Two receptor tyrosine phosphatases dictate the depth of axonal stabilizing layer in the visual system
- Tokyo Institute of Technology, Japan
Figures
Figure 1
Defects of R7 axons in the Lar, Ptp69D double mutant.
(A–D) Horizontal image of the adult medulla; WT (A), LAR mutant (B), Ptp69D mutant (C) and LAR, Ptp69D double mutant (D). Photoreceptor axons were labeled with mAb24B10 (red), R7 photoreceptor axons …
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Figure 1—source data 1
Excel file compiling source data for the Figure 1H.
- https://doi.org/10.7554/eLife.31812.003
Figure 2 with 1 supplement
Retraction of R7 axons in the LAR, Ptp69D double mutant.
(A–G’) The R7 axons of control (A–G) and LAR, Ptp69D double heterozygote with double RNAi (A’–G’) during the pupal stage were visualized with mCD8GFP (green) and counterstained with mAb24B10 (red) …
Figure 2—figure supplement 1
Temporal requirement of LAR and Ptp69D.
(A–H) The expression level of LAR and Ptp69D was manipulated temporally using LAR Ptp69D double RNAi. When LAR and Ptp69D RNAi was expressed simultaneously by GMR-Gal4 together with tub-Gal80[ts], …
Figure 3 with 1 supplement
Final stabilizing layer determinations by LAR and Ptp69D.
(A–E) Genetic manipulation using a temperature sensitive Gal80ts system driving RNAi expression was employed to manipulate the cumulative expression level of LAR and Ptp69D. (A–C) LAR, Ptp69D RNAi …
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Figure 3—source data 1
Excel file compiling source data for the Figure 3F.
- https://doi.org/10.7554/eLife.31812.008
Figure 3—figure supplement 1
The expression level of LAR and Ptp69D in the fly strain used in Figure 3.
Genetic manipulation using a temperature sensitive Gal80ts system driving RNAi expression was employed to manipulate the cumulative expression level of LAR and Ptp69D. The expression level of LAR …
Figure 4 with 2 supplements
Specificity of RPTP signaling in R7 stabilization.
(A–L) Horizontal images of the medulla of LAR, Ptp69D adult double mutant that also harbored the indicated transgene expressed using the GMR-Gal4 driver. Photoreceptor axons are labeled with …
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Figure 4—source data 1
Excel file compiling source data for the Figure 4M and N.
- https://doi.org/10.7554/eLife.31812.012
Figure 4—figure supplement 1
The expression level of LAR and Ptp69D transgenes used in Figure 4.
The expression level of transgenes used for rescue experiments in Figure 4 was evaluated. Each UAS-transgene was crossed to GMR-Gal4 and larval eye-discs were stained with anti-LAR antibody (green …
Figure 4—figure supplement 2
Layer-specific binding of LAR and Ptp69D extracellular domain.
(A) The extracellular domain of LAR fused to PLAP sequence was applied to the agarose section of 40hrAPF wild type pupae. LAR-AP staining was detected in R7 temporary layer and the layer between R7 …
Figure 5 with 1 supplement
Specificity of RPTP signaling in R8 axons.
(A–F) Horizontal images of adult medulla of LAR, Ptp69D double mutants also harboring the indicated transgene expressed under the GMR-Gal4 driver. Photoreceptor axons were labeled with mAb24B10 …
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Figure 5—source data 1
Excel file compiling source data for the Figure 5G.
- https://doi.org/10.7554/eLife.31812.015
Figure 5—figure supplement 1
Overexpression of LAR and Ptp69D has no effect on R8 axons.
(A–C) Overexpression of LAR (A) as well as Ptp69D (B) did not cause any dominant effect on R8 axons. The quantifications are shown in (C).
Figure 6 with 1 supplement
Specificity of RPTP ectodomains in R7 axons.
(A–F) Horizontal images of adult medulla of LAR (A–C) or Ptp69D (D–F) single mutants carrying the indicated transgene expressed by the GMR-Gal4. Photoreceptor axons are labeled with mAb24B10 (red), …
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Figure 6—source data 1
Excel file compiling source data for the Figure 6G.
- https://doi.org/10.7554/eLife.31812.018
Figure 6—figure supplement 1
Overexpression of LAR and Ptp69D.
(A–C) Overexpression of LAR (A) as well as Ptp69D (B) did not cause any dominant effect on R7 axons. The quantifications are shown in (C).
Figure 7 with 1 supplement
Cell-autonomous function of LAR and Ptp69D.
(A–C) Single R7 clones of LAR, Ptp69D double mutant. Ptp69D mutation was introduced by creating the clone with MARCM system using GMR-FLP, whereas LAR expression was down-regulated by combining LAR …
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Figure 7—source data 1
Excel file compiling source data for the Figure 7C and F and 7I.
- https://doi.org/10.7554/eLife.31812.021
Figure 7—figure supplement 1
Specificity of the PM181-Gal4 and 2–80 Gal4.
(A–B) The expression pattern of R7-specific PM181-Gal4 (A) and R8-specific 2–80-Ga4 (B) was assessed at 24 hr after puparium formation (APF) by crossing to UAS-mCD8GFP (Green). Photoreceptor axons …
Figure 8 with 1 supplement
Genetic interactions between LAR, Ptp69D and Abl, Ena, and Trio in R7 targeting.
(A–F) The expression of Abl, Ena, and Trio was downregulated using RNAi or upregulated by overexpression of transgenes in LAR, Ptp69D double RNAi background. All the RNAi and transgenes were …
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Figure 8—source data 1
Excel file compiling source data for the Figure 8G.
- https://doi.org/10.7554/eLife.31812.024
Figure 8—figure supplement 1
RNAi and overexpression of Abl, Ena and Trio.
(A–C) Downregulation of Abl (A), Ena (B) and Trio (C) using RNAi driven by GMR-Gal4 did not show any defects on R7 axons. (D–F) Overexpression of Abl (D), Ena (E) and Trio (F) did not cause any …
Figure 9 with 1 supplement
A model for common and distinct functions of LAR and Ptp69D in R7Axons.
(A) Both LAR and Ptp69D are expressed in R7 axon terminals. Their ectodomains bind to distinct ligands whereas intracellular domains share a common downstream pathway. (B) Around 24hrAPF, R7 axons …
Figure 9—figure supplement 1
R7 axon stabilization is not dependent on neuronal activity.
(A–D) R7 axons normally project to M6 layer (A) but they do not innervate medulla and terminate inside lamina in LAR and Ptp69D double knock-out created by combining heterozygous double mutant and …
Tables
Key resource table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers |
|---|---|---|---|
| gene (Drosophila) | LAR | NA | |
| Ptp69D | NA | ||
| strain, strain background (Drosophila) | D. melanogaster: ey-FLP2 | (Newsome et al., 2000a) | BDRC5580 |
| D. melanogaster: Ptp69D[D1689] | (Newsome et al., 2000a) | DGRC109897 | |
| D. melanogaster: LAR[2127] | (Maurel-Zaffran et al., 2001) | BDRC63796 | |
| D. melanogaster: UAS-LAR | (Maurel-Zaffran et al., 2001) | N/A | |
| D. melanogaster: UAS-LARΔC | (Maurel-Zaffran et al., 2001) | N/A | |
| D. melanogaster: UAS-LARΔIg1-3ΔFn1-6 | (Hofmeyer and Treisman, 2009) | N/A | |
| D. melanogaster: Rh4-mCD8GFP | (Berger et al., 2001) | N/A | |
| D. melanogaster: Rh6-mCD8GFP | (Berger et al., 2001) | N/A | |
| D. melanogaster: 20C11-FLP | (Chen et al., 2014) | BDRC63796 | |
| D. melanogaster: UAS-Trio | (Newsome et al., 2000b) | N/A | |
| D. melanogaster: UAS-Abl | (Fogerty et al., 1999) | N/A | |
| D. melanogaster: UAS-Ena | (Wills et al., 1999) | N/A | |
| D. melanogaster: UAS-FRT-stop-FRT-mcd8GFP | Bloomington Drosophila Stock Center | BDRC30032 | |
| D. melanogaster: tub-Gal80[ts] | Bloomington Drosophila Stock Center | BDRC7108 | |
| D. melanogaster: UAS-Lar.ΔIg123 | Bloomington Drosophila Stock Center | BDRC8586 | |
| D. melanogaster: UAS-Lar.ΔFn123 | Bloomington Drosophila Stock Center | BDRC8587 | |
| D. melanogaster: UAS-Lar.ΔFn456 | Bloomington Drosophila Stock Center | BDRC8588 | |
| D. melanogaster: UAS-Lar.ΔFn789 | Bloomington Drosophila Stock Center | BDRC8589 | |
| D. melanogaster: UAS-Lar.ΔPTP-D2 | Bloomington Drosophila Stock Center | BDRC8590 | |
| D. melanogaster: UAS-Lar.C1638S | Bloomington Drosophila Stock Center | BDRC8591 | |
| D. melanogaster: UAS-Lar.C1929S | Bloomington Drosophila Stock Center | BDRC8592 | |
| D. melanogaster: UAS-Lar.CSX2 | Bloomington Drosophila Stock Center | BDRC8593 | |
| D. melanogaster: UAS-Ptp69D Δintra | KYOTO Stock Center | DGRC109088 | |
| D. melanogaster: UAS-Ptp69D DA1 | KYOTO Stock Center | DGRC109089 | |
| D. melanogaster: UAS- Ptp69D DA3(DA1DA2) | KYOTO Stock Center | DGRC109090 | |
| D. melanogaster: UAS-Ptp69D | KYOTO Stock Center | DGRC109091 | |
| D. melanogaster: UAS-LAR RNAi | Vienna Drosophila Resource Center | VDRC36269 | |
| D. melanogaster: UAS-ptp69D RNAi | Vienna Drosophila Resource Center | VDRC27090 | |
| D. melanogaster: UAS-abl RNAi | Bloomington Drosophila Stock Center | BDRC28325, 35327 | |
| D. melanogaster: UAS-ena RNAi | Bloomington Drosophila Stock Center | BDRC31582, 39034 | |
| D. melanogaster: UAS-trio RNAi | Bloomington Drosophila Stock Center | BDRC27732, 43549 | |
| D. melanogaster:<LAR < | This paper | Harvard Exelixis collection; e04149, e00822 | |
| D. melanogaster:<Ptp69D< | This paper | Harvard Exelixis collection; f03442, e00274 | |
| antibody | mAb24B10 | Developmental Studies Hybridoma Bank | 24B10 |
| rat antibody to CadN(N-Cad) | Developmental Studies Hybridoma Bank | DN-Ex #8 | |
| rat antibody to elav | Developmental Studies Hybridoma Bank | 7E8A10 | |
| mouse antibody to LAR | Developmental Studies Hybridoma Bank | 9D8 | |
| mouse antibody to Ptp69D | Developmental Studies Hybridoma Bank | 3 F11 | |
| GFP Tag Polyclonal Antibody, Alexa Fluor 488 | Life technologies | Cat #A-21311 | |
| Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 | Life technologies | Cat #A-11011 | |
| Goat anti-Rat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 633 | Life technologies | Cat #A-21094 | |
| Anti-Placental alkaline phosphatase (PLAP) | Abcam | Cat #ab118856 | |
| Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) preabsorbed | Abcam | Cat #ab150085 | |
| rabbit antibody to HA | Abcam | Cat #ab9110 | |
| chemical compound, drug | Paraformaldehyde 16% | Nisshin EM | Cat#15710 |
| Agar powder (gelling temperature 30–31°C) | Nacalai tesque | Cat#01059–85 | |
| Vectashield mouting medium | Vector Laboratories | Funakoshi #H-1000 | |
| software, algorithm | NIS-Elements AR | Nikon | |
| Adobe Photoshop CS6 | Adobe |
Additional files
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Supplementary file 1
Table showing the list of all full genotypes used.
- https://doi.org/10.7554/eLife.31812.027
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Transparent reporting form
- https://doi.org/10.7554/eLife.31812.028
