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First published online 20 July 2005
doi: 10.1242/dev.01934

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The cell adhesion molecule NrCAM is crucial for growth cone behaviour and pathfinding of retinal ganglion cell axons

Department of Developmental Neurobiology, University of Heidelberg, 69120 Heidelberg, Im Neuenheimer Feld 232, Germany

View larger version (78K): [in a new window]   Fig. 1. Localisation of NrCAM on RGCs. (A,B) Double labelling of a retina section stained by (A) NrCAM serum and (B) the nuclear marker DAPI. In the E6 retina, NrCAM is exclusively present in the optic fibre layer (OFL) and the optic nerve (ON), both formed by RGC axons. The ganglion cell layer (GCL), containing the RGC somata, is clearly NrCAM negative. The as yet undifferentiated neuroepithelial cells (NEC) are almost entirely NrCAM negative, with only a minor staining in the ventricular region near the pigment epithelium. (C) Single-cell cultures stained for NrCAM show that NrCAM is present on the entire RGC axon and is highly concentrated in its distal region. (D) NrCAM is strongly present in the central part of the RGC growth cone, and to a lesser degree on the lamellipodia and filopodia. Scale bars: (A,B) 100 µm; (C) 50 µm; (D) 10 µm.

View larger version (114K): [in a new window]   Fig. 2. Presence of NrCAM in the embryonic retina. Laser scanning micrograph of a flat-mounted retina (E4) stained for NrCAM.(A) NrCAM is selectively present on RGC axons. Because of the developmental gradient from the central to the peripheral retina, only single axons and thin axon bundles are present in the periphery, converging to increasingly thicker bundles towards the optic fissure. The inset shows an overview of the retina and position of the micrograph; the upper left square delineates the peripheral region of the retina shown enlarged in B; the lower right square the central region shown in C. (B,C) Top and corresponding side views of (B) peripheral and (C) central retina regions. NrCAM is present on the very young, still short RGC axons in the peripheral retina, and is already restricted to the axonal compartment in this early phase of axon formation. In the central retina, NrCAM is exclusively present on axons of RGCs; their somata (forming the GCL) are not stained. OFL, optic fibre layer; GCL, ganglion cell layer; NEC, neuroepithelial cells. Scale bars: (A) 100 µm, (inset) 500 µm; (B,C) 25 µm.

View larger version (21K): [in a new window]   Fig. 3. Effect of NrCAM on RGC axons and growth cones.(A) Overall length of RGC axons of E6 retinal single-cell cultures grown on the indicated substrates. Average length of axons grown on NrCAM/PLL is significantly increased compared with those grown on PLL. Axons grown on NrCAM/Laminin/PLL are significantly longer than those on Laminin/PLL. (B) Proportions of RGC axons grown on the indicated substrates plotted as percentages falling into four length classes. (C) The average growth cone size (i.e. area covered) is not significantly different between axons extending on NrCAM/Laminin/PLL and those on Laminin/PLL. (D) NrCAM causes a small but highly significant increase in the growth cone perimeters on NrCAM/Laminin/PLL compared with Laminin/PLL. (E) Growth cone perimeter divided by the square root of its area, as a measure of protrusions and indentations formed, shows a highly significant increase on NrCAM/Laminin/PLL compared with Laminin/PLL. Error bars represent s.d. * P0.05; *** P0.01.

View larger version (98K): [in a new window]   Fig. 4. Preference of RGC axons for NrCAM. RGC axons extending from E6 retinal explant strips on alternating substrate lanes coated with Laminin (dark lanes) and Laminin plus the indicated protein, which is stained by the respective antibody (green lanes).(A) Axons showed a preference for NrCAM in 77% of the NrCAM lanes, i.e. they did not cross the borders to the Laminin lanes (3307 borders evaluated; n=56 independent experiments). (B) By contrast, axons did not cross the substrate borders in only 7% of immunoglobulin-containing lanes; most exhibited random growth (1341 borders; n=20).(C) Substrate pre-incubation with NrCAM F(ab) fragments decreases axonal preference for NrCAM lanes to 15% of the lanes, and axon extension is almost completely random (2143 borders; n=38). (D) By contrast, pre-incubation with non-specific F(ab) fragments results in an only slightly reduced axonal preference for NrCAM; 62% of the lanes show axon preference (1351 borders; n=27). IgG, immunoglobulin; LN, Laminin. Scale bar: 200 µm.

View larger version (53K): [in a new window]   Fig. 5. NrCAM inhibition modulates growth cone morphology. (A-D) Two examples each of growth cones in flat-mounted retinae (E4.5) in the presence of non-specific F(ab) fragments (A,B) or NrCAM F(ab) fragments (C,D); top and side view of each growth cone is depicted; orientation of the growth cones in the retina is as indicated in D. Under control conditions, growth cones typically have a simple slim `torpedo-like' shape (A,B). Under NrCAM inhibition, growth cones acquire a complex morphology with a surplus of protrusions and a shorter, irregular form (C,D); in C, four protrusions (1-4) of a growth cone are indicated. Protrusions are directed towards deeper layers of the retina (3,4 in C; D) or to the left and right of the growth direction (1,2 in C). Occasionally the entire growth cone bends away from the correct growth direction; in contrast to the protrusions, it does not dive into the retina but stays in contact with the basal lamina (D). (E-H) Quantification of changes in growth cone morphology. (E) When NrCAM is inhibited, the proportion of complex growth cones is significantly increased compared with under control conditions. (F) The number of growth cones with two and more protrusions is significantly increased under NrCAM inhibition compared with controls. (G) The surface area of growth cones under NrCAM inhibition is significantly larger than those under control conditions. (H) NrCAM inhibition also causes a significant increase in growth cone volume P0.01. OF, optic fissure; VIT, vitreal side of the retina; VEN, ventricular side of the retina. Scale bar: 10 µm. (See also the 3D reconstructions and rotations in Movies 1 and 2 in the supplementary material.)

View larger version (65K): [in a new window]   Fig. 6. Axon navigation in the retina under NrCAM inhibition. (A-D) Axons growing towards the optic fissure in a retina flat-mount under NrCAM inhibition (top view). (A) Four axons (marked 1-4) grow on pre-existing axons and have large, complex growth cones. Axon 1 forms a growth cone with wide, laterally exploring protrusions (B), detaches from the other axons and turns away at an almost rectangular angle (C). This aberrant growth direction, perpendicular to the pathway to the optic fissure, is maintained for more than 20 minutes (D). Axon 2 turns away from the other axons at a smoother angle (B,C); later it performs a compensatory turn, returning to the correct direction (D). At 54 minutes, axon 3 also starts to deflect (D). Axon 4 grows towards the optic fissure during the observation period, displaying a growth behaviour typically found under control conditions. (E,F) Growth cone kinetic plots of two axons each, growing in retina flat-mounts in control conditions (E) and with NrCAM inhibition (F); each dot represents the position of the growth cone neck localised every two minutes (observation time in brackets). The lower plot in F corresponds to axon 2 shown in A-D. Under control conditions, axons grow rather straight and steadily towards the optic fissure (E). Under NrCAM inhibition the pathway is more crooked and deviating from the correct direction; in addition long pauses and retractions are observed (F). The overall distance covered is considerably shorter under NrCAM inhibition; note that the observation period is about 20% longer in F than in E. Scale bar: 20 µm.

View larger version (57K): [in a new window]   Fig. 7. Axon dynamics in the retina under NrCAM inhibition. (A,B) Time plots of RGC axon growth in E4.5 flat-mounted retinae in the presence of (A) non-specific F(ab) fragments (axons C1-C25) or (B) NrCAM F(ab) fragments (axons N1-N30). Growth (forward movement >3 µm), pauses (movement <3 µm), and retractions (backward movement >3 µm) were determined every 2 minutes. Under NrCAM inhibition, the frequency of long pauses (>12 minutes) almost doubled compared with under control conditions (7 and 12, respectively, in 50 minutes). The proportion of axons that retract is much higher under NrCAM inhibition compared with under control conditions (15 and 1, respectively). (C) The overall growth cone advance (observed over a period of 50 minutes) is significantly reduced by NrCAM inhibition when compared with controls. The elongation rate itself (measured during the growth phases of the axons) is not significantly changed. (D) The duration of growth phases under NrCAM inhibition is decreased compared with controls, and the duration of pauses under NrCAM inhibition is increased compared with controls. Error bars represent s.d. P0.05.

View larger version (92K): [in a new window]   Fig. 8. Misrouting at the optic nerve head under NrCAM inhibition. Two typical examples of E4.5 eyes organ-cultured in presence of non-specific F(ab) fragments (A,B) and two in presence of NrCAM F(ab) fragments (C,D). Inset in A shows three DiO crystals placed on a retina flat-mount, which label groups of RGC axons. Dashed line indicates the position of the optic fissure (OF); arrow indicates the optic nerve head. The majority of axons had already reached the OF and dived into the optic nerve head before the inhibition treatment. (A,B) Under control conditions, all RGC axons grow towards the OF, enter the OF, and leave the retina at the optic nerve head. (C,D) In presence of NrCAM F(ab) fragments, axons fail to enter the optic nerve; they stray away from the pre-existing axons (which leave the retina) and extend onto the opposite side of the retina towards the periphery. Scale bars: (A-D) 50 µm; (inset) 500 µm.

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