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Heparan sulfate endosulfatases shape axonal patterns by regulating axon guidance protein distribution
e10
Abstracts / Neuroscience Research 71S (2011) e6–e44
ance between research and clinical medicine, and the duties to inform, warn and care. Research fund: NIH/NIMH, NIHR, CFI, BCKDF. doi:10.1016/j.neures.2011.07.033
S2-D-2-1 Heparan sulfate endosulfatases shape axonal patterns by regulating axon guidance protein distribution Masayuki Masu 1 , Takuya Okada 1 , Satoshi Nagamine 1 , Tatsuyuki Ohto 1,2 , Fuyuki Kametani 4 , Masato Hasegawa 4 , Satoshi Kunita 3 , Satoru Takahashi 3 , Kazuko Keino-Masu 1 Dept. Mol. Neurobiol., Univ. of Tsukuba, Tsukuba 2 Dept. Pediatrics, Univ. of Tsukuba, Tsukuba 3 Laboratory Animal Resource Center, Univ. of Tsukuba, Tsukuba 4 Dept. Mol. Neurobiol., Tokyo Institute of Psychiatry, Tokyo
Therefore, growth cones may be able to polarize themselves and cell and tissue polarity signaling may contribute significantly to the highly organized patterns of axonal connections throughout the nervous system. Reference (1) Lyuksyutova, A.I., Lu, C.C., Milanesio, N., King, L.A., Guo, N., Wang, Y., Nathans, J., Tessier-Lavigne, M., Zou, Y., 2003. Anterior-posterior guidance of commissural axons by Wnt-frizzled signaling. Science 302, 1984–8. (2) Shafer, B., Onishi, K., Lo, C., Colakoglu, G., Zou, Y., 2011. Vangl2 promotes Wnt/planar cell polarity-like signaling by antagonizing Dvl1-mediated feedback inhibition in growth cone guidance. Dev. Cell 20, 177–91.
1
The brain consists of numerous neuronal cells that are connected to each other to exchange information. During development, growing axons are navigated towards their targets by chemoattractants and repellents. Previous studies have identified a number of axon guidance molecules and their receptors, which act cooperatively to establish the complex neuronal network. As a molecular device to generate further diversity in axon guidance signaling, our group is interested in heparan sulfate (HS). HS is a long linear polysaccharide attached to the core proteins of proteoglycans. It shows enormous structural heterogeneities because its sugar chain is extensively and unequally modified by epimerization and sulfation. HS binds to a wide variety of proteins, including growth factors, morphogens, and axon guidance proteins, thereby regulating cell proliferation, differentiation, migration, and axonal patterning. Biochemical studies have demonstrated that sulfation motifs in HS give rise to specific binding sites, and that 6-O-sulfation plays an important role in ligand binding and receptor activation. Sulfation patterns are created by several sulfotransferases and subsequently modified by extracellular HS endosulfatases, Sulf1 and Sulf2. They remove 6-O-sulfates from mature HS specifically and modulate cellular signaling. We found that Sulf1/2 double knockout mice have defects in the corticospinal tract. Cortical axons are defasciculated and dorsally displaced on the midbrain surface. Electroporation-mediated rescue experiments reveal that expression of Sulf1/Sulf2 genes in the third ventricle of the posterior hypothalamus, one of their high overlapping expression regions in wild-type brains, is important for navigating corticospinal axons properly. In this symposium, we would like to discuss the roles of Sulf1/2 in axon guidance, especially in the context of regulation of axon guidance protein distribution in the extracellular matrix. Research fund: KAKENHI (22103006). doi:10.1016/j.neures.2011.07.034
S2-D-2-2 Cell polarity signaling and growth cone guidance Keisuke Onishi , Beth Shafer, Charles Lo, Gulsen Colakglu, Yimin Zou University of California, San Diego The signal transduction and the cell biological mechanisms leading to directed navigation of the axon steering apparatus, the growth cone, remain vaguely understood. Wnt family morphogens, presented in concentration gradients along the main body axes, are now well-accepted conserved axon guidance cues. One of the Wnt-Frizzled signaling pathways is the highly conserved planar cell polarity (PCP) pathway, which establishes polarized cellular morphology along the plane of many different types of epithelial tissues. We found the core PCP components are required for Wnt5a-stimulated outgrowth and anterior-posterior guidance of commissural axons in the spinal cord. Dishevelled1 can inhibit PCP signaling by increasing hyperphosphorylation of Frizzled3 and preventing its internalization. Vangl2 antagonizes that by reducing Frizzled3 phosphorylation and promotes internalization. In commissural axon growth cones, Vangl2 is predominantly localized on the plasma membrane and is highly enriched on the tips of the filopodia as well as in patches of membrane where new filopodia emerge. Taken together, we propose that the antagonistic functions of Vangl2 and Dvl1 (over Frizzled3 hyperphosphorylation and endocytosis) allows sharpening of PCP signaling locally on the tips of the filopodia for sensing directional cues, Wnts, eventually causing turning of growth cones (1) (2). Our previous studies identified that atypical protein kinase C, a key component of the apical-basal polarity pathway which sets up cell polarity perpendicular to PCP in many epithelial tissues, is also essential for mediating Wnt-Frizzled attraction and axon guidance. Wnt/PCP signaling also controls anterior-posterior axon organization in the midbrain and hindbrain.
doi:10.1016/j.neures.2011.07.035
S2-D-2-3 Intra-axonal compartmentalization of axon guidance receptors: Its mechanism and roles in axon guidance Yasushi Hiromi 1,2 1
National Institute of Genetics, Mishima, Japan SOKENDAI, Mishima, Japan
2
Department of Genetics,
The major function attributed to axon guidance receptors is the detection and interpretation of the extracellular guidance information at the growth cone, to steer the axon to proper directions. In addition to such cell autonomous function, several axon guidance receptors posses cell non-autonomous guidance activity—influencing behavior of other cells. It is likely that this activity is mediated by the ability of receptors to affect the distribution of their ligands, and is executed by receptors localized to the axon shaft. Indeed many axon guidance receptors are localized to specific segments of the axon shaft, raising a possibility that such localization patterns provide intricate guidance information that is not possible by a simple gradient of the guidance molecule created by passive diffusion. We are analyzing how such intra-axonal localization of guidance receptors can be achieved, and its potential roles during nervous system development. Using primary culture of Drosophila embryonic neurons, we have shown that intra-axonal localization of multiple axon guidance receptors can be established in the absence of cell–cell contacts. Thus the distribution of guidance cues may originate, at least in part, from cell-intrinsic patterning ability of individual neurons. The delivery of guidance receptors to specific intra-axonal segments involves regulated membrane traffic. A diffusion barrier that restricts movements of membrane proteins is found at the midpoint of the axon, dividing the axon into proximal and distal compartments. I will discuss how such compartmentalization of the axonal membrane might be utilized for cell-autonomous and non-autonomous functions of axon guidance receptors during in neural circuit formation. Research fund: KAKENHI 21370100. doi:10.1016/j.neures.2011.07.036
S2-D-2-4 Wnt signals and Frizzled receptors regulate dendrite formation in C. elegans Massimo A. Hilliard , Leonie Kirszenblat, Divya Pattabiraman, Brent Neumann Queensland Brain Institute, The University of Queensland, Brisbane, Australia Neurons exhibit distinct morphological domains, axons and dendrites, which are essential for functional wiring of the nervous system. To understand how dendrites develop we focused on the C. elegans PQR oxygen sensory neuron. PQR has its cell body positioned in the left lumbar ganglion on the posterior-lateral side of the body. A single dendrite extends posterior with sensory cilia at its tip, while the axon extends anterior along the ventral nerve cord. PQR is born post-embryonically allowing easy visualization of dendrite development using the gcy-36::GFP transgene. In a genetic screen for dendrite defective mutants we isolated a previously uncharacterized mutation in lin-17, a C. elegans Frizzled receptor gene. We found that in lin17(vd002), the PQR dendrite was absent, shortened or misrouted anterior. Similar dendrite defects were also observed in other known alleles of lin-17. Cell-specific expression of wild-type LIN-17 in PQR indicated a cell-autonomous role of this molecule in regulating dendrite development. LIN-44 is a Wnt ligand known to bind the Frizzled receptor LIN-17 and is expressed by four hypodermal cells in the tip of the tail. We found that lin-44 mutants presented PQR dendrite defects similar to those observed in lin-17 mutants. Remarkably, the PQR axonal morphology of lin-44 mutants remained unaltered, suggesting a dendrite specific effect for LIN-44. We expressed LIN-44 ectopically from more anterior regions of the body and found that it worsened the PQR dendrite defects of lin-44 mutants, indicating LIN-44 functions as an instructive cue. Analysis of the lin-17 lin-44 double mutant indicated a genetic interac-
Abstracts / Neuroscience Research 71S (2011) e6–e44
ance between research and clinical medicine, and the duties to inform, warn and care. Research fund: NIH/NIMH, NIHR, CFI, BCKDF. doi:10.1016/j.neures.2011.07.033
S2-D-2-1 Heparan sulfate endosulfatases shape axonal patterns by regulating axon guidance protein distribution Masayuki Masu 1 , Takuya Okada 1 , Satoshi Nagamine 1 , Tatsuyuki Ohto 1,2 , Fuyuki Kametani 4 , Masato Hasegawa 4 , Satoshi Kunita 3 , Satoru Takahashi 3 , Kazuko Keino-Masu 1 Dept. Mol. Neurobiol., Univ. of Tsukuba, Tsukuba 2 Dept. Pediatrics, Univ. of Tsukuba, Tsukuba 3 Laboratory Animal Resource Center, Univ. of Tsukuba, Tsukuba 4 Dept. Mol. Neurobiol., Tokyo Institute of Psychiatry, Tokyo
Therefore, growth cones may be able to polarize themselves and cell and tissue polarity signaling may contribute significantly to the highly organized patterns of axonal connections throughout the nervous system. Reference (1) Lyuksyutova, A.I., Lu, C.C., Milanesio, N., King, L.A., Guo, N., Wang, Y., Nathans, J., Tessier-Lavigne, M., Zou, Y., 2003. Anterior-posterior guidance of commissural axons by Wnt-frizzled signaling. Science 302, 1984–8. (2) Shafer, B., Onishi, K., Lo, C., Colakoglu, G., Zou, Y., 2011. Vangl2 promotes Wnt/planar cell polarity-like signaling by antagonizing Dvl1-mediated feedback inhibition in growth cone guidance. Dev. Cell 20, 177–91.
1
The brain consists of numerous neuronal cells that are connected to each other to exchange information. During development, growing axons are navigated towards their targets by chemoattractants and repellents. Previous studies have identified a number of axon guidance molecules and their receptors, which act cooperatively to establish the complex neuronal network. As a molecular device to generate further diversity in axon guidance signaling, our group is interested in heparan sulfate (HS). HS is a long linear polysaccharide attached to the core proteins of proteoglycans. It shows enormous structural heterogeneities because its sugar chain is extensively and unequally modified by epimerization and sulfation. HS binds to a wide variety of proteins, including growth factors, morphogens, and axon guidance proteins, thereby regulating cell proliferation, differentiation, migration, and axonal patterning. Biochemical studies have demonstrated that sulfation motifs in HS give rise to specific binding sites, and that 6-O-sulfation plays an important role in ligand binding and receptor activation. Sulfation patterns are created by several sulfotransferases and subsequently modified by extracellular HS endosulfatases, Sulf1 and Sulf2. They remove 6-O-sulfates from mature HS specifically and modulate cellular signaling. We found that Sulf1/2 double knockout mice have defects in the corticospinal tract. Cortical axons are defasciculated and dorsally displaced on the midbrain surface. Electroporation-mediated rescue experiments reveal that expression of Sulf1/Sulf2 genes in the third ventricle of the posterior hypothalamus, one of their high overlapping expression regions in wild-type brains, is important for navigating corticospinal axons properly. In this symposium, we would like to discuss the roles of Sulf1/2 in axon guidance, especially in the context of regulation of axon guidance protein distribution in the extracellular matrix. Research fund: KAKENHI (22103006). doi:10.1016/j.neures.2011.07.034
S2-D-2-2 Cell polarity signaling and growth cone guidance Keisuke Onishi , Beth Shafer, Charles Lo, Gulsen Colakglu, Yimin Zou University of California, San Diego The signal transduction and the cell biological mechanisms leading to directed navigation of the axon steering apparatus, the growth cone, remain vaguely understood. Wnt family morphogens, presented in concentration gradients along the main body axes, are now well-accepted conserved axon guidance cues. One of the Wnt-Frizzled signaling pathways is the highly conserved planar cell polarity (PCP) pathway, which establishes polarized cellular morphology along the plane of many different types of epithelial tissues. We found the core PCP components are required for Wnt5a-stimulated outgrowth and anterior-posterior guidance of commissural axons in the spinal cord. Dishevelled1 can inhibit PCP signaling by increasing hyperphosphorylation of Frizzled3 and preventing its internalization. Vangl2 antagonizes that by reducing Frizzled3 phosphorylation and promotes internalization. In commissural axon growth cones, Vangl2 is predominantly localized on the plasma membrane and is highly enriched on the tips of the filopodia as well as in patches of membrane where new filopodia emerge. Taken together, we propose that the antagonistic functions of Vangl2 and Dvl1 (over Frizzled3 hyperphosphorylation and endocytosis) allows sharpening of PCP signaling locally on the tips of the filopodia for sensing directional cues, Wnts, eventually causing turning of growth cones (1) (2). Our previous studies identified that atypical protein kinase C, a key component of the apical-basal polarity pathway which sets up cell polarity perpendicular to PCP in many epithelial tissues, is also essential for mediating Wnt-Frizzled attraction and axon guidance. Wnt/PCP signaling also controls anterior-posterior axon organization in the midbrain and hindbrain.
doi:10.1016/j.neures.2011.07.035
S2-D-2-3 Intra-axonal compartmentalization of axon guidance receptors: Its mechanism and roles in axon guidance Yasushi Hiromi 1,2 1
National Institute of Genetics, Mishima, Japan SOKENDAI, Mishima, Japan
2
Department of Genetics,
The major function attributed to axon guidance receptors is the detection and interpretation of the extracellular guidance information at the growth cone, to steer the axon to proper directions. In addition to such cell autonomous function, several axon guidance receptors posses cell non-autonomous guidance activity—influencing behavior of other cells. It is likely that this activity is mediated by the ability of receptors to affect the distribution of their ligands, and is executed by receptors localized to the axon shaft. Indeed many axon guidance receptors are localized to specific segments of the axon shaft, raising a possibility that such localization patterns provide intricate guidance information that is not possible by a simple gradient of the guidance molecule created by passive diffusion. We are analyzing how such intra-axonal localization of guidance receptors can be achieved, and its potential roles during nervous system development. Using primary culture of Drosophila embryonic neurons, we have shown that intra-axonal localization of multiple axon guidance receptors can be established in the absence of cell–cell contacts. Thus the distribution of guidance cues may originate, at least in part, from cell-intrinsic patterning ability of individual neurons. The delivery of guidance receptors to specific intra-axonal segments involves regulated membrane traffic. A diffusion barrier that restricts movements of membrane proteins is found at the midpoint of the axon, dividing the axon into proximal and distal compartments. I will discuss how such compartmentalization of the axonal membrane might be utilized for cell-autonomous and non-autonomous functions of axon guidance receptors during in neural circuit formation. Research fund: KAKENHI 21370100. doi:10.1016/j.neures.2011.07.036
S2-D-2-4 Wnt signals and Frizzled receptors regulate dendrite formation in C. elegans Massimo A. Hilliard , Leonie Kirszenblat, Divya Pattabiraman, Brent Neumann Queensland Brain Institute, The University of Queensland, Brisbane, Australia Neurons exhibit distinct morphological domains, axons and dendrites, which are essential for functional wiring of the nervous system. To understand how dendrites develop we focused on the C. elegans PQR oxygen sensory neuron. PQR has its cell body positioned in the left lumbar ganglion on the posterior-lateral side of the body. A single dendrite extends posterior with sensory cilia at its tip, while the axon extends anterior along the ventral nerve cord. PQR is born post-embryonically allowing easy visualization of dendrite development using the gcy-36::GFP transgene. In a genetic screen for dendrite defective mutants we isolated a previously uncharacterized mutation in lin-17, a C. elegans Frizzled receptor gene. We found that in lin17(vd002), the PQR dendrite was absent, shortened or misrouted anterior. Similar dendrite defects were also observed in other known alleles of lin-17. Cell-specific expression of wild-type LIN-17 in PQR indicated a cell-autonomous role of this molecule in regulating dendrite development. LIN-44 is a Wnt ligand known to bind the Frizzled receptor LIN-17 and is expressed by four hypodermal cells in the tip of the tail. We found that lin-44 mutants presented PQR dendrite defects similar to those observed in lin-17 mutants. Remarkably, the PQR axonal morphology of lin-44 mutants remained unaltered, suggesting a dendrite specific effect for LIN-44. We expressed LIN-44 ectopically from more anterior regions of the body and found that it worsened the PQR dendrite defects of lin-44 mutants, indicating LIN-44 functions as an instructive cue. Analysis of the lin-17 lin-44 double mutant indicated a genetic interac-