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This study unveils a redundant function for the endocytic proteins Eps15 and Eps15L1 in mouse embryo development and erythropoiesis, and a unique nonredundant role for Eps15L1 in the nervous system., EPS15 and its homologous EPS15L1 are endocytic accessory proteins. Studies in mammalian cell lines suggested that EPS15 and EPS15L1 regulate endocytosis in a redundant manner. However, at the organismal level, it is not known to which extent the functions of the two proteins overlap. Here, by exploiting various constitutive and conditional null mice, we report redundant and nonredundant functions of the two proteins. EPS15L1 displays a unique nonredundant role in the nervous system, whereas both proteins are fundamental during embryo development as shown by the embryonic lethality of -Eps15/Eps15L1-double KO mice. At the cellular level, the major process redundantly regulated by EPS15 and EPS15L1 is the endocytosis of the transferrin receptor, a pathway that sustains the development of red blood cells and controls iron homeostasis. Consequently, hematopoietic-specific conditional Eps15/Eps15L1-double KO mice display traits of microcytic hypochromic anemia, due to a cell-autonomous defect in iron internalization.

Summary Background Integrin-mediated adhesion of cells to the extracellular matrix (ECM) relies on the dynamic formation of focal adhesions (FAs), which are biochemical and mechanosensitive platforms composed of a large variety of cytosolic and transmembrane proteins. During migration, there is a constant turnover of ECM contacts that initially form as nascent adhesions at the leading edge, mature into FAs as actomyosin tension builds up, and are then disassembled at the cell rear, thus allowing for cell detachment. Although the mechanisms of FA assembly have largely been defined, the molecular circuitry that regulates their disassembly still remains elusive. Results Here, we show that RN-tre, a GTPase-activating protein (GAP) for Rabs including Rab5 and Rab43, is a novel regulator of FA dynamics and cell migration. RN-tre localizes to FAs and to a pool of Rab5-positive vesicles mainly associated with FAs undergoing rapid remodeling. We found that RN-tre inhibits endocytosis of β1, but not β3, integrins and delays the turnover of FAs, ultimately impairing β1-dependent, but not β3-dependent, chemotactic cell migration. All of these effects are mediated by its GAP activity and rely on Rab5. Conclusions Our findings identify RN-tre as the Rab5-GAP that spatiotemporally controls FA remodeling during chemotactic cell migration.

Insulin regulates glycaemia, lipogenesis and increases mRNA translation. Cells with reduced eukaryotic initiation factor 6 (eIF6) do not increase translation in response to insulin. The role of insulin-regulated translation is unknown. Here we show that reduction of insulin-regulated translation in mice heterozygous for eIF6 results in normal glycaemia, but less blood cholesterol and triglycerides. eIF6 controls fatty acid synthesis and glycolysis in a cell autonomous fashion. eIF6 acts by exerting translational control of adipogenic transcription factors like C/EBPβ, C/EBPδ and ATF4 that have G/C rich or uORF sequences in their 5′ UTR. The outcome of the translational activation by eIF6 is a reshaping of gene expression with increased levels of lipogenic and glycolytic enzymes. Finally, eIF6 levels modulate histone acetylation and amounts of rate-limiting fatty acid synthase (Fasn) mRNA. Since obesity, type 2 diabetes, and cancer require a Fasn-driven lipogenic state, we propose that eIF6 could be a therapeutic target for these diseases., Insulin enhances mRNA translation via the translation initiation factor eIF6. Here, Brina et al. show that insulin-mediated activation of eIF6 is associated with the selective translation of genes involved in glycolysis and lipid synthesis with characteristic G/C-rich and uORF sequences in their mRNA.

SUMMARY Dynamic modulation of the actin cytoskeleton is critical for synaptic plasticity, abnormalities of which are thought to contribute to mental illness and addiction. Here we report that mice lacking Eps8, a regulator of actin dynamics, are resistant to some acute intoxicating effects of ethanol and show increased ethanol consumption. In the brain, the N-methyl-Daspartate (NMDA) receptor is a major target of ethanol. We show that Eps8 is localized to postsynaptic structures and is part of the NMDA receptor complex. Moreover, in Eps8 null mice, NMDA receptor currents and their sensitivity to inhibition by ethanol are abnormal. In addition, Eps8 null neurons are resistant to the actin-remodeling activities of NMDA and ethanol. We propose that proper regulation of the actin cytoskeleton is a key determinant of cellular and behavioral responses to ethanol.

Sos-1, a guanine nucleotide exchange factor (GEF), eps8 and Abi1, two signaling proteins, and the lipid kinase phosphoinositide 3-kinase (PI3-K), assemble in a multimolecular complex required for Rac activation leading to actin cytoskeletal remodeling. Consistently, eps8 –/– fibroblasts fail to form membrane ruffles in response to growth factor stimulation. Surprisingly, eps8 null mice are healthy, fertile, and display no overt phenotype, suggesting the existence of functional redundancy within this pathway. Here, we describe the identification and characterization of a family of eps8-related proteins, comprising three novel gene products, named eps8L1, eps8L2, and eps8L3. Eps8Ls display collinear topology and 27–42% identity to eps8. Similarly to eps8, eps8Ls interact with Abi1 and Sos-1; however, only eps8L1 and eps8L2 activate the Rac-GEF activity of Sos-1, and bind to actin in vivo. Consistently, eps8L1 and eps8L2, but not eps8L3, localize to PDGF-induced, F-actin–rich ruffles and restore receptor tyrosine kinase (RTK)-mediated actin remodeling when expressed in eps8 –/– fibroblasts. Thus, the eps8Ls define a novel family of proteins responsible for functional redundancy in the RTK-activated signaling pathway leading to actin remodeling. Finally, the patterns of expression of eps8 and eps8L2 in mice are remarkably overlapping, thus providing a likely explanation for the lack of overt phenotype in eps8 null mice.

The assembly of 80S ribosomes requires joining of the 40S and 60S subunits, which is triggered by the formation of an initiation complex on the 40S subunit. This event is rate-limiting for translation, and depends on external stimuli and the status of the cell. Here we show that 60S subunits are activated by release of eIF6 (also termed p27BBP). In the cytoplasm, eIF6 is bound to free 60S but not to 80S. Furthermore, eIF6 interacts in the cytoplasm with RACK1, a receptor for activated protein kinase C (PKC). RACK1 is a major component of translating ribosomes, which harbour significant amounts of PKC. Loading 60S subunits with eIF6 caused a dose-dependent translational block and impairment of 80S formation, which were reversed by expression of RACK1 and stimulation of PKC in vivo and in vitro. PKC stimulation led to eIF6 phosphorylation, and mutation of a serine residue in the carboxy terminus of eIF6 impaired RACK1/PKC-mediated translational rescue. We propose that eIF6 release regulates subunit joining, and that RACK1 provides a physical and functional link between PKC signalling and ribosome activation.

Eps15 and Eps15R are related tyrosine kinase substrates, which have been implicated in endocytosis and synaptic vesicle recycling. Through the protein:protein interaction abilities of their EH domains, they establish a complex network of interactions with several proteins, including Numb, a protein necessary for neuronal cell fate specification. We analyzed the expression of Eps15 and Eps15R during murine development, at the time of active neurogenesis. The most striking difference was at the level of subcellular localization, with Eps15 present in the cytosol and on the plasma membrane, while Eps15R exhibited mainly a nuclear localization. Interesting topographical differences also emerged. In the 12.5 days post coitum neuroepithelium, Eps15 was expressed in the ventricular zone, which contains proliferating neuroblasts, whereas Eps15R was found only in postmitotic neurons. Conversely, both proteins were expressed in sensory and cranial ganglia. At later times, the expression of Eps15 and Eps15R was widely maintained in neuronal structures. In other tissues, Eps15 was first seen in the liver primordium and at low levels in choroid plexus, lung, kidney and intestine; later on the expression was maintained at high levels in epithelia. Nuclear staining of Eps15R was present in kidney, intestine, lung and liver, as well as in heart and pancreas.

Mechanotransduction in the mammalian auditory system depends on mechanosensitive channels in the hair bundles that project from the apical surface of the sensory hair cells. Individual stereocilia within each bundle contain a core of tightly packed actin filaments, whose length is dynamically regulated during development and in the adult. We show that the actin-binding protein epidermal growth factor receptor pathway substrate 8 (Eps8)L2, a member of the Eps8-like protein family, is a newly identified hair bundle protein that is localized at the tips of stereocilia of both cochlear and vestibular hair cells. It has a spatiotemporal expression pattern that complements that of Eps8. In the cochlea, whereas Eps8 is essential for the initial elongation of stereocilia, Eps8L2 is required for their maintenance in adult hair cells. In the absence of both proteins, the ordered staircase structure of the hair bundle in the cochlea decays. In contrast to the early profound hearing loss associated with an absence of Eps8, Eps8L2 null-mutant mice exhibit a late-onset, progressive hearing loss that is directly linked to a gradual deterioration in hair bundle morphology. We conclude that Eps8L2 is required for the long-term maintenance of the staircase structure and mechanosensory function of auditory hair bundles. It complements the developmental role of Eps8 and is a candidate gene for progressive age-related hearing loss.

Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220⁺ bone marrow cells, CD19⁻ thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis.

The tyrosine kinase receptor trkB is thought to mediate the biological actions of brain-derived neurotrophic factor. This receptor is expressed by a large variety of neurons during development. Truncated trkB molecules lacking the tyrosine kinase domain have also been described, but their functions remain elusive. In order to gain insight into their role, we studied the pattern of expression and properties of these truncated receptors in the chick embryo. mRNA coding for truncated trkB was detected already early during neurogenesis and in situ hybridisation experiments indicated that the expression was in non-neuronal cells, as previously observed in the brain of adult rodents. Ependymal and leptomeningeal cells expressing high levels of truncated trkB were found to completely surround the developing brain and the spinal cord throughout development. In the otic vesicle, mesenchymal cells expressing truncated trkB surround cells producing brain-derived neurotrophic factor, as well as neurons expressing trkB with its tyrosine kinase domain. Non-neuronal cells were found not to express trkB mRNA coding for the tyrosine kinase domain. Studies with radioiodinated brain-derived neurotrophic factor performed on frozen sections of the chick embryo revealed that non-neuronal cells expressing truncated trkB bind brain-derived neurotrophic factor with high affinity and selectivity. In addition, experiments with dissociated leptomeningeal cells revealed that binding is rapidly followed by selective internalisation of the ligand. These results suggest that truncated trkB molecules form an efficient and selective barrier preventing the diffusion of brain-derived neurotrophic factor and eliminating it by internalisation. This barrier is in place early during neu-rogenesis and might be necessitated by the multiplicity of developing structures producing brain-derived neu-rotrophic factor, as well as by the large number of different neuronal populations responding to brain-derived neu-rotrophic factor.

Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220(+) bone marrow cells, CD19(-) thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis.

Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells., Author Summary Mammalian hearing depends on mechanosensory inner and outer hair cells within the inner ear that convert sound vibrations into electrical signals. While inner hair cells are the primary sensory receptors, outer hair cells improve auditory sensitivity. Although we know that sensory cells of the auditory, visual and olfactory systems undergo a series of regulated physiological and morphological changes during development, relatively little is known about the molecular mechanisms that regulate the development of these cells. In this study, we find that the protein Eps8, which binds to the key structural protein actin and regulates cell growth and neural development, is an essential component of auditory hair cell development and function. We show that mice lacking Eps8 are profoundly deaf and that their mechanically sensitive hair bundles do not fully grow. However, we also show that the bundles retain their ability to transduce mechanical stimuli. Further study revealed that Eps8 has additional functions in the physiological maturation of inner hair cells and in their ability to transmit electrical information to the brain. Combined, our results provide evidence for the complex physiological role of Eps8 in hair cells and the reason why its absence causes profound deafness.

Stable expression of the tyrosine kinase receptor ErbB4 confers increased migratory behavior to the neuronal progenitor cell line ST14A, in response to neuregulin 1 (NRG1) stimulation. We used gene expression profiling analysis to identify transcriptional changes associated with higher migratory activity caused by the activation of a specific ErbB4 isoform, and found constitutive up-regulation of the epidermal growth factor receptor pathway substrate 8 (Eps8), a multimodular regulator of actin dynamics. We confirmed the increase of Eps8, both at the mRNA and at the protein level, in stable clones expressing two different ErbB4 isoforms, both characterized by high migratory activity. Using Transwell assays and experimental manipulation of Eps8 expression level, we demonstrated that Eps8 synergizes with ErbB4 to increase both basal and ligand induced cell migration, whereas siRNA mediated Eps8 silencing strongly impairs cell motility and NRG1 induced actin cytoskeleton remodeling. By transient knockdown of Eps8 through in vivo siRNA electroporation, followed by explant primary cultures, we demonstrated that Eps8 down-regulation affects migration of normal neuronal precursors. In conclusion, our data demonstrate that Eps8 is a key regulator of motility of neuronal progenitor cells expressing ErbB4, both in basal conditions and in response to external motogenic cues.

The unusual dual functions of the actin-binding protein EPS8 as an actin capping and actin bundling factor are mapped to distinct structural features of the protein and to distinct physiological activities in vivo., Actin capping and cross-linking proteins regulate the dynamics and architectures of different cellular protrusions. Eps8 is the founding member of a unique family of capping proteins capable of side-binding and bundling actin filaments. However, the structural basis through which Eps8 exerts these functions remains elusive. Here, we combined biochemical, molecular, and genetic approaches with electron microscopy and image analysis to dissect the molecular mechanism responsible for the distinct activities of Eps8. We propose that bundling activity of Eps8 is mainly mediated by a compact four helix bundle, which is contacting three actin subunits along the filament. The capping activity is mainly mediated by a amphipathic helix that binds within the hydrophobic pocket at the barbed ends of actin blocking further addition of actin monomers. Single-point mutagenesis validated these modes of binding, permitting us to dissect Eps8 capping from bundling activity in vitro. We further showed that the capping and bundling activities of Eps8 can be fully dissected in vivo, demonstrating the physiological relevance of the identified Eps8 structural/functional modules. Eps8 controls actin-based motility through its capping activity, while, as a bundler, is essential for proper intestinal morphogenesis of developing Caenorhabditis elegans., Author Summary One of the key components of the cytoskeleton of cells is actin, which allows cells to move. Actin-based motility is involved in many biological processes, such as intestinal development, intracellular trafficking and cell migration. Actin monomers are individual building blocks that can be linked together to form actin filaments. Numerous actin-binding proteins are involved in controlling the higher order architecture and dynamics of these actin filaments within cells. For example, actin capping proteins regulate actin dynamics by controlling the number of growing filament ends, and actin cross-linking or bundling proteins determine how to organize these filaments into higher order structures. The protein Eps8 is capable of capping as well as bundling actin filaments. However, the structural basis of this dual role of Eps8 remains unknown. In this study, we use a combination of techniques to unravel the molecular and structural basis of Eps8 interactions with actin filaments. We show that distinct structural modules of Eps8 are responsible for capping versus bundling activity, and we determine the contributions of these modules in vitro and in vivo. At the functional level, we find that Eps8 regulates actin-based motility and cellular trafficking through its capping activity, whereas Eps8-mediated bundling is essential for intestinal morphogenesis.

The regulation of filopodia plays a crucial role during neuronal development and synaptogenesis. Axonal filopodia, which are known to originate presynaptic specializations, are regulated in response to neurotrophic factors. The structural components of filopodia are actin filaments, whose dynamics and organization are controlled by ensembles of actin-binding proteins. How neurotrophic factors regulate these latter proteins remains, however, poorly defined. Here, using a combination of mouse genetic, biochemical, and cell biological assays, we show that genetic removal of Eps8, an actin-binding and regulatory protein enriched in the growth cones and developing processes of neurons, significantly augments the number and density of vasodilator-stimulated phosphoprotein (VASP)-dependent axonal filopodia. The reintroduction of Eps8 wild type (WT), but not an Eps8 capping-defective mutant, into primary hippocampal neurons restored axonal filopodia to WT levels. We further show that the actin barbed-end capping activity of Eps8 is inhibited by brain-derived neurotrophic factor (BDNF) treatment through MAPK-dependent phosphorylation of Eps8 residues S624 and T628. Additionally, an Eps8 mutant, impaired in the MAPK target sites (S624A/T628A), displays increased association to actin-rich structures, is resistant to BDNF-mediated release from microfilaments, and inhibits BDNF-induced filopodia. The opposite is observed for a phosphomimetic Eps8 (S624E/T628E) mutant. Thus, collectively, our data identify Eps8 as a critical capping protein in the regulation of axonal filopodia and delineate a molecular pathway by which BDNF, through MAPK-dependent phosphorylation of Eps8, stimulates axonal filopodia formation, a process with crucial impacts on neuronal development and synapse formation.

Truncated trkB.T1 is a splice variant of the neurotrophin receptor trkB. In spite of its abundance, and ability to bind and internalize BDNF, it is not clear whether it can transmit BDNF signaling. We tested this hypothesis by searching for proteins binding the evolutionarily conserved cyto-domain of trkB.T1, and by studying BDNF-induced changes of gene expression through DNA microarrays. Cells bearing trkB.T1 receptors presented morphological changes. However, no cytoplasmic interactors of trkB.T1 were found. In addition, BDNF-dependent modulation of gene expression was detected in cells bearing trkB.TK but not trkB.T1 receptors. These results suggest that the main function of trkB.T1 is to regulate local availability of neurotrophins and that it is unable to sense changes in BDNF availability.

During avian embryonic development, terminal erythroid differentiation occurs in the circulation. Some of the key events, such as the induction of erythroid 2,3-bisphosphoglycerate (2,3-BPG), carbonic anhydrase (CAII), and pyrimidine 5′-nucleotidase (P5N) synthesis are oxygen dependent (Baumann R, Haller EA, Schöning U, and Weber M, Dev Biol 116: 548–551, 1986; Dragon S and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 280: R870–R878, 2001; Dragon S, Carey C, Martin K, and Baumann R, J Exp Biol202: 2787–2795, 1999; Dragon S, Glombitza S, Götz R, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 271: R982–R989, 1996; Dragon S, Hille R, Götz R, and Baumann R, Blood 91: 3052–3058, 1998; Million D, Zillner P, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 261: R1188–R1196, 1991) in an indirect way: hypoxia stimulates the release of norepinephrine (NE)/adenosine into the circulation (Dragon et al., J Exp Biol 202: 2787–2795, 1999; Dragon et al., Am J Physiol Regulatory Integrative Comp Physiol 271: R982–R989, 1996). This leads via erythroid β-adrenergic/adenosine A2receptor activation to a cAMP signal inducing several proteins in a transcription-dependent manner (Dragon et al., Am J Physiol Regulatory Integrative Comp Physiol 271: R982–R989, 1996; Dragon et al., Blood 91: 3052–3058, 1998; Glombitza S, Dragon S, Berghammer M, Pannermayr M, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 271: R973–R981, 1996). To understand how the cAMP-dependent processes are initiated, we screened an erythroid cDNA library for cAMP-regulated genes. We detected three genes that were strongly upregulated (>5-fold) by cAMP in definitive and primitive red blood cells. They are homologous to the mammalian Tob, Ifr1, and Fos proteins. In addition, the genes are induced in the intact embryo during short-term hypoxia. Because the genes are regulators of proliferation and differentiation in other cell types, we suggest that cAMP might promote general differentiating processes in erythroid cells, thereby allowing adaptive modulation of the latest steps of erythroid differentiation during developmental hypoxia.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) selectively bind to distinct members of the Trk family of tyrosine kinase receptors, but all three bind with similar affinities to the neurotrophin receptor p75 (p75NTR). The biological significance of neurotrophin binding to p75NTR in cells that also express Trk receptors has been difficult to ascertain. In the absence of TrkA, NGF binding to p75NGR activated the transcription factor nuclear factor kappa B (NF-kappa B) in rat Schwann cells. This activation was not observed in Schwann cells isolated from mice that lacked p75NTR. The effect was selective for NGF; NF-kappa B was not activated by BDNF or NT-3.

In order to gain insight into potential roles of neurotrophins in Schwann cell biology, the expression of neurotrophin receptors of the trk gene family was investigated in rat sciatic nerve development. This analysis revealed differential regulation of truncated and full-length receptors. TrkA was undetectable even when analysed with a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) method. TrkB was present at the mRNA as well as protein level only in its truncated form. Surprisingly, multiple isoforms of trkC, including full-length forms, were detected in early postnatal nerve. Specific antibodies detected truncated and full-length trkC proteins in Western blotting, and RT-PCR revealed the presence of two full-length isoforms, one of them containing the 14 amino acid kinase insert. In situ hybridisation localized the expression of trkC to a subpopulation of Schwann cells. TrkC receptors are expressed already in nerves from day-16 embryos. In contrast to early postnatal stages, full-length trkC receptors are no longer expressed in adult nerves, which, however, maintain expression of truncated trkC transcripts. The presence of trkC kinases in peripheral nerve suggests a role for neurotrophin-3, the only known trkC ligand, in peripheral nerve development.