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First published online 24 July 2008
doi: 10.1242/dev.025049

Development 135, 2865-2871 (2008)
Published by The Company of Biologists 2008
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Pathfinding in a large vertebrate axon tract: isotypic interactions guide retinotectal axons at multiple choice points

Andrew J. Pittman1,2,*, Mei-Yee Law1,2,* and Chi-Bin Chien1,2,3,{dagger}

1 Program in Neuroscience, University of Utah Medical Center, 20 North 1900 East, Salt Lake City, UT 84132, USA.
2 Department of Neurobiology and Anatomy, University of Utah Medical Center, 20 North 1900 East, Salt Lake City, UT 84132, USA.
3 Brain Institute, University of Utah Medical Center, 20 North 1900 East, Salt Lake City, UT 84132, USA.


Figure 1
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  		Fig. 1. ath5 MO blocks differentiation of early- but not late-born RGCs. (A) Normally, early RGCs differentiate in a wave across central retina (blue arrow); late RGCs are then added centrifugally (red arrows). (B,C) 6 dpf isl2b:GFP, lateral views, anterior towards the left. Insets schematize cell bodies, axons and optic nerve head (star). (B) Wild-type eye shows GFP+ RGCs throughout central retina; axons are obscured by cell bodies. (C) A high dose of ath5MO blocks differentiation of early RGCs, but late RGCs still form (arrows). Without central RGCs, peripheral axons are visible (arrowheads). (D) Dose-response curve showing timing of RGC formation with different doses of ath5MO. In wild type and with 3 ng control MO, GFP+ RGCs appear by 33 hpf. Increasing concentrations of ath5MO increasingly delay the appearance of the first RGCs. A, anterior; D, dorsal; P, posterior; V, ventral. Scale bar: 50 µm.

 

Figure 2
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  		Fig. 2. Early RGCs are necessary for axons to exit eye. (A) Diagram showing field of view for B,C. (B,C) 5 dpf isl2b:GFP, confocal z-projections; dorsal views, anterior upwards. Insets show cell bodies in eye. (B) Wild-type axons exit eye (arrowheads), project through the optic chiasm (broken lines) and to the optic tecta. (C) With high dose of ath5 MO, axons do not exit eyes (arrowheads) or innervate tecta (outlined). (D) Retinal exit in dose-response experiment of Fig. 1D, plotting percentage of embryos in which axons exit the eye against the time at which their first isl2b:gfp-positive RGCs were born. When RGCs are born by 42 hpf, axons usually exit the eye; when delayed after 42 hpf, axons rarely exit. Number of embryos is indicated at base of each bar. (E) 72 hpf isl2b:GFP, confocal z-projection; dorsal upwards. In a high-dose ath5 morphant, axons from peripheral RGCs remain trapped in the RGC layer without entering the optic nerve. To better appreciate 3D structure, see volume reconstruction in Movie 1 in the supplementary material. Arrowhead indicates the optic nerve head, stained by anti-Pax2 (not shown). Scale bars: 100 µm in C; 50 µm in E.

 

Figure 3
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  		Fig. 3. ath5 MO does not appear to affect the molecular and cellular environment of the optic nerve head or the intraretinal optic nerve. Coronal sections through 48-49 hpf isl2b:GFP (A,B) and 36 hpf non-transgenic (C,D) eyes, from either uninjected embryos (A,C) or embryos injected with high dose ath5 TMO (B,D). (A,B) Pax2a antibody staining (magenta) labels presumptive glial cells which line the intraretinal region of the optic nerve (arrows) in both wild type (A) and ath5 morphants (B). RGCs and their axons (green) are present in wild type (A), but not in ath5 morphants (B). The trigeminal ganglion (arrowheads) is also labeled by isl2b:gfp and serves as a staining control. (C,D) In situ hybridization shows that netrin1a expression (arrows) surrounds the optic nerve and optic nerve head in wild type (C), and is unchanged in ath5 morphants (D). Scale bars: 50 µm.

 

Figure 4
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  		Fig. 4. Transplanted WT central RGCs rescue retinal exit in ath5 morphants. (A) Blastula transplants from isl2b:mCherry donors resupply ath5 morphant hosts, labeled with isl2b:gfp, with early RGCs. RhDx, rhodamine-dextran cell lineage marker. (B) Resupplied WT RGCs and axons (magenta) are sufficient to rescue host axons in morphants (green). Pigment cell autofluorescence is seen around eyes and at dorsal midline (magenta). Retinal axons project across chiasm (broken lines); donor axons terminate in central tectum (asterisk), while host axons terminate in peripheral tectum (arrowheads). Dorsal view, 5 dpf. (C) Lateral view of 5 dpf eye showing peripheral host axons (green) fasciculating with resupplied WT axons (magenta). (C') Donor and (C'') host axons, reverse contrast. Scale bars: 100 µm in B; 50 µm in C.

 

Figure 5
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  		Fig. 5. Axon-axon interactions strongly influence retinotectal pathfinding. (A) Transplants yield GFP-expressing donor RGCs in host eyes. (B-E) Dorsal views, 5 dpf, rostral upwards. Donor axons labeled with brn3c:GFP; pathfinding errors indicated by yellow stars. (B'-E') Error quantitation. (B,B') In wild-type> wild-type transplants, donor axons pathfind perfectly. (C,C') By contrast, when transplanted into ast hosts, wild-type axons make significantly more errors. (D,D') As expected, ast donor axons make many errors in ast hosts. (E,E') However, when transplanted into wild-type hosts, ast axons make significantly fewer errors. Scale bar: 50 µm.

 

Figure 6
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  		Fig. 6. Pioneer axons influence pathfinding of follower axons. (A) Transplants resupply early RGCs to brn3c:GFP hosts injected with ath5 MO. (B-E) Dorsal views at 5 dpf, rostral upwards. Host axons terminate peripherally on the tectum; errors indicated by stars. (B'-E') Error quantitation. (B,B') In wild type>wild type;ath5 MO transplants, host follower axons pathfind normally. (C,C') When pioneers are replaced with ast cells, wild type follower axons make significantly more mistakes. (D,D') In ast>ast;ath5MO transplants, host follower axons make many errors. (E,E') When pioneers are replaced with wild-type cells, follower ast axons make fewer errors but are not completely rescued. Scale bar: 50 µm.

 

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