Ephrin-A/EphA specific co-adaptation as a novel mechanism in topographic axon guidance
- Karlsruhe Institute of Technology, Zoological Institute, Germany
Figures
Figure 1
The retinotectal projection.
The topographic projection in the chicken visual system connects RGCs from the retina to the midbrain's optic tectum. The temporal/nasal (t/n) axis of the retina is mapped onto the …
Figure 2 with 1 supplement
Growth cone desensitization towards soluble ephrin-A5 and EphA3.
(A) Temporal GCs initially collapse upon application of 250 ng/ml ephrin-A5-Fc (eA5 20 min: 77.1%), but recover their morphology within 120 min in the presence of the cue (eA5 120 min: 32.9% …
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Figure 2—source data 1
Original data underlying bar charts of Figure 2A, C.
- https://doi.org/10.7554/eLife.25533.005
Figure 2—figure supplement 1
EphA3 collapse assays and ephrin-A5/EphA3 dissociation constants.
(A) Moderate concentrations of EphA3-Fc (EA3) do not trigger a collapse of nasal or temporal GCs, neither in dimeric nor in an antibody clustered form (Fc, 1 µg/ml, 20 min: 16.1% collapsed; EA3, 2 …
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Figure 2—figure supplement 1—source data 1
Original data underlying bar charts of Figure 2—figure supplement 1.
- https://doi.org/10.7554/eLife.25533.007
Figure 3
Growth cone adaptation towards substrate-bound ephrin-A5 and EphA3.
Subfigures in (A) and (B) each display a cartoon (left) illustrating the experimental setup consisting of explant (thick black strip), axons and printed guidance protein (colored field(s)), the …
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Figure 3—source data 1
Original data underlying bar chart of Figure 3C.
- https://doi.org/10.7554/eLife.25533.009
Figure 4 with 1 supplement
Modeling growth cone adaptation and topographic mapping.
(A) Fiber terminals are modeled as circular discs bearing Gaussian-shaped distributions of EphAs (RF, blue) and ephrin-As (LF, red), according to their retinal origin, moving on a rectangular target …
Figure 4—figure supplement 1
Inclusion of co-adaptation does not alter the explanatory power of the computational model.
For purposes of comparison, this figure, produced using the updated model that includes co-adaptation presented in this paper is structured in parallel to figure 7 in Gebhardt et al. (2012), which …
Figure 5 with 1 supplement
Co-adaptation of retinal growth cones in double-cue gap assays.
(A–C) Naïve axons stop in front of a homogeneous field of EphA3-Fc (A; EA3, blue; nasal axons), ephrin-A5-Fc (B; eA5, red; temporal axons), or Sema3A-Fc (C; S3A, green; temporal axons). Scale: 100 …
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Figure 5—source data 1
Original data underlying bar chart of Figure 5D.
- https://doi.org/10.7554/eLife.25533.013
Figure 5—figure supplement 1
Growth cone adaptation towards Sema3A.
(A) Naïve axons stop in front of a homogeneous field of Sema3A-Fc (S3A, green), but predominantly ignore a similar field in gap assays with(B) 65 µm, (C) 115 µm and (D) 215 µm wide gaps. Axons …
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Figure 5—figure supplement 1—source data 1
Original data underlying bar chart of Figure 5—figure supplement 1H.
- https://doi.org/10.7554/eLife.25533.015
Figure 6 with 1 supplement
Endocytosis of guidance sensors upon growth cone desensitization.
(A) SNAP–ephrin-A5 surface signal is strongly reduced on GCs growing on either homogeneous EphA3-Fc (A') or ephrin-A5-Fc (A'') compared to controls (on Fc), indicating clearance of sensors from the …
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Figure 6—source data 1
Original data underlying the bar charts of Figure 6B—D.
- https://doi.org/10.7554/eLife.25533.017
Figure 6—figure supplement 1
SNAP–ephrin-A5 expression constructs pSNAP–ephrin-A5–IRES-FP contains the coding sequence of a fusion protein consisting of the signal sequence of chick ephrin-A5 (60 bp), the SNAP-tag and full-length chick ephrin-A5 downstream of the CAG enhancer/promoter and upstream of an IRES-FP sequence.
The FP is EGFP in pSNAP–ephrin-A5–IRES-EGFP and dTomato in pSNAP–ephrin-A5–IRES–dTom.
Figure 7 with 1 supplement
Dynamics of SNAP–ephrin-A5 during growth cone re-sensitization.
(A) Exemplary trajectories of five GCs (combined from N = 3 independent experiments) on ephrin-A5 gap patterns (red) in the presence of 40 µM AIM. Positions of GCs (black dots) were marked at the …
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Figure 7—source data 1
Original data underlying the bar chart in Figure 7D.
- https://doi.org/10.7554/eLife.25533.020
Figure 7—figure supplement 1
Staining procedure for recycled SNAP–ephrin-A5.
Extracellular SNAP–ephrin-A5 was blocked by SNAP surface block (gray), before the cell-permeant SNAP cell fluorescein was applied to the living cells (green). After washing, axons were allowed to …
Figure 8
Colocalization of SNAP–ephrin-A5 and Rab11-positive endosomes.
(A) eGFP–Rab11 localization in a transfected RGC axon (outline drawn in the actin channel indicated as white, dotted line). Rab11-positive vesicular structures are predominantly observed in the axon …
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Figure 8—source data 1
Original data underlying the bar chart of Figure 8D.
- https://doi.org/10.7554/eLife.25533.023
Figure 9 with 1 supplement
Disintegration of lipid microdomains induces co-adaptive growth cone desensitization.
(A) Ephrin-A5-Fc and (B) EphA3-Fc collapse assays. Methyl-beta-cyclodextrin (MβCD, 2 mg/ml)-treated GCs show enhanced desensitization (reduced collapse rates) when exposed to soluble ephrin-A5-Fc …
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Figure 9—source data 1
Original data underlying the bar charts in Figure 9A, B, E.
- https://doi.org/10.7554/eLife.25533.025
Figure 9—figure supplement 1
Disintegration of lipid microdomains by sphingomyelinase desensitizes growth cones towards soluble ephrin-A5 and EphA3.
(A) Ephrin-A5-Fc and (B) EphA3-Fc collapse assays. In accordance with MβCD experiments, Sphingomyelinase (SMase, 400mU/ml)-treated GCs show reduced collapse rates when exposed to soluble …
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Figure 9—figure supplement 1—source data 1
Original data underlying the bar charts of Figure 9—figure supplement 1.
- https://doi.org/10.7554/eLife.25533.027
Figure 10
Modeling tectal innervation.
Co-adaptation enables fiber terminals to enter a tectal target field initially and allows correct mapping therein (gray circles), whereas non-adapted terminals (white circles) fail to enter a tectal …
Figure 11
Proposed mechanism of co-adaptation.
(A) EphAs (blue) and ephrin-As (red), located in membrane lipid microdomains (dark gray), signal in trans due to external cues. Trans forward (FWD) and reverse (REV) signals integrate into the …
