Article Figures & Data
Figures
- Figure 1.
PTPRO expression pattern in the developing TG. A, Representative images of PTPRO and Tuj1 immunostainings of sagittal sections of E10.5, E11.5, and E12.5 TG. Arrows point to cell bodies and arrowheads to axons. Scale bar, 200 μm (E10.5 and E11.5) and 400 μm (E12.5). B, Graph shows mean ± SEM of the percentage of TrkA+, TrkB+, TrkC+, and Ret+ neurons expressing PTPRO at E12.5, E15.5, and P0. For each data point, n = 3 embryos (9 images/embryo). For each group (TrkA, TrkB, TrkC, and Ret), the percentages of neurons expressing PTPRO at different developmental stages were compared using one-way ANOVA, followed by Bonferroni's post hoc comparison test (*p < 0.05, **p < 0.01). C–F, Confocal images showing colocalization in E12.5 and P0 TG of PTPRO with TrkA (C), TrkB (D), TrkC (E), and Ret (F). Scale bar, 100 μm. Arrowheads point to neurons coexpressing PTPRO and either Trks or Ret.
- Figure 2.
PTPRO−/− embryos show exuberant arborization of the ophthalmic branch of the trigeminal nerve and defasciculation of the maxillary branch. A, D, Representative pictures of trigeminal nerve branches from whole-mount neurofilament stained E11.5 and E12.5 embryos. Red and blue dashed lines encircle the area of ophthalmic and maxillary arbors, respectively. The inset shows a higher magnification of the arbor of the ophthalmic branch that was analyzed (A). Bottom panels in D show tracings of the ophthalmic arbors. B, Graph represents the mean ± SEM area of 18 wild-type, 21 PTPRO+/−, and 15 PTPRO−/− ophthalmic and maxillary arbors. C, Graph represents the mean ± SEM ratio of the areas covered by the ophthalmic and maxillary arbors. Numbers of embryos analyzed were the same as in B. E, Sholl analysis of the ophthalmic arbor at E12.5 was done on 32 wild-type and PTPRO+/− and 24 PTPRO−/− TG. F, Tuj1 immunostaining on sagittal sections of E12.5 wild-type and PTPRO−/− TG. The inset displays a higher magnification of the maxillary nerve. Red arrowheads point to defasciculated axons. G, Graph represents the percentage of sections with defasciculated axons (mean ± SEM, n = 16 embryos per genotype). Statistical analysis was done using two-tailed Student's t test (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bar, 500 μm.
- Figure 3.
Cultured PTPRO−/− trigeminal neurons are more sensitive to BDNF and GDNF but not NGF stimulation. A, Representative pictures of E12.5 trigeminal neurons, stimulated with growth factors as indicated. Scale bar, 100 μm. Quantification of the length of the axons (B) or the number of branching points (C) of neurons stimulated as indicated on the x-axis. Graphs represent mean ± SEM. Numbers of trigeminal neurons analyzed from at least three independent cultures: for NGF stimulation, 200 neurons (wild type) and 195 neurons (PTPRO−/−); for BDNF and GDNF stimulation, 150 neurons per genotype. D, E, The length of axons and the number of branching points in response to increasing amounts of NGF, BDNF, and GDNF in the presence of caspase inhibitors (10 μm Q-Oph-VD) were measured in dissociated trigeminal cultures. Numbers of trigeminal neurons analyzed: no stimulation, 550 neurons; NGF stimulation, 150 neurons; BDNF and GDNF stimulation, 200 neurons per genotype. Statistical analysis was done as for Figure 2.
- Figure 4.
PTPRO−/− embryos do not have defects in neuronal differentiation, but there is a loss of TrkA+ and TrkC+ neurons at P0. A, C, Immunostainings for TrkA, TrkB, TrkC, Ret, and NeuN on cryosections from E12.5 (A) and newborn (C) wild-type and PTPRO−/− TG. Scale bars, 50 μm. B, D, Graphs represent the average number (mean ± SEM, n = 3–4 embryos, 9–20 images/embryo) of TrkA+, TrkB+, TrkC+,Ret+, and NeuN+ neurons per section. Statistical analysis was done as for Figure 2.
- Figure 5.
PTPRO dephosphorylates TrkB. A, HeLa cells transfected with TrkB and PTPRO and immunostained to detect surface expression of PTPRO (PTPRO surface) and TrkB (TrkB surface) and total expression of TrkB (TrkB total). B, C, HeLa cells transfected with TrkB with (C) or without (B) mPTPRO–Flag and immunostained for Flag (PTPRO), TrkB, and phosphotyrosine (pTyr). Cells outlines are labeled with Cell Mask Blue. Insets are higher-magnification images of the areas marked with a box. Scale bar, 20 μm. D, Graph represents the intensity of phosphotyrosine (pTyr) staining normalized by the intensity of TrkB staining (mean ± SEM). Number of cells analyzed: 26 cells for TrkB and 29 cells for TrkB and PTPRO from 3 independent experiments. E, Western blots of HEK293 cells transfected with TrkB with or without mPTPRO–Flag and stimulated as indicated. Total cell lysates (TCL) were probed against phospho-ERK (pERK), ERK1/2, and Flag (PTPRO). Immunoprecipitates of TrkB (IP αTrkB) were probed against pTyr and TrkB. F, G, Graphs represent TrkB autophosphorylation levels (F) and ERK phosphorylation (G). Three independent experiments were performed, and the intensities of the phospho bands were quantified using NIH ImageJ and normalized by the total levels of the proteins. Statistical analysis was done as for Figure 2.
- Figure 6.
PTPRO dephosphorylates Ret. A, HeLa cells transfected with Ret51 and PTPRO and immunostained to detect surface expression of PTPRO (PTPRO surface) and Ret (Ret surface) and total expression of Ret (Ret total). B–D, HeLa cells transfected with Ret51 and with (B, D) or without (C) mPTPRO–Flag and stimulated as indicated. Fixed cells were stained with anti-Flag (PTPRO), anti-Ret, and pTyr antibodies and marked with Cell Mask Blue. Scale bar, 20 μm. E, Graph represents the degree of colocalization of Ret and PTPRO (mean ± SEM) before and after GDNF stimulation, with (total) or without (surface) cell permeabilization. Numbers of cells analyzed after permeabilization (total staining): 23 cells before and 14 cells after stimulation from at least 3 independent experiments. Numbers of cells analyzed without permeabilization (surface staining): 13 cells before and 23 cells after stimulation from 3 independent experiments. F, Graph represents pTyr staining intensity normalized by the intensity of Ret staining (mean ± SEM). Numbers of cells analyzed: 48 cells before and 26 cells after stimulation for Ret alone, and 36 cells before and 26 cells after stimulation for Ret and PTPRO, from at least 3 independent experiments. G, Western blots of HEK293 cells transfected with Ret, with or without mPTPRO–Flag, and stimulated as indicated. TCL were probed against phosphotyrosine (pTyr), Ret phosphotyrosine 1062 (Ret pY1062), Ret, pERK, ERK1/2, and Flag (PTPRO). H–J, Graphs represent Ret autophosphorylation levels (H, I) or ERK phosphorylation (J). Quantification of phosphorylation was done as for Figure 5, and statistical analysis was done as for Figure 2.
- Figure 7.
Eph signaling is not affected in PTPRO−/− embryos. A, Representative pictures of trigeminal neuron explant cultures stimulated with 0.5 μg/ml preclustered Fc (negative control) or preclustered ephrinA5. Explants were stained with Phalloidin-568. Arrows point to non-collapsed growth cones, and arrowheads show collapsed growth cones. Scale bar, 100 μm. B, Graph represents the percentage of collapsed growth cones. Four to six explants per condition were analyzed from three embryos per genotype. C, Representative pictures for the analysis of the retinocollicular projection in wild-type and PTPRO−/− mice at P8. Injection of DiI into a small area of nasal retina (drawings on left side) result in labeling of topographically appropriate termination zones in the caudal part of the superior colliculus. Numbers of animals analyzed for the termination zone were 7 wild-type, 12 PTPRO+/−, and 5 PTPRO−/−. Numbers of animals analyzed for ectopic branching were three wild-type, four PTPRO+/−, and four PTPRO−/−. D, Representative pictures of motor neuron explant cultures stimulated with Fc (as a control) or ephrins. Arrows and arrowheads point to non-collapsed and collapsed growth cones, respectively. In green is the Hb9–GFP and in red the Phalloidin-568 staining. Scale bar, 200 μm. E, Graph represents the percentage of collapsed growth cones. Three to six explants per condition were analyzed from four embryos per genotype. Compared with the respective controls, all the ephrinA2/A5 stimulated explants show a statistically significant increase in the percentage of growth cone collapse (0.1 μg/ml ephrin-Fc on wild-type cultures, p = 0.038; 0.5 μg/ml on wild type, p = 0.008; 0.1 μg/ml on PTPRO−/−, p = 0.007; 0.5 μg/ml on PTPRO−/−, p = 0.002). F, Representative pictures of neurofilament stained E12.5 whole-mount embryos. Arrowheads point to the peroneal nerve; arrows point to the tibial nerve. G, Graph represents the ratios between the diameter of the peroneal and the tibial nerves of 16 wild-type, 12 PTPRO+/−, and 15 PTPRO−/− hindlimbs (mean ± SEM). H, Single confocal plane of ventral retrograde tracings in E12.5 embryos. RD was injected in the ventral shank of the hindlimb, and sections were stained with Islet1 and Lim1 to label the medial (LMCM) and lateral (LMCL) cohorts (populations are delimited by a dashed lines), respectively. Scale bar is 50 μm. I, Graph represents the percentage of misprojections (neurons positive for RD and Lim1 staining) in relation to all RD-labeled cells in ventral retrograde tracings of six wild-type or PTPRO+/− and eight PTPRO−/− embryos. J, Western blots of HEK293 cells cotransfected with EphA4 and mPTPRO or cPTPRO isoforms of PTPRO–Flag and stimulated as indicated. Total cell lysates (TCL) were probed against phospho- and total EphA4 and Flag. Asterisks indicate PTPRO bands; mPTPRO runs at 140 kDa and cPTPRO at 160 kDa. K, Graph represents the levels of EphA4 autophosphorylation (mean ± SEM) after control Fc and ephrinA4-Fc stimulation. The experiment was done in triplicate, and the intensities of the phospho bands were quantified using NIH ImageJ and normalized by the total level of the proteins. Statistical analysis was done as for Figure 2.
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