Filopodia are required for cortical neurite initiation

© 2007 Nature Publishing Group

ART ICLES

Filopodia are required for cortical neurite initiationErik W. Dent1,4, Adam V. Kwiatkowski1,5, Leslie M. Mebane1, Ulrike Philippar1, Melanie Barzik1, Douglas A. Rubinson1, Stephanie Gupton1, J. Edward Van Veen1, Craig Furman1, Jiangyang Zhang2, Arthur S. Alberts3, Susumu Mori2 and Frank B. Gertler1,6

Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VASP-null cortical neurons lack filopodia and fail to elaborate neurites. Here, we report that neuritogenesis in Ena/VASP-null neurons can be rescued by restoring filopodia formation through ectopic expression of the actin nucleating protein mDia2. Conversely, wild-type neurons in which filopodia formation is blocked fail to elaborate neurites. We also report that laminin, which promotes the formation of filopodia-like actin-rich protrusions, rescues neuritogenesis in Ena/VASP-deficient neurons. Therefore, filopodia formation is a key prerequisite for neuritogenesis in cortical neurons. Neurite initiation also requires microtubule extension into filopodia, suggesting that interactions between actin-filament bundles and dynamic microtubules within filopodia are crucial for neuritogenesis.

Neurite extension from a symmetrical cell body is a hallmark of nervous system development. When grown in culture, hippocampal1 or corti-cal neurons2 will initially attach to the substrate and form lamellipodia tipped by filopodia3. The lamellipodia will then coalesce to form neurites, which differentiate into the axon and dendrites4. Although the molecular pathways driving axon specification are well studied5, less is known about how the first morphological step of neurite formation occurs.

F-actin and microtubules are both important for axon outgrowth and guidance6, but their roles in neurite formation are less clear. Microtubules extend into and retract from the actin-rich lamellipodia and microtubule bundling within the lamellipodia of stage 1 neurons is important for neu-rite initiation7. The linkage between actin and microtubules is thought to be coordinated during neuritogenesis, within both lamellipodia and filopodia3,8,9. Although it has been suspected that filopodia are important for neuritogenesis, formal proof of this has been lacking.

Ena/VASP proteins regulate actin dynamics by antagonizing actin-filament capping and promoting filament bundling10,11. These proteins are critical for filopodia formation in axonal growth cones and function downstream of several axon-guidance cues12–14. We recently completed the first analysis of mice lacking all three Ena/VASP proteins – Mena, VASP and EVL (mmvvee) and found that, surprisingly, Ena/VASP defi-ciency disrupts axon-tract formation in the cortex and blocks neurite formation in culture15. Here, we show that filopodia are necessary for neurite formation in cortical neurons and that the requirement for

Ena/VASP in this process can be bypassed by either intrinsic or extrinsic pathways that drive formation of filopodia-like processes. We propose that filopodia are crucial for neurite formation, allowing microtubule orientation along their underlying F-actin bundles.

RESULTSFilopodia can dilate to form neuritesRecently15, Ena/VASP-deficient (mmvvee) cortical neurons were shown to lack actin bundles and filopodia when cultured on poly-d-lysine and usually failed to elaborate neurites (see Supplementary Information, Fig. S1), although they expressed the neuronal-specific markers β3-tubu-lin (Fig. 1a, b) and neurofilament protein (data not shown). To investigate how filopodia formation was related to neurite initiation, control and mmvvee neurons were imaged by short- and long-term time-lapse phase microscopy. Time-lapse imaging revealed that membrane dynamics in mmvvee neurons were markedly different from control neurons. Instead of normal extensions and retractions of filopodia and veils (Fig. 1c and see Supplementary Information, Movie 1), mmvvee neurons showed little membrane protrusion or retraction over 10 min, but continued to exhibit retrograde membrane movement (Fig. 1d and see Supplementary Information, Movie 2). Therefore, deletion of Ena/VASP proteins mark-edly inhibits filopodia and veil dynamics in stage 1 cortical neurons.

Interestingly, a small percentage (5–20%) of mmvvee neurons did form neurites. To determine how these mmvvee ‘escapers’ formed

1Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 2Department of Radiology, Division of Nuclear Magnetic Resonance Research, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 3Laboratory of Cell Structure and Signal Integration, Van Andel Research Institute, Grand Rapids, MI 49503, USA. 4Current address: Department of Anatomy, University of Wisconsin, Madison, WI 53706, USA.5Current address: Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.6Correspondence should be addressed to F.B.G. (e-mail: [email protected])

Received 3 September 2007; accepted 29 October 2007; Published online 18 November 2007; DOI: 10.1038/ncb1654

nature cell biology volume 9 | number 12 | DeCember 2007 1347

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neurites, wild-type or mmvvee neurons were imaged with phase time-lapse microscopy over several days. Control neurons exhibited characteristic filopodia activity followed by membrane segmentation and subsequent neurite extension (Fig. 1e and see Supplementary Information, Movie 3). By imaging many mmvvee neurons (n >50), it was observed that the few neurons that were able to form neurites (n = 6) always formed them by the dilation of a single stable filopodium (Fig. 1f, g and see Supplementary Information, Movies 4 and 5). The dilation of a single filopodium into a neurite was surprising given that control neurons generally form neurites in regions of multiple, dynamic filopodia (Fig. 1e).

Elevated actin-filament capping inhibits filopodia and neuritogenesisWe next asked whether filopodia were required for neuritogenesis. The balance between actin capping and anti-capping activity within cells can modulate filopodia formation16. Therefore, we attempted to block filopodia formation by modestly increasing actin-filament cap-ping activity within neurons. Treatment of wild-type cortical neurons with the actin capping drug cytochalasin D (100 nM) for 44 h resulted in significantly decreased filopodia (Fig. 2a) and impaired neuritogen-esis (Fig. 2b). Culturing cortical neurons in 100 nM cytochalasin D also induced morphology similar to mmvvee neurons (Fig. 2e).

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Figure 1 Filopodia can dilate to form neurites. (a, b) Representative images of control and mmvvee neurons cultured for 48 h, fixed and labelled for the neuron-specific marker β3-tubulin. (c, d) Single phase images of a control (c) and mmvvee (d) stage 1 neuron cultured on poly-d-lysine for 16 h. Single pixel kymographs (x dimension is distance, y dimension is time) were compiled from the positions (1–4) marked in the upper panel in c over a period of 10 min. The bottom panels in c and d show a single-frame composite (maximum projection) of 121

images (10 min at 5 s intervals) of the neurons in the upper panels. Note the robust extension and retraction of filopodia and veils in the control versus mmvvee neurons. (e) Selected frames from a phase-contrast time-lapse series that show how neurites emerge from the periphery of a control stage 1 neuron over a period of many hours. (f, g) Two examples of neurites forming, over a period of hours, from the dilation of a single filopodium in mmvvee neurons. The scale bars represent 50 µm in a and b, and 10 µm in c–g.

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This cytochalasin D concentration is well below levels (1 µM) in which growth-cone translocation is still observed17. Overexpressing both the α and β subunits of heterodimeric capping protein in wild-type neu-rons also inhibited filopodia formation (Fig. 2c) and arrested neurons in stage 1 (Fig. 2d). Thus, treatments that inhibit filopodia formation also inhibit neuritogenesis.

Re-expression of Ena/VASP proteins rescues filopodia and neuritogenesis We asked whether the filopodia and neuritogenesis defect in mmvvee neurons could be rescued by re-expression of Ena/VASP. Normally, embryonic cortical neurons contain all three Ena/VASP proteins (Mena, VASP, EVL), as well as a neuron-enriched higher molecular mass

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Figure 2 Capping actin filaments in control neurons with cytochalasin D or capping protein inhibits filopodia formation and neuritogenesis. (a) Histogram of filopodia number per stage 1 wild-type neuron exposed to 100 nM cytochalasin D for 43 h (cytochalasin D added 1 h after plating). Cytochalasin D markedly inhibited filopodia formation. The triple asterisk indicates P <0.001 (two-tailed unpaired t-test with Welch’s correction) (b) Stacked histogram showing that addition of 100 nM cytochalasin D causes significantly more wild-type cortical neurons to arrest in stage 1 compared with controls, at the expense of stage 3 neurons. The triple asterisk indicates P <0.001 (two-way ANOVA with Bonferroni post hoc comparisons). (c) Histogram of filopodia number per stage 1 wild-type neuron transfected with capping protein α and β. Capping-protein transfection significantly decreased the number of filopodia in stage 1 neurons in culture for 44 h.

The triple asterisk indicates P <0.001 (two-tailed unpaired t-test with Welch’s correction). (d) Stacked histogram showing overexpression of capping protein α and β significantly increases the number of neurons arrested in stage 1 at the expense of stage 3 neurons. The single asterisk indicates P <0.05 and the triple asterisk indicates P <0.001 (two-way ANOVA with Bonferroni post hoc comparisons). (e) DIC images of a fixed control stage 1 neuron and a neuron treated with cytochalasin D (added after 1 h in culture) after 44 h in culture. Note the lack of filopodia on the cytochalasin D-treated neuron. Fluorescent images of the same neurons in the DIC panels stained for F-actin with fluorescent phalloidin are also shown. Note the lack of actin bundles in the cytochalasin D-treated neuron. The scale bar represents 10 µm in e. The numbers over the bars in a–d indicate the number of neurons examined. All error bars represent ± s.e.m.



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isoform of Mena (Mena+)18,19. In mmvvee neurons transfected with EGFP–Mena, EGFP–VASP or EGFP–EVL (see Supplementary Information, Fig. S2), all three proteins rescued filopodia and neuritogenesis

substantially, with Mena consistently rescuing slightly better than VASP or EVL (see Supplementary Information, Fig. S2). Morphologically and ultrastructurally, rescued filopodia were indistinguishable from

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Figure 3 mDia2 and myosin X expression rescue filopodia and neuritogenesis in mmvvee neurons. (a) Histogram of filopodia number per stage 1 mmvvee neuron transfected with different proteins. EGFP–Mena, EGFP–myosin X (MyoX), EGFP–mDia2 and constitutively active mDia2 (EGFP–mDia2M1041A) rescue filopodia to similar levels. P values are indicated (Kruskal-Wallis one-way ANOVA with Dunn’s post hoc comparisons). (b) Stacked histogram showing EGFP–mDia2 and MyoX rescue neuritogenesis to similar levels as EGFP–Mena (all stages P >0.05). Constitutively active EGFP–mDia2M1041A does not rescue neuritogenesis. The single asterisk indicates P <0.05 and the double asterisk indicates P <0.01 compared with EGFP (two-way

ANOVA with Bonferroni post hoc comparisons). (c) Images of representative mmvvee neurons transfected with indicated EGFP-labelled proteins, fixed and stained with a marker for dynamic microtubules (Tyr–MTs) and stable microtubules (Glu–MTs). Mena, MyoX and mDia2 do not induce stabilization of microtubules (increase in Glu–MT staining) in stage 1 neurons compared with untransfected control neurons (arrows in EGFP–mDia2M1041A and EGFP–MyoX images), but the constitutively active form of mDia2M1041 does increase the ratio of Glu–MTs to Tyr–MTs. The scale bar represents 10 µm in c. The numbers at top of bars in a and b indicate the number of neurons examined. All error bars in figure represent ± s.e.m.



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wild-type filopodia (data not shown). Nevertheless, there are sev-eral splice variants of Mena in cortical neurons20, but the broadly expressed Mena isoform rescued both filopodia formation and neuri-togenesis as well as, or better than, any of the other Mena splice variants (see Supplementary Information, Fig. S2).

mDia2 and myosin X rescue filopodia and neuritogenesis in the absence of Ena/VASPWe next examined whether induction of actin bundles and filopodia by expression of other factors could support neuritogenesis without Ena/VASP. Filopodia contain different ‘tip-complex’ proteins that can regulate filopodial initiation and dynamics21. mDia2 is a formin protein that can nucleate linear actin filaments and remain attached processively to the growing filament, protecting it from being capped22. Expression of mDia2 induces filopodia formation in a variety of non-neuronal cell types23–25. mDia2 was not detectable in E14 cortical neurons (see Supplementary Information, Fig. S3); therefore, ectopic induction of this protein or a con-stitutively active point mutant (mDia2M1041A)26 might result in filopodia formation in mmvvee neurons. Expression of EGFP–mDia2 and EGFP–mDia2M1041A rescued filopodia formation in mmvvee neurons, indicating that mDia2 can induce filopodia independently of Ena/VASP (Fig. 3a). EGFP–mDia2 rescued neuritogenesis in mmvvee neurons, but surpris-ingly EGFP–mDia2M1041A did not (Fig. 3b). Ectopic expression of myosin X, an unconventional myosin tip-complex protein that can drive filopodia formation independently of Ena/VASP27, also rescued filopodia formation and neuritogenesis in mmvvee neurons (Fig. 3a, b). Therefore, filopodia formed by three different types of molecules — Ena/VASP, mDia2, and myosin X — were all capable of supporting neurite initiation.

Dynamic microtubules are required for cortical neuritogenesisWe wondered why mDia2M1041A failed to rescue neuritogenesis (Fig. 3b). Previous work indicated that constitutively active mDia2 stabilized micro-tubules when overexpressed28. To determine whether EGFP–mDia2M1041A stabilized microtubules in stage 1 cortical neurons, EGFP–mDia2M1041A-expressing neurons were labelled with antibodies against dynamic tyro-sinated microtubules (Tyr–MTs) and stable detyrosinated microtubules (Glu–MTs). Stage 1 neurons that expressed low levels of EGFP–mDi-a2M1041A seemed to have higher ratios of stable:dynamic microtubules than untransfected (Fig. 3c) or EGFP–Mena-, EGFP–mDia2- and EGFP–myosin X-transfected stage 1 neurons (Fig. 3c). Most of the EGFP–mDia2

M1041A-transfected neurons analysed (21/29) seemed to have high levels of detyrosinated and acetylated microtubules (data not shown). The levels of stable microtubules seemed to be proportional to the level of EGFP–mDia2M1041A transfection. Surprisingly, when EGFP–mDia2M1041A was expressed at high levels it stabilized microtubules to such an extent that they formed hairpin loops in filopodia (see Supplementary Information, Fig. S3). Therefore, hyperstabilized microtubules in stage 1 neurons seem to inhibit neurite initiation, even in the presence of filopodia.

We hypothesized that pharmacological inhibition of microtubule dynamics should also reduce neurite formation. Nanomolar concentra-tions of taxol and nocodazole were used to inhibit microtubule dynam-ics, while leaving total microtubule polymer levels intact17,29,30. Treatment of wild-type cortical neurons with taxol and nocodazole significantly inhibited neuritogenesis (Fig. 4b). Labelling Tyr–MTs and Glu–MTs also showed decreased numbers of dynamic microtubules and increased numbers of stable microtubules after nocodazole and taxol treatment

(Fig. 4c). Neurons treated with these drugs also had few microtubules extending into peripheral lamellipodia and filopodia (Fig. 4c). Instead, both stable and dynamic microtubules were concentrated in the peri-nuclear region. However, these treatments did not inhibit filopodia formation (Fig. 4a). This phenotype was strikingly similar to EGFP–mDia2M1041A overexpression (Fig. 3c). Taken together, these data indicate that filopodia are necessary but not sufficient for neurite formation, and that microtubule dynamics in stage 1 cortical neurons are required for neurite formation.

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Figure 4 Filopodia are necessary but not sufficient for neuritogenesis. (a) Histogram of filopodia number per stage 1 wild-type neuron exposed to 50 nM nocodazole or 20 nM taxol for 43 h (drugs added 1 h after plating). Nocodazole and taxol leave filopodia number intact. (b) Stacked histogram showing addition of nocodazole or taxol causes significantly more wild-type cortical neurons to arrest in stage 1 compared with controls, at the expense of stage 3 neurons. The single asterisk indicates P <0.05 and the triple asterisk indicates P <0.001 compared with control (two-way ANOVA with Bonferroni post hoc comparisons). (c) Images of representative stage 1 wild-type cortical neurons stained for F-actin (phalloidin), Tyr–MTs and Glu–MTs. The overlaid images are magnifications of the boxed section of each neuron shown in the Tyr–MTs column, and show F-actin in red, Tyr–MTs in green and Glu–MTs in blue. Nocodazole- and taxol-treated neurons exhibit control levels of actin bundles. However, the newly polymerized Tyr–MTs in nocodazole- and taxol-treated neurons are confined to more central regions of the cell (arrowheads indicate individual microtubules in the cell periphery) and the levels of Glu–MTs are increased slightly with these treatments. The scale bar represents 10 µm in c. The numbers above the bars in a and b indicate the number of neurons examined. The control bars in a and b are the same as those in Fig. 2. The error bars represent ± s.e.m.



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Figure 5 Filopodia are targeted by dynamic microtubules. (a) A section of a wild-type stage 1 neuron cotransfected with mCherry–β-actin and EGFP–α-tubulin showing prominent actin bundles in filopodia (red in overlay) and microtubules (green in overlay) extending into filopodia. Two filopodia (boxed regions) in a 145 s time-series montage show EGFP–α-tubulin only. Several microtubules target each filopodium during the 145 s by polymerization, and are transported and depolymerize out of filopodia (arrowheads point to individual microtubules and each colour denotes a different microtubule). (b) A section of an mmvvee neuron cotransfected with actin and tubulin as in a. Note the microtubules polymerize to the membrane and then often turn parallel to the membrane and are transported rearward in the retrograde actin flow. Labelling of microtubules is the same as in a. (c) Histogram showing that microtubules in mmvvee neurons traverse more lateral membrane area than wild-type controls.

The wild-type microtubules are constrained in their lateral movement when they polymerize into filopodia. The triple asterisk indicates P = 0.0004 (two-tailed unpaired t-test with Welch’s correction). The numbers over bars indicate number of microtubules examined. (d) Kymographs of actin speckles taken from the two boxed regions in a and b. The slope of the yellow lines indicates the speed of retrograde actin flow. Note that the slope is steeper in the wild-type neuron compared with the mmvvee neuron, indicating that actin retrograde flow is slower in the mmvvee neuron. (e) Histogram showing that actin retrograde flow is significantly slower in mmvvee neurons compared with wild-type controls. The triple asterisk indicates P <0.001 (two-tailed unpaired t-test with Welch’s correction). The numbers over bars indicate number of actin-speckle paths examined in greater than 10 cells per condition. All error bars in c and e represent ± s.e.m. The scale bar in a represents 5 µm.



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To examine how filopodial actin bundles affect microtubule dynam-ics in stage 1 neurons, control and mmvvee neurons were cotransfected with mCherry–β-actin and EGFP–α-tubulin, and time-lapse record-ings of stage 1 neurons were made. Both control (Fig. 5a) and mmvvee (Fig. 5b) neurons contained dynamic actin filaments and microtubules (see Supplementary Information, Movie 6). However, the lack of actin bundles in mmvvee neurons caused growing microtubules that reached the membrane to turn parallel to it and then be swept back toward the nucleus (Fig. 5b and see Supplementary Information, Movie 6). Microtubule clearing from the peripheral domain occurred despite the fact that retrograde actin flow was significantly slower in mmvvee stage 1 neurons compared with wild-type controls (Fig. 5d, e). In contrast, microtubules in control neurons often polymerized along actin filament bundles in the core of filopodia, but were subsequently removed from filopodia by a combination of retrograde actin flow and microtubule shrinkage (Fig. 5a and see Supplementary Information, Movie 6). Also, several microtubules polymerized sequentially into a single wild-type filopodium over a 2 min interval (Fig. 5a). Because they were laterally constrained by the filopodium, microtubules tips in control neurons exhibited much less lateral spread than those near the peripheral mem-brane of mmvvee neurons (Fig. 5c).

Laminin rescues Ena/VASP neuritogenesis defectDeletion of all Ena/VASP proteins causes a marked decrease in axon-tract formation in the cortex and also causes cobblestone cortex15, where cortical neurons migrate through breaks in the pial membrane and form ectopias outside the brain (Fig. 6a–c). These ectopias were heavily labelled with a neuron-specific antibody against β3-tubulin (Fig. 6b), indicating they contain mostly neurons. Curiously, analysis of mmvvee embryos revealed increased staining of the axonal marker Tau-1 within, and immediately ventral to, ectopias — presumably from ectopic neu-ron axon formation (Fig. 6a). Formation of these fibre tracts (Fig. 6c)15 suggested that signals absent from the cortical plate, but present within ectopias, supported axonogenesis independently of Ena/VASP. To exam-ine this hypothesis, control and mmvvee cortical neurons were plated onto mmvvee meningeal fibroblasts, the primary source of extracellular matrix (ECM) components in regions lining the cortex31. As previously noted, mmvvee neurons extend very few neurites when plated on poly-d-lysine (Fig. 1b)15. Strikingly, plating mmvvee neurons onto mmvvee meningeal fibroblasts rescued neuritogenesis completely, compared with littermate controls (Fig. 6h–l).

The primary ECM protein produced by meningeal fibroblasts is laminin. Ectopic cortical (Fig. 6a–c), retinal ganglion, hippocampal and DRG neurons are exposed to laminin during development31,32. The majority of mmvvee embryos are exencephalic (86%), hindering analysis of axon tract development15; therefore, diffusion weighted MRI (diffu-sion tensor microimaging, µDTI)33 was used to non-invasively image fibre tracts in a single mmvvee brain. µDTI measures the anisotropic diffusion of water in fixed samples with a resolution of 60 µm, and allows estimation of axonal organization because water diffuses more easily along an axonal tract than perpendicular to it. Interestingly, axonal out-growth and fibre-tract formation was observed in specific regions of the mmvvee brain — most notably the hippocampus and optic nerve (Fig. 6d–g). Although the hippocampus was poorly organized in the mmvvee brain (data not shown), the ventral hippocampal fibre tract was formed (Fig. 6d). However, consistent with the axon-guidance defects we

reported in Mena/VASP double-knockout (mmvvEE) mice34, the tract failed to reach the midline to form the commissure (data not shown). μDTI also revealed stunted and thin optic nerves in the mmvvee embryo that extended into the brain but failed to form the optic chiasm (Fig. 6f, g). Finally, µDTI and immunohistochemistry revealed that dorsal root ganglia (DRG) axons formed in mmvvee embryos (A.V.K. and F.B.G., unpublished observations). Taken together, these data suggest that neu-rons growing in laminin-rich regions formed axons in mmvvee embryos, whereas neurons occupying the laminin-poor cortical plate did not. Thus, laminin may rescue neuritogenesis in mmvvee neurons. To test this hypothesis, mmvvee neurons were plated onto coverslips coated with laminin, fibronectin or collagen. Strikingly, laminin rescued neu-ritogenesis almost completely, whereas collagen and fibronectin had no effect (Fig. 6m). These results suggested Ena/VASP proteins and laminin function in independent pathways to promote neuritogenesis.

We searched for additional extrinsic factors that might restore neu-ritogenesis in mmvvee neurons. A variety of growth factors and com-pounds known to induce axon outgrowth in neurons or enhance neurite formation in neuroblastoma cells were tested. Activation of protein kinase A (PKA) with forskolin and inhibition of Rho-associated kinase (ROCK) with Y-27632 both failed to rescue neuritogenesis in mmvvee neurons (Fig. 7a). Similarly, the guidance cue netrin-1 and the neuro-trophins brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and nerve growth factor (NGF) did not rescue neuritogenesis (Fig. 7a). Of the factors tested, only laminin was capable of significantly rescuing neuritogenesis and filopodia-like extensions in mmvvee neu-rons (see below). However, neurite and filopodia-like extension number in mmvvee neurons did not reach those of control neurons on laminin (Fig. 7a, b). Nevertheless, there was a direct correlation between filopo-dia number and neurite formation (Fig. 7a, b).

Laminin-induced filopodia-like extensions promote neuritogenesis in the absence of Ena/VASPTime-lapse microscopy was used to further examine how laminin rescued neuritogenesis in mmvvee neurons and showed that lam-inin induced dynamic filopodia-like extensions around the periph-ery of mmvvee neurons (Fig. 7c and see Supplementary Information, Movie 7). These laminin-induced extensions were often curved or kinked (Fig. 7c), and sometimes seemed to adhere to the substrate only at their tips. In addition to filopodia-like extensions, laminin induced segmented lamellipodial regions soon after plating (Fig. 7c). Regions enriched in segmented lamellar and filopodia-like extensions formed neurites and subsequently axons over a period of 15–25 h (Fig. 7e and see Supplementary Information, Movie 8).

We then investigated the properties of the filopodia-like extensions in greater detail. The filopodia-like extensions that were induced by culturing mmvvee neurons on laminin were rich in bundled actin fila-ments, similar to controls (Fig. 7d). Platinum-replica electron micro-scopy revealed that laminin-induced extensions contained bundled F-actin (Fig. 7f). Interestingly, in these laminin-induced extensions, the bulk of actin filaments often originated on one side of the filo-podium and were aligned parallel to the membrane before bending into the filopodium (Fig. 7g, h). In control neurons, actin filaments usually coalesced from both sides of the filopodium (Fig. 7f)15. The difference in actin architecture (bilateral actin filaments coalescing into a filopodium versus bundles entering primarily from one side)



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Figure 6 Laminin rescues neurite initiation in mmvvee mutants. (a) Tau-1 immunostaining of an E16.5 mmvvee brain (coronal section, 10 µm) reveals a marked increase in axonal labelling (arrowheads) extending from ectopias. Dashed white line marks the pial membrane. Inset is a littermate control brain sectioned and stained similarly. (b) Laminin and neuron-specific β3-tubulin immunolabelling of an E16.5 mmvvee brain (coronal section, 10 µm). β3-tubulin staining is prominent in regions dorsal to breaks in the pia (white dashes). Haematoxylin and eosin stain of a sagittal section (5 µm) through an E18.5 mmvvee head reveals fibre tracts (arrows) extending ventrally from the ectopic neurons that have migrated through a pial break. (d, e) µDTI imaging of horizontal sections reveals hippocampal fibre tracts (arrow) in both the control and mmvvee brain. Tissue orientation is denoted by the coloured arrows in d. L, lateral; M, medial; A, anterior; P, posterior. (f, g) µDTI imaging of horizontal sections reveals optic nerves projecting from the retinas of both control and mmvvee embryos. The optic nerves were thinner in the mmvvee embryo and never reached the midline to form the x-shaped optic chiasm (g). Tissue orientation is denoted by the coloured

arrows in f. The second panels in f and g show areas of the optic chiasm (white arrow (Control, f) points to the optic chiasm; white arrow (mmvvee, g) points to the area where the optic chiasm should be). (h–k) Cortical neurons from control and mmvvee mutant littermates were cultured on meningeal fibroblasts (MFs) from a separate mmvvee brain for 30 h, fixed and stained for β3-tubulin. Representative fields of cells are shown: fluorescence alone (h, j) and fluorescence overlays on DIC images (i, k) to visualize labelled neurons on top of fibroblasts. (l) Scoring for stage development reveals mmvvee meningeal fibroblasts restore cortical stage development in mmvvee neurons to levels of control neurons (P >0.05). (m) mmvvee cortical neurons were plated on poly-d-lysine (PdL) or PdL supplemented with 20 µg ml–1 laminin-1 (+Ln), 20 µg ml–1 fibronectin (+Fn) or 20 µg ml–1 collagen-1 (+Cn) and scored for developmental stage after 48 h in culture. The triple asterisk indicates P <0.001 (two-way ANOVA with Bonferroni post hoc comparisons). The numbers over bars in l and m indicate number of neurons examined and the error bars represent ± s.e.m. The scale bars represent 100 µm in a–c, 500 µm in d–g and 20 µm in h–k.



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and morphology (straight versus curved) of control and laminin-induced filopodia indicate they are likely to be initiated through distinct mechanisms.

To determine whether laminin-induced extensions contained a canonical marker for filopodia, neurons were stained for fascin35. The filopodia-like extensions produced by mmvvee neurons plated on laminin were fascin positive (see Supplementary Information, Fig. S4). Interestingly, the few filopodia produced by mmvvee neu-rons plated on poly-d-lysine were also labelled for fascin. Finally, we asked whether microtubules explored the laminin-induced extensions. Immunofluorescence microscopy showed that microtubules seemed

to enter the filopodia-like extensions (see Supplementary Information, Fig. S4). Therefore, laminin-induced extensions are indeed filopodia-like as they contain bundled F-actin and fascin, and seem to support microtubule exploration.

Myosin II inhibition also rescues filopodia-like extensions and neuritogenesis in mmvvee neuronsA study using cultured superior cervical ganglion neurons demonstrated that myosin II functioned downstream of laminin-induced outgrowth36. Inhibiting myosin II activity caused neurons growing on poly-ornithine to extend long axons, similar in length to those on laminin36. Therefore,

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Figure 7 Laminin rescues neuritogenesis through formation of actin-rich filopodia-like extensions. (a) Laminin addition (20 µg ml–1) partially rescues the neuritogenesis in mmvvee neurons. The triple asterisk indicates P <0.001 (two-way ANOVA with Bonferroni post hoc comparisons). Forskolin (5 µM), Y-27632 (10 µM), netrin-1 (100 ng ml–1), BDNF (100 ng ml–1), NT3 (100 ng ml–1) or NGF (100 ng ml–1) had no effect. (b) Number of filopodia-like extensions per stage 1 neuron after 40 h in culture. The triple asterisk indicates P <0.001 (Kruskal-Wallis one-way ANOVA with Dunn’s post hoc comparisons). The numbers over the bars in a and b indicate the number of neurons examined, and the error bars represent ± s.e.m. (c) Image of an mmvvee neuron plated on poly-d-lysine and laminin (20 µg ml–1) after 16 h in culture. The white arrowheads (1–4) indicate regions where kymographs were taken. Kymographs were compiled from a one pixel wide line from each time-lapse image (121 images) and stacked horizontally. The x-axis is time (10 min) and the y-axis is distance (15 µm). The bottom frame is a maximum projection of 121 images. Laminin treatment occasionally resulted

in a long, labile filopodia-like extension (white arrow in bottom panel). (d) Fluorescent images of a control and mmvvee neuron plated on poly-d-lysine, and an mmvvee neuron plated on poly-d-lysine and laminin. All neurons were fixed and labelled for F-actin with fluorescent phalloidin. Note that the laminin-induced filopodia-like extensions are rich in actin filaments. (e) Phase-contrast images from a time-lapse series of a single mmvvee neuron plated on poly-d-lysine and laminin (20 µg ml–1). (f) Platinum-replica electron microscopy images of a control and mmvvee neuron plated on poly-d-lysine, and an mmvvee neuron plated on poly-d-lysine and laminin. The boxed region is magnified in h. (g, h) Platinum-replica electron microscopy images of filopodia-like extensions from stage 1 mmvvee neurons treated with laminin (20 µg ml–1). Laminin induced curved filopodia-like extensions that contain actin filaments originating from one side of the extension (arrowheads). Magnified sections of the regions in yellow boxes are also shown. The arrows point to individual actin filaments. The scale bars represent 10 µm in c–e and 0.75 µm in f and h.



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Figure 8 Myosin II inhibition rescues neuritogenesis through formation of actin-rich filopodium-like extensions. (a) Number of filopodium-like extensions per stage 1 neuron after 40 h in culture. Blebbistatin (50 µM) increases the number of filopodia per cell in mmvvee neurons compared with the same concentration of inactive enantiomer (Blebb φ). The triple asterisk indicates P <0.001 (two-tailed unpaired t-test with Welch’s correction). The numbers above the bars in a and b indicate the numbers of neurons examined and the error bars indicate ± s.e.m.. (b) Blebbistatin addition substantially rescues the defect in neuritogenesis in mmvvee neurons. The triple asterisk indicates P <0.001 (two-way ANOVA with Bonferroni post hoc comparisons). (c) Platinum-replica electron microscopy of filopodia induced by blebbistatin addition to mmvvee neurons. The filopodium-like extensions are curved like the laminin-induced extensions, but unlike laminin actin bundles extend into the extension from both sides. Magnifications (5x) of the

boxed regions are also shown. The arrows point to individual actin filaments. (d) A group of mmvvee neurons after 40 h in blebbistatin (50 µM). Neurons are double labelled for F-actin (phalloidin) and tyrosinated tubulin (Tyr). The arrows point to axons. (e) Fluorescent images from a time-lapse series of a single mmvvee neuron cotransfected with mCherry–β-actin and EGFP-α-tubulin. Within 30 min of blebbistatin (50 µM) addition, mmvvee neurons sprout actin-rich bundles and form filopodia-like extensions (arrowheads). Microtubules (arrows) target the filopodia. (f) DIC images from a time-lapse series of an Ena/VASP-inactivated neuron exposed to 50 µM blebbistatin (drug addition at 0 min). The neuron sprouts a filopodia-like extension soon after addition of blebbistatin. This filopodium engorges and lengthens to become a neurite. (g) Schematic representation showing how actin bundle–microtubule (MT) interactions may initiate neurite outgrowth. The scale bars represent 0.75 µm in c and 10 µm in d–f.



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we asked whether inhibition of myosin II could rescue filopodia and neuritogenesis in mmvvee neurons growing on poly-lysine. In this set of experiments both mmvvee neurons and neurons in which Ena/VASP pro-teins were functionally inactivated through expression of a construct that sequestered all Ena/VASP proteins to the mitochondria37 were used. The latter method markedly inhibited filopodia and neurite formation, identi-cal to mmvvee neurons (see Supplementary Information, Fig. S5).

Similarly to laminin, inhibiting myosin II by addition of 50 µM blebbistatin resulted in a significant increase in filopodia (Fig. 8a) and rescued neuritogenesis (Fig. 8b). To determine how myosin II inhibi-tion gave rise to filopodia-like extensions, and if these extensions con-tained actin-filament bundles, mmvvee neurons were transfected with mCherry–β-actin and time-lapse fluorescent images were collected before and after addition of 50 µM blebbistatin. Surprisingly, within 35 min of blebbistatin addition, actin-rich filopodia-like extensions emerged from an otherwise lamellar stage 1 mmvvee neuron (Fig. 8e and see Supplementary Information, Movie 9). Interestingly, micro-tubules rapidly targeted these filopodia-like extensions (Fig. 8e). We confirmed that blebbistatin-induced extensions from mmvvee neurons contained many bundled actin filaments with platinum-replica electron microscopy. These filaments coalesced from both sides of the filopodia-like extensions, similarly to classical filopodia, but the structures were often curved like laminin-induced extension (Fig. 8c). We also noted that circumferential actin arcs, known to result from compaction of the actin-filament network in myosin II-based actin retrograde flow in growth cones38, diminished in prominence (Fig. 8e). These data suggest that myosin II activity in mmvvee neurons contributes to the formation of actin arcs in stage 1 neurons, much like it does in growth cones38. The increase in actin arcs in mmvvee neurons (Fig. 7)15 could be due to increased levels of myosin activity in mmvvee neurons. Control and mmvvee stage 1 neurons were labelled with an antibody against phos-phorylated (activated) myosin light chain. Similar levels of labelling were observed in both populations of neurons, indicating myosin activity is not increased in mmvvee neurons (data not shown).

With longer-term imaging, blebbistatin-induced filopodia in stage 1 mmvvee neurons were observed to dilate and form neurites directly within 3 h of drug addition (Fig. 8f and see Supplementary Information, Movie 10). Furthermore, after incubation in 50 µM blebbistatin for 40 h, many mmvvee neurons progressed to stage 3, forming a single axon (Fig. 8d). This blebbistatin-induced neurite formation is reminiscent of neurite formation in ‘escaper’ mmvvee neurons that occurs by dilation of the rare filopodium. However, the mmvvee escaper neurites formed over a period of several days, whereas blebbistatin addition could induce neu-rite formation in a matter of hours. Inhibition of myosin II in mmvvee neurons accelerated the process of neuritogenesis by an order of magni-tude, indicating that myosin II activity inhibits filopodia and/or process formation, and neurite formation, in mmvvee neurons.

DISCUSSIONWe have found that actin-bundle and filopodia formation, together with dynamic microtubules that extend into filopodia, are critical early steps in neurite formation that can be regulated by multiple factors (Fig. 8g). Loss of Ena/VASP results in a striking reduction in filopodia formation in primary cortical neurons15. In wild-type neurons, increased actin-filament barbed-end capping by cytochalasin D, or expression of cap-ping protein, also suppresses filopodia formation. Ena/VASP promotes

filopodia formation by mechanisms that may include antagonism of actin-filament capping activity and filament clustering near filopodial tips10,11,16,39,40. Interestingly, expression of mDia2 (and myosin X) rescues filopodia formation independently of Ena/VASP in neurons. This is in contrast to Dictyostelium, which requires both DdVASP41 and DdDia2 for filopodia formation39,42, and indicates there are likely to be multiple cell-type and perhaps species-specific mechanisms that give rise to filopodia.

It has been proposed that the first step in CNS neuritogenesis is breaking of the neuronal sphere by localized budding43 or formation of segmented lamellipodia from an initial broad lamellipodium7. Ena/VASP-null neurons can form broad lamellipodia, and even segmented lamellipodia (data not shown); however, they are rarely able to form neurites. The capacity of mDia2 and myosin X (intrinsic) or laminin (extrinsic) to rescue neuritogenesis in the absence of Ena/VASP indi-cates that the filopodia–actin bundle architecture itself is the essential component for this aspect of neurite initiation, rather than some other Ena/VASP-specific function (Fig. 8g).

As laminin can induce filopodia-like extensions and neurites inde-pendently of Ena/VASP, its presence may explain how mmvvee neurons can form neurites in regions other than the laminin-poor cortical plate15. The existence of an extrinsic cue that supports Ena/VASP-independent neuritogenesis may also explain why this neuritogenesis defect is not observed in mutants for the invertebrate Ena/VASP orthologues13,14,44,45. We speculate that in non-cortical neurons (and invertebrates), cell-extrinsic factors (such as laminin) activate signalling pathways that induce Ena/VASP-independent filopodia formation, thereby supporting limited neuritogenesis and subsequent axon outgrowth. Alternatively, other cell intrinsic factors (such as mDia2 or myosin X) may support neurite formation in regions outside the cortex.

A small percentage of mmvvee neurons produced neurites and sub-sequently an axon. These neurons always produced a neurite from a prominent filopodium (Fig. 1f, g). Thus, neurons can form neurites from the dilation and extension of a limited number of individual filopodia. Consistent with this, inhibition of myosin II in mmvvee neurons can induce filopodia-like processes that directly dilate and extend, forming a neurite and subsequent axon.

Actin filaments and microtubules must be coordinated during neuri-togenesis. Actin filament bundles and/or filopodia were not sufficient for neuritogenesis — neurons transfected with the constitutively active form of mDia2 (mDia2M1040A) formed filopodia but not neurites, most likely because microtubules were hyperstabilized and could not engage actin-filament bundles. Consistent with this, the ratio of stable to dynamic microtubules seemed higher in these neurons and, significantly, damp-ening microtubule dynamics with either taxol or nocodazole inhibited formation of neurites in wild-type cortical neurons. Premature stabiliza-tion of microtubules in stage 1 neurons seemed to limit their interac-tions with peripheral actin-filament bundles and caused them to assume an orientation not conducive to new neurite formation. These stable microtubules are similar to looped microtubules that are observed in paused growth cones46.

What prevents other cell types from sprouting neurites, which also contain actin bundles and dynamic microtubules, is not known. We speculate that microtubule engagement with actin bundles is an early step in neurite formation. A new growth cone must also form and its proximal region must consolidate into a neurite shaft — a process that is likely to require proteins that stabilize and crosslink microtubules.



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Microtubule-associated proteins (MAPs, such as MAP2c and MAP1B) and the dynein-associated protein Tctex-1 are thought to be important for neurite formation and elongation3,8,9. Actin-filament bundles may provide a scaffold that increases the probability of several microtubules polymerizing along one axis and crosslinking into a bundle — perhaps an intermediate in the formation of a neurite shaft. The inherent high concentrations of neuronal MAPs might then crosslink and stabilize several microtubules in parallel, eventually producing a neurite. This is consistent with the initiation of neurite-like processes by MAPs in non neuronal cells3,47. Notably, MAP2c can also promote formation of cell protrusions in COS7 cells by a process that depends on dynein-driven microtubule transport, but not actin48. The full complement of proteins required for physiological neurite formation is unknown, and a major goal is to understand the microtubule–F-actin crosstalk in filopodia that triggers neurite initiation and growth-cone formation.

Note added in proof: a related manuscript by Burnette et al. (Nature Cell Biol. 9, doi: 10.1038/ncb1655; 2007) is also published in this issue.

METHODSPlasmids and reagents. Full-length mouse Ena/VASP (Mena, Mena+, VASP and EVL) cDNA was subcloned in frame with EGFP or mCherry (gift from R. Tsien, UCSD, San Diego, CA) and EGFP–FP4–Mito and EGFP–AP4–Mito into the pCAX vector49. The pCAX vector contains a β-actin promoter and a CMV-IE enhancer. RT-PCR of additional mouse Mena exons (Mena2+, Mena3+ and Mena3+/2+) was performed using standard protocols. In some instances, EGFP was replaced with mCherry. Full-length mouse mDia2 sequence and the con-stitutively active form of mDia2M1041A were subcloned into an EGFP-C1 vector (Clontech, Mountain View, CA) in frame with EGFP or mCherry. Human β-actin cDNA was subcloned into a C1 vector (Clontech) in frame with mCherry resulting in mCherry–β-actin. Human cDNA for α-tubulin was subcloned into the pCAX vector in frame with EGFP. Mouse EGFP–Capping protein (and EGFP–capping protein (subunits were in C1 vectors and cotransfected. Bovine MyoX cDNA in a C2–EGFP vector was a gift from R. Cheney (UNC, Chapel Hill, NC).

Neurotrophin-3 (NT3), brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), laminin-1, cytochalasin D, paxitaxel (taxol) and nocoda-zole were purchased from Sigma (St. Louis, MO). The ROCK inhibitor Y-27632 and forskolin were purchased from EMD Chemicals (San Diego, CA). Antibodies against Mena were generated as previously described12. Rabbit anti-mDia2 anti-bodies (p158) were used at 1:1000. Rat anti-tyrosinated tubulin at 1:1000 (YL1/2 clone, Millipore, Burlington, MA), rabbit anti-detyrosinated tubulin at 1:1000 (Millipore) and rabbit anti-β3-tubulin at 1:1000 (Promega, Madison, WI) were used to label microtubules. All Alexa-conjugated secondary antibodies and Alexa-conjugated phalloidin, used to label actin filaments, were purchased from Invitrogen (Carlsbad, CA ) and used at 1:500-1000.

Cortical-neuron culture and transfection. Generation of mouse knockouts and chimaeras was previously described15. All mouse procedures were approved by the Massachusetts Institute of Technology (MIT) Committee on Animal Care. Cortical neuron cultures were prepared from embryonic day (E) 14.5 mice essen-tially as previously described15. Briefly, cortices were dissected, trypsinized and dissociated. Most mmvvee embryos were exencephalic with the cortices inverted over the eyes; therefore, the most anterior region of cortex was removed and dissected free of meninges. The morphology and development of these neurons was indistinguishable from closed-head mmvvee cortical neurons and GFP+ mmvvee neurons removed from chimaeric cortices15. Dissociated cortical neu-rons from individual mmvvee corticies were resuspended in Nucleofector solu-tion (mouse neuron kit; Amaxa Biosystems, Cologne, Germany) and transfected with an Amaxa Nucleofector, according to the manufacturer’s directions, with up to six different plasmids per cortex. In some rescue experiments, Mena3+ was used in place of Mena with similar results. Transfected neurons were plated at densities of 5–10 × 10–3 neurons cm–2 on poly-d-lysine (Sigma)-coated glass coverslips adhered to the bottom of 35 mm plastic culture dishes that had a 15 mm hole drilled through the bottom of the chamber. Neurons were plated in

plating medium. After 1 h, this medium was replaced with serum-free medium containing neurobasal medium with B27 and glutamine. All pharmacological compounds were added when the plating medium was replaced with serum-free medium. As a single allele of Mena (Mmvvee) was sufficient for viability and a grossly normal CNS15, neurons containing one or more Mena alleles from knockout animals were used as controls.

Immunocytochemistry and imaging. Neurons were fixed in 4% paraformal-dehyde–KREBs–sucrose or 4% paraformaldehyde–PHEM medium50 at 37 °C. Cultures were rinsed in PBS and blocked with 10% BSA–PBS, permeabilized in 0.2% Triton X-100–PBS and labelled with primary (see above) and secondary antibodies at 1:500 (Millipore). Phalloidin coupled to Alexa 488, Alexa 568 or Alexa 647 (Millipore) was used to label actin filaments. Neurons were imaged with 100× 1.45NA Plan Apo (DIC–fluor), 100× 1.4NA Plan Apo Ph3 (phase), 60× 1.4NA Plan Apo (DIC–fluor), 40× 1.3NA Plan Apo (DIC–fluor), 20× 0.75NA Ph2 (phase–fluor) Nikon lenses depending on the experiment. Living or fixed neurons were imaged with an ORCA-ER camera (Hamamatsu, Hamamatsu City, Japan) attached to either a Nikon TE300 microscope with dual Ludl filter wheels and a mercury light source or a Nikon TE2000 micro-scope with dual Sutter filter wheels, a spinning disk confocal head (Yokogawa, Tokyo, Japan) and a Coherent 70C 2 watt multi-line laser. All images were col-lected, measured and compiled with the aid of Metamorph imaging software (Molecular Devices, Sunnyvale, CA), Adobe Photoshop (images) and Adobe Premiere (movies). During time-lapse microscopy, neurons were kept at 37 °C in an incubation chamber (Solent, Segensworth, UK) fitted for each micro-scope. For all fluorescent live-cell imaging, a 1:100 dilution of Oxyrase (Oxyrase, Mansfield, OH) in culture medium was added and the chamber was closed with a glass ring, coverslip and silicone grease.

Platinum-replica electron microscopy. Platinum-replica and correlative plati-num-replica microscopy was performed essentially as previously described12.

Micro-diffusion tensor imaging (µDTI). Before imaging, Bouin’s-fixed embryos were washed in PBS for more than 24 h to remove the fixation solution and trans-ferred into home-built magnetic resonance-compatible tubes. The tubes were then filled with fomblin (fomblin profludropolyether; Ausimont, Thorofare, NJ) to prevent dehydration.

Imaging was performed using an 11.7 Tesla spectrometer with microimaging gradient (300 Gauss cm–1 maximum). A saddle coil (10 mm diameter; Bruker Biospin, Billerica, MA) was used as both the radio frequency signal transmitter and receiver33. The MRI sequence was based on a three-dimensional fast-echo sequence with navigator-echo phase-correction scheme and segmented k-space acquisition with an echo-train length of four. Three-dimensional diffusion-weighted images were acquired with a repetition time of 0.9 s, an echo time of 25 ms and four signal averages. The field of view was 16 mm × 9 mm × 9 mm and the native imaging resolution was approximately 0.09 mm × 0.09 mm × 0.09 mm. At least six diffusion-weighted images with b values of 1000–1200 s mm–2 were acquired. Diffusion sensitizing gradients were applied along six different orien-tations: [0.707, 0.707, 0], [0.707, 0, 0.707], [0, 0.707, 0.707], [−0.707, 0.707, 0], [0.707, 0, −0.707], [0, −0.707, 0.707]. We also acquired at least one image with a b value of 150 s mm–2. The imaging time for µDTI was approximately 20 h.

The diffusion tensor was calculated using a multivariate linear fitting method, and three pairs of eigenvalues and eigenvectors were calculated for each pixel. The eigenvector associated with the largest eigenvalue was referred to as the primary eigenvector. For the quantification of anisotropy, fractional anisotropy was used. Colour-map images were generated by combining the images of primary eigen-vector and fractional anisotropy into red–green–blue (RGB) images. In the colour map images, the ratio between RGB components in each pixel was defined by the ratio of the absolute values of x, y and z components of the primary eigenvector, and the intensity was proportional to the fractional anisotropy. Red was assigned to the anterior–posterior axis, green to the medial–lateral axis, and blue to the superior–inferior axis.

Statistics. All statistical tests were performed with Graphpad Prism 4.0c. For each data set, parametric or non-parametric statistical tests and post-hoc tests were performed depending on the variance of the data sets.

Note: Supplementary Information is available on the Nature Cell Biology website.


http://dx.doi.org/10.1038/ncb1515

http://dx.doi.org/10.1038/ncb1515

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ACKNOWLEDGEMENTSWe thank: R. Cheney (UNC Chapel Hill) for EGFP–MyoX and MyoX antibodies; R. Tsien (UC San Diego) for mCherry; R. Makar for generous assistance with the mouse colony and advice; M. Wold and N. Enzer; and E. Pinheiro and K. O’Brien for help during early phases of the work. We appreciate the helpful advice, comments and discussion from all the Gertler lab members. E.W.D. was supported by a National Institutes of Health (NIH) grant (F32-NS45366). A.V.K. was supported by an Anna Fuller Predoctoral Fellowship. S.G. was supported by a Jane Coffin Childs fellowship. U.P. was supported by funds from ICBP (Integrative Cancer Biology Program). D.A.R. was supported by a Ludwig Fellowship. C.F. was supported by NIH grant F32-GM071156. This work was supported by NIH grant GM68678 and funds from the Stanley Medical Research Institute (F.B.G).

AUThOR CONTRiBUTiONSE.W.D. and F.B.G. conceived the project. E.W.D. performed all of the cell biological experiments and wrote the manuscript with F.B.G. A.V.K. generated the EVL–/– mice and contributed substantively to revising the manuscript. A.V.K and D.A.R. established the mmvvee colony, helped with initial experiments and provided advice and guidance. L.M. executed the platinum-replica electron microscopy. M.B. generated mDia2 subclones and helped in planning all mDia2 experiments. U.P. cloned Mena2+, Mena3+ and Mena3+2+ isoforms and performed RT–PCR on prenatal cortex. J.E.V provided much help with the mouse colony, and J.E.V. and S.G. performed experiments resulting in Supplementary Fig. S7. C.F. generated the capping-protein constructs and provided much advice during the project. A.A. provided mDia2 reagents and advice. J.Z and S.M. performed all µDTI on mmvvee embryos. F.B.G provided advice, overall direction and supervised the execution of the project. All authors read and edited the manuscript.

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S U P P L E M E N TA RY I N F O R M AT I O N

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Figure S1. Loss of Ena/VASP proteins inhibits filopodia and neuritogenesis. (a) Bar graph of filopodia number per stage 1 control and mmvvee (Mena/VASP/EVL-null) neurons at 40hrs in culture. Loss of all three Ena/VASP family members markedly inhibits filopodia formation (***, p<0.001, two tailed unpaired T-test with Welch’s correction, numbers over bars indicate number of neurons examined). All error bars in figure are +/-SEM. (b) Stacked bar graph showing stage progression in control and mmvvee neurons. Ena/VASP deletion causes many more neurons to remain in stage 1 (no neurites), compared to controls (***p<0.001, two way ANOVA with

Bonferroni post hoc comparisons). (c) Representative stage 1 and stage 2 cortical neurons transfected with EGFP-Mena and fixed at one day in culture. Mena is concentrated at the cell periphery (like VASP and EVL – data not shown). The overlay image shows that EGFP-Mena concentrates at the ends of actin filament bundles in the periphery of stage 1 neurons and at the tips of filopodia in stage 2 neuron growth cones. Endogenous protein shows the same concentration at the periphery, especially at the end of peripheral actin bundles12, 18.



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Figure S2. Transfection of Ena/VASP proteins rescues filopodia and neuritogenesis in mmvvee neurons. (a) Domain diagram of Ena/VASP family members. (b) Bar graph showing that Mena, VASP and EVL transfection into mmvvee neurons rescues the number of filopodia in stage 1 neurons (p values given for Kruskal-Wallis one way ANOVA with Dunn’s post hoc comparisons). All error bars in figure are +/-SEM. (c) Stacked bar graph showing transfection of Mena, VASP and EVL all substantially rescue neuritogenesis in stage 1 neurons. (*p<0.05, ***p<0.001, two way ANOVA with Bonferroni post hoc comparisons). Numbers at bottom (filopodia)

or top (stages) of bars indicate number of neurons examined. (d) Domain diagram of Mena with indicated regions where +, 2+ and 3+ exons are inserted. (e) Bar graphs of number of filopodia per stage 1 neuron. Each isoform rescued filopodia to similar levels (p values indicated, Kruskal-Wallis one way ANOVA with Dunn’s post hoc comparisons). (f) Stacked bar graph showing efficiency to which Mena isoforms rescue neuritogenesis. Only the Mena3+2+ isoform does not rescue neuritogenesis (*p<0.05, ***p<0.001, two way ANOVA with Bonferroni post hoc comparisons). Numbers at bottom (filopodia) or top (stages) of bars indicate number of neurons examined.




Figure S3. mDia2 is not detected in E14.5 cortex or adult cerebellum and overexpression of mDia2M1041A hyperstabilizes microtubules, causing them to form hairpin loops in filopodia. (a) A single mmvvee neuron transfected with high levels of EGFP-mDia2M1041A. Note that mDia2M1041A signal is concentrated at the tips of filopodia but is also present at high levels throughout the cytoplasm. (b) In this cell newly polymerized, dynamic microtubules (Tyr-MTs) extend to the tips of filopodia. (c) Older, stable microtubules (Glu-MTs) also extend well into filopodia and form hairpin loops (arrowheads in (e)). (d) Differential interference contrast (DIC) image of neuron. (e) Overlay of panels b (red) and c (green). (f) Overlay of panels a (blue), b (red) and c (green).

Arrowheads indicate hairpin microtubule loops at tips of filopodia. (g) Western blot of RIPA extracts from HeLa cells, E14.5 cortex, adult cerebellum and E14.5 cortical neurons plated for 1 day in culture and labeled for mDia2. The blot was stripped and reprobed for α-tubulin to show that lanes containing neuronal extracts were overloaded compared to the HeLa cell extract. Note that there is little to no mDia2 in E14.5 cortex or when E14.5 cortical neurons are cultured for 1 day. This antibody was shown to react with RIPA extracts from mouse cell lines indicating absence of labeling is not due to lack of immunoreactivity with mouse tissue (data not shown). Standards (in middle) are 250, 150, 100, 75 and 50kd. Scale bar is 10µm.




Figure S4. Filopodia and actin-rich filopodia-like extensions that form after laminin addition contain fascin and are invaded by microtubules. (a-c) An example of a control neuron (prominent growth cone shown) cultured for 24hrs on PdL, fixed and labeled for F-actin (phalloidin) and fascin, a filopodium-enriched protein. (d-f) An example of a stage 1 mmvvee neuron cultured for 24hrs on PdL that has extended three filopodia. These filopodia are well labeled with anti-fascin antibody. (g-i) An example of a stage 1 neuron cultured on PdL and laminin (20µg/ml) that has extended several

prominent filopodia enriched in fascin. (j-r’) Three examples of E14.5 mmvvee cortical neurons plated on PdL and laminin (20µg/ml) for 8 hours, fixed and labeled for F-actin (phalloidin) (j, m, p) and β3-Tubulin (k, n, q). F-actin (red) and β3-Tubulin (green) images are shown in overlay (l, o, r). Boxed regions in l, o and r are digitally magnified five times in l’, o’ and r’. Arrowheads indicate microtubules extending into filopodia. These images also show that neurons express a marker for differentiated neurons (β3-Tubulin) soon after plating. Scale bar is 10µm except 2µm for l’, o’ and r’.




Figure S5. Ena/VASP-null phenotype occurs in Ena/VASP inactivated cortical neurons. (a) Expression of EGFP-FP4-Mito blocks Ena/VASP function by depleting Ena/VASP proteins from their sites of function and sequestering them to the mitochondrial surface 12. Stacked column graph illustrating the percentage of neurons in each stage after 44hrs in culture. First bar represent untransfected neurons, second bar (AP4-Mito) represents neurons transfected with a control construct that targets the mitochondria but does not sequester Ena/VASP proteins, and the third bar (FP4-Mito) represents neurons transfected with a construct that sequesters all Ena/VASP proteins to the mitochondria, effectively inactivating them. Note the marked increase

in stage 1 neurons at the expense of stage 3 neurons between AP4-Mito and FP4-Mito for stage 1 and stage 3 neurons (**p<0.01 compared to both AP4-Mito and untransfected, two way ANOVA with Bonferroni post hoc comparisons). Numbers under graph indicate number of neurons examined. All error bars in figure are +/-SEM. (b) Two cortical neurons labeled with phalloidin to label F-actin (red) and an antibody to β3-Tubulin to label neuron specific microtubules (blue). The stage 1 neuron is transfected with EGFP-FP4-Mito so the mitochondria fluoresce green, while the stage 3 neuron is not transfected. Scale bar is 10µm.




SUPPLEMENTAL MOVIES

Movie 1. A phase contrast movie of a newly plated control cortical neuron on a poly-D-lysine substrate. Note the continued extension and retraction of filopodia. Time = 10 minutes, images captured every 5 seconds.

Movie 2. A phase contrast movie a newly plated Ena/VASP-null (mmvvee) cortical neuron on a poly-D-lysine substrate. Note the lack of membrane protrusion but continued retrograde flow throughout the lamellipodium. Time = 10 minutes, images captured every 5 seconds.

Movie 3. A phase contrast movie of a control neuron forming a neurite. Note that there are many filopodia in the region where the neurite forms with no one dominating to give rise to the neurite. Rather, over a period of several hours filopodial and veil protrusions give rise to a growth cone that consolidates into a neurite.

Movie 4. A phase contrast movie of an mmvvee neuron forming a neurite. In contrast to the control neuron in movie 7 the mmvvee neuron is able to extend a filopodium that is stabilized over a period of several hours. This filopodium dilates to form a neurite that continues to extend, forming an axon over a period of several days.

Movie 5. Another example of an mmvvee neuron forming a neurite through extension, stabilization and dilation of a single filopodium.

Movie 6. A movie of a control and mmvvee neuron transfected with mCherry-β-actin and EGFP-α-tubulin. Only the control cell has extensive actin bundles that give rise to filopodia. Note the extensive microtubule invasion into the periphery of each neuron. However, in the mmvvee neuron microtubules turn parallel to the membrane and are brought back in retrograde flow. Time = 10 minutes, images captured every 5 seconds.

Movie 7. A phase contrast movie of a newly plated mmvvee neuron on a poly-D-lysine and laminin substrate. Note the extensive dynamic filopodia along the segmented periphery of the cell. Time = 10 minutes, images captured every 5 seconds.

Movie 8. A phase contrast movie of an mmvvee neuron plated on a poly-D-lysine and laminin substrate. Note the extensive filopodia and segmented regions around the periphery of the cell that give rise to neurites and subsequently a single axon. Time = 25 hours, images captured every 2 minutes.

Movie 9. A movie of an mmvvee neuron transfected with mCherry-β-actin and EGFP-α-tubulin (not shown) and treated with blebbistatin (time of blebbistatin addition indicated on upper right of movie). Note that within 30 minutes a number of actin-rich filopodia emerge from an otherwise lamellar periphery. Time = 1:26 hrs, images captured every 20 seconds.

Movie 10. A DIC movie of an Ena/VASP-inhibited neuron that was treated with blebbistatin (a specific myosin II inhibitor) at 40 minutes. A filopodium emerges from a lamellar region and continually elongates and dilates, resulting in a neurite after several hours. Time = 2:55 hrs, images captured every 20 seconds.


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Filopodia are required for cortical neurite initiation