Manitt C et al. (2009), Netrin participates in the development of retin...

XB-ART-40390

J Neurosci 2009 Sep 09;2936:11065-77. doi: 10.1523/JNEUROSCI.0947-09.2009.

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Netrin participates in the development of retinotectal synaptic connectivity by modulating axon arborization and synapse formation in the developing brain.

Manitt C , Nikolakopoulou AM , Almario DR , Nguyen SA , Cohen-Cory S .

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Netrin has been implicated in retinal ganglion cell (RGC) axon pathfinding in a number of species. In Xenopus laevis, RGC axons reaching their target in the optic tectum can be repelled by a netrin-1 gradient in vitro, suggesting that netrin may also function in wiring events that follow successful axon pathfinding. Here, we examined the contribution of netrin to RGC axon arborization and synapse formation at the target. Time-lapse confocal microscopy imaging of individual RGC axons coexpressing GFP-synaptobrevin and DsRed in the intact Xenopus brain demonstrated a role for deleted in colorectal cancer (DCC)-mediated netrin signaling. Microinjection of netrin-1 into the tectum induced a rapid and transient increase in presynaptic site addition that resulted in higher presynaptic site density over a 24 h observation period. Moreover, netrin induced dynamic axon branching, increasing branch addition and retraction; a behavior that ultimately increased total branch number. In contrast, microinjection of DCC function-blocking antibodies prevented the increase in presynaptic site number normally observed in control axons as well as the associated increase in branch number and axon arbor growth. Dynamic analysis of axon arbors demonstrated that the effects of anti-DCC on axon morphology and presynaptic connectivity were attributable to a specific decrease in new synapse and branch additions, without affecting the stability of existing synapses and branches. Together, these results indicate that, in the absence of DCC signaling, RGC axons fail to branch and differentiate, and support a novel role for netrin in later phases of retinotectal development.

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Species referenced: Xenopus laevis
Genes referenced: dcc map2 ntn1 psd vamp1
???displayArticle.antibodies??? Dcc Ab1 Dcc Ab2 Dlg4 Ab1 Neuronal Ab4 Ntn1 Ab1

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	Figure 1. Netrin is present in tectal neurons and is identified at synaptic sites in the stage 44â€“45 Xenopus optic tectum. A, B, Localization of netrin immunoreactivity in the tectal midbrain. B, Schematic diagram of a stage 44â€“45 Xenopus tectal midbrain (horizontal view). Neuronal precursors, born in rows adjacent to the ventricle in the right and left sides of the optic tectum, follow a lateral and rostral migratory path while extending a primary dendrite that will eventually terminate in the tectal neuropil and begin to elaborate a dendritic arbor. In this diagram, the boxed region corresponds to the micrograph shown in A. A, This horizontal view of the optic tectum demonstrates that a large, evenly distributed subset of cells is immunopositive for netrin-1. Punctate immunostaining is also observed throughout the tectal neuropil (n). Câ€“F, Confocal micrographs of a horizontal section through the stage 44â€“45 Xenopus optic tectum coimmunostained with antibodies to netrin (green immunofluorescence) and the dendritic marker MAP2 (red immunofluorescence). C, Note that MAP2 immunostaining identifies optic tectal neuron dendritic processes projecting to and branching in the tectal neuropil (n). Dâ€“F, The high-magnification confocal image reveals that netrin immunoreactivity is localized to cell bodies (asterisk) and proximal dendrites (arrow) of neurons within the medial portion of the optic tectum that are also immunoreactive for MAP2. G, Confocal micrograph of the tectal neuropil in stage 44â€“45 Xenopus optic tectum coimmunostained with antibodies to netrin (red immunofluorescence) and the presynaptic marker SNAP-25 (green immunofluorescence). Note the punctate distribution of netrin (red) and SNAP-25 (green) in the tectal neuropil; the netrin-immunoreactive puncta are in direct apposition to SNAP-25-labeled presynaptic sites. H, Confocal micrograph of stage 44â€“45 Xenopus optic tectum coimmunostained with antibodies to netrin (red immunofluorescence) and the postsynaptic marker, PSD-95 (green immunofluorescence). Netrin-immunoreactive puncta in the tectal neuropil colabel with PSD-95-positive postsynaptic densities. Scale bars: A, 50 Î¼m; C, 100 Î¼m; Dâ€“F, 10 Î¼m; G, H, 5 Î¼m.
	Figure 2. RGC axons branching the optic tectum are immunopositive for DCC at presynaptic sites. A, Schematic illustration of the retinotectal circuit in stage 44â45 Xenopus optic tectum (horizontal view). RGC axons project to the neuropil in the lateral tectum, in which they elaborate an arbor and form synapses with tectal neuron dendrites. Confocal micrographs of the tectal neuropil shown in CâH correspond to regions demarcated by the gray box. BâE, After unilateral right eye ablation, sections through the tectal midbrain were coimmunostained with antibodies to DCC and a marker for RGC axons (neurofilament-associated protein, 3A10). B, Horizontal section showing the localization of 3A10 (red)- and DCC (green)-immunoreactive fiber bundles in the two sides of the tectal neuropil. A large bundle of double-labeled fibers in the right side of the optic tectum is absent from the left side, which normally would receive innervation from the right, ablated eye. This manipulation identifies the missing axonal fibers as RGC axons. Note that a smaller, more medial population of DCC-positive fibers remains intact after eye ablation (BâE, G, arrows), indicating the presence of a distinct population of DCC-positive fibers in addition to RGCs. CâE, High-power micrographs of the RGC axon bundle shown in B illustrates the colocalization of DCC (green) and 3A10 (red) immunoreactivities in the axonal fibers [double labeling (C); 3A10 immunoreactivity alone (D); DCC labeling alone (E)]. A high degree of colocalization in DCC and 3A10 immunoreactivity in RGC axons is observed. Note, however, that a small subset of cell bodies and their dendrites are also positive for 3A10 but negative for DCC (BâE, small arrowheads). FâI, Micrographs of stage 44â45 Xenopus tectal neuropil coimmunostained with antibodies to DCC and netrin. DCC immunoreactivity (green) localizes to RGC axonal fibers projecting and branching in the tectal neuropil. Netrin immunoreactivity (red) is distributed in a punctate pattern in the tectal neuropil, in close proximity to DCC-positive axon fibers. I, Higher power micrograph of the horizontal section shown in F better illustrates how the netrin-immunoreactive puncta (red) are in close apposition to DCC-positive fibers (green). JâM, The subcellular localization of DCC in stage 44â45 optic tectum was determined by preembedding immunoelectron microscopy. The silver enhancement of secondary antibody-conjugated 1 nm gold particles shows that DCC immunoreactivity localizes to vesicles at presynaptic specializations (J, K, arrows), as well as presynaptic membranes (L, arrow) in the tectal neuropil. The presence of a synapse is indicated by the arrowheads. M, Discrete silver-enhanced DCC-immunoreactive clusters were also observed on presynaptic filopodia (arrow). Scale bars: B, 100 Î¼m; CâE, 50 Î¼m; I, 5 Î¼m; J, K, 0.2 Î¼m.
	Figure 3. DCC-mediated netrin signaling contributes to RGC axon presynaptic differentiation during the development of retinotectal connectivity. A, Sample RGC axons coexpressing Ds-Red (red) and GFP-synaptobrevin (green) imaged by time-lapse confocal microscopy over a 24 h period after microinjection of recombinant netrin-1 (+ netrin) or function-blocking antibodies to DCC (â DCC). Projections on the right show the GFP-synaptobrevin fluorescence only. Scale bar, 50 Î¼m. B, C, Manipulations in netrin signaling alter the number of GFP-synaptobrevin-labeled presynaptic sites and influence RGC axon arbor morphology. B, As RGC axons branch and differentiate, the absolute number of GFP-synaptobrevin-labeled presynaptic sites increases over time (0â4, 0â8, 0â24 h). Microinjection of recombinant netrin-1 induced a significantly higher increase in the number of presynaptic sites relative to controls by 4 h, an effect that persisted for the remainder of the 24 h observation period. Microinjection of DCC function-blocking antibodies led to a smaller increase in presynaptic site number relative to controls from 8 h onward. C, The increase in the number of total branches was significantly higher in RGC axons 8 h after netrin treatment relative to controls, whereas RGC axons had fewer branches 24 h after anti-DCC treatment. *Significance with p â¤ 0.05. #Trend toward significance with 0.05 > p < 0.10. Error bars indicate SEM.
	Figure 4. DCC-mediated netrin signaling contributes to RGC axon presynaptic differentiation during the development of retinotectal connectivity. Changes in RGC presynaptic differentiation and in axon arborization were measured and expressed as percentage of initial values for each individual axon. A, Microinjection of recombinant netrin-1 into the optic tectum induced a significant increase in GFP-synaptobrevin-labeled presynaptic sites when compared with controls over a 24 h observation period. In contrast, microinjection of DCC function-blocking antibodies prevented the normal increase in presynaptic site number observed in controls over the 24 h observation period. B, Even though netrin induces a significant net increase in branches 8 h after treatment (Fig. 3C), the increase in branch number in RGC axons in netrin-treated tadpoles relative to the initial branch number was significantly different from controls by 24 h only. In contrast, anti-DCC treatment prevented the increase in branch number observed in controls at 8 and 24 h. C, The effect of anti-DCC treatment on axon arbor growth is also demonstrated by measuring the change in total arbor branch length. Total arbor branch length in RGC axons increased by 24 h in both control and netrin-treated tadpoles, whereas this measure was unchanged in the anti-DCC-treated tadpoles. D, E, The number of GFP-synaptobrevin-labeled presynaptic sites per unit arbor length and per branch number provided a measure of presynaptic site density. Netrin treatment significantly increased presynaptic site density in RGC axons from 4 h onward, whereas anti-DCC treatment resulted in RGC axons with lower presynaptic site density relative to controls by 24 h. F, We obtained a comparative measure of branch length by calculating average axon segment length (length/branch) at each observation interval and expressing it as percentage of initial value for each axon. This measure revealed that, on average, axon branch segments in RGC axon arbors in netrin-treated tadpoles became shorter than controls from 8 to 24 h after treatment. *Significance with p â¤ 0.05. #Trend toward significance with 0.05 > p < 0.10. Error bars indicate SEM.
	Figure 5. Perturbations in netrin signaling alter presynaptic site and axon branch dynamics. AâD, Netrin and anti-DCC influence presynaptic site dynamics. A, The number of GFP-synaptobrevin-labeled presynaptic sites added was significantly higher in RGCs axons in tadpoles treated with netrin-1 at all observation intervals (y-axis; absolute values). The number of newly added GFP-synaptobrevin-labeled presynaptic sites, however, was decreased after anti-DCC treatment, an effect that became significant in the 8â24 h observation interval. B, A small and gradual increase in the number of stabilized presynaptic clusters was observed after netrin treatment, with the number of stabilized presynaptic sites becoming significantly higher than controls in the 8â24 h interval. C, When expressed as percentage of initial value, the number of presynaptic clusters added after netrin treatment was significantly different from controls at the 0â4 h observation interval only. This suggests that the rate of presynaptic cluster addition was rapidly increased after netrin treatment, to then be maintained at a rate that matched controls. In contrast, when compared with its initial value (percentage of total), the number of presynaptic clusters added was significantly lower in RGC axons in anti-DCC-treated tadpoles both at 4â8 and 8â24 h when compared with controls. Anti-DCC had no effect, however, on the number of GFP-synaptobrevin clusters stabilized (B). D, The rates of increase in total presynaptic site (top), and branch number (bottom), in RGC axons treated with netrin (red) relative to controls (green) are also illustrated by the line graphs. EâH, Netrin and DCC influence branch addition but not stabilization. E, Netrin-1 increased the number of branches added throughout the imaging period, an effect that was significant from 8 to 24 h. The number of branches added in RGC axons in tadpoles treated with anti-DCC, in contrast, was significantly lower at the 8â24 h observation interval when compared with controls. F, Netrin and anti-DCC did not alter the number of branches stabilized. G, H, The rates of branch addition (G), and elimination (H), were significantly higher at all observation intervals after netrin treatment and, conversely, the rates of branch addition were significantly lower after anti-DCC treatment when compared with controls (Î4 h: 0â4 h, 4â8 h data combined). Anti-DCC did not affect the rate of branch elimination (H). *Significance with p â¤ 0.05. #Trend toward significance with 0.05 > p < 0.10. Error bars indicate SEM.
	Figure 6. Dynamic changes in presynaptic structure in RGC axon arbors in response to alterations in netrin signaling. A, Confocal projections of representative RGC axons coexpressing Ds-Red (red) and GFP-synaptobrevin (green) in tadpoles microinjected with netrin-1 (+ netrin) or DCC function-blocking antibodies (â DCC) right after the first imaging session. Note the significantly higher number of GFP-synaptobrevin-labeled presynaptic sites in the morphologically more complex arbors after netrin treatment. In contrast, the RGC axon arbors that received anti-DCC treatment did not change their morphology or presynaptic connectivity significantly within a 24 h period (Fig. 7). Scale bar, 50 Î¼m. B, Enlarged projections of single branches for the sample control-, netrin-, and anti-DCC-treated axons shown in A (gray boxes) illustrate branch and presynaptic site dynamics. Sample branches that were added (white arrows), eliminated (blue arrows), or added and then eliminated (magenta arrows) are shown for each experimental group. More branches were added and then eliminated (magenta arrows) in RGC arbors of netrin-treated tadpoles compared with controls. Although fewer in number, newly added branches in controls tended to remain stable for the remainder of the observation period (white arrows). Preexisting branches were eliminated (blue arrows) after anti-DCC treatment, whereas no new branches were added during the 24 h observation period. Sample GFP-synaptobrevin puncta added (green asterisks) or eliminated (red asterisks) highlight presynaptic site dynamics in the individual axon branches. Note that more GFP-synaptobrevin puncta were added per axon branch within the first 4 h after netrin treatment, and then the number continued to increase more gradually during the remainder of the 24 h observation period. Here, the green asterisks highlight a few examples. In comparison, axon branches in control-treated tadpoles underwent a slower increase in the number of GFP-synaptobrevin puncta across time points, whereas axons added fewer GFP-synaptobrevin puncta after anti-DCC treatment relative to controls. The relative rate of disassembly of GFP-synaptobrevin puncta (red asterisks) in axon branches was similar to controls for both netrin and anti-DCC-treated tadpoles.
	Figure 7. Schematic representation of changes in presynaptic differentiation of RGC axon arbors induced by alterations in DCC-mediated netrin signaling. Top, Control, RGC axons become more complex over time by the dynamic addition, elimination, and stabilization of presynaptic sites (Synapse Dynamics) and axon branches (Branch Dynamics). As more branches are added than eliminated, the arbor gradually increases its complexity over time. Middle, + netrin, Excess netrin induces rapid, novel presynaptic site addition, which gradually results in more stabilized presynaptic sites. The effects of netrin on presynaptic site addition are followed by new branch additions with a time delay. Note that, even though RGC axons exposed to netrin add more new branches, the relative number of branches that are stabilized remains constant. Bottom, â DCC, In contrast to netrin, anti-DCC prevents normal presynaptic addition without influencing presynaptic site stabilization (compare with axon projections shown in Fig. 6). Similarly, blockade of DCC signaling interferes with new branch addition without affecting branch stabilization. Thus, altering DCC-mediated netrin signaling interferes with the normal morphological and synaptic maturation of RGC axon arbors by preventing growth rather than by influencing their stability overall.

References [+] :

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