NORMAL GENOMIC

• Honigberg L, Kenyon C
• Establishment of left/right asymmetry in neuroblast migration by UNC-40/DCC, UNC-73/Trio and DPY-19 proteins in C. elegans.
• Development. 2000; 127: 4655-68
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• The bilateral C. elegans neuroblasts QL and QR are born in the same anterior/posterior (A/P) position, but polarize and migrate left/right asymmetrically: QL migrates toward the posterior and QR migrates toward the anterior. After their migrations, QL but not QR switches on the Hox gene mab-5. We find that the UNC-40/netrin receptor and a novel transmembrane protein DPY-19 are required to orient these cells correctly. In unc-40 or dpy-19 mutants, the Q cells polarize randomly; in fact, an individual Q cell polarizes in multiple directions over time. In addition, either cell can express MAB-5. Both UNC-40 and DPY-19, as well as the Trio/GTPase exchange factor homolog UNC-73, are required for full polarization and migration. Thus, these proteins appear to participate in a signaling system that orients and polarizes these migrating cells in a left/right asymmetrical fashion during development. The C. elegans netrin UNC-6, which guides many cells and axons along the dorsoventral axis, is not involved in Q cell polarization, suggesting that a different netrin-like ligand serves to polarize these cells along the anteroposterior axis.

• Yin Y, Sanes JR, Miner JH
• Identification and expression of mouse netrin-4.
• Mech Dev. 2000; 96: 115-9
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• Netrins are secreted proteins that serve as potent axon guidance molecules in vertebrates and invertebrates. We report the identification of a novel mammalian member of this family. Netrin-4 is similar in predicted size and secondary structure to the other three netrins; all contain, in order, an amino-terminal signal sequence, a laminin-type globular domain of the 'VI' type, three laminin-type epidermal growth factor (EGF) repeats, and a carboxyl-terminal 'netrin module'. In terms of primary sequence, however, netrin-4 is a distant relative of netrins-1-3, and its globular domain is more closely related to those of laminins than to those of other netrins. Netrin-4 is broadly expressed in both neural and non-neural tissues of embryonic and adult mice. In embryonic spinal cord, it is selectively expressed by cells at the lateral margins of the floor plate. In postnatal brain, it is selectively expressed in subsets of neurons, including cerebellar granule and hippocampal pyramidal cells.

• Su M et al.
• Regulation of the UNC-5 netrin receptor initiates the first reorientation of migrating distal tip cells in Caenorhabditis elegans.
• Development. 2000; 127: 585-94
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• Cell migrations play a critical role in animal development and organogenesis. Here, we describe a mechanism by which the migration behaviour of a particular cell type is regulated temporally and coordinated with over-all development of the organism. The hermaphrodite distal tip cells (DTCs) of Caenorhabditis elegans migrate along the body wall in three sequential phases distinguished by the orientation of their movements, which alternate between the anteroposterior and dorsoventral axes. The ventral-to-dorsal second migration phase requires the UNC-6 netrin guidance cue and its receptors UNC-5 and UNC-40, as well as additional, UNC-6-independent guidance systems. We provide evidence that the transcriptional upregulation of unc-5 in the DTCs is coincident with the initiation of the second migration phase, and that premature UNC-5 expression in these cells induces precocious turning in an UNC-6-dependent manner. The DAF-12 steroid hormone receptor, which regulates developmental stage transitions in C. elegans, is required for initiating the first DTC turn and for coincident unc-5 upregulation. We also present evidence for the existence of a mechanism that opposes or inhibits UNC-5 function during the longitudinal first migration phase and for a mechanism that facilitates UNC-5 function during turning. The facilitating mechanism presumably does not involve transcriptional regulation of unc-5 but may represent an inhibition of the phase 1 mechanism that opposes or inhibits UNC-5. These results, therefore, reveal the existence of two mechanisms that regulate the UNC-5 receptor that are critical for responsiveness to the UNC-6 netrin guidance cue and for linking the directional guidance of migrating distal tip cells to developmental stage advancements.

• Goodwin RG
• CDw137.
• J Biol Regul Homeost Agents. 2000; 14: 139-41

• Roy PJ, Zheng H, Warren CE, Culotti JG
• mab-20 encodes Semaphorin-2a and is required to prevent ectopic cell contacts during epidermal morphogenesis in Caenorhabditis elegans.
• Development. 2000; 127: 755-67
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• The Semaphorins are a family of secreted and transmembrane proteins known to elicit growth cone repulsion and collapse. We made and characterized a putative null mutant of the C. elegans gene semaphorin-2a (Ce-sema-2a). This mutant failed to complement mutants of mab-20 (Baird, S. E., Fitch, D. H., Kassem, I. A. A. and Emmons, S. W. (1991) Development 113, 515-526). In addition to low-frequency axon guidance errors, mab-20 mutants have unexpected defects in epidermal morphogenesis. Errant epidermal cell migrations affect epidermal enclosure of the embryo, body shape and sensory rays of the male tail. These phenotypic traits are explained by the formation of inappropriate contacts between cells of similar type and suggest that Ce-Sema-2a may normally prevent formation or stabilization of ectopic adhesive contacts between these cells.

• Meyerhardt JA et al.
• Netrin-1: interaction with deleted in colorectal cancer (DCC) and alterations in brain tumors and neuroblastomas.
• Cell Growth Differ. 1999; 10: 35-42
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• Netrins, a family of laminin-related secreted proteins, have critical roles in axon guidance and cell migration during development. The deleted in colorectal cancer (DCC) protein has been implicated as a netrin-1 receptor component. The expression and function of netrins in adult tissues remain unknown, and direct interaction of netrin-1 with DCC has not been demonstrated. We cloned the human netrin-1 (NTN1L) gene, mapped it to chromosome 17p12-13, and found that it encodes a 604 amino acid protein with 98% identity to mouse netrin-1 and 50% identity with the Caenorhabditis elegans UNC-6 protein. NTN1L transcripts were detected in essentially all normal adult tissues studied, and markedly reduced or absent NTN1L expression was seen in approximately 50% of brain tumors and neuroblastomas. In one neuroblastoma, missense mutations at highly conserved NTN1L codons were found. Netrin-1 protein could be cross-linked to DCC protein on the cell surface, but it did not immunoprecipitate with DCC in the absence of cross-linking and it failed to bind to a soluble fusion protein containing the entire DCC extracellular domain. Our findings demonstrating NTN1L loss of expression and mutations suggest that NTN1L alterations may contribute to the development of some cancers. Furthermore, the binding of netrin-1 to DCC appears to depend on the presence of a coreceptor or accessory proteins.

• Hong K, Hinck L, Nishiyama M, Poo MM, Tessier-Lavigne M, Stein E
• A ligand-gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion.
• Cell. 1999; 97: 927-41
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• Netrins are bifunctional: they attract some axons and repel others. Netrin receptors of the Deleted in Colorectal Cancer (DCC) family are implicated in attraction and those of the UNC5 family in repulsion, but genetic evidence also suggests involvement of the DCC protein UNC-40 in some cases of repulsion. To test whether these proteins form a receptor complex for repulsion, we studied the attractive responses of Xenopus spinal axons to netrin-1, which are mediated by DCC. We show that attraction is converted to repulsion by expression of UNC5 proteins in these cells, that this repulsion requires DCC function, that the UNC5 cytoplasmic domain is sufficient to effect the conversion, and that repulsion can be initiated by netrin-1 binding to either UNC5 or DCC. The isolated cytoplasmic domains of DCC and UNC5 proteins interact directly, but this interaction is repressed in the context of the full-length proteins. We provide evidence that netrin-1 triggers the formation of a receptor complex of DCC and UNC5 proteins and simultaneously derepresses the interaction between their cytoplasmic domains, thereby converting DCC-mediated attraction to UNC5/DCC-mediated repulsion.

• Ren XC, Kim S, Fox E, Hedgecock EM, Wadsworth WG
• Role of netrin UNC-6 in patterning the longitudinal nerves of Caenorhabditis elegans.
• J Neurobiol. 1999; 39: 107-18
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• The nervous system of Caenorhabditis elegans comprises circumferential and longitudinal axon tracts. Netrin UNC-6 is required for the guidance of circumferential axon migrations and is expressed by ventral neuroglia and neurons in temporally and spatially regulated patterns. Migrating axons mediate the UNC-6 signal through the UNC-5 and UNC-40 receptors. It is thought that UNC-6 is secreted and becomes associated with basement membranes and cell surfaces to form gradients that direct circumferentially migrating axons toward or away from the ventral UNC-6 sources. Little is known about the effects of UNC-6 on longitudinally migrating axons. In unc-6, unc-5, and unc-40 null mutants, some longitudinal nerves are dorsally or ventrally misdirected. Furthermore, the organization of axons are disrupted within nerves. We show that cells ectopically expressing UNC-6 can redirect the migrations of some neighboring longitudinal axons, suggesting that the gradients postulated to direct circumferential migration also help specify the dorsoventral positions of these longitudinal nerves. We also manipulated the temporal and spatial expression pattern of UNC-6 by two different means. First, we removed the PVT midline neuron which expresses UNC-6 for a short time during axon outgrowths. Second, we expressed UNC-6 uniformly in the nervous system throughout development. The results suggest that changing UNC-6 expression patterns modify the distribution of the cue by providing new localized sources. This new guidance information is critical for organizing the axons of longitudinal nerves.

• Kim S, Ren XC, Fox E, Wadsworth WG
• SDQR migrations in Caenorhabditis elegans are controlled by multiple guidance cues and changing responses to netrin UNC-6.
• Development. 1999; 126: 3881-90
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• The netrin guidance cue, UNC-6, and the netrin receptors, UNC-5 and UNC-40, guide SDQR cell and axon migrations in C. elegans. In wild-type larvae, SDQR migrations are away from ventral UNC-6-expressing cells, suggesting that UNC-6 repels SDQR. In unc-6 null larvae, SDQR migrations are towards the ventral midline, indicating a response to other guidance cues that directs the migrations ventrally. Although ectopic UNC-6 expression dorsal to the SDQR cell body would be predicted to cause ventral SDQR migrations in unc-6 null larvae, in fact, more migrations are directed dorsally, suggesting that SDQR is not always repelled from the dorsal source of UNC-6. UNC-5 is required for dorsal SDQR migrations, but not for the ventral migrations in unc-6 null larvae. UNC-40 appears to moderate both the response to UNC-6 and to the other cues. Our results show that SDQR responds to multiple guidance cues and they suggest that, besides UNC-6, other factors influence whether an UNC-6 responsive cell migrates toward or away from an UNC-6 source in vivo. We propose that multiple signals elicited by the guidance cues are integrated and interpreted by SDQR and that the response to UNC-6 can change depending on the combination of cues encountered during migration. These responses determine the final dorsoventral position of the SDQR cell and axon.

• PuschelPuschel AW
• Divergent properties of mouse netrins.
• Mech Dev. 1999; 83: 65-75
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• The netrins are a small but highly conserved family of axonal guidance signals found throughout the animal kingdom. This sequence conservation was used to isolated cDNAs for two mouse netrins. Analysis of their expression patterns and functional properties showed that mouse netrin-1 is in most respects similar to its orthologs in other vertebrates while the properties of netrin-3 differ markedly from those of other members of this protein family. In contrast to netrin-1 which is widely expressed both in the developing nervous system and in mesodermal tissues, netrin-3 transcripts are largely restricted to dorsal root ganglia and the developing limb buds. Netrin-3 binds with a significantly lower affinity to the netrin receptor DCC (deleted in colorectal cancer) and is also ineffective in eliciting the outgrowth of commissural axons in vitro. These results demonstrate that, although the netrins are highly conserved signals that guide axons to or away from the midline of the developing nervous system, at the same time they show a surprising degree of divergence in vertebrates.

• Ackerman SL, Knowles BB
• Cloning and mapping of the UNC5C gene to human chromosome 4q21-q23.
• Genomics. 1998; 52: 205-8
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• The vertebrate Unc5 genes, like their Caenorhabditis elegans counterpart, define a family of putative netrin receptors. One member of this family, Unc5h3, has been shown to have an important role during cell migration in the developing murine cerebellum. Mice homozygous for mutations in Unc5h3 are ataxic and have cerebellar hypoplasia and laminar structure defects. In addition, these mice have ectopic granule and Purkinje cells in the midbrain and brainstem. We have identified the human homologue of this gene, UNC5C, and shown it to have a restricted expression pattern in adult human tissues. By radiation hybrid analysis, we have determined that UNC5C localizes to chromosome 4q21-q23 between markers D4S1557 and D4S836 and is closely linked to the Parkinson disease gene.

• Hapner SJ, Boeshore KL, Large TH, Lefcort F
• Neural differentiation promoted by truncated trkC receptors in collaboration with p75(NTR).
• Dev Biol. 1998; 201: 90-100
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• trkC receptors, which serve critical functions during the development of the nervous system, are alternatively spliced to yield isoforms containing the catalytic tyrosine kinase domain (TK+) and truncated isoforms which lack this domain (TK-). To test for potential differences in their roles during early stages of neural development, TK+ and TK- isoforms were ectopically expressed in cultures of neural crest, the stem cell population that gives rise to the vast majority of the peripheral nervous system. NT-3 activation of ectopically expressed trkC TK+ receptors promoted both proliferation of neural crest cells and neuronal differentiation. Strikingly, the trkC TK- isoform was significantly more effective at promoting neuronal differentiation, but had no effect on proliferation. Furthermore, the trkC TK- response was dependent on a conserved receptor cytoplasmic domain and required the participation of the p75(NTR) neurotrophin receptor. Antibody-mediated receptor dimerization of TK+ receptors, but not TK- receptors, was sufficient to stimulate differentiation. These data identify a phenotypic response to activation of the trkC TK- receptor and demonstrate a functional interaction with p75(NTR), indicating there may be multiple trkC receptor-mediated systems guiding neuronal differentiation.

• Byk T, Ozon S, Sobel A
• The Ulip family phosphoproteins--common and specific properties.
• Eur J Biochem. 1998; 254: 14-24
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• The search for intracellular phosphoproteins implicated in the regulation of neuronal differentiation led to the identification of Ulip1, a mammalian protein related to the Caenorhabditis elegans unc-33 gene product [Byk, T., Dobransky, T., Cifuentes-Diaz, C. & Sobel, A. (1996) J. Neurosc. 16, 688-701]. The expression level and phosphorylation pattern of Ulip1 were shown to be strongly regulated during development and neuronal differentiation. We have isolated three additional complete coding sequences for members of the Ulip family in the mouse, Ulips 2-4, all preferentially expressed in the nervous system. Furthermore, two Ulip sequences, Ulips A and Ulips B, could be identified in C. elegans. The Ulip family is highly conserved throughout evolution (more than 96 % for Ulips 1-3 and 92.5 % for Ulip4 between mouse and human) and the various members of the family within a single species display about 75% similarity. Sequence comparisons further reveal several highly similar domains and subdomains, including a 32-amino-acid region highly conserved from a bacterial hydantoinase to human Ulips. Two-dimensional immunoblot analysis of in vitro translated Ulips 1-4 demonstrates the existence, for each Ulip protein, of several, most probably differentially phosphorylated forms, in agreement with the presence of conserved phosphorylation consensus sites within their sequences. The expression of Ulips 1-4 mRNAs is differentially regulated during development and nerve-growth-factor-induced neuronal differentiation of PC12 cells. Our results indicate a differential, possibly complementary role of phosphoproteins of the highly conserved Ulip family in the control of neuronal differentiation, in relation with the development and plasticity of the nervous system.

• Colavita A, Culotti JG
• Suppressors of ectopic UNC-5 growth cone steering identify eight genes involved in axon guidance in Caenorhabditis elegans.
• Dev Biol. 1998; 194: 72-85
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• The UNC-5 guidance receptor, in response to the UNC-6/netrin path cue, orients growing axons in a dorsal direction along the epidermis of Caenorhabditis elegans. When ectopically expressed in the touch neurons, which normally extend ventrally or longitudinally, UNC-5 is able to reorient their axons toward the dorsal side in an UNC-6-dependent manner. This forms the basis of a genetic screen to identify other mutations that, like unc-6 mutations, suppress unc-5-induced growth cone guidance. These mutations may identify new components required for pioneer axon guidance by unc-5. In this paper, we describe eight genes that are required for ectopic unc-5-induced growth cone steering. Mutations in four of these identify the previously known axon guidance genes unc-6, unc-40, unc-34, and unc-44 and mutations in four others identify the novel genes unc-129, seu-1, seu-2, and seu-3. Several of these mutations cause axon guidance defects similar to those found in unc-5 mutants. We propose that some or all of these genes may function in a developmentally important unc-5 signaling pathway.

• van Kesteren RE et al.
• Early evolutionary origin of the neurotrophin receptor family.
• EMBO J. 1998; 17: 2534-42
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• Neurotrophins and their Trk receptors play a crucial role in the development and maintenance of the vertebrate nervous system, but to date no component of this signalling system has been found in invertebrates. We describe a molluscan Trk receptor, designated Ltrk, from the snail Lymnaea stagnalis. The full-length sequence of Ltrk reveals most of the characteristics typical of Trk receptors, including highly conserved transmembrane and intracellular tyrosine kinase domains, and a typical extracellular domain of leucine-rich motifs flanked by cysteine clusters. In addition, Ltrk has a unique N-terminal extension and lacks immunoglobulin-like domains. Ltrk is expressed during development in a stage-specific manner, and also in the adult, where its expression is confined to the central nervous system and its associated endocrine tissues. Ltrk has the highest sequence identity with the TrkC mammalian receptor and, when exogenously expressed in fibroblasts or COS cells, binds human NT-3, but not NGF or BDNF, with an affinity of 2.5 nM. These findings support an early evolutionary origin of the Trk family as neuronal receptor tyrosine kinases and suggest that Trk signalling mechanisms may be highly conserved between vertebrates and invertebrates.

• Bredesen DE et al.
• p75NTR and the concept of cellular dependence: seeing how the other half die.
• Cell Death Differ. 1998; 5: 365-71
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• Cells depend on specific stimuli, such as trophic factors, for survival and in the absence of such stimuli, undergo apoptosis. How do cells initiate apoptosis in response to the withdrawal of trophic factors or other dependent stimuli? Recent studies of apoptosis induction by neurotrophin withdrawal argue for a novel form of pro-apoptotic signal transduction - 'negative signal transduction' - in which the absence of ligand-receptor interaction induces cell death. We have found that the prototype for this form of signaling - the common neurotrophin receptor, p75NTR - creates a state of cellular dependence (or addiction) on neurotrophins, and that this effect requires an 'addiction/dependence domain' (ADD) in the intracytoplasmic region of p75NTR. We have recently found other receptors that include dependence domains, arguing that dependence receptors, and their associated dependence domains, may be involved in a rather general mechanism to create cellular states of dependence on trophic factors, cytokines, adhesion, electrical activity and other dependent stimuli.

• Colavita A, Krishna S, Zheng H, Padgett RW, Culotti JG
• Pioneer axon guidance by UNC-129, a C. elegans TGF-beta.
• Science. 1998; 281: 706-9
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• The unc-129 gene, like the unc-6 netrin gene, is required to guide pioneer motoraxons along the dorsoventral axis of Caenorhabditis elegans. unc-129 encodes a member of the transforming growth factor-beta (TGF-beta) superfamily of secreted signaling molecules and is expressed in dorsal, but not ventral, rows of body wall muscles. Ectopic expression of UNC-129 from ventral body wall muscle disrupts growth cone and cell migrations that normally occur along the dorsoventral axis. Thus, UNC-129 mediates expression of dorsoventral polarity information required for axon guidance and guided cell migrations in C. elegans.

• Martin SC, Sandell JH, Heinrich G
• Zebrafish TrkC1 and TrkC2 receptors define two different cell populations in the nervous system during the period of axonogenesis.
• Dev Biol. 1998; 195: 114-30
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• We identified previously five distinct trk genes in the zebrafish. The structures of two of these, TrkC1 and TrkC2, are most similar to mammalian TrkC. Detailed sequence comparisons reported here indicate that although the similarities to TrkC are greatest in those regions of the extracellular domain implicated in ligand binding, the two sequences also differ significantly in these regions. Whole-mount in situ hybridization experiments in the early embryo revealed full-length trkC1 but no trkC2 transcripts in the cranial ganglia and in a subset of Rohon-Beard neurons. At the same time, full-length trkC2 but no trkC1 transcripts were detected laterally in the spinal cord, in the caudal hindbrain, in reticulospinal neurons of rhombomeres 4, 5, and 6, and in the midbrain. Both types of transcripts were expressed in clusters of cells in the dorsal telencephalon and the nucleus of the tract of the postoptic commissure. These results suggest distinct functions of trkC1 and trkC2 in nervous system development. The expression patterns define two different neuronal populations in the zebrafish.

• Nocentini G, Giunchi L, Ronchetti S, Bartoli A, Migliorati G, Riccardi C
• Glucocorticoids: regulation of gene expression and apoptosis.
• J Chemother. 1998; 10: 187-91

• Kamata T et al.
• Isolation and characterization of a bovine neural specific protein (CRMP-2) cDNA homologous to unc-33, a C. elegans gene implicated in axonal outgrowth and guidance.
• Brain Res Mol Brain Res. 1998; 54: 219-36
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• We have cloned the cDNA encoding bovine CRMP-2 from bovine brains. A full length cDNA encoding bovine CRMP-2 was isolated and sequenced. The deduced amino acid sequence reveals that the gene encodes a protein of 572 amino acids and is highly homologous to Caenorhabditis elegans unc-33, which controls the guidance and outgrowth of neuronal axons. The CRMP-2 transcript was present in bovine brains but not non-neural tissues, and its protein product existed in both soluble and membrane-bound forms. The expression of CRMP-2 protein and mRNA was upregulated during neuronal differentiation of rat PC12 cells. Immunoprecipitation of PC12 cell extracts shows that CRMP-2 was co-immunoprecipitated with a 190 kDa protein (p190). Both CRMP-2 and p190 were phosphorylated on serine residues in vivo and in vitro in a kinase assay of CRMP-2 immune complexes. Immunocytochemistry shows that CRMP-2 was exclusively localized in both the central and peripheral nervous systems in mouse embryos and detectable in the adult brain although the level of CRMP-2 decreased. The protein was expressed in the axon, dendrite, and cytoplasm of postmitotic neurons and in the cytoplasm of oligodendrocytes and astrocytes. The CRMP-2 gene maps to the region of mouse chromosome 14 syntenic with human chromosome 8p21. Taken together, these data suggest multi-functional roles for CRMP-2 in developing and adult nervous systems, and the biological activity of CRMP-2 could be regulated by phosphorylation reaction.

• Peterson DA, Leppert JT, Lee KF, Gage FH
• Basal forebrain neuronal loss in mice lacking neurotrophin receptor p75.
• Science. 1997; 277: 837-9

• Mandich P et al.
• Comments on Davar et al., Pain, 67 (1996) 135-139.
• Pain. 1997; 72: 287-8

• Ninkina N, Grashchuck M, Buchman VL, Davies AM
• TrkB variants with deletions in the leucine-rich motifs of the extracellular domain.
• J Biol Chem. 1997; 272: 13019-25
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• We have isolated two novel variants involving the extracellular domain of TrkB from developing sensory neurons. These variants are generated by alternative splicing and lack two or all three of the leucine-rich motifs. Each of these variants is expressed as isoforms that possess or lack the intracellular tyrosine kinase domain. Fibroblast cell lines stably expressing these variants do not bind any of the TrkB ligands (brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5) and neither survive nor undergo morphological transformation in response to neurotrophins. These results demonstrate that the leucine-rich motifs in TrkB are essential for ligand binding and signaling and indicate that the extracellular immunoglobulin-like domains alone are insufficient to confer neurotrophin binding to TrkB.

• Livesey FJ, Hunt SP
• Netrin and netrin receptor expression in the embryonic mammalian nervous system suggests roles in retinal, striatal, nigral, and cerebellar development.
• Mol Cell Neurosci. 1997; 8: 417-29
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• The netrins are laminin-like axon guidance molecules that are conserved among Caenorhabditis elegans, Drosophila, and vertebrates and that have chemoattractive and chemorepellant properties. To study the possible actions of this gene family in the developing and adult mammalian nervous systems, we have cloned a partial cDNA which corresponds to a region conserved among chick netrin-1, netrin-2, and unc-6 and studied its expression and that of a netrin receptor, dcc, the deleted in colorectal cancer gene, in the developing and adult rat CNS. The localization of cells expressing netrin or dcc suggests that these genes, in addition to their actions in defining the ventral midline, may act in controlling retinal ganglion cell axon guidance in the optic nerve, cell migration in the developing cerebellum and olfactory epithelium, and development and maintenance of connections to the substantia nigra and corpus striatum.

• Yeo TT et al.
• Deficient LAR expression decreases basal forebrain cholinergic neuronal size and hippocampal cholinergic innervation.
• J Neurosci Res. 1997; 47: 348-60
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• A role in neural development for protein tyrosine phosphatase (PTPase) receptors has been suggested by the finding of aberrant neurite outgrowth in Drosophila mutants lacking functional leukocyte common antigen-related (LAR) PTPase receptors; however, PTPase functions in the mammalian nervous system remain to be established. In transgenic mice containing a gene trap in the LAR gene, only trace expression of full-length LAR transcripts was found. In these mice, the size of basal forebrain cholinergic neurons was significantly reduced and cholinergic innervation of the dentate gyrus was markedly decreased. These findings constitute the first demonstration of an aberrant neuronal phenotype in a mammalian PTPase mutant and support the hypothesis that LAR-type PTPase receptors function to establish and/or maintain neuronal networks.

• Drescher U
• Netrins find their receptor.
• Nature. 1996; 384: 416-7

• Byk T, Dobransky T, Cifuentes-Diaz C, Sobel A
• Identification and molecular characterization of Unc-33-like phosphoprotein (Ulip), a putative mammalian homolog of the axonal guidance-associated unc-33 gene product.
• J Neurosci. 1996; 16: 688-701
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• The control of neuritic extension and guidance is critical for the development, maturation, and regeneration of functional neuronal circuits. We identified a neuronal 64-85 kDa phosphoprotein, the expression of which in mouse brain is regulated during development, reaching a peak at approximately 5 d postnatal, when maturation of neurons and synaptic connections is highly active. The amino acid sequence of the mouse protein deduced from its cloned cDNA reveals similarities with that of the neuritic outgrowth- and guidance-related product of the unc-33 gene in Caenorhabditis elegans. The regulation of its phosphorylation in response to nerve growth factor, as well as its localization in neurites and growth cones and at the neuromuscular junction, further indicates that Ulip (for Unc-33-like phosphoprotein) is not only a structural but likely is also a functional mammalian homolog of Unc-33, potentially involved in the control of neuritic outgrowth and axonal guidance.

• Wadsworth WG, Bhatt H, Hedgecock EM
• Neuroglia and pioneer neurons express UNC-6 to provide global and local netrin cues for guiding migrations in C. elegans.
• Neuron. 1996; 16: 35-46
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• Netrins are laminin-related proteins that guide circumferential migrations on the ectoderm. To understand how netrin cues direct cell movements, we examined the expression of nematode netrin UNC-6 from embryo to adult. UNC-6 is expressed in 12 types of neuroglia and neurons, creating a hierarchy of netrin cues in the developing nervous system. Comparing gene expression pattern with in vivo phenotypes, we suggest how multiple netrin cues, each with a characteristic role, guide cells and axons during development. We also present the molecular analysis of selective loss-of-function and null alleles. The results indicate that the biological activities of netrins are mediated through distinct protein domains. Subtle mutations in domain VI can produce selective defects in both direction- and tissue-specific guidance. EGF-like module V-2 is essential for dorsal guidance activity; we infer this module is important for interactions between UNC-6 and the dorsal guidance receptor UNC-5.

• Parhad IM, Scott JN, Cellars LA, Bains JS, Krekoski CA, Clark AW
• Axonal atrophy in aging is associated with a decline in neurofilament gene expression.
• J Neurosci Res. 1995; 41: 355-66
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• Neurofilaments (Nfs) are major determinants of axonal caliber. Nf transcript levels increase during development and maturation, and are associated with an increase in Nf protein, Nf numbers, and caliber of axons. With aging there is axonal atrophy. In this study we asked whether the axonal atrophy of aging was associated with a decline in Nf transcript expression, Nf protein levels, and Nf numbers. Expression of transcripts for the three Nf subunits was evaluated in dorsal root ganglia (DRG) of Fischer-344 rats aged 3-32 months by Northern and in situ hybridization. There was an approximately 50% decrease in Nf subunit mRNA levels in DRG of aged (> 23 months) as compared to young and mature (3 and 12 months) rats, whereas expression of another neuronal mRNA, GAP-43, showed no decline. Western analysis showed a corresponding decrease in Nf subunit proteins and no decline in GAP-43. Morphometric analysis showed a 50% decrease in Nf numbers within axons. The decrease in Nf gene expression and Nf numbers was accompanied by a decrease in cross-sectional area and circularity of all myelinated fibers, with the largest fibers showing the most marked changes, and a shrinkage in the perikaryal area of large neurons. Furthermore, we found a concomitant decrease in the expression of transcripts for the nerve growth factor receptors trkA and p75 with aging. Although the mechanisms leading to the decrease in Nf gene expression with aging are not known, a decrease in the availability of growth factors, or the neuron's ability to respond to them, may play a role in this process.

• Martin SC, Marazzi G, Sandell JH, Heinrich G
• Five Trk receptors in the zebrafish.
• Dev Biol. 1995; 169: 745-58
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• Using a homology-based cloning strategy we have identified five members of the Trk family in the zebrafish Danio rerio. They are homologous to the three mammalian Trk receptors in their conserved intracellular kinase regions and the organization of their extracellular regions. The five trk genes are differentially expressed in the developing brain, spinal cord, cranial ganglia, and retina. Full-length forms of three of the trk genes are expressed when neurons pioneer the major axon tracts, whereas the two other trk genes have a later onset of expression. Truncated transcripts and forms containing an extracellular juxtamembrane region insert were found. The degree of sequence variation and expression differences within the family suggest that each of the five zebrafish Trk receptors have a functionally distinct role. These findings demonstrate that the vertebrate Trk family is larger than previously appreciated.

• Culotti JG
• Axon guidance mechanisms in Caenorhabditis elegans.
• Curr Opin Genet Dev. 1994; 4: 587-95
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• Genetic studies have identified an extracellular path cue molecule, UNC-6, and a neuronal receptor, UNC-5, that act to guide migrating pioneer growth cones along the dorsoventral coordinate of the Caenorhabditis elegans body wall. Ectopic expression studies and characterization of mutants have demonstrated directly the instructive action of these molecules, suggesting a molecular model for how they perform their guidance functions. Recent evidence suggests that these and other genetically identified axon guidance molecules are likely to have vertebrate counterparts.

• Hamelin M, Zhou Y, Su MW, Scott IM, Culotti JG
• Expression of the UNC-5 guidance receptor in the touch neurons of C. elegans steers their axons dorsally.
• Nature. 1993; 364: 327-30
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• Growth cones in developing nervous systems encounter a sequence of extracellular cues during migration. In theory, a growth cone can navigate by selectively expressing or activating surface receptor(s) that recognize extracellular cues appropriate to each migratory phase. Using the simple Caenorhabditis elegans nervous system, we attempted to demonstrate that path selection by migrating growth cones can be predictably altered by ectopic expression of a single receptor. The unc-5 gene of C. elegans encodes a unique receptor of the immunoglobulin superfamily (UNC-5), required cell-autonomously to guide growth cone and mesodermal cell migrations in a dorsal direction on the epidermis. We report here that the UNC-5 receptor induces dorsally oriented axon trajectories when ectopically expressed in the touch receptor neurons which normally extend pioneer axons longitudinally or ventrally on the epidermis. These errant trajectories depend on unc-6, which encodes a putative epidermal path cue, just as normal dorsally oriented axon trajectories do (such as those of certain motor neurons), suggesting that UNC-5 acts to reorient the touch cell growth cones by using its normal guidance mechanisms. These results support previous evidence that UNC-5 and UNC-6 play instructive rules in guiding growth cone migrations on the epidermis in C. elegans, and indicate that pioneering growth cones, which normally migrate in different directions, may use equivalent intracellular signalling mechanisms for guidance.

• Hedgecock EM, Culotti JG, Hall DH
• The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans.
• Neuron. 1990; 4: 61-85
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• Three known genes guide circumferential migrations of pioneer axons and mesodermal cells on the nematode body wall. unc-5 affects dorsal migrations, unc-40 primarily affects ventral migrations, and unc-6 affects migrations in both directions. Circumferential movements still occur, but are misdirected whereas longitudinal movements are normal in these mutants. Pioneer growth cones migrating directly on the epidermis are affected; growth cones migrating along established axon fascicles are normal. Thus these genes affect cell guidance and not cell motility per se. We propose that two opposite, adhesive gradients guide circumferential migrations on the epidermis. unc-5, unc-6, and unc-40 may encode these adhesion molecules or their cellular receptors. Neurons have access to the basal lamina and the basolateral surfaces of the epidermis, but mesodermal cells contact only the basal lamina. These genes probably identify molecular cues on the basal lamina that guide mesodermal migrations. The same basal lamina cues, or perhaps related molecules on the epidermal cell surfaces, guide pioneer neurons.

• van den Pol AN, Decavel C, Levi A, Paterson B
• Hypothalamic expression of a novel gene product, VGF: immunocytochemical analysis.
• J Neurosci. 1989; 9: 4122-37
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• VGF is the designation for a new 712 amino acid protein, regulated by nerve growth factor (NGF) in PC12 cells, that has not been previously described in the CNS. Northern blot analysis with a nick-translated VGF cDNA probe revealed a single band of mRNA in the brain with a molecular weight identical to that found in PC12 cells. The current paper presents a series of immunocytochemical studies of VGF expression with a focus on the hypothalamus. Two different antisera were raised against nonoverlapping amino acid sequences of a bacterial-expressed protein from the VGF gene cloned from PC12 cells. VGF immunoreactivity is strongly expressed in the rat suprachiasmatic nucleus (SCN), particularly in the dorsomedial part of the nucleus. The administration of colchicine to block axonal transport facilitates detection of the VGF immunoreactivity also in the ventrolateral suprachiasmatic nucleus. This protein appears to be the first one of limited neuronal distribution which is found in both dorsomedial SCN and ventrolateral SCN. Immunostaining of serial 1 micron SCN sections reveals co-localization of VGF in cells which also contain vasopressin or vasoactive intestinal polypeptide. Weaker immunoreactivity is also found in the magnocellular paraventricular and supraoptic nuclei, where the VGF immunoreactivity co-localizes with oxytocin or vasopressin. Mutant Brattleboro rats which do not express vasopressin showed strong VGF immunoreactivity both in the dorsomedial SCN and in cells of the magnocellular neuronal systems, including cells which normally express vasopressin. When axonal transport of the protein is blocked by colchicine, VGF-immunoreactive cells in the hypothalamic arcuate, parvocellular paraventricular, and tuberomammillary nuclei can also be detected, in addition to weakly immunoreactive scattered cells in the hippocampus, amygdala, thalamus, and cortex. VGF immunoreactivity is strong in the axonal projections of SCN and weak in the axons of the paraventricular and supraoptic nuclei. With ultrastructural studies, VGF immunoreactivity is found in presynaptic boutons in the SCN and in axons in the neurohypophysis. Weak axonal staining is present in some regions of the hypothalamus and in the external and internal zones of the median eminence. Immunoreactivity is absent from the intermediate lobe of the hypophysis. In neonatal rats strong VGF immunoreactivity is found throughout the SCN at postnatal day 4 but not in the adjacent hypothalamus. VGF immunoreactivity is also seen in other areas of the brain in neonatal rats, including the lateral geniculate nucleus; while the staining in the dorsal lateral geniculate disappears in the adult, that in the intergeniculate leaflet, a visual center which projects to the SCN, remains.(ABSTRACT TRUNCATED AT 400 WORDS)