Abstract
The formation of neuronal networks in the central nervous system (CNS) requires precise control of axonal branch development and stabilization. Here we show that cell-specific ablation of the murine gene Ptk2 (more commonly known as fak), encoding focal adhesion kinase (FAK), increases the number of axonal terminals and synapses formed by neurons in vivo. Consistent with this, fak mutant neurons also form greater numbers of axonal branches in culture because they have increased branch formation and reduced branch retraction. Expression of wild-type FAK, but not that of several FAK variants that prevent interactions with regulators of Rho family GTPases including the p190 Rho guanine nuclear exchange factor (p190RhoGEF), rescues the axonal arborization phenotype observed in fak mutant neurons. In addition, expression of a mutant p190RhoGEF that cannot associate with FAK results in a phenotype very similar to that of neurons lacking FAK. Thus, FAK functions as a negative regulator of axonal branching and synapse formation, and it seems to exert its actions, in part, through Rho family GTPases.
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Acknowledgements
We thank D. Ilic and P. Soriano for the faknull and R26R strains; J. Oberdick for the L7 minigen; G. Martin for the Cre plasmid; B. Sauer for the Cre-EGFP plasmid; T. Parsons for the FRNK, Myc-FAKwt, Myc-FAKY397F, Myc-FAKP878A and Myc-FAKL1034S constructs; W.H. Moolenaar for p190RhoGEF; O. Marín, S. Bamji, M. Stryker and C. Damsky for comments on the manuscript; members of L.F.R.'s laboratory for discussions; C. Mason, H. Heuer and S. Bamji for advice on cultures; S. Huling for assistance with electron microscopy; and O. Marín and S. Martínez for infrastructure support. This work was supported by a grant from the NIH and by the Howard Hughes Medical Institute (HHMI). B.R. was supported by a grant from the Ministerio de Educación y Cultura of Spain and by the HHMI. L.F.R. is an Investigator of the HHMI. B.R. is a Ramón y Cajal Investigator from Ministerio de Ciencia y Tecnología of Spain.
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Supplementary Fig. 1
Tg(L7cre)22LFR transgene construct and loss of FAK in the cerebellum of fak conditional mutant mice. The Tg(L7cre)22LFR transgenic line was generated by insertion of a cre cassette into the exon 4 of the L7 promoter. In brief, Tg(L7cre)22LFR mice were generated by microinjection into male pronuclei of fertilized oocytes from a C57/Bl6xDBA/2 mixed background of a SfiI/SfiI fragment containing a cre cassette (Kozac sequence–nuclear localization signal–Cre recombinase (creNLS) –Protamine–1 poly A signal) inserted into exon 4 of the L7 minigene. The L7 minigen (Oberdick et al., Neuron 1(5): 367–376, 1988), and creNLS (Lewandoski et al., Curr. Biol. 7(2): 148–151, 1997) have been previously described. Southern blots using a probe recognizing cre and PCR were used to screen the different transgenic lines generated. Of the 20 lines analyzed, 2 were positive by both Southern blot and PCR. To analyze the spatio–temporal distribution of recombination, positive F1 mice of Tg(L7–cre)LFR lines were crossed with R26R reporter mice (Soriano, Nat. Genet. 21(1): 70–71, 1999). The fak conditional allele used in this study is described in detail elsewhere (Beggs et al., Neuron 40: 501–514, 2003). To generated Purkinje cell– specific targeted mice, we first crossed the L7cre mice onto a fak null/+ background (Illic et al., Nature 377 (6549): 539–544, 1995). The L7cre;faknull;fak+ mice were then crossed to fakflox;fakflox animals. 50% of the progeny of this cross will be fakflox;faknull, so a single recombination event will remove FAK activity completely. As controls for our experiments we used either wild–type, fakflox;fakflox, or fakflox;faknull littermates. Similar results were obtained with each of the control mice used. (a) cre cassette containing cre recombinase with an optimized Kozac sequence, nuclear localization signal (NLS) and a protamine–1 polyA 3' UTR was inserted into a BamH1 site in Exon 4 of the L7 minigen. BglII sites were generated by PCR reaction flanking the cre cassette. Presence of the transgenic allele was verified on a genomic Southern blot by the presence of a 3.7 kb XbaI fragment which was absent from controls. (b) Immunoblot analyses of FAK in the cerebellum of P35–P40 fakflox;faknull and L7cre;fakflox;faknull. Protein extracts were prepared from cerebella of fakflox;fakflox, fakflox;faknull, and L7cre;fakflox;faknull. We used 20 μg of protein per lane for SDS–PAGE and immunoblots. Antibodies against the kinase domain of FAK (Upstate, 1:1000) and anti–β–tubulin (Sigma, 1:400) were used sequentially on the same blot for normalization. Blots were quantified by measuring the mean intensity using NIH image software. Despite the small contribution of Purkinje cells to the mass of the cerebellum, western–blot analysis revealed a trend to reduction in the concentration of FAK in the cerebellum 20 ± 13% (n = 3) of FAK protein in the conditional mutant cerebellar extracts in comparison with the fakflox;faknull extracts. (JPG 44 kb)
Supplementary Fig. 2
Motor coordination in L7cre;fakflox;faknull conditional mutants. An accelerating rotorod treadmill (Ugo Basile; Stoelting, Chicago, Illinois) was used to test the effects of cerebellar FAK loss on motor coordination. Young male mice with similar body weights between P30–P50 were chosen for these experiments. The rotorod accelerated from a rate of 3.7 to 37.5 rpm over a period of 300 s. Mice performance in seconds was recorded as described47. Asterisks denote significant differences between the genotypes (n = 8, P < 0.05 by ANOVA repeated measures). (GIF 14 kb)
Supplementary Fig. 3
Increase in the total area labeled by GAD65. (a, b) Confocal plane images illustrating GAD65 immunohistochemistry and their corresponding binary images using NIH software in the DCN. Scale bar, 25 μm (a, b). For GAD65–immunostaining light microscopy analysis, we examined and quantified GAD65–immunoreactive boutons with a Bio–Rad MRC 1,000 confocal microscope (100X, n.a. 1.30 Plan–Neofluor) as described before by Rico and collaborators (Nat. Neurosci. 5(3): 225–233, 2002). Conditional mutants and their control littermates were processed in parallel in each experimental group (n = 4, 4 controls and 4 mutants). In order to standard our results we focused our analysis on the fastigial nucleus in the deep cerebellar nuclei. We selected the same fields for all genotypes. This selection was blind to the phenotype. Two images spaced 1–μm apart were used to quantify the fluorescence intensity from each sample. All images were taken by Kalman averaging (four times) by excitation at 568 nm (laser power 3% iris 2.4). To obtain binary images, we used the background threshold of the control sections for the corresponding sections from the L7Cre;fakflox/faknull littermates. For particle analysis, the threshold was set at 8 pixels. We measured the fluorescent intensities (gray levels) and the immunolabeled regions in an area of 8,600 μm2, using NIH Image software as describe before by Fukuda and collaborators (J.Comp.Neurol. 399: 424–426, 1998). (JPG 88 kb)
Supplementary Fig. 4
Cre expression in wildtype mouse hippocampal culture. (a–b) Fluorescence images and representative drawings of widltype neurons transfected with control pCDNA3 (a) or cre plasmid (b) at 6 DIV. (c) Quantification of branches at increasing branch orders per neuron. (d) Quantification of the total number of branches. (e) Quantification of the total axon length per neuron expressed in mm. (mice hippocampal cultures: 48 neurons over 3 cultures by t–test 2–tailed, n.s., not significance). Data shown are the mean ± s.e.m. Scale bar, 200 μm (a, b). (JPG 79 kb)
Supplementary Fig. 5
Rat hippocampal cultures expressing the dominant negative FRNK. (a–f) Fluorescence images and drawings of neurons transfected with control pCDNA3 plasmid (a–c) or FRNK plasmid (d–f) at 5 DIV and 6 DIV. (g,h) Quantification of the number of increasing branch order per neuron. (i), Quantification of the total axon length per neuron expressed in μm. (j), Quantification of the total number of branches per μm of axon. Asterisks in the graphs denote significant differences from controls (rat hippocampal cultures: 82–5 DIV– and 56–6 DIV– neurons over 3 cultures P < 0.01– P < 0.01–P < 0.0001 (g), P < 0.0005–P < 0.0001 (h), control 1,174.60 ± 101.32–5 DIV, 1,784.59 ± 160.18–6 DIV, mutant 1,963.29 ± 139.45–5 DIV, 2,657.01 ± 179.37–6 DIV P < 0.0005–P < 0.0001 (i), control 0.00040 ± 0.0005–5 DIV, 0.0029 ± 0.0002–6 DIV, mutant 0.0055 ± 0.0004–5 DIV, 0.0051 ± 0.0004–6 DIV P < 0.05–P < 0.0001 (j), by t–test 2–tailed). Data shown are the mean ± s.e.m. Scale bar, 200 μm (a–f). (JPG 122 kb)
Supplementary Fig. 6
Model for the role of FAK signaling in axonal branch formation and synaptogenesis. FAK negatively regulates axonal branching and facilitates the disassembly of axons from inappropriate contacts. (a) Neurons expressing FAK (blue) are able to control the extension and retraction of their axons. Neurons where the FAK function is compromise (red), have an increase in axonal branching and a decrease in retraction, resulting in a dramatic increase in arborization. The boxed area in the arbor of wild–type and FAK mutant neurons is magnified on the right, showing a representative fine–structure of impinging axonal terminals and synapses onto a dendrite. In the presence of FAK neurons branch normally and make an appropriate number of synapses. In the absence of FAK function, this exuberant arborization results in an increase in the number of synapses. The individual efficiency of these synapses may be reduced due to the decreased number of docked vesicles observed. (b) Extracellular signals like integrins or ephrins may activate FAK. FAK controls the cytoskeleton by activation of RhoGTPases, and 190RhoGEF mediate its function. Graf may mediate also FAK function by regulation of Rho or another proteins. This event leads to disengagement of the axons from previously established adhesion contacts, and consequently promotes retraction (c) Loss of FAK blocks RhoGTPases activation preventing the disassembly of point contacts and therefore pruning of these axons. (JPG 82 kb)
Supplementary Table 1
Statistics among experiments. n.s., not significant, s., significant from graphs located in Fig. 6g, j, k. (PDF 300 kb)
Supplementary Video 1
Time-lapse fluorescence images of growth cones expressing DsRed/pCDNA. Each frame is acquired every 15 minutes. (MOV 70 kb)
Supplementary Video 2
Time-lapse fluorescence images of growth cones expressing DsRed/EGFP-cre. Each frame is acquired every 15 minutes. (MOV 95 kb)
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Rico, B., Beggs, H., Schahin-Reed, D. et al. Control of axonal branching and synapse formation by focal adhesion kinase. Nat Neurosci 7, 1059–1069 (2004). https://doi.org/10.1038/nn1317
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DOI: https://doi.org/10.1038/nn1317
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