Santiago-Medina M , Myers JP , Gomez TM .

NS41564 NINDS NIH HHS , R01 NS041564 NINDS NIH HHS , R01 NS041564-11 NINDS NIH HHS , R56 NS041564 NINDS NIH HHS , T32 GM008692 NIGMS NIH HHS

	Figure 1. Identifying neuronal types in cell culture. A–C: Confocal microscope images of a ∼24 hpf live Xenopus embryo that was previously injected at the 8‐cell stage with mRNA encoding GFP into a single blastomere fated to make dorsal spinal cord. Note many ventrally projecting CIs (commissural interneurons; arrowhead in C). D: DIC image of a spinal cord explant in cross‐section from the same spinal cord as in (A–C) cultured onto PDL‐LN coverslips for 24 h. E: GFP fluorescence shows many labeled RB (Rohon‐Beard) and CIs extending from the dorsal‐lateral region of this explant. Also note many presumed CIs that extend toward the floor plate and stay within the explant (arrowhead). F: Fixed explant that was immunofluorescently labeled with an anti‐neurofilament antibody (3A10) that is specific for CIs and the peripheral process of RB neurons in vivo. Note dorsal lateral labeled axons emerge from both sides of spinal explant G. Merged image of fluorescent and DIC channels with dorsal‐ventral and right‐left quadrants indicated. Presumed neural crest cells (arrow) are often observed migrating from the dorsal aspect of spinal cord explants. Scale bar, 62 μm (in A–C) and 100 μm (in D–E). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
	Figure 2. Quantification of active Src in growth cones by ICC. A: Control growth cone immunolabeled with antibodies against phospho‐tyrosine 418 Src (P‐Y418 Src). Note intense labeling in the axon shaft (white arrow), peripheral veil (green arrow) and some labeled filopodial tips (arrowhead). B: A growth cone treated with the Src inhibitor PP2 for 5 min before immunolabeling for P‐Y418 Src. Note reduced labeling throughout this growth cone compared to control. A′, B′: The growth cones from (A, B) were colabeled for total protein (pseudocolored red) with Alexa Fluor 647 carboxylic acid, succinimidyl ester. All images were collected with a confocal microscope. C: Normalized total fluorescent intensity measurements of P‐Y418 Src immunolabeling, and the normalized P‐Y418 Src/total protein ratio. PP2 treatment results in a significant decrease in P‐Y418 Src by both measures. *p < 0.05. Scale bar, 10 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
	Figure 3. Analysis of cell signaling and structure with live cell imaging. A, B: Total Internal Reflection Fluorescence (TIRF) microscopy images of paxillin‐GFP and mCherry dual‐Src homology 2 domain (mCh‐dSH2) fluorescent images of a growth cone on PDL‐LN. Note that paxillin and phosphotyrosine (PY), as revealed with mCh‐dSH2, colocalize at adhesion sites (arrowheads in A, B), whereas the tip of a growing filopodium has PY, without paxillin (arrow in B). C: Merged image of the growth cone in (A, B) shows colocalization at several peripheral adhesions. Note that mCh‐dSH2 puncta within the central domain are mobile vesicles (green arrow in B). D: Single line kymograph (see text for details) constructed from region between the arrowheads (C). Note a stable adhesion (arrow) that disassembles after a new protrusion extends forward, followed by the formation of a second adhesion (arrowhead), which stabilizes the receding protrusion. Scale bar, 10 μm in all images and as indicated in kymographs. E, F: GFP‐WASP binding domain (GFP‐WBD; active Cdc42 binding) and tetramethylrhodamine‐dextran (TMR‐D; volume marker) fluorescent images of a growth cone on LN captured with a confocal microscope. The average pixel intensities (8‐bit scale) within regions at the growth cone periphery (red), central domain (blue) and axon (green) show that GFP‐WBD is selectively enriched in the peripheral lamellipodia of this growth cone. G: A pseudo‐colored ratio image of the growth cone in (E, F) shows the GFP‐WBD/TMR‐D ratio is elevated throughout peripheral lamellipodia and filopodia (arrows). H: Single line kymograph constructed from region indicated by the white line in (G). Note an elevated WBD/TMR‐D ratio during lamellipodial protrusion. I, J: GFP‐Utrophin (GFP‐Utr; F‐actin probe) and TMR‐Kabiramide C (TMR‐KabC; F‐actin barbed end binding) fluorescent images of a growth cone on PDL‐LN collected with a TIRF microscope. Note bright TMR‐KabC fluorescence at the periphery (arrow in J), indicating a high concentration of F‐actin barbed ends. K: Merged image of the growth cone in (I, J) shows strong colocalization of GFP‐Utr and TMR‐KabC in central domain foci, but primarily TMR‐KabC in the peripheral domain. Weak labeling of peripheral actin with GFP‐Utr is consistent with the slow association rate of GFP‐Utr onto recently polymerized F‐actin (Rybakova et al., 2006). L: Single line kymograph constructed from region indicated by the white line in (K). The slope of the diagonal bands of GFP‐Utr and TMR‐KabC fluorescence (arrows) indicates a retrograde flow rate of ∼4 μm/min. Scale bars, 5 μm or as indicated. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
	Figure 4. Heat maps of point contact lifetime. A, B: Heat maps indicating point contact lifetime generated by applying a series of filters (see the text for details). Images were generated from of a Xenopus retinal ganglion cell growth cone expressing paxillin‐GFP before (A) and after (B) the addition of 1 μg/mL EphrinA1. The arrows highlight point contacts. Note the stabilization of point contacts and partial collapse of the growth cone after addition of EphrinA1. Scale bar, 5 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
	Figure 5. Labeling and measurement of CI axons. A: Maximum projection of a confocal z‐series of a ventral view of a 27 hpf neural tube immunolabeled with 3A10 (neurofilament in CIs) antibody (blue) and expressing GFP (green) in crossed CIs. Anterior is up in all ventral views. B: Traced trajectories of GFP‐labeled CIs (from A) at various points before, during and after midline crossing. Red lines indicate the position of the ventral fascicles. Note that CIs directly cross the midline and turn to ascend in the contralateral spinal cord after midline crossing. C: Ventral view of a 27 hpf neural tube immunolabeled with 3A10 antibody. D: Same embryo as (C) showing GFP‐FRNK (dominant‐negative FAK) expression targeted to the left side. E: Merge of 3A10 (blue) and GFP‐FRNK (green) fluorescence. F: Traced trajectories of FRNK‐expressing axons in (D). One axon at the floor plate prematurely turned posteriorly (black asterisk), while two others aberrantly reoriented back towards the ipsilateral side (red asterisks). G, H: Confocal maximum projection of a ventral view of a 27 hpf neural tube that was electroporated at 21 hpf with mRNA encoding GFP and TMR‐Dextran. Note that GFP expression occurs in the dorsal CIs that project their axons ventrally on one side (G), while TMR‐D was electroporated into ventral cells on the opposite side of the spinal cord (H). This distribution is likely due to charge difference between mRNA (negative) and TMR‐D (positive). I: ß‐tubulin (ß‐tub) labeling of the spinal cord from (G, H). Note that ß‐tub labels all neurons in the spinal cord, including the neurons that form the ventral fascicles (blue arrowheads). J: Merged image of the spinal cord from (G–I) showing GFP (green), TMR‐D (red) and ß‐tub (blue). K: Optical cross sectional views of confocal image stacks of the spinal cord from (G–J) taken at the position indicated by the white arrowheads in (J). Scale bar, 50 μm (in A–J) and 40 μm (K). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]

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