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. 2013 Jun 5;78(5):813-26.
doi: 10.1016/j.neuron.2013.04.001.

Interactions between a receptor tyrosine phosphatase and a cell surface ligand regulate axon guidance and glial-neuronal communication

Hyung-Kook Peter Lee  1 , Amy Cording, Jost Vielmetter, Kai Zinn
Affiliations

Interactions between a receptor tyrosine phosphatase and a cell surface ligand regulate axon guidance and glial-neuronal communication

Hyung-Kook Peter Lee et al. Neuron. .

Abstract

We developed a screening method for orphan receptor ligands, in which cell-surface proteins are expressed in Drosophila embryos from GAL4-dependent insertion lines and ligand candidates identified by the presence of ectopic staining with receptor fusion proteins. Stranded at second (Sas) binds to the receptor tyrosine phosphatase Ptp10D in embryos and in vitro. Sas and Ptp10D can interact in trans when expressed in cultured cells. Interactions between Sas and Ptp10D on longitudinal axons are required to prevent them from abnormally crossing the midline. Sas is expressed on both neurons and glia, whereas Ptp10D is restricted to CNS axons. We conducted epistasis experiments by overexpressing Sas in glia and examining how the resulting phenotypes are changed by removal of Ptp10D from neurons. We find that neuronal Ptp10D restrains signaling by overexpressed glial Sas, which would otherwise produce strong glial and axonal phenotypes.

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Figures

Figure 1
Figure 1. Identification of Sas in an ectopic expression screen for binding to 10D-AP
(A) Flowchart of the screen. (B–K) Staining of live-dissected late stage 16 embryos with 10D-AP. The top row of images shows double-labeling of CNS longitudinal tracts and motor axons with mAb 1D4 (Alexa 488, green) and 10D-AP (Alexa 568, magenta); the bottom row shows 10D-AP labeling alone, in white. (B–C) A whole filleted embryo. Note that 10D-AP prominently stains VNC axons (arrowhead) and the brain (white spot; arrow). (D–K) Each image shows two hemisegments, including the CNS and the ventral and lateral body walls. Anterior is to the left, and dorsal is up. (D–E) Wild-type (yw). The axon tracts of the CNS (out of focus) are stained by 10D-AP, but there is no clear pattern in the body wall. (F–G) 24B>Sas. The ventral, ventrolateral, and lateral muscle fibers are brightly stained. Arrowhead indicates muscle 12. (H–I) Repo>Sas. Peripheral glia surrounding the motor and sensory axon tracts are stained (arrowhead). The CNS is brighter due to staining of CNS glia (out of focus; arrow). (J–K) 5053A>Sas. This driver expresses only in muscle 12, which is brightly labeled (arrowhead). Scale bars in (A) and (D), 20 μm.
Figure 2
Figure 2. Sas-Fc binds to Ptp10D in embryos
Live-dissected late stage 16 embryos double-stained with anti-Ptp10D mAb (top row) and Sas-Fc (bottom row) using immunofluorescence for detection (Alexa 488 for Ptp10D, Alexa 568 for Sas-Fc). In (A–H), four segments of the CNS are shown in each panel; anterior is up. In (I–L), two segments of the CNS, ventral, and ventrolateral muscles are shown in each panel; anterior is to the left and dorsal is up. (A–B) Wild-type. Sas-Fc weakly stains CNS axons. (C–D) Ptp4E1. Sas-Fc staining intensity is increased relative to (B). CNS axons are labeled, in addition to staining at the midline (arrow) and on cell bodies, which are located on the sides of the axon ladder. (E–F) Ptp4E1 Ptp10D1. No Ptp10D staining is observed in (E). Sas-Fc staining intensity is reduced relative to (D). (G–H) Ptp4E1, Elav>Ptp10D. The intensity of Ptp10D axonal staining is increased relative to (C), and some cell body staining is observed. Sas-Fc staining intensity is increased relative to (D), and both axons and cell bodies are stained. (I–J) Ptp4E1. Anti-Ptp10D stains ganglionic tracheal branches (arrow). Sas-Fc labeling has no clear pattern in the periphery. (K–L) Ptp4E1, Tub>Ptp10D. The edges of the ventral and ventrolateral muscles are brightly labeled by anti-Ptp10D in (K) and by Sas-Fc in (L). Arrowhead indicates muscle 6. One brightly labeled, out-of-focus CNS longitudinal tract (bracket) is seen in each panel. This tract is much brighter in (K) vs. (I), and in (L) vs. (J), reflecting Ptp10D overexpression and increased Sas-Fc staining on axons. Scale bar in (A): 10 μm.
Figure 3
Figure 3. Sas-Ptp10D interactions in vitro and in cultured cells
The bar graphs show the absorbance at 370 nm obtained using the modified ELISA assay for the indicated pairs of proteins. The height of each bar represents the mean of 8 replicate assays. (A) Sas-Fc binds to 10D-AP, but not to 69D-AP or Lar-AP. Actual values for the four pairs are: Sas-Fc::10D-AP: 0.340±0.040 (S.D.); Sas-Fc::Lar-AP: 0.007±0.001; Sas-Fc::69D-AP: 0.010±0.002; Sas-Fc::blank: 0.017±0.004. The limit of quantification (LOQ) is given by: LOQ = (mean blank signal) + (10 × S.D. for the blank). If a signal is above the LOQ it is very unlikely that the difference between the signal and the blank arose by chance. Here LOQ is 0.053, signal is 0.340, and signal/LOQ is >6. (B) 10D-AP binds to Sas-Fc, but not to Unc5-Fc or FasII-Fc. Actual values are: Sas-Fc::10D-AP: 0.375±0.104; Sas-Fc::Lar-AP: 0.010±0.004; Unc5-Fc::10D-AP: 0.012±0.002; FasII-Fc::10D-AP: 0.028±0.008. LOQ for Unc5-Fc::10D-AP is 0.031, and signal/LOQ is 12; LOQ for FasII-Fc::10D-AP is 0.111, and signal/LOQ is 3.4. (C) A field of Ptp10D-expressing stably transfected S2 cells (magenta). A typical cluster is circled (dotted outline). Scale bar, 20 μm. (D) A field of Sas-expressing stably transfected S2 cells (green). No clusters form when these cells are incubated alone. (E) A field of a mixture of Ptp10D and Sas-expressing cells. The outlined cluster of Ptp10D-expressing cells has several green Sas cells associated with it.
Figure 4
Figure 4. Expression patterns of Ptp10D and Sas at late stage 16
Embryos were double-stained with anti-Ptp10D mAb and rabbit anti-Sas, and visualized by immunofluorescence. (A) Ptp10D. Note that CNS axons are brightly stained, and cell outlines are almost invisible. (B) Sas. The intersegmental (arrowhead) and segmental (double arrowhead) nerve roots are brightly stained. Two longitudinal bundles express Sas at higher levels within each longitudinal tract (arrows). Cell outlines are clearly visible (ring around asterisk). (C) Ptp10D. The apical surfaces of tracheal branches are brightly labeled. Arrowheads in (C) and (D) indicate the dorsal trunk. (D) Sas. The tracheal pattern of Sas expression is essentially identical to that of Ptp10D. Scale bars in (A) and (C): 10 μm.
Figure 5
Figure 5. sas LOF CNS phenotypes
Embryos were stained with mAb 1D4 or mAb BP102, and HRP immunohistochemistry was used for detection. Each panel shows three segments of the VNC, which was dissected out of the embryos. (A, B, G, H): stage 14. (C, D, I, J): early stage 16. (E, F, K, L): late stage 16. (G, H) sas15/Df transheterozygotes are indistinguishable from wild-type (yw) (A, B) at stage 14 using either marker. (I, J) Slight irregularities in the 1D4 and BP102 patterns are observed at early stage 16 in sas15/Df. (E) In late stage 16 wild-type embryos, 1D4 labels three longitudinal bundles on each side of the CNS, and does not stain commissural axons. (K) In sas15/Df at late stage 16, the outer 1D4 bundle is interrupted (arrowhead), and there are occasional axon bundles crossing the midline (arrow). (F) In late stage 16 wild-type embryos, a regular BP102 ladder is observed. (L) In sas15/Df at late stage 16, the ladder is slightly irregular and narrowed. One segment has an abnormal anterior commissure (bracket).
Figure 6
Figure 6. Ptp10D and Sas work together in neurons to regulate axon guidance across the midline
1D4 (A–E) and BP102 (F–J) staining at late stage 16 is shown. (B, G) Ptp10D, sas. The 1D4 and BP102 patterns are like that of sas (Figure 5) with occasional axon bundles crossing the midline (arrow) and interruptions in the outer 1D4 bundle (arrowhead). The BP102 ladder has an abnormal anterior commissure (arrowhead). (C, H) Ptp10D, Ptp69D. In (C), thick axon bundles cross the midline in each segment (bracket), and there is only one distinct longitudinal bundle. In (H), the commissures are completely fused in all three segments (bracket), and the longitudinal tracts are thinned (arrowhead). (D, I) sas, Ptp69D. In (D), two bundles cross the midline in each segment (bracket), and the outer longitudinal tract is often missing (arrowhead). In (I), commissural organization is disrupted (bracket), and longitudinal tracts are thinned (arrowhead). (E, J) sas, Ptp69D, FasII>Sas. The 1D4 and BP102 patterns are almost like wild-type. Mutant genotypes shown in panels are: Ptp10D1, sas15/Df(3R)ED5221 (B, G); Ptp10D1, Ptp69D1/Df(3L)8ex25 (C, H); sas15/sas15, Ptp69D1/Df(3L)8ex25 (D); sas15/Df(3R)ED5221, Ptp69D1/Df(3L)8ex25 (I); sas15/Df(3R)ED5221, Ptp69D1/Df(3L)8ex25, FasII-GAL4Mz507, UAS-Sas cDNA (E, J). (K) Bar graph quantitating the % of segments exhibiting ectopic midline crossing of 1D4 axons. Brown bar, Ptp10D Ptp69D; gray bars, sas15 genotypes; black bars, sas15/Df genotypes. n, number of segments scored (n=162 for wt (yw); 128 for Ptp10D; 738 for Ptp69D; 864 for sas; 585 for sas/Df; 702 for Ptp10D, sas; 522 for Ptp10D, sas/Df; 225 for Ptp10D Ptp69D; 324 for sas, Ptp69D; 685 for sas/Df, Ptp69D, 567 for sas, Ptp69D, FasII>Sas; 284 for sas/Df, Ptp69D, FasII>Sas). p<.00001 for the differences in penetrance between sas/Df and sas/Df Ptp69D, and between sas/Df Ptp69D and sas/Df Ptp69D, FasII>Sas.
Figure 7
Figure 7. Interactions between glial Sas and axonal Ptp10D affect glial organization and axon guidance
(A–D) 1D4 staining at late stage 16. In (A–C), a normal 1D4 pattern is observed, although the 1D4 bundles are slightly wavy in Repo>Sas (C), and one segment has an axon bundle crossing the midline (arrow). (D) In Ptp10D1, Repo>Sas, a strong axon guidance phenotype is observed. The inner 1D4 bundle crosses the midline in all three segments (arrow), and the longitudinal bundles are partially fused and disrupted (arrowhead). (E) Bar graph quantitating the % of segments exhibiting ectopic midline crossing of 1D4 axons. n=162 for wt; 128 for Ptp10D−; 414 for Repo>Sas; 424 for Ptp10D−, Repo>Sas. p<.00001 for the difference in penetrance between Repo>Sas and Ptp10D−, Repo>Sas. (F–I) Glial nuclei, visualized with anti-dsRed. Repo-GAL4 was used to drive RedStinger (a nuclear version of dsRed) in the indicated genotypes. The focal plane is at the level of the interface glia. Note the organized glial lattice in (F–H), with a double vertical row of nuclei along each longitudinal tract and a band of nuclei crossing the midline in each segment. The vertical rows are slightly irregular in (H). (I) In Ptp10D1, Repo>Sas, the glial lattice is disrupted. The vertical rows have become aggregations of varying width, and there is no regular pattern of glial nuclei near the midline.
Figure 8
Figure 8. Models for signaling by Ptp10D and Sas
Triangles, VWC domains; blue circles, FN3 repeats (Ptp10D actually has 12); rectangles, Ig domains; red circles, PTP domains; double green lines, cell membranes. (A) Models for signaling in neurons in wild-type. The sas Ptp69D phenotype is rescued by Sas expression in longitudinal tract neurons, suggesting that either: 1) Sas interacts with Ptp10D in the same cell, or, 2) ECM-bound or transmembrane Sas on other longitudinal axons interacts with Ptp10D. Ptp10D/Sas and Ptp69D may both affect the activity of a common downstream signaling protein(s) that helps prevent longitudinal axons from crossing the midline. Ptp69D is cleaved as indicated. (B) Model for signaling between glia and neurons in embryos overexpressing Sas in glia. Overexpressed Sas produces a signal in glia (top) that alters their presentation of axon guidance cues to neurons (bottom). Generation of the signal is suppressed (⊥ symbol) by interactions between neuronal Ptp10D and glial Sas.
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