Reference: Forscher, P., Kaczmarek, L.K., Buchanan, J. and Smith, S.J. (1987) Cyclic AMP induces changes in distribution and transport of organelles

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1 Reference: Forscher, P., Kaczmarek, L.K., Buchanan, J. and Smith, S.J. (1987) Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cell neurons. J. Neurosci. 7:

2 Movie 1. Axonal growth cone motility of a 3 day-old hippocampal neuron transfected with soluble EGFP and imaged after 8 hrs of expression. Fluorescence images were acquired every 5 sec over a period of 10 min

3 Figure 1: This drawing of Cajal illustrates axonal growth cones in the spinal cord of the E4 chick embryos. Note that Cajal represents a diversity of shapes corresponding to growth cones in different situations. Those with the simplest forms 'C' belong to axons traveling through the prospective white matter. In contrast, growth cones with more complex forms are within the ventral commissure 'B' or passing through the gray matter 'A'. It is of interest to note that this diversity corresponds to what we know today as the sensitivity of growth cones to cues in their micro-environment: in straight paths, the cones have simple forms, whereas in decision points the exhibit much more complex morphologies. Copyright Herederos de Santiago Ramón y Cajal. 3

4 F-actin and microtubule distribution in a hippocampal growth cone. (A) In this typical mouse hippocampal growth cone, labeled with fluorescent phalloidin, the F-actin is concentrated in filopodia (bundled F-actin) and lamellipodia (meshwork of F-actin), with relatively little F-actin in the axon shaft. (B) Microtubules, labeled with an antibody to tyrosinated tubulin, are concentrated as a bundle in the axon shaft but also splay apart in the growth cone, extending into distal peripheral regions. (C) A false-color overlay of images in A and B. Microtubules are in red and F-actin is in green. (D) A magnified view of the boxed region in C. Note the close apposition of an F-actin bundle (closed arrowheads) at the base of a filopodium and an individual microtubule (open arrowheads). At this magnification, the dendritic (D) actin meshwork can also be discerned.

5 (A) Organisation of the growth-cone cytoskeleton. The growth cone is an expanded, motile structure at the tip of the axon and is divided into several morphological regions (as indicated by dashed red lines). In the axon shaft, microtubules (green lines) are organised into parallel bundles by MAPs (purple dumbbells), whereas in the C-domain they become de-fasciculated and extend individually through the T-zone and into the P-domain, where they become aligned alongside the F- actin bundle (black lines) in filopodia. In the P-domain, the F-actin in filopodia is organised into parallel bundles that extend rearwards across the P-domain to terminate in the T-zone, where they are severed into short filaments by a mechanism that is incompletely understood. F-actin in the lamellipodia of the P-domain is organised into a branched dendritic network. In the T-zone and at the periphery of the C-domain there are so-called actin arcs (blue lines) composed of anti-parallel bundles of F-actin and myosin II. Actin arcs produce compressive forces in the C-domain that coral the unbundled microtubules and thereby facilitate microtubule bundling in the growth-cone wrist.

6 Structural characteristics of F-actin and actin-associated proteins. (A) Actin filaments are polar polymers composed of a barbed end, where the bulk of actin monomer addition occurs, and a pointed end, where dissociation of actin monomers occur. The nucleotide state of the actin changes as the filaments age (ATP ADPpi ADP). (B) F-actin in a filopodium forms bundles due to the action of bundling proteins. Actin monomers add onto existing filaments at the tip of the filopodium through the action of barbed-end binding proteins. Actin filaments are constantly undergoing retrograde flow (large vertical arrows) and are disassembled near their pointed ends by severing proteins. Motor proteins use the bundled F-actin to transport cargo both anterogradely and retrogradely. In contrast, F-actin in the lamellipodium forms a dendritic network through the action of dendritic nucleator proteins and capping proteins. Addition of actin monomers also occurs near the membrane, and disassembly occurs more proximally in the growth cone.

7 Structural characteristics of microtubules and microtubule-associated proteins. (A) Microtubules are polar polymers composed of a plus end, where dimer addition and dissociation occur, and a minus end where dimer dissociation can occur. In neurons the minus end of microtubules is often stabilized. Microtubule dynamics occur primarily through polymerization and depolymerization at the plus end. The conversion of microtubule growth to shrinkage is termed catastrophe and the conversion from shrinkage to growth is termed rescue in this figure. The nucleotide state of tubulin also changes soon after dimer addition (GTP GDP). (B) As microtubules polymerize, they bind +TIP proteins at their plus ends. There are many structural microtubule-associated proteins (MAPs) that usually act to stabilize the microtubule. Motor proteins, such as the kinesin family of proteins and cytoplasmic dynein, also transport cargos along microtubules. Several proteins aid in the depolymerization of microtubules, while others can sever microtubules.

8 A working model of cytoskeletal dynamics in a growth cone exposed to a gradient of a positive guidance cue. (A) A schematic of a growth cone that includes actin bundles (green), an actin meshwork (blue), and microtubules (red). The guidance cue gradient (gray) is high in the upper right of the figure, toward which the growth cone is turning. Boxed regions of the growth cone are magnified in subsequent panels. (B) This region of the growth cone is undergoing protrusion. Protrusion is due to activation of barbed-end binding proteins and actin nucleators, resulting in protrusion of filopodia and lamellipodia, respectively. Actin severing can also occur, resulting in new barbed ends for growth. (C) This region of the growth cone is where the actin and microtubule cytoskeleton coordinate their activities, resulting in directed outgrowth. F-actin bundles can guide microtubules. The increased microtubule polymerization/stabilization on one side of the growth cone may favor polarized delivery of materials, which would subsequently favor growth in a particular direction. (D) This region of the growth cone is undergoing retraction. Actin bundles and dendritic networks are disassembled, potentially through the inactivation of barbed-end binding and actin bundling proteins, increased severing of F-actin without subsequent polymerization and continued myosin-

9 driven retrograde actin flow. Microtubules may undergo increased catastrophe or decreased rescue, resulting in their departure from this side of the growth cone. Importantly, several of the cytoskeletal interactions outlined in this model are suggestions based on the current literature but have yet to be documented experimentally. 8

10 Switching sensitivity at the midline. As they cross the floor plate, vertebrate commissural axons lose sensitivity to the midline attractant, netrin, and acquire sensitivity to Slit and semaphorin repellents. This switch may be mediated in part by silencing of netrin attraction by Slit. Drosophila commissural axons also become sensitive to Slit only after crossing. This appears to reflect Comm's role in regulating the intracellular trafficking of Robo. 9

11 Left: Unrestrained netrin-1 does not turn SCN growth cones. A 30x real-time DIC video of a netrin-1 coated, 1.5μm bead presented to a SCN growth cone using optical tweezers. The bead is released upon contact with a filopodia at the 58s time point. Timer is hh:mm:ss. Right: Another example of a SCN axon turning towards an immobilized netrin-1 coated, 1.5μm bead. Timer is hh:mm:ss. 10

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13 Movie 1.Inhibition of PI3K by LY induces a transient growth cone collapse. PTEN couples Sema3A signalling to growth cone collapse doi: / jcs JCS March 1, 2006 vol. 119 no

14 Growth cone collapse and neurite retraction in NG108-EphB2 expressi Repelling class discrimination: ephrin-a5 binds to and activates EphB2 receptor signaling Juha-Pekka Himanen, Michael J Chumley, Martin Lackmann, Chen Li, William A Barton, Phillip D Jeffrey, Christopher Vearing, Detlef Geleick, David A Feldheim, Andrew W Boyd, Mark Henkemeyer & Dimitar B Nikolov Nature Neuroscience 7, (2004) Published online: 25 April 2004 doi: /nn1237 ng cells following treatment with pre-clustered ephrin-b1-fc. 13

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19 Regional specification of the developing brain. (A) Early in gestation the neural tube becomes subdivided into the prosencephalon (at the anterior end of the embryo), mesencephalon, and rhombencephalon. The spinal cord differentiates from the more posterior region of the neural tube. The initial bending of the neural tube at its anterior end leads to a cane shape. Below is a longitudinal section of the neural tube at this stage, showing the position of the major brain regions. (B) Further development distinguishes the telencephalon and diencephalon from the prosencephalon; two other subdivisions the metencephalon and myelencephalon derive from the rhombencephalon. These subregions give rise to the rudiments of the major functional subdivisions of the brain, while the spaces they enclose eventually form the ventricles of the mature brain. Below is a longitudinal section of the embryo at the developmental stage shown in (B). (C) The fetal brain and spinal cord are clearly differentiated by the end of the second trimester. Several major subdivisions, including the cerebral cortex and cerebellum, are clearly seen from the lateral surfaces. 18

20 Flat-mount view of the HH21 chick rhombencephalon illustrating its early segmentation into 8 rhombomeres (r1-r8). The hox code specific for each rhombomere or odd- and even-numbered pair of rhombomeres is indicated by a color code. The different cranial motornuclei and nerve root are also represented. Abbreviations: IV, trochlear nucleus; V, trigeminal nucleus; VI, abducens nucleus; VII, facial nucleus; IX, glossopharyngeal nucleus; X, vagus nucleus; XII, hypoglossus nucleus; MHB midbrain hindbrain boundary; Mes, mesencephalon; FP, floorplate. Adapted from Kiecker and Lumsden,