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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 epidermal cells underlying the developing axon.(A) Epidermal expression of lon-2/glypican is sufficient for function. Providing wild-type lon-2(+) in the hypodermis (under the heterologous hypodermal promoters Pdpy-7 and Pelt-3) rescues the function of lon-2 in the double mutants lon-2 slt-1, as it brings the defects down to the level of slt-1 single mutants. In contrast, expression of lon-2(+) in other epidermal cells (seam cells), the migrating neuron AVM, the intestine, or the body wall muscles fails to rescue the function of lon-2. For each rescued transgenic line, transgenic animals were compared to nontransgenic sibling controls (see S2 and S3 Tables). Data for wild type, lon-2, slt-1, and lon-2 slt-1 are the same as in Fig 1B and 1C. (B) Expression of sdn-1/syndecan in the migrating neuron is sufficient for function. Providing wild-type copies of sdn-1(+) in AVM (expressed under the heterologous promoter Pmec-7) rescues the axon guidance function of sdn-1 in a lon-2 sdn-1 double mutant. We assayed rescue of sdn-1 function using the double mutant lon-2 sdn-1 since it is easier to rescue defects that are 33% penetrant (as in the double lon-2(e678) sdn-1(zh20)) than to rescue defects that are 12% penetrant (as in the single mutant sdn-1(zh20)). For each transgenic line, transgenic animals were compared to nontransgenic sibling controls (see S2 Table). Data for wild type, lon-2, sdn-1, and lon-2 sdn-1 are the same as in Fig 1B–1D. Scale bar, 5 μm. 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.g004: lon-2/glypican functions in the epidermal cells underlying the developing axon.(A) Epidermal expression of lon-2/glypican is sufficient for function. Providing wild-type lon-2(+) in the hypodermis (under the heterologous hypodermal promoters Pdpy-7 and Pelt-3) rescues the function of lon-2 in the double mutants lon-2 slt-1, as it brings the defects down to the level of slt-1 single mutants. In contrast, expression of lon-2(+) in other epidermal cells (seam cells), the migrating neuron AVM, the intestine, or the body wall muscles fails to rescue the function of lon-2. For each rescued transgenic line, transgenic animals were compared to nontransgenic sibling controls (see S2 and S3 Tables). Data for wild type, lon-2, slt-1, and lon-2 slt-1 are the same as in Fig 1B and 1C. (B) Expression of sdn-1/syndecan in the migrating neuron is sufficient for function. Providing wild-type copies of sdn-1(+) in AVM (expressed under the heterologous promoter Pmec-7) rescues the axon guidance function of sdn-1 in a lon-2 sdn-1 double mutant. We assayed rescue of sdn-1 function using the double mutant lon-2 sdn-1 since it is easier to rescue defects that are 33% penetrant (as in the double lon-2(e678) sdn-1(zh20)) than to rescue defects that are 12% penetrant (as in the single mutant sdn-1(zh20)). For each transgenic line, transgenic animals were compared to nontransgenic sibling controls (see S2 Table). Data for wild type, lon-2, sdn-1, and lon-2 sdn-1 are the same as in Fig 1B–1D. Scale bar, 5 μm. 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: The AVM growth cone extends along a basement membrane that is located between the epidermis, which is referred to as the hypodermis, and body wall muscles [11]. lon-2/glypican is expressed in the hypodermis and the intestine [28]. We asked in which cell type lon-2/glypican needs to be produced to guide AVM. We found that wild-type lon-2(+) transgenes expressed under the heterologous epidermal promoters Pdpy-7 and Pelt-3 (that drive expression in the hypodermis underlying the AVM growth cone, hyp7) rescued lon-2 slt-1 double mutants back to slt-1 single mutant levels, as efficiently as when expressed under the endogenous promoter Plon-2 (Fig 4A, S3 Table). Rescue was not observed when we expressed lon-2/glypican in other epidermal cells (seam cells, Pgrd-10), in the migrating neuron itself (Pmec-7), in the intestine (Pelt-2), or in body wall muscles (Pmyo-3) (Fig 4A, S3 Table). Our results suggest that lon-2/glypican is produced by the hypodermis underlying the growth cone of AVM to function in axon guidance.


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 epidermal cells underlying the developing axon.(A) Epidermal expression of lon-2/glypican is sufficient for function. Providing wild-type lon-2(+) in the hypodermis (under the heterologous hypodermal promoters Pdpy-7 and Pelt-3) rescues the function of lon-2 in the double mutants lon-2 slt-1, as it brings the defects down to the level of slt-1 single mutants. In contrast, expression of lon-2(+) in other epidermal cells (seam cells), the migrating neuron AVM, the intestine, or the body wall muscles fails to rescue the function of lon-2. For each rescued transgenic line, transgenic animals were compared to nontransgenic sibling controls (see S2 and S3 Tables). Data for wild type, lon-2, slt-1, and lon-2 slt-1 are the same as in Fig 1B and 1C. (B) Expression of sdn-1/syndecan in the migrating neuron is sufficient for function. Providing wild-type copies of sdn-1(+) in AVM (expressed under the heterologous promoter Pmec-7) rescues the axon guidance function of sdn-1 in a lon-2 sdn-1 double mutant. We assayed rescue of sdn-1 function using the double mutant lon-2 sdn-1 since it is easier to rescue defects that are 33% penetrant (as in the double lon-2(e678) sdn-1(zh20)) than to rescue defects that are 12% penetrant (as in the single mutant sdn-1(zh20)). For each transgenic line, transgenic animals were compared to nontransgenic sibling controls (see S2 Table). Data for wild type, lon-2, sdn-1, and lon-2 sdn-1 are the same as in Fig 1B–1D. Scale bar, 5 μm. 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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getmorefigures.php?uid=PMC4493048&req=5

pbio.1002183.g004: lon-2/glypican functions in the epidermal cells underlying the developing axon.(A) Epidermal expression of lon-2/glypican is sufficient for function. Providing wild-type lon-2(+) in the hypodermis (under the heterologous hypodermal promoters Pdpy-7 and Pelt-3) rescues the function of lon-2 in the double mutants lon-2 slt-1, as it brings the defects down to the level of slt-1 single mutants. In contrast, expression of lon-2(+) in other epidermal cells (seam cells), the migrating neuron AVM, the intestine, or the body wall muscles fails to rescue the function of lon-2. For each rescued transgenic line, transgenic animals were compared to nontransgenic sibling controls (see S2 and S3 Tables). Data for wild type, lon-2, slt-1, and lon-2 slt-1 are the same as in Fig 1B and 1C. (B) Expression of sdn-1/syndecan in the migrating neuron is sufficient for function. Providing wild-type copies of sdn-1(+) in AVM (expressed under the heterologous promoter Pmec-7) rescues the axon guidance function of sdn-1 in a lon-2 sdn-1 double mutant. We assayed rescue of sdn-1 function using the double mutant lon-2 sdn-1 since it is easier to rescue defects that are 33% penetrant (as in the double lon-2(e678) sdn-1(zh20)) than to rescue defects that are 12% penetrant (as in the single mutant sdn-1(zh20)). For each transgenic line, transgenic animals were compared to nontransgenic sibling controls (see S2 Table). Data for wild type, lon-2, sdn-1, and lon-2 sdn-1 are the same as in Fig 1B–1D. Scale bar, 5 μm. 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: The AVM growth cone extends along a basement membrane that is located between the epidermis, which is referred to as the hypodermis, and body wall muscles [11]. lon-2/glypican is expressed in the hypodermis and the intestine [28]. We asked in which cell type lon-2/glypican needs to be produced to guide AVM. We found that wild-type lon-2(+) transgenes expressed under the heterologous epidermal promoters Pdpy-7 and Pelt-3 (that drive expression in the hypodermis underlying the AVM growth cone, hyp7) rescued lon-2 slt-1 double mutants back to slt-1 single mutant levels, as efficiently as when expressed under the endogenous promoter Plon-2 (Fig 4A, S3 Table). Rescue was not observed when we expressed lon-2/glypican in other epidermal cells (seam cells, Pgrd-10), in the migrating neuron itself (Pmec-7), in the intestine (Pelt-2), or in body wall muscles (Pmyo-3) (Fig 4A, S3 Table). Our results suggest that lon-2/glypican is produced by the hypodermis underlying the growth cone of AVM to function in axon guidance.

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