transient membrane protrusions. These are highly dynamic, actin-enriched structures, collectively called filopodia, which can mature in neurons to form stable branches. Consequently, the generation of filopodia protrusions is crucial during the formation of neuronal circuits and involves the precise control of an interplay between the plasma membrane and actin dynamics. In this issue of PLOS Biology, Hou and colleagues identify a Ca²⁺/CaM-dependent molecular machinery in dendrites that ensures proper targeting of branch formation by activation of the actin nucleator Cobl.     

Regulated tyrosine phosphorylation at the tips of growth cone filopodia

Several types of evidence suggest that protein-tyrosine phosphorylation is important during the growth of neuronal processes, but few specific roles, or subcellular localizations suggestive of such roles, have been defined. We report here a localization of tyrosine-phosphorylated protein at the tips of growth cone filopodia. Immunocytochemistry using a mAb to phosphorylated tyrosine residues revealed intense staining of the tips of most filopodia of Aplysia axons growing slowly on a polylysine substrate, but of few filopodia of axons growing rapidly on a substrate coated with Aplysia hemolymph, which has growth-promoting material. Cytochalasin D, which causes F-actin to withdraw rapidly from the growth cone, caused the tyrosine-phosphorylated protein to withdraw rapidly from filopodia, suggesting that the protein associates or interacts with actin filaments. Phosphotyrosine has previously been found concentrated at adherens junctions, where bundles of actin filaments terminate, but video-enhanced contrast-differential interference contrast and confocal interference reflection microscopy demonstrated that the filopodial tips were not adherent to the substrate. Acute application of either hemolymph or inhibitors of protein-tyrosine kinases to neurons on polylysine resulted in a rapid loss of intense staining at filopodial tips concomitant with a lengthening of the filopodia (and their core bundles of actin filaments). These results demonstrate that tyrosine-phosphorylated protein can be concentrated at the barbed ends of actin filaments in a context other than an adherens junction, indicate an association between changes in phosphorylation and filament dynamics, and provide evidence for tyrosine phosphorylation as a signaling mechanism in the filopodium that can respond to environmental cues controlling growth cone dynamics.     

Filopodial focal complexes direct adhesion and force generation towards filopodia outgrowth

Filopodia are key structures within many cells that serve as sensors constantly probing the local environment. Although filopodia are involved in a number of different cellular processes, their function in migration is often analyzed with special focus on early processes of filopodia formation and the elucidation of filopodia molecular architecture. An increasing number of publications now describe the entire life cycle of filopodia, with analyses from the initial establishment of stable filopodium-substrate adhesion to their final integration into the approaching lamellipodium. We and others can now show the structural and functional dependence of lamellipodial focal adhesions as well as of force generation and transmission on filopodial focal complexes and filopodial actin bundles. These results were made possible by new high resolution imaging techniques as well as by recently developed elastomeric substrates and theoretical models. The data additionally provide strong evidence that formation of new filopodia depends on previously existing filopodia through a repetitive filopodial elongation of the stably adhered filopodial tips. In this commentary we therefore hypothesize a highly coordinated mechanism that regulates filopodia formation, adhesion, protein composition and force generation in a filopodia dependent step by step process.     

Drebrin coordinates the actin and microtubule cytoskeleton during the initiation of axon collateral branches

Drebrin is a cytoskeleton‐associated protein which can interact with both actin filaments and the tips of microtubules. Its roles have been studied mostly in dendrites, and the functions of drebrin in axons are less well understood. In this study, we analyzed the role of drebrin, through shRNA‐mediated depletion and overexpression, in the collateral branching of chicken embryonic sensory axons. We report that drebrin promotes the formation of axonal filopodia and collateral branches in vivo and in vitro. Live imaging of cytoskeletal dynamics revealed that drebrin promotes the formation of filopodia from precursor structures termed axonal actin patches. Endogenous drebrin localizes to actin patches and depletion studies indicate that drebrin contributes to the development of patches. In filopodia, endogenous drebrin localizes to the proximal portion of the filopodium. Drebrin was found to promote the stability of axonal filopodia and the entry of microtubule plus tips into axonal filopodia. The effects of drebrin on the stabilization of filopodia are independent of its effects on promoting microtubule targeting to filopodia. Inhibition of myosin II induces a redistribution of endogenous drebrin distally into filopodia, and further increases branching in drebrin overexpressing neurons. Finally, a 30 min treatment with the branch‐inducing signal nerve growth factor increases the levels of axonal drebrin. This study determines the specific roles of drebrin in the regulation of the axonal cytoskeleton, and provides evidence that drebrin contributes to the coordination of the actin and microtubule cytoskeleton during the initial stages of axon branching. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1092–1110, 2016     

A Highlights from MBoC Selection: Cell type–dependent mechanisms for formin-mediated assembly of filopodia

Filopodia are finger-like protrusions from the plasma membrane and are of fundamental importance to cellular physiology, but the mechanisms governing their assembly are still in question. One model, called convergent elongation, proposes that filopodia arise from Arp2/3 complex–nucleated dendritic actin networks, with factors such as formins elongating these filaments into filopodia. We test this model using constitutively active constructs of two formins, FMNL3 and mDia2. Surprisingly, filopodial assembly requirements differ between suspension and adherent cells. In suspension cells, Arp2/3 complex is required for filopodial assembly through either formin. In contrast, a subset of filopodia remains after Arp2/3 complex inhibition in adherent cells. In adherent cells only, mDia1 and VASP also contribute to filopodial assembly, and filopodia are disproportionately associated with focal adhesions. We propose an extension of the existing models for filopodial assembly in which any cluster of actin filament barbed ends in proximity to the plasma membrane, either Arp2/3 complex dependent or independent, can initiate filopodial assembly by specific formins.     

Regulation of Dendritic Filopodial Interactions by ZO-1 and Implications for Dendrite Morphogenesis

Neuronal dendrites dynamically protrude many fine filopodia in the early stages of neuronal development and gradually establish complex structures. The importance of the dendritic filopodia in the formation of axo-dendritic connections is established, but their role in dendrite morphogenesis remains unknown. Using time-lapse imaging of cultured rat hippocampal neurons, we revealed here that many filopodia dynamically protruded from dendrites and transiently interacted with each other to form dendritic filopodia-filopodia contacts in the early stages of neuronal development. The MAGUK family member, Zonula Occludens-1 (ZO-1), which is known to be associated with the nectin and cadherin cell adhesion systems, was concentrated at these dendritic filopodia-filopodia contact sites and also at the tips of free dendritic filopodia. Overexpression of ZO-1 increased the formation of dendritic filopodia and their interactions, and induced abnormal dendrite morphology. Conversely, knockdown of ZO-1 decreased the formation of dendritic filopodia and their interactions, and induced abnormal dendrite morphology which was different from that induced by the overexpression of ZO-1. The components of the nectin and cadherin systems were co-localized with ZO-1 at the dendritic filopodia-filopodia contact sites, but not at the tips of free dendritic filopodia. Overexpression of ZO-1 increased the accumulation of these cell adhesive components at the dendritic filopodia-filopodia contact sites and stabilized their interactions, whereas knockdown of ZO-1 reduced their accumulation at the dendritic filopodia-filopodia contact sites. These results indicate that ZO-1 regulates dendritic filopodial dynamics, which is implicated in dendrite morphogenesis cooperatively with the nectin and cadherin systems in cultured neurons.     

Robust patterns in the stochastic organization of filopodia

Background  

Filopodia are actin-based cellular projections that have a critical role in initiating and sustaining directional migration in vertebrate cells. Filopodia are highly dynamic structures that show a rich diversity in appearance and behavior. While there are several mathematical models of filopodia initiation and growth, testing the capacity of these theoretical models in predicting empirical behavior has been hampered by a surprising shortage of quantitative data related to filopodia. Neither is it clear how quantitatively robust the cellular filopodial network is and how perturbations alter it.     

Autonomous regulation of growth cone filopodia

The fan-shaped array of filopodia is the first site of contact of a neuronal growth cone with molecules encountered during neuronal pathfinding. Filopodia are highly dynamic structures, and the “action radius” of a growth cone is strongly determined by the length and number of its filopodia. Since interactions of filopodia with instructive cues in the vicinity of the growth cone can have effects on growth cone morphology within minutes, it has to be assumed that a large part of the signaling underlying such morphological changes resides locally within the growth cone proper. In this study, we tested the hypothesis that two important growth cone parameters namely, the length and number of its filopodiaare regulated autonomously in the growth cone. We previously demonstrated in identified neurons from the snail Helisoma trivolvis that filopodial length and number are regulated by intracellular calcium. Here, we investigated filopodial dynamics and their regulation by the second-messenger calcium in growth cones which were physically isolated from their parent neuron by neurite transection. Our results show that isolated growth cones have longer but fewer filopodia than growth cones attached to their parent cell. These isolated growth cones, however, are fully capable of undergoing calcium-induced cytoskeletal changes, suggesting that the machinery necessary to perform changes in filopodial length and number is fully intrinsic to the growth cone proper. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 179–192, 1998     

Cdc42 participates in the regulation of ADF/cofilin and retinal growth cone filopodia by brain derived neurotrophic factor

Rho family GTPases have important roles in mediating the effects of guidance cues and growth factors on the motility of neuronal growth cones. We previously showed that the neurotrophin BDNF regulates filopodial dynamics on growth cones of retinal ganglion cell axons through activation of the actin regulatory proteins ADF and cofilin by inhibiting a RhoA-dependent pathway that phosphorylates (inactivates) ADF/cofilin. The GTPase Cdc42 has also been implicated in mediating the effects of positive guidance cues. In this article we investigated whether Cdc42 is involved in the effects of BDNF on filopodial dynamics. BDNF treatment increases Cdc42 activity in retinal neurons, and neuronal incorporation of constitutively active Cdc42 mimics the increases in filopodial number and length. Furthermore, constitutively active and dominant negative Cdc42 decreased and increased, respectively, the activity of RhoA in retinal growth cones, indicating crosstalk between these GTPases in retinal growth cones. Constitutively active Cdc42 mimicked the activation of ADF/cofilin that resulted from BDNF treatment, while dominant negative Cdc42 blocked the effects of BDNF on filopodia and ADF/cofilin. The inability of dominant negative Cdc42 to block ADF/cofilin activation and stimulation of filopodial dynamics by the ROCK inhibitor Y-27632 indicate interaction between Cdc42 and RhoA occurs upstream of ROCK. Our results demonstrate crosstalk occurs between GTPases in mediating the effects of BDNF on growth cone motility, and Cdc42 activity can promote actin dynamics via activation of ADF/cofilin.     

Ras regulates neuronal polarity via the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway

The establishment of a polarized morphology is an essential event in the differentiation of neurons into a single axon and dendrites. We previously showed that glycogen synthase kinase-3beta (GSK-3beta) is critical for specifying axon/dendrite fate by the regulation of the phosphorylation of collapsin response mediator protein-2 (CRMP-2). Here, we found that the overexpression of the small GTPase Ras induced the formation of multiple axons in cultured hippocampal neurons, whereas the ectopic expression of the dominant negative form of Ras inhibited the formation of axons. Inhibition of phosphatidylinositol-3-kinase (PI3-kinase) or extracellular signal-related kinase (ERK) kinase (MEK) suppressed the Ras-induced formation of multiple axons. The expression of the constitutively active form of PI3-kinase or Akt (also called protein kinase B) induced the formation of multiple axons. The overexpression of Ras prevented the phosphorylation of CRMP-2 by GSK-3beta. Taken together, these results suggest that Ras plays critical roles in establishing neuronal polarity upstream of the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway and mitogen-activated protein kinase cascade.     

Isoform-specific palmitoylation of JNK regulates axonal development

The c-jun N-terminal kinase (JNK) proteins are encoded by three genes (Jnk1-3), giving rise to 10 isoforms in the mammalian brain. The differential roles of JNK isoforms in neuronal cell death and development have been noticed in several pathological and physiological contexts. However, the mechanisms underlying the regulation of different JNK isoforms to fulfill their specific roles are poorly understood. Here, we report an isoform-specific regulation of JNK3 by palmitoylation, a posttranslational modification, and the involvement of JNK3 palmitoylation in axonal development and morphogenesis. Two cysteine residues at the COOH-terminus of JNK3 are required for dynamic palmitoylation, which regulates JNK3's distribution on the actin cytoskeleton. Expression of palmitoylation-deficient JNK3 increases axonal branching and the motility of axonal filopodia in cultured hippocampal neurons. The Wnt family member Wnt7a, a known modulator of axonal branching and remodelling, regulates the palmitoylation and distribution of JNK3. Palmitoylation-deficient JNK3 mimics the effect of Wnt7a application on axonal branching, whereas constitutively palmitoylated JNK3 results in reduced axonal branches and blocked Wnt7a induction. Our results demonstrate that protein palmitoylation is a novel mechanism for isoform-specific regulation of JNK3 and suggests a potential role of JNK3 palmitoylation in modulating axonal branching.     

Characterization of an extremely motile cellular network in the rotifer Asplanchna spp.

The pseudocoelomic body cavity of the rotifer Asplanchna spp. contains free cells that form a highly dynamic, three-dimensional polygonal network of filopodia. Using video-enhanced differential interference contrast microscopy, we have qualitatively and quantitatively characterized the motion types involved with network motility: (1) filopodial junctions are displaced laterally at 10.52 +/- 0.46 microns/s; (2) free-ending filopodia form and extend at rates of 8.77 +/- 0.40 microns/s, until they retract again at 7.23 +/- 0.87 microns/s; (3) filopodial strands fuse either laterally or tip to the lateral side. The combination of these motion types results in enlargements, diminutions, and extinctions of filopodial polygons, and in the formation of new polygons. Moreover, there is intense and fast (5.11 +/- 0.28 microns/s) particle transport within the filopodial strands. The organization of the cytoskeleton in filopodia was examined by electron microscopy and by labeling with fluorescent-tagged phalloidin. Filopodia contain several microtubules that are often organized in a bundle. Moreover, F-actin is present within the filopodia. To characterize which of these cytoskeletal systems is involved with cell and organelle motility, we have examined cell dynamics after incubations with colchicine or cytochalasin D. The results of these pharmacological experiments provide evidence that microtubules are required for both cell and organelle motility, but that actin filaments contribute to these phenomena and are required for the structural maintenance of slender filopodia.     

Induction of filopodia by direct local elevation of intracellular calcium ion concentration.

In neuronal growth cones, cycles of filopodial protrusion and retraction are important in growth cone translocation and steering. Alteration in intracellular calcium ion concentration has been shown by several indirect methods to be critically involved in the regulation of filopodial activity. Here, we investigate whether direct elevation of [Ca2+]i, which is restricted in time and space and is isolated from earlier steps in intracellular signaling pathways, can initiate filopodial protrusion. We raised [Ca2+]i level transiently in small areas of nascent axons near growth cones in situ by localized photolysis of caged Ca2+ compounds. After photolysis, [Ca2+]i increased from approximately 60 nM to approximately 1 microM within the illuminated zone, and then returned to resting level in approximately 10-15 s. New filopodia arose in this area within 1-5 min, and persisted for approximately 15 min. Elevation of calcium concentration within a single filopodium induced new branch filopodia. In neurons coinjected with rhodamine-phalloidin, F-actin was observed in dynamic cortical patches along nascent axons; after photolysis, new filopodia often emerged from these patches. These results indicate that local transient [Ca2+]i elevation is sufficient to induce new filopodia from nascent axons or from existing filopodia.     

Development of the dendritic branching pattern of the Medial Giant Interneuron in the grasshopper embryo

The embryonic development of the grasshopper's Medial Giant Interneuron (MGI) was examined by injecting the cell with the fluorescent dye Lucifer Yellow at a series of stages in its growth. Particular attention was given to the way in which this neuron constructs its stereotyped dendritic branching pattern. The MGI's dendrites originate as secondary processes which sprout at characteristic points along the neurite after the primary growth cone has passed. These processes then arborize to form a miniature version of their adult branching pattern before the end of embryonic life. While growing, the dendritic branches are covered with a radiant profusion of filopodia; however, these filopodia are ephemeral structures and disappear once the cell matures. By contrast there is no significant reduction in either the number or the spatial extent of the actual dendrites at any embryonic stage. This implies that the stereotyped branching pattern of the mature MGI is primarily determined by a precise pattern of initial growth, and that secondary pruning of branches does not play an important role in shaping the final form of this cell. The coordinate ingrowth of the first cercal sensory axons was examined by cobalt filling the embryonic nerve, and the means by which these sensory axons make their initial contacts with the MGI's dendrites is herein discussed. The following paper considers the degree to which this sensory innervation regulates dendritic growth and branching.