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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

unc-6/netrin signaling via the unc-5/UNC5 receptor requires lon-2/glypican.(A) The axon of PVM normally migrates ventrally in the wild type, but it can be forced to migrate dorsally by misexpressing the repulsive receptor unc-5/UNC5. We quantified PVM since AVM could not be reliably identified (both AVM and neighboring ALMR axons project dorsally in Pmec-7::unc-5 transgenic animals.) (B) Upon misexpression of unc-5/UNC5 in PVM, using the transgene Pmec-7::unc-5, the axon of PVM projects dorsally in an unc-6/netrin-, unc-40/DCC-, and unc-34/enabled-dependent manner. Loss of lon-2/glypican partially suppresses this forced dorsal migration, indicating that unc-6/netrin signaling depends on lon-2/glypican. Scale bar, 5 μm. Error bars are standard error of the proportion. Asterisks denote significant difference: *** p ≤ 0.001 (z-tests, p-values were corrected by multiplying by the number of comparisons). ns, not significant. Wild type (without evIs25) is the same as in Fig 1B.
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pbio.1002183.g003: unc-6/netrin signaling via the unc-5/UNC5 receptor requires lon-2/glypican.(A) The axon of PVM normally migrates ventrally in the wild type, but it can be forced to migrate dorsally by misexpressing the repulsive receptor unc-5/UNC5. We quantified PVM since AVM could not be reliably identified (both AVM and neighboring ALMR axons project dorsally in Pmec-7::unc-5 transgenic animals.) (B) Upon misexpression of unc-5/UNC5 in PVM, using the transgene Pmec-7::unc-5, the axon of PVM projects dorsally in an unc-6/netrin-, unc-40/DCC-, and unc-34/enabled-dependent manner. Loss of lon-2/glypican partially suppresses this forced dorsal migration, indicating that unc-6/netrin signaling depends on lon-2/glypican. Scale bar, 5 μm. Error bars are standard error of the proportion. Asterisks denote significant difference: *** p ≤ 0.001 (z-tests, p-values were corrected by multiplying by the number of comparisons). ns, not significant. Wild type (without evIs25) is the same as in Fig 1B.

Mentions: To complement the above loss-of-function approach, we next used a gain-of-function strategy to test the model that lon-2/glypican functions in the unc-6/netrin signaling pathway. We focused on the axon of the PVM neuron instead of AVM, because it could reliably be identified (AVM cannot be distinguished from ALMR in these experiments). In wild-type animals, PVM, like AVM, expresses the receptor unc-40/DCC, and its axon grows ventrally towards unc-6/netrin (Fig 3A). In mutants lacking unc-6/netrin signaling, PVM axons that fail to extend ventrally instead extend anteriorly (never dorsally, see S4 Table). The PVM axon normally does not express the receptor unc-5/UNC5 that mediates repulsive guidance away from ventral unc-6/netrin [6], but misexpression of the receptor unc-5/UNC5 (using transgene Pmec-7::unc-5 [21]) in PVM forces its axon to extend dorsally in an unc-6/netrin- and unc-40/DCC-dependent manner (Fig 3A and 3B, [21]). We used this unc-6/netrin-dependent unc-5/UNC5-mediated abnormal dorsal migration to further investigate the function of lon-2/glypican in netrin signaling. By analyzing lon-2/glypican mutants carrying Pmec-7::unc-5, we found that compete loss of lon-2/glypican function significantly suppressed the unc-6/netrin-dependent unc-5-mediated abnormal dorsal migration of the PVM axon, indicating that unc-6/netrin signaling is lon-2/glypican dependent (Fig 3B). In contrast, the complete loss of sdn-1/syndecan, of slt-1/Slit, or of sax-3/Robo function did not suppress these PVM abnormal dorsal migrations (Fig 3B, see S4 Table), highlighting the specificity of lon-2/glypican action on unc-6/netrin signaling. As expected, lon-2 sdn-1 double mutants lacking both lon-2/glypican and sdn-1/syndecan and expressing unc-5/UNC5 in PVM did not further suppress the abnormal unc-5/UNC-5-mediated dorsal migration of PVM as compared to lon-2 single mutants, further supporting the specificity of lon-2/glypican on unc-6/netrin signaling.


Glypican Is a Modulator of Netrin-Mediated Axon Guidance.

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

unc-6/netrin signaling via the unc-5/UNC5 receptor requires lon-2/glypican.(A) The axon of PVM normally migrates ventrally in the wild type, but it can be forced to migrate dorsally by misexpressing the repulsive receptor unc-5/UNC5. We quantified PVM since AVM could not be reliably identified (both AVM and neighboring ALMR axons project dorsally in Pmec-7::unc-5 transgenic animals.) (B) Upon misexpression of unc-5/UNC5 in PVM, using the transgene Pmec-7::unc-5, the axon of PVM projects dorsally in an unc-6/netrin-, unc-40/DCC-, and unc-34/enabled-dependent manner. Loss of lon-2/glypican partially suppresses this forced dorsal migration, indicating that unc-6/netrin signaling depends on lon-2/glypican. Scale bar, 5 μm. Error bars are standard error of the proportion. Asterisks denote significant difference: *** p ≤ 0.001 (z-tests, p-values were corrected by multiplying by the number of comparisons). ns, not significant. Wild type (without evIs25) is the same as in Fig 1B.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493048&req=5

pbio.1002183.g003: unc-6/netrin signaling via the unc-5/UNC5 receptor requires lon-2/glypican.(A) The axon of PVM normally migrates ventrally in the wild type, but it can be forced to migrate dorsally by misexpressing the repulsive receptor unc-5/UNC5. We quantified PVM since AVM could not be reliably identified (both AVM and neighboring ALMR axons project dorsally in Pmec-7::unc-5 transgenic animals.) (B) Upon misexpression of unc-5/UNC5 in PVM, using the transgene Pmec-7::unc-5, the axon of PVM projects dorsally in an unc-6/netrin-, unc-40/DCC-, and unc-34/enabled-dependent manner. Loss of lon-2/glypican partially suppresses this forced dorsal migration, indicating that unc-6/netrin signaling depends on lon-2/glypican. Scale bar, 5 μm. Error bars are standard error of the proportion. Asterisks denote significant difference: *** p ≤ 0.001 (z-tests, p-values were corrected by multiplying by the number of comparisons). ns, not significant. Wild type (without evIs25) is the same as in Fig 1B.
Mentions: To complement the above loss-of-function approach, we next used a gain-of-function strategy to test the model that lon-2/glypican functions in the unc-6/netrin signaling pathway. We focused on the axon of the PVM neuron instead of AVM, because it could reliably be identified (AVM cannot be distinguished from ALMR in these experiments). In wild-type animals, PVM, like AVM, expresses the receptor unc-40/DCC, and its axon grows ventrally towards unc-6/netrin (Fig 3A). In mutants lacking unc-6/netrin signaling, PVM axons that fail to extend ventrally instead extend anteriorly (never dorsally, see S4 Table). The PVM axon normally does not express the receptor unc-5/UNC5 that mediates repulsive guidance away from ventral unc-6/netrin [6], but misexpression of the receptor unc-5/UNC5 (using transgene Pmec-7::unc-5 [21]) in PVM forces its axon to extend dorsally in an unc-6/netrin- and unc-40/DCC-dependent manner (Fig 3A and 3B, [21]). We used this unc-6/netrin-dependent unc-5/UNC5-mediated abnormal dorsal migration to further investigate the function of lon-2/glypican in netrin signaling. By analyzing lon-2/glypican mutants carrying Pmec-7::unc-5, we found that compete loss of lon-2/glypican function significantly suppressed the unc-6/netrin-dependent unc-5-mediated abnormal dorsal migration of the PVM axon, indicating that unc-6/netrin signaling is lon-2/glypican dependent (Fig 3B). In contrast, the complete loss of sdn-1/syndecan, of slt-1/Slit, or of sax-3/Robo function did not suppress these PVM abnormal dorsal migrations (Fig 3B, see S4 Table), highlighting the specificity of lon-2/glypican action on unc-6/netrin signaling. As expected, lon-2 sdn-1 double mutants lacking both lon-2/glypican and sdn-1/syndecan and expressing unc-5/UNC5 in PVM did not further suppress the abnormal unc-5/UNC-5-mediated dorsal migration of PVM as compared to lon-2 single mutants, further supporting the specificity of lon-2/glypican on 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