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

Stepanek L, Sun QL, Wang J, Wang C, Bixby JL - J. Cell Biol. (2001)

Bottom Line: We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons.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.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, University of Miami School of Medicine, Miami, FL 33136, USA.

ABSTRACT
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.

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Localization of cPTPRO binding sites and cPTPRO in E7 chick retina. 10-μm sections of chick retina were cut on a cryostat. (A and B) 5 μg/ml of cPTPRO–Fc was allowed to bind to E7 tissue sections (B). 10 μg/ml of mIgG-1 was used as the negative control (A). Staining was most noticeable in the RGC layer, suggesting that this is the predominant location of binding partner(s) for cPTPRO. (C and D) Sections adjacent to those above were used to compare cPTPRO location. The optic fiber layer (OFL) and RGC layer were stained most strongly by anti-cPTPRO antibody (D). The IgG portion of preimmune serum was used as a control (C). Similar results were obtained in multiple experiments. INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer; PR, photoreceptors. The dark stained layer in all panels is pigment epithelium. Bar, 100 μm.
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fig3: Localization of cPTPRO binding sites and cPTPRO in E7 chick retina. 10-μm sections of chick retina were cut on a cryostat. (A and B) 5 μg/ml of cPTPRO–Fc was allowed to bind to E7 tissue sections (B). 10 μg/ml of mIgG-1 was used as the negative control (A). Staining was most noticeable in the RGC layer, suggesting that this is the predominant location of binding partner(s) for cPTPRO. (C and D) Sections adjacent to those above were used to compare cPTPRO location. The optic fiber layer (OFL) and RGC layer were stained most strongly by anti-cPTPRO antibody (D). The IgG portion of preimmune serum was used as a control (C). Similar results were obtained in multiple experiments. INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer; PR, photoreceptors. The dark stained layer in all panels is pigment epithelium. Bar, 100 μm.

Mentions: Previous experiments demonstrated that cPTPRO is expressed in the embryonic chick retina. In particular, cPTPRO is strongly expressed in RGCs and their axons from E6 to E10 (when axons are growing to the tectum), in various fiber layers of the retina by E10, and becomes downregulated after this time (Ledig et al., 1999b). We used the cPTPRO–Fc fusion protein to localize potential binding partners for cPTPRO in the retina. Sections of E7 chick retina were stained by binding cPTPRO–Fc to the sections, followed by anti-Fc immunohistochemistry to localize the bound fusion protein. cPTPRO binding sites were found in most of the neural retina (except the photoreceptor layer), with the strongest staining in the RGC layer and the weakest in the outer plexiform layer (Fig. 3 B). The optic fiber layer, containing the axons of the RGCs, was nearly devoid of staining. Adjacent sections stained with antibodies to cPTPRO confirmed that the majority of cPTPRO immunoreactivity is found both in the optic fiber layer and the ganglion cell layer at this time (Fig. 3 D). Control sections stained with mIgG alone demonstrated the specificity of binding (Fig. 3, A and C). These results demonstrate the presence of at least one cPTPRO binding partner in the retina, which apparently is expressed in several cell types. Because the distribution of cPTPRO and its binding partner overlap but are not identical, our results suggest, but do not prove, that cPTPRO has at least one heterophilic binding partner in the retina during embryogenesis.


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

Stepanek L, Sun QL, Wang J, Wang C, Bixby JL - J. Cell Biol. (2001)

Localization of cPTPRO binding sites and cPTPRO in E7 chick retina. 10-μm sections of chick retina were cut on a cryostat. (A and B) 5 μg/ml of cPTPRO–Fc was allowed to bind to E7 tissue sections (B). 10 μg/ml of mIgG-1 was used as the negative control (A). Staining was most noticeable in the RGC layer, suggesting that this is the predominant location of binding partner(s) for cPTPRO. (C and D) Sections adjacent to those above were used to compare cPTPRO location. The optic fiber layer (OFL) and RGC layer were stained most strongly by anti-cPTPRO antibody (D). The IgG portion of preimmune serum was used as a control (C). Similar results were obtained in multiple experiments. INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer; PR, photoreceptors. The dark stained layer in all panels is pigment epithelium. Bar, 100 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196468&req=5

fig3: Localization of cPTPRO binding sites and cPTPRO in E7 chick retina. 10-μm sections of chick retina were cut on a cryostat. (A and B) 5 μg/ml of cPTPRO–Fc was allowed to bind to E7 tissue sections (B). 10 μg/ml of mIgG-1 was used as the negative control (A). Staining was most noticeable in the RGC layer, suggesting that this is the predominant location of binding partner(s) for cPTPRO. (C and D) Sections adjacent to those above were used to compare cPTPRO location. The optic fiber layer (OFL) and RGC layer were stained most strongly by anti-cPTPRO antibody (D). The IgG portion of preimmune serum was used as a control (C). Similar results were obtained in multiple experiments. INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer; PR, photoreceptors. The dark stained layer in all panels is pigment epithelium. Bar, 100 μm.
Mentions: Previous experiments demonstrated that cPTPRO is expressed in the embryonic chick retina. In particular, cPTPRO is strongly expressed in RGCs and their axons from E6 to E10 (when axons are growing to the tectum), in various fiber layers of the retina by E10, and becomes downregulated after this time (Ledig et al., 1999b). We used the cPTPRO–Fc fusion protein to localize potential binding partners for cPTPRO in the retina. Sections of E7 chick retina were stained by binding cPTPRO–Fc to the sections, followed by anti-Fc immunohistochemistry to localize the bound fusion protein. cPTPRO binding sites were found in most of the neural retina (except the photoreceptor layer), with the strongest staining in the RGC layer and the weakest in the outer plexiform layer (Fig. 3 B). The optic fiber layer, containing the axons of the RGCs, was nearly devoid of staining. Adjacent sections stained with antibodies to cPTPRO confirmed that the majority of cPTPRO immunoreactivity is found both in the optic fiber layer and the ganglion cell layer at this time (Fig. 3 D). Control sections stained with mIgG alone demonstrated the specificity of binding (Fig. 3, A and C). These results demonstrate the presence of at least one cPTPRO binding partner in the retina, which apparently is expressed in several cell types. Because the distribution of cPTPRO and its binding partner overlap but are not identical, our results suggest, but do not prove, that cPTPRO has at least one heterophilic binding partner in the retina during embryogenesis.

Bottom Line: We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons.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.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, University of Miami School of Medicine, Miami, FL 33136, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus