ADAM13 disintegrin and cysteine-rich domains bind to the second heparin-binding domain of fibronectin

ADAM13 is a member of the disintegrin and metalloprotease protein family that is expressed on cranial neural crest cells surface and is essential for their migration. ADAM13 is an active protease that can cleave fibronectin in vitro and remodel a fibronectin substrate in vivo. Using a recombinant secreted protein containing both disintegrin and cysteine-rich domains of ADAM13, we show that this "adhesive" region of the protein binds directly to fibronectin. Fibronectin fusion proteins corresponding to the various functional domains were used to define the second heparin-binding domain as the ADAM13 binding site. Mutation of the syndecan-binding site (PPRR --> PPTM) within this domain abolishes binding of the recombinant disintegrin and cysteine-rich domains of ADAM13. We further show that the adhesive disintegrin and cysteine-rich domain of ADAM13 can promote cell adhesion via beta(1) integrins. This adhesion requires integrin activation and can be prevented by antibodies to the cysteine-rich domain of ADAM13 and beta(1) integrin. Finally, wild type, but not the E/A mutant of ADAM13 metalloprotease domain, can be shed from the cell surface, releasing the metalloprotease domain associated with the disintegrin and cysteine-rich domains. This suggests that ADAM13 shedding may involve its own metalloprotease activity and that the released protease may interact with both integrins and extracellular matrix proteins.     

Invariant Sema5A inhibition serves an ensheathing function during optic nerve development

Retinal axon pathfinding from the retina into the optic nerve involves the growth promoting axon guidance molecules L1, laminin and netrin 1, each of which governs axon behavior at specific regions along the retinal pathway. In identifying additional molecules regulating this process during embryonic mouse development, we found that transmembrane Semaphorin5A mRNA and protein was specifically expressed in neuroepithelial cells surrounding retinal axons at the optic disc and along the optic nerve. Given that growth cone responses to a specific guidance molecule can be altered by co-exposure to a second guidance cue, we examined whether retinal axon responses to Sema5A were modulated by other guidance signals axons encountered along the retinal pathway. In growth cone collapse, substratum choice and neurite outgrowth assays, Sema5A triggered an invariant inhibitory response in the context of L1, laminin, or netrin 1 signaling, suggesting that Sema5A inhibited retinal axons throughout their course at the optic disc and nerve. Antibody-perturbation studies in living embryo preparations showed that blocking of Sema5A function led to retinal axons straying out of the optic nerve bundle, indicating that Sema5A normally helped ensheath the retinal pathway. Thus, development of some CNS nerves requires inhibitory sheaths to maintain integrity. Furthermore, this function is accomplished using molecules such as Sema5A that exhibit conserved inhibitory responses in the presence of co-impinging signals from multiple families of guidance molecules.     

The receptor tyrosine phosphatase CRYPalpha affects growth cone morphology

During development of the nervous system receptor tyrosine kinases and receptor protein tyrosine phosphatases act in a coordinate way during axon growth and guidance. In the developing avian retinotectal system, many different receptor protein tyrosine phosphatases are expressed. Most of them have unknown functions. Retinal ganglion cells express at least three different members of this receptor family on their axons and growth cones: CRYPalpha, CRYP-2 and PTPmu. CRYPalpha interacts heterophilically with at least two different ligands found in the basal membranes of the retina and the optic tectum. To analyze the role of the CRYPalpha-ligand interaction, retinal ganglion cell axons were grown on retinal basal membranes (inner limiting membrane) and the receptor-ligand interaction was blocked from both the receptor side (by receptor specific antibodies) and from the ligand side by using a receptor-alkaline phosphatase fusion protein. Both of these treatments reduced average retinal axon length and induced a dramatic change in morphology of retinal ganglion cell growth cones on basal membranes, but not on other substrates like laminin, N-cadherin, matrigel- and detergent-treated basal membranes. These results suggest that CRYPalpha and its ligand act as growth-promoting molecules during intraretinal axon growth.     

CRYP-2/cPTPRO is a neurite inhibitory repulsive guidance cue for retinal neurons in vitro

Receptor protein tyrosine phosphatases (RPTPs) are implicated as regulators of axon growth and guidance. Genetic deletions in the fly have shown that type III RPTPs are important in axon pathfinding, but nothing is known about their function on a cellular level. Previous experiments in our lab have identified a type III RPTP, CRYP-2/cPTPRO, specifically expressed during the period of axon outgrowth in the chick brain; cPTPRO is expressed in the axons and growth cones of retinal and tectal projection neurons. We constructed a fusion protein containing the extracellular domain of cPTPRO fused to the Fc portion of mouse immunoglobulin G-1, and used it to perform in vitro functional assays. We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons. In addition, cPTPRO had potent growth cone collapsing activity in vitro, and locally applied gradients of cPTPRO repelled growing retinal axons. This chemorepulsive effect could be regulated by the level of cGMP in the growth cone. Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina. Taken together, our results indicate that cPTPRO may act as a guidance cue for retinal ganglion cells during vertebrate development.     

The axonal glycoprotein TAG-1 is an immunoglobulin superfamily member with neurite outgrowth-promoting activity

Pathfinding of axons in the developing nervous system is thought to be mediated by glycoproteins expressed on the surface of embryonic axons and growth cones. One molecule suggested to play a role in axonal growth is TAG-1, a 135 kd glycoprotein expressed transiently on the surface of subsets of neurons in the developing mammalian nervous system. We isolated a full-length cDNA clone encoding rat TAG-1. TAG-1 has six immunoglobulin-like domains and four fibronectin type III-like repeats and is structurally similar to other immunoglobulin-like proteins expressed on developing axons. Neurons maintained in vitro on a substrate of TAG-1 extend long neurites, suggesting that this protein plays a role in the initial growth and guidance of axons in vivo. TAG-1 is anchored to the neuronal membrane via a glycosyl phosphatidylinositol linkage and is also released from neurons, suggesting that TAG-1 also functions as a substrate adhesion molecule when released into the extracellular environment.     

Heparan sulfate proteoglycans are ligands for receptor protein tyrosine phosphatase sigma

RPTPsigma is a cell adhesion molecule-like receptor protein tyrosine phosphatase involved in nervous system development. Its avian orthologue, known as cPTPsigma or CRYPalpha, promotes intraretinal axon growth and controls the morphology of growth cones. The molecular mechanisms underlying the functions of cPTPsigma are still to be determined, since neither its physiological ligand(s) nor its substrates have been described. Nevertheless, a major class of ligand(s) is present in the retinal basal lamina and glial endfeet, the potent native growth substrate for retinal axons. We demonstrate here that cPTPsigma is a heparin-binding protein and that its basal lamina ligands include the heparan sulfate proteoglycans (HSPGs) agrin and collagen XVIII. These molecules interact with high affinity with cPTPsigma in vitro, and this binding is totally dependent upon their heparan sulfate chains. Using molecular modelling and site-directed mutagenesis, a binding site for heparin and heparan sulfate was identified in the first immunoglobulin-like domain of cPTPsigma. HSPGs are therefore a novel class of heterotypic ligand for cPTPsigma, suggesting that cPTPsigma signaling in axons and growth cones is directly responsive to matrix-associated cues.     

RGM and its receptor neogenin regulate neuronal survival

Repulsive guidance molecule (RGM) is an axon guidance protein that repels retinal axons upon activation of the neogenin receptor. To understand the functions of RGM-neogenin complexes in vivo, we used gene transfer technology to perturb their expression in the developing neural tube of chick embryos. Surprisingly, neogenin over-expression or RGM down-expression in the neural tube induces apoptosis. Neogenin pro-apoptotic activity in immortalized neuronal cells and in the neural tube is associated with the cleavage of its cytoplasmic domain by caspases. Thus neogenin is a dependence receptor inducing cell death in the absence of RGM, whereas the presence of RGM inhibits this effect.     

Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system.

During development, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. In Drosophila, the Slit protein regulates midline axon crossing through repulsion. To determine the role of Slit proteins in RGC axon guidance, we disrupted Slit1 and Slit2, two of three known mouse Slit genes. Mice defective in either gene alone exhibited few RGC axon guidance defects, but in double mutant mice a large additional chiasm developed anterior to the true chiasm, many retinal axons projected into the contralateral optic nerve, and some extended ectopically-dorsal and lateral to the chiasm. Our results indicate that Slit proteins repel retinal axons in vivo and cooperate to establish a corridor through which the axons are channeled, thereby helping define the site in the ventral diencephalon where the optic chiasm forms.     

MICALs,a family of conserved flavoprotein oxidoreductases,function in plexin-mediated axonal repulsion

Members of the semaphorin family of secreted and transmembrane proteins utilize plexins as neuronal receptors to signal repulsive axon guidance. It remains unknown how plexin proteins are directly linked to the regulation of cytoskeletal dynamics. Here, we show that Drosophila MICAL, a large, multidomain, cytosolic protein expressed in axons, interacts with the neuronal plexin A (PlexA) receptor and is required for Semaphorin 1a (Sema-1a)-PlexA-mediated repulsive axon guidance. In addition to containing several domains known to interact with cytoskeletal components, MICAL has a flavoprotein monooxygenase domain, the integrity of which is required for Sema-1a-PlexA repulsive axon guidance. Vertebrate orthologs of Drosophila MICAL are neuronally expressed and also interact with vertebrate plexins, and monooxygenase inhibitors abrogate semaphorin-mediated axonal repulsion. These results suggest a novel role for oxidoreductases in repulsive neuronal guidance.     

Nel positively regulates the genesis of retinal ganglion cells by promoting their differentiation and survival during development

For correct functioning of the nervous system, the appropriate number and complement of neuronal cell types must be produced during development. However, the molecular mechanisms that regulate the production of individual classes of neurons are poorly understood. In this study, we investigate the function of the thrombospondin-1–like glycoprotein, Nel (neural epidermal growth factor [EGF]-like), in the generation of retinal ganglion cells (RGCs) in chicks. During eye development, Nel is strongly expressed in the presumptive retinal pigment epithelium and RGCs. Nel overexpression in the developing retina by in ovo electroporation increases the number of RGCs, whereas the number of displaced amacrine cells decreases. Conversely, knockdown of Nel expression by transposon-mediated introduction of RNA interference constructs results in decrease in RGC number and increase in the number of displaced amacrine cells. Modifications of Nel expression levels do not appear to affect proliferation of retinal progenitor cells, but they significantly alter the progression rate of RGC differentiation from the central retina to the periphery. Furthermore, Nel protects RGCs from apoptosis during retinal development. These results indicate that Nel positively regulates RGC production by promoting their differentiation and survival during development.     

Receptor protein tyrosine phosphatases regulate retinal ganglion cell axon outgrowth in the developing Xenopus visual system

Receptor protein tyrosine phosphatases (RPTPs) are regulators of axon outgrowth and guidance in a variety of different vertebrate and invertebrate systems. Three RPTPs, CRYP-alpha, PTP-delta, and LAR, are expressed in overlapping but distinct patterns in the developing Xenopus retina, including expression in retinal ganglion cells (RGCs) as they send axons to the tectum (Johnson KG, Holt CE. 2000. Expression of CRYP-alpha, LAR, PTP-delta, and PTP-rho in the developing Xenopus visual system. Mech Dev 92:291-294). In order to examine the role of these RPTPs in visual system development, putative dominant negative RPTP mutants (CS-CRYP-alpha, CS-PTP-delta, and CS-LAR) were expressed either singly or in combination in retinal cells. No effect was found on either retinal cell fate determination or on gross RGC axon guidance to the tectum. However, expression of these CS-RPTP constructs differentially affected the rate of RGC axon outgrowth. In vivo, expression of all three CS-RPTPs or CS-PTP-delta alone inhibited RGC axon outgrowth, while CS-LAR and CS-CRYP-alpha had no significant effect. In vitro, expression of CS-CRYP-alpha enhanced neurite outgrowth, while CS-PTP-delta inhibited neurite outgrowth in a substrate-dependent manner. This study provides the first in vivo evidence that RPTPs regulate retinal axon outgrowth.     

Ncam1a and Ncam1b: two carriers of polysialic acid with different functions in the developing zebrafish nervous system

Polysialic acid (polySia) is mainly described as a glycan modification of the neural cell adhesion molecule NCAM1. PolySia-NCAM1 has multiple functions during the development of vertebrate nervous systems including axon extension and fasciculation. Phylogenetic analyses reveal the presence of two related gene clusters, NCAM1 and NCAM2, in tetrapods and fishes. Within the ncam1 cluster, teleost fishes express ncam1a (ncam) and ncam1b (pcam) as duplicated paralogs which arose from a second round of ray-finned fish-specific genome duplication. Tetrapods, in contrast, express a single NCAM1 gene. Using the zebrafish model, we identify Ncam1b as a novel major carrier of polySia in the nervous system. PolySia-Ncam1a is expressed predominantly in rostral regions of the developing nervous system, whereas polySia-Ncam1b prevails caudally. We show that ncam1a and ncam1b have different expression domains which only partially overlap. Furthermore, Ncam1a and Ncam1b and their polySia modifications serve different functions in axon guidance. Formation of the posterior commissure at the forebrain/midbrain junction requires polySia-Ncam1a on the axons for proper fasciculation, whereas Ncam1b, expressed by midbrain cell bodies, serves as an instructive guidance cue for the dorso-medially directed growth of axons. Spinal motor axons, on the other hand, depend on axonally expressed Ncam1b for correct growth toward their target region. Collectively, these findings suggest that the genome duplication in the teleost lineage has provided the basis for a functional diversification of polySia carriers in the nervous system.     

Rearranging Exosites in Noncatalytic Domains Can Redirect the Substrate Specificity of ADAMTS Proteases

ADAMTS proteases typically employ some combination of ancillary C-terminal disintegrin-like, thrombospondin-1, cysteine-rich, and spacer domains to bind substrates and facilitate proteolysis by an N-terminal metalloprotease domain. We constructed chimeric proteases and substrates to examine the role of C-terminal domains of ADAMTS13 and ADAMTS5 in the recognition of their physiological cleavage sites in von Willebrand factor (VWF) and aggrecan, respectively. ADAMTS5 cleaves Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds in bovine aggrecan but does not cleave VWF. Conversely, ADAMTS13 cleaves the Tyr(1605)-Met(1606) bond of VWF, which is exposed by fluid shear stress but cannot cleave aggrecan. Replacing the thrombospondin-1/cysteine-rich/spacer domains of ADAMTS5 with those of ADAMTS13 conferred the ability to cleave the Glu(1615)-Ile(1616) bond of VWF domain A2 in peptide substrates or VWF multimers that had been sheared; native (unsheared) VWF multimers were resistant. Thus, by recombining exosites, we engineered ADAMTS5 to cleave a new bond in VWF, preserving physiological regulation by fluid shear stress. The results demonstrate that noncatalytic thrombospondin-1/cysteine-rich/spacer domains are principal modifiers of substrate recognition and cleavage by both ADAMTS5 and ADAMTS13. Noncatalytic domains may perform similar functions in other ADAMTS family members.     

Kinase independent function of EphB receptors in retinal axon pathfinding to the optic disc from dorsal but not ventral retina

Optic nerve formation requires precise retinal ganglion cell (RGC) axon pathfinding within the retina to the optic disc, the molecular basis of which is not well understood. At CNS targets, interactions between Eph receptor tyrosine kinases on RGC axons and ephrin ligands on target cells have been implicated in formation of topographic maps. However, studies in chick and mouse have shown that both Eph receptors and ephrins are also expressed within the retina itself, raising the possibility that this receptor-ligand family mediates aspects of retinal development. Here, we more fully document the presence of specific EphB receptors and B-ephrins in embryonic mouse retina and provide evidence that EphB receptors are involved in RGC axon pathfinding to the optic disc. We find that as RGC axons begin this pathfinding process, EphB receptors are uniformly expressed along the dorsal-ventral retinal axis. This is in contrast to the previously reported high ventral-low dorsal gradient of EphB receptors later in development when RGC axons map to CNS targets. We show that mice lacking both EphB2 and EphB3 receptor tyrosine kinases, but not each alone, exhibit increased frequency of RGC axon guidance errors to the optic disc. In these animals, major aspects of retinal development and cellular organization appear normal, as do the expression of other RGC guidance cues netrin, DCC, and L1. Unexpectedly, errors occur in dorsal but not ventral retina despite early uniform or later high ventral expression of EphB2 and EphB3. Furthermore, embryos lacking EphB3 and the kinase domain of EphB2 do not show increased errors, consistent with a guidance role for the EphB2 extracellular domain. Thus, while Eph kinase function is involved in RGC axon mapping in the brain, RGC axon pathfinding within the retina is partially mediated by EphB receptors acting in a kinase-independent manner.     

Nr-CAM promotes neurite outgrowth from peripheral ganglia by a mechanism involving axonin-1 as a neuronal receptor.

Nr-CAM is a neuronal cell adhesion molecule (CAM) belonging to the immunoglobulin superfamily that has been implicated as a ligand for another CAM, axonin-1, in guidance of commissural axons across the floor plate in the spinal cord. Nr-CAM also serves as a neuronal receptor for several other cell surface molecules, but its role as a ligand in neurite outgrowth is poorly understood. We studied this problem using a chimeric Fc-fusion protein of the extracellular region of Nr-CAM (Nr-Fc) and investigated potential neuronal receptors in the developing peripheral nervous system. A recombinant Nr-CAM-Fc fusion protein, containing all six Ig domains and the first two fibronectin type III repeats of the extracellular region of Nr-CAM, retains cellular and molecular binding activities of the native protein. Injection of Nr-Fc into the central canal of the developing chick spinal cord in ovo resulted in guidance errors for commissural axons in the vicinity of the floor plate. This effect is similar to that resulting from treatment with antibodies against axonin-1, confirming that axonin-1/Nr-CAM interactions are important for guidance of commissural axons through a spatially and temporally restricted Nr-CAM positive domain in the ventral spinal cord. When tested as a substrate, Nr-Fc induced robust neurite outgrowth from dorsal root ganglion and sympathetic ganglion neurons, but it was not effective for tectal and forebrain neurons. The peripheral but not the central neurons expressed high levels of axonin-1 both in vitro and in vivo. Moreover, antibodies against axonin-1 inhibited Nr-Fc-induced neurite outgrowth, indicating that axonin-1 is a neuronal receptor for Nr-CAM on these peripheral ganglion neurons. The results demonstrate a role for Nr-CAM as a ligand in axon growth by a mechanism involving axonin-1 as a neuronal receptor and suggest that dynamic changes in Nr-CAM expression can modulate axonal growth and guidance during development.     

UNC-71, a disintegrin and metalloprotease (ADAM) protein,regulates motor axon guidance and sex myoblast migration in C. elegans

The migration of cells and growth cones is a process that is guided by extracellular cues and requires the controlled remodeling of the extracellular matrix along the migratory path. The ADAM proteins are important regulators of cellular adhesion and recognition because they can combine regulated proteolysis with modulation of cell adhesion. We report that the C. elegans gene unc-71 encodes a unique ADAM with an inactive metalloprotease domain. Loss-of-function mutations in unc-71 cause distinct defects in motor axon guidance and sex myoblast migration. Many unc-71 mutations affect the disintegrin and the cysteine-rich domains, supporting a major function of unc-71 in cell adhesion. UNC-71 appears to be expressed in a selected set of cells. Genetic mosaic analysis and tissue-specific expression studies indicate that unc-71 acts in a cell non-autonomous manner for both motor axon guidance and sex myoblast migration. Finally, double mutant analysis of unc-71 with other axon guidance signaling molecules suggests that UNC-71 probably functions in a combinatorial manner with integrins and UNC-6/netrin to provide distinct axon guidance cues at specific choice points for motoneurons.     

Modulation of EphA receptor function by coexpressed ephrinA ligands on retinal ganglion cell axons

The Eph family is thought to exert its function through the complementary expression of receptors and ligands. Here, we show that EphA receptors colocalize on retinal ganglion cell (RGC) axons with EphA ligands, which are expressed in a high-nasal-to-low-temporal pattern. In the stripe assay, only temporal axons are normally sensitive for repellent axon guidance cues of the caudal tectum. However, overexpression of ephrinA ligands on temporal axons abolishes this sensitivity, whereas treatment with PI-PLC both removes ephrinA ligands from retinal axons and induces a striped outgrowth of formerly insensitive nasal axons. In vivo, retinal overexpression of ephrinA2 leads to topographic targeting errors of temporal axons. These data suggest that differential ligand expression on retinal axons is a major determinant of topographic targeting in the retinotectal projection.     

Repulsive guidance molecule/neogenin: a novel ligand-receptor system playing multiple roles in neural development

The repulsive guidance molecule (RGM) is a membrane-bound protein originally isolated as an axon guidance molecule in the visual system. Recently, the transmembrane protein, neogenin, has been identified as the RGM receptor. In vitro analysis with retinal explants showed that RGM repels temporal retinal axons and collapses their growth cones through neogenin-mediated signaling. However, RGM and neogenin are also broadly expressed at the early embryonic stage, suggesting that they do not only control the guidance of visual axons. Gene expression perturbation experiments in chick embryos showed that neogenin induces cell death, and its ligand, RGM, blocks the pro-apoptotic activity of neogenin. Thus, RGM/neogenin is a novel dependence ligand/receptor couple as well as an axon guidance molecular complex.