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Glypican Is a Modulator of Netrin-Mediated Axon Guidance.

Blanchette CR, Perrat PN, Thackeray A, Bénard CY - PLoS Biol. (2015)

Bottom Line: However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown.Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor.We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor-expressing cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor-expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.

No MeSH data available.


Related in: MedlinePlus

lon-2/glypican functions in the attractive unc-6/netrin guidance pathway.(A) During the first larval stage of C.elegans, the pioneer neuron AVM extends ventrally along the body wall until it reaches the ventral nerve cord. Its migration results from the combined attractive response to UNC-6/netrin (secreted at the ventral midline) via the UNC-40/DCC receptor and the repulsive response to SLT-1/Slit (secreted by the dorsal muscles) via its SAX-3/Robo receptor. We visualized the morphology of the AVM axon using the transgene Pmec-4::gfp. (B) The heparan sulfate proteoglycans lon-2/glypican and sdn-1/syndecan cooperate to guide the axon of AVM, as their simultaneous loss enhances guidance defects. The role of lon-2/glypican in axon guidance is specific, as the loss of lon-2/glypican, but not the loss of the other C. elegans glypican, gpn-1, enhances the defects of sdn-1/syndecan mutants. (C) Complete loss of lon-2/glypican enhances the axon guidance defects resulting from disrupted slt-1/Slit signaling in mutants for slt-1/Slit or its receptor sax-3/Robo, as well as in animals misexpressing slt-1 in all body wall muscles (using a Pmyo-3::slt-1 transgene). Data for wild type and lon-2 are the same as in (B). (D) Complete loss of lon-2/glypican does not enhance the AVM guidance defects of unc-6/netrin mutants or of mutants for its receptor unc-40/DCC, suggesting that lon-2/glypican functions in the same genetic pathway as unc-6/netrin. Data for wild type and lon-2 are the same as in (B). (E) Loss of sdn-1/syndecan function does not enhance the defects of slt-1/Slit or sax-3/Robo mutants but enhances the defects of unc-40/DCC mutants. Data for wild type, sdn-1, slt-1, sax-3, and unc-40 are the same as in (B–D). Error bars are standard error of the proportion. Asterisks denote significant difference\: *** p ≤ 0.001,** p ≤ 0.01, and * p ≤ 0.05 (z-tests, p-values were corrected by multiplying by the number of comparisons). ns, not significant.
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pbio.1002183.g001: lon-2/glypican functions in the attractive unc-6/netrin guidance pathway.(A) During the first larval stage of C.elegans, the pioneer neuron AVM extends ventrally along the body wall until it reaches the ventral nerve cord. Its migration results from the combined attractive response to UNC-6/netrin (secreted at the ventral midline) via the UNC-40/DCC receptor and the repulsive response to SLT-1/Slit (secreted by the dorsal muscles) via its SAX-3/Robo receptor. We visualized the morphology of the AVM axon using the transgene Pmec-4::gfp. (B) The heparan sulfate proteoglycans lon-2/glypican and sdn-1/syndecan cooperate to guide the axon of AVM, as their simultaneous loss enhances guidance defects. The role of lon-2/glypican in axon guidance is specific, as the loss of lon-2/glypican, but not the loss of the other C. elegans glypican, gpn-1, enhances the defects of sdn-1/syndecan mutants. (C) Complete loss of lon-2/glypican enhances the axon guidance defects resulting from disrupted slt-1/Slit signaling in mutants for slt-1/Slit or its receptor sax-3/Robo, as well as in animals misexpressing slt-1 in all body wall muscles (using a Pmyo-3::slt-1 transgene). Data for wild type and lon-2 are the same as in (B). (D) Complete loss of lon-2/glypican does not enhance the AVM guidance defects of unc-6/netrin mutants or of mutants for its receptor unc-40/DCC, suggesting that lon-2/glypican functions in the same genetic pathway as unc-6/netrin. Data for wild type and lon-2 are the same as in (B). (E) Loss of sdn-1/syndecan function does not enhance the defects of slt-1/Slit or sax-3/Robo mutants but enhances the defects of unc-40/DCC mutants. Data for wild type, sdn-1, slt-1, sax-3, and unc-40 are the same as in (B–D). Error bars are standard error of the proportion. Asterisks denote significant difference\: *** p ≤ 0.001,** p ≤ 0.01, and * p ≤ 0.05 (z-tests, p-values were corrected by multiplying by the number of comparisons). ns, not significant.

Mentions: We addressed these questions using the nematode C. elegans, which has been instrumental for discovering major conserved axon guidance pathways. During larval development, the axon of the mechanosensory neuron AVM migrates ventrally as its growth cone integrates signals from two complementary guidance cues (Fig 1A) [1,4–6, 11–13]: (1) UNC-6/netrin is secreted at the ventral midline and attracts the growth cone ventrally via the receptor UNC-40/DCC [5,14], and (2) SLT-1/Slit is secreted by the dorsal muscles and repels the growth cone away from the dorsal side via the receptor SAX-3/Robo [12,13]. Animals for the guidance cues unc-6/netrin or slt-1/Slit exhibit partial AVM ventral axon guidance defects, and loss of both cues in unc-6 slt-1 double mutants results in fully penetrant guidance defects (S1 Fig, [13]). AVM axons defective in guidance fail to extend ventrally and instead migrate laterally in the anterior direction (Fig 1). In this study, we use the AVM axon as a model to elucidate mechanisms that regulate UNC-6/netrin signaling.


Glypican Is a Modulator of Netrin-Mediated Axon Guidance.

Blanchette CR, Perrat PN, Thackeray A, Bénard CY - PLoS Biol. (2015)

lon-2/glypican functions in the attractive unc-6/netrin guidance pathway.(A) During the first larval stage of C.elegans, the pioneer neuron AVM extends ventrally along the body wall until it reaches the ventral nerve cord. Its migration results from the combined attractive response to UNC-6/netrin (secreted at the ventral midline) via the UNC-40/DCC receptor and the repulsive response to SLT-1/Slit (secreted by the dorsal muscles) via its SAX-3/Robo receptor. We visualized the morphology of the AVM axon using the transgene Pmec-4::gfp. (B) The heparan sulfate proteoglycans lon-2/glypican and sdn-1/syndecan cooperate to guide the axon of AVM, as their simultaneous loss enhances guidance defects. The role of lon-2/glypican in axon guidance is specific, as the loss of lon-2/glypican, but not the loss of the other C. elegans glypican, gpn-1, enhances the defects of sdn-1/syndecan mutants. (C) Complete loss of lon-2/glypican enhances the axon guidance defects resulting from disrupted slt-1/Slit signaling in mutants for slt-1/Slit or its receptor sax-3/Robo, as well as in animals misexpressing slt-1 in all body wall muscles (using a Pmyo-3::slt-1 transgene). Data for wild type and lon-2 are the same as in (B). (D) Complete loss of lon-2/glypican does not enhance the AVM guidance defects of unc-6/netrin mutants or of mutants for its receptor unc-40/DCC, suggesting that lon-2/glypican functions in the same genetic pathway as unc-6/netrin. Data for wild type and lon-2 are the same as in (B). (E) Loss of sdn-1/syndecan function does not enhance the defects of slt-1/Slit or sax-3/Robo mutants but enhances the defects of unc-40/DCC mutants. Data for wild type, sdn-1, slt-1, sax-3, and unc-40 are the same as in (B–D). Error bars are standard error of the proportion. Asterisks denote significant difference\: *** p ≤ 0.001,** p ≤ 0.01, and * p ≤ 0.05 (z-tests, p-values were corrected by multiplying by the number of comparisons). ns, not significant.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493048&req=5

pbio.1002183.g001: lon-2/glypican functions in the attractive unc-6/netrin guidance pathway.(A) During the first larval stage of C.elegans, the pioneer neuron AVM extends ventrally along the body wall until it reaches the ventral nerve cord. Its migration results from the combined attractive response to UNC-6/netrin (secreted at the ventral midline) via the UNC-40/DCC receptor and the repulsive response to SLT-1/Slit (secreted by the dorsal muscles) via its SAX-3/Robo receptor. We visualized the morphology of the AVM axon using the transgene Pmec-4::gfp. (B) The heparan sulfate proteoglycans lon-2/glypican and sdn-1/syndecan cooperate to guide the axon of AVM, as their simultaneous loss enhances guidance defects. The role of lon-2/glypican in axon guidance is specific, as the loss of lon-2/glypican, but not the loss of the other C. elegans glypican, gpn-1, enhances the defects of sdn-1/syndecan mutants. (C) Complete loss of lon-2/glypican enhances the axon guidance defects resulting from disrupted slt-1/Slit signaling in mutants for slt-1/Slit or its receptor sax-3/Robo, as well as in animals misexpressing slt-1 in all body wall muscles (using a Pmyo-3::slt-1 transgene). Data for wild type and lon-2 are the same as in (B). (D) Complete loss of lon-2/glypican does not enhance the AVM guidance defects of unc-6/netrin mutants or of mutants for its receptor unc-40/DCC, suggesting that lon-2/glypican functions in the same genetic pathway as unc-6/netrin. Data for wild type and lon-2 are the same as in (B). (E) Loss of sdn-1/syndecan function does not enhance the defects of slt-1/Slit or sax-3/Robo mutants but enhances the defects of unc-40/DCC mutants. Data for wild type, sdn-1, slt-1, sax-3, and unc-40 are the same as in (B–D). Error bars are standard error of the proportion. Asterisks denote significant difference\: *** p ≤ 0.001,** p ≤ 0.01, and * p ≤ 0.05 (z-tests, p-values were corrected by multiplying by the number of comparisons). ns, not significant.
Mentions: We addressed these questions using the nematode C. elegans, which has been instrumental for discovering major conserved axon guidance pathways. During larval development, the axon of the mechanosensory neuron AVM migrates ventrally as its growth cone integrates signals from two complementary guidance cues (Fig 1A) [1,4–6, 11–13]: (1) UNC-6/netrin is secreted at the ventral midline and attracts the growth cone ventrally via the receptor UNC-40/DCC [5,14], and (2) SLT-1/Slit is secreted by the dorsal muscles and repels the growth cone away from the dorsal side via the receptor SAX-3/Robo [12,13]. Animals for the guidance cues unc-6/netrin or slt-1/Slit exhibit partial AVM ventral axon guidance defects, and loss of both cues in unc-6 slt-1 double mutants results in fully penetrant guidance defects (S1 Fig, [13]). AVM axons defective in guidance fail to extend ventrally and instead migrate laterally in the anterior direction (Fig 1). In this study, we use the AVM axon as a model to elucidate mechanisms that regulate UNC-6/netrin signaling.

Bottom Line: However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown.Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor.We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor-expressing cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor-expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.

No MeSH data available.


Related in: MedlinePlus