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Fig. 2. R8 and R7 growth cones reach their destined layers during the second
layer-selection stage. (A-D') R7 and R8 layer-specific targeting
assessed at 40% (A,A'), 50% (B,B'), 60% (C,C'), and 70% APF
(D,D'). Approximately 70% of R7 axons were labeled using a flipase-based
method (see Materials and methods) and visualized with the anti-GFP antibody
(green). R8 axons (red) were marked with RFP but not GFP. The neuropils were
stained with the anti-HRP antibody (purple). (A') At 40% APF, R7
(yellow, large arrow) and R8 (red, large arrowhead) growth cones terminate at
their temporary layers. (B') At 50% APF, R7 growth cones (yellow, large
arrow) extend 
3 µm further with filapodia (small arrow) leaving behind
in the R7-temporary layer. R8 growth cones (red) extend filapodia (small
arrowhead) deeper into the medulla while leaving their growth cone propers
(large arrowhead) in the R8-temporary layer. (C') At 60% APF, both R8
(large arrowhead) and R7 (large arrow) growth cones have reached their final
target layers. Note that R7 axon shafts expanded in the R7-temporary layer
(small arrow). (D') At 70% APF, both R8 (large arrowhead) and R7 growth
cones retract their filapodia and consolidate into axonal terminals. Two types
of R7 terminals, straight (large arrow) and horseshoe (double-arrow) are
noticeable (as seen in adult flies). (E-G') Layer-specific connections
of R7 (C,C'), R8 (D,D') and L2 (E,E') assessed at the adult
stage. (E,E') R7 axons (green) were labeled with Rh3 Rh4-Gal4,
UAS-synb-GFP. Glass-lacZ marker labeled both R7 and R8 axons (red).
(F,F') R8 axons (green) were labeled with Rh5 Rh6-Gal4, UAS-synb-GFP.
(G,G') L2 axons (green) were labeled with L2-Gal4 UAS-mCD8-GFP. R7 and
R8 axons were visualized with Mab24B10. Note that L2 axons terminate at the M2
layer, just above the R8-recipient layer (M3). (A'-G') High
magnification views of A-G, respectively. Scale bars: in A, 30 µm for A-D;
in A' 5 µm for A'-D'; in E, 20 µm for E-G; in
E', 10 µm for E'-G'. (H) A schematic diagram showing the
progression of R7 and R8 axonal targeting during the second layer-selection
stage. For clarity, most L1-L5 neurons are omitted in the figure for the pupal
stages. Abbreviations as in Fig.
1.
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