Chapter 5 Wiring Up the Brain Axon Navigation

Fig. 5. 1 (A) Part of the fly medulla reconstructed at Janelia Farm Research Campus from transmission electron microscopy (TEM) images of serially sectioned 40 -nm thick slices of tissue. (B) Confocal microscopic image of the dentate gyrus of the hippocampus in an adult brainbow mouse. The brainbow technique is an ingenious genetic recombination strategy using a palette of genetically engineered fluorescent proteins that can be used to randomly label neurons in the brain with myriad colors (Livet et al. , 2007). Here one sees the cell body layer in the middle while above and below a myriad of axons and dendrites interweave. © 2019 Elsevier Inc. All rights reserved. 2

Fig. 5. 2 A Purkinje cell after 5 weeks of culture has sent out

Fig. 5. 3 In tissue culture, a hippocampal neuron begins by putting out several minor processes that are basically equivalent. One of these, the future axon, then begins to grow faster than the other processes and collects axon-specific components. After the axon has elongated, dendrites begin to grow and express dendrite-specific components. This figure shows three young hippocampal neurons in culture stained for microtubules (red) and actin (green). At this stage, one process (the future axon) is elongating while the shorter processes are on the way to becoming dendrites. If, at this stage, the emerging axon is cut, then a minor process, which would have otherwise become a dendrite, begins to grow more rapidly and becomes the axon. © 2019 Elsevier Inc. All rights reserved. 4

Fig. 5. 4 Top panel shows successive images over the course of a few hours of an isolated zebrafish retinal ganglion cell in vitro encountering a laminin-coated bead with one of its emerging neurites, which then becomes the axon and develops a large growth cone. Bottom panel shows a retinal ganglion cell in vivo in a retina where the laminin has been knocked out. In such animals retinal ganglion cells do not polarize efficiently. In this case, a laminin-coated bead helps the cell put out an axon which then develops a growth cone. © 2019 Elsevier Inc. All rights reserved. 5

Fig. 5. 5 The increasing complexity of fiber tracts in the developing vertebrate brain. Antibodies against axons in the embryonic zebrafish brain at successive stages of development over the course of just 20 h reveal that a variety of new axons are added at each stage. © 2019 Elsevier Inc. All rights reserved. 6

Fig. 5. 6 The growth of the Ti 1 pioneers is aided by guidepost or stepping stone cells. (A) A grasshopper embryo showing the developing legs. (B) The Ti 1 pioneers in red reach from one of the guidepost neurons(blue) to the next, successively contacting fe 1, Tr 1, and Cx 1 on their way to the CNS. (C) When the Cx 1 cells are ablated, the Ti 1 cells lose their way and do not cross into the coxal segment of the embryonic leg. © 2019 Elsevier Inc. All rights reserved. 7

Fig. 5. 7 Mauthner cells grow posteriorly in the hindbrain due to local cues. (A) At the neural plate stage, a segment of the hindbrain region of a salamander embryo is removed, rotated 180 degrees, and reimplanted. (B) Shows a dorsal view of a larval brain of such an animal. (C) The bilaterally symmetric giant Mauthner neurons in the normal unoperated larval brain. (D) The trajectory of Mauthner axons in an experimental animal in which a segment of the hindbrain containing the Mauthner primordia is rotated. (E) Photo of host and graft Mauthner cell axons in the same animal. © 2019 Elsevier Inc. All rights reserved. 8

Fig. 5. 8 (A) An axon growing to its target is like B, a

Fig. 5. 9 Axons grow from the retina to the tectum using their growth cones to guide them. (A) A dorsolateral view of the embryonic frog brain. (B) Images of single retinal ganglion cell axons through the plane of the section indicated in (A) shows that as they grow to the tectum, they are always tipped by active growth cones. (C) When the axon is separated from the cell body by cutting the optic stalk, time-lapse imaging shows that isolated growth cones still grow along the correct pathway. © 2019 Elsevier Inc. All rights reserved. 10

Fig. 5. 10 Early observations of growth cones. (A) In the late 1800 s, Ramón y Cajal saw expansions of axons near the ventral midline of the chick neural tube. (B) In the 1930 s, Speidel observed growing nerve fibers tipped with axons in the frog tail. (C) In the early 1900 s, Harrison grew neural explants in culture and watched them extend axons, or leader axons of the opposite side, tipped with motile growth cones. © 2019 Elsevier Inc. All rights reserved. 11

Fig. 5. 11 Leaders and followers. A schematic drawing shows leader axons and follower axons (blue and black) growing through the midline. The leading axon, being the first, is completely exposed to the guidance cues in the environment. Its growth cone must sense all the positive and negative midline cues and interpret them accordingly, which results in slow progress and complex morphology of leader growth cones at the midline where these cues are found. Time-lapse observations show that by growing along the leader, follower axons are less exposed to midline cues, have more simply shaped growth cones, and grow faster. © 2019 Elsevier Inc. All rights reserved. 12

Fig. 5. 12 Microtubules are added at the growing end. (A) A growing axon is labeled with fluorescent tubulin, and then some of this fluorescence is bleached by a beam of light (circle) focused on the axon near the growth cone. As the axon elongates distally, the bleached spot stays in approximately the same place (bottom panel), implying that the microtubules along the axon shaft do not move forward but rather that new microtubules are assembled at the distal tip. (B) Two beads placed on an axon move further apart from each other as the axon grows, with the front bead moving further forward than the rear bead. (C) Filamentous actin is assembled in the filopodia of the growth cone, and some of this is left behind in the submembranous cortex of the axon as part of the axon’s cytoskeleton. © 2019 Elsevier Inc. All rights reserved. 13

Fig. 5. 13 Views of an Aplysia growth cone. (A) Nomarski image showing the growth cone. The bulging central domain and the thin peripheral domain containing actin cables are visible. (B) Labeling the actin filaments with a fluorescent probe reveals they are concentrated in the peripheral domain and the filopodia. (C) Labeling the microtubules reveals that these structures are in the central domain. (D) Pseudo-colored merged image of actin filaments (red) and microtubules (green). © 2019 Elsevier Inc. All rights reserved. 14

Fig. 5. 14 The structure of the growth cone. (A) Actin bundles fill filopodia, which are bounded by membranes with cell adhesion molecules and various receptors, poke out at the advancing edge, and are retracted at the trailing edge of the growth cone. Between the filopodia are sheets of lamellipodia that extend forward. They are filled with an actin meshwork that is continuous with that in the main body of the growth cone. Here, microtubules also push forward and carry cargo to and from the cell body along the axon shaft as they enter the growth cone and fan out toward the filopodia. Some dynamic microtubules can be seen entering into filopodia (B–F). Close-ups of various regions show some of the molecular components of the cytoskeletal network that are localized in the growth cone. © 2019 Elsevier Inc. All rights reserved. 15

Fig. 5. 15 Actin filaments are necessary to guide growth cones. (A) In the grasshopper limb, the Ti 1 growth cones are hairy with active filopodia (top). If the growth cones are treated with the actin-depolymerizing agent cytochalasin, the axon fails to navigate (bottom). (B) In the vertebrate visual system, axons enter the brain from the optic nerve and grow toward the tectum by growing dorsally and turning posteriorly (top). When these axons are treated with cytochalasin, the axons fail to make the appropriate posterior turn, and most axons miss the tectum (bottom). © 2019 Elsevier Inc. All rights reserved. 16

Fig. 5. 16 Filopodium growth and retrograde f-actin flow are inversely proportional. (A) To compare the rates of f-actin flow before and after treatments that inhibited myosin activity, 200 nm beads were positioned at the same location (box) using the laser trap. Bar represents 5 mm. (B) Robust retrograde f-actin flow (black dashed line) and little, if any, filopodium outgrowth (white dashed line) were observed under control conditions. (C) After application of 10 m. M BDM (which inhibits myosin), filopodium elongation (white dashed line) occurred along with slowing of retrograde f-actin flow (black dashed line). Bar represents 1 mm. © 2019 Elsevier Inc. All rights reserved. 17

Fig. 5. 17 Single filopodia can direct growth cones. (A) A single filopodium from a growth cone exerts tension and pulls on an axon it contacts in culture. (B) A single filopodium touches a laminin-coated spot in a culture dish and reorients. (C) A single filopodium of a Ti 1 cell contacts a guidepost cell, and by the process of microtubule invasion becomes the new leading edge. (D) The clutch mechanism: myosin at the base of filopodia pulls on actin cables that are attached to the substrate through a transmembrane clutch and so pulls the main body of the growth cone forward. © 2019 Elsevier Inc. All rights reserved. 18

Fig. 5. 18 Actin and microtubules steer growth cones. (A) Local depolymerization of actin on one side of a growth cone causes it to turn the other way. (B) Local stabilization of microtubules on one side of a growth cone causes it to turn toward that side. (C) Destabilization of microtubules on one side causes it to turn the other way. © 2019 Elsevier Inc. All rights reserved. 19

Fig. 5. 19 (A) Image of a growth cone showing actin (in red) and Ena/Vasp (in green) located at the tips of the filopodia where these proteins act as anticapping agents, preventing the binding of actin capping proteins and thus encouraging plus-end elongation. (B) In wildtype neurons, growth cones are highly dynamic structures. Numerous filopodia and lamellipodia are extended and retracted quickly at the surface of the growth cone. (C) Growth cones of neurons expressing FP 4 -Mito, which sequesters Ena/Vasp to the mitochondria instead of the growth cone periphery, have nearly lost their capacity to generate filopodia yet frequently develop lamellipodia and ruffles. © 2019 Elsevier Inc. All rights reserved. 20

Fig. 5. 20 Axons may follow mechanical pathways. (A) The axons of neurons on a dried collagen matrix growing through the cracks. (B) Axons of the corpus callosum can use an artificial sling to grow from one side of the brain to the other. © 2019 Elsevier Inc. All rights reserved. 21

Fig. 5. 21 Mechanosensitivity of RGC axons in vitro. (A, B) Cultures of Xenopus eye primordia (asterisks) on (A) soft (0. 1 k. Pa) and (B) stiff (1 k. Pa) substrates. Arrows indicate axons. (C) Eye primordium grown on a stiff substrate and treated with spider venom component Gs. MTx 4 which blocks stretch activated mechanical sensing channels in the growth cone. Scale bar: 200 μm. © 2019 Elsevier Inc. All rights reserved. 22

Fig. 5. 22 Growth cones and adhesion. (A) On a very adhesive substrate growth cones are flattened, have lots of filopodia, and do not move rapidly (top). On a less adhesive substrate, growth cones are more compact, rounded, have fewer processes, and often move more quickly. (B) Neurites in culture given a choice between an adhesive and a nonadhesive substrate will tend to follow the adhesive trails. © 2019 Elsevier Inc. All rights reserved. 23

Fig. 5. 23 Differential adhesion of growth cones. (A) To quantitate adhesivity, a measured blast of culture medium is directed at the growth cone. At a particular time, the growth cone becomes detached. (B) Growth is quantified by axon length increase over an interval time. (C) By using such tests, it can be shown that the neurons tested show a particular adhesion profile and tend to grow more slowly on more adhesive substrates. © 2019 Elsevier Inc. All rights reserved. 24

Fig. 5. 24 The main classes of adhesion molecules expressed on the growth cone. Cadherins are calcium-dependent adhesion molecules, most are homophilic. Some members of the Ig. G superfamily of CAMs bind homophilically; others are heterophilic. Integrins are composed of various alpha and beta subunits that bind to a variety of different extracellular matrix components with distinct affinity profiles. © 2019 Elsevier Inc. All rights reserved. 25

Fig. 5. 25 Homophilic adhesion is regulated by polysialic acid. (A) The brachial plexus region in the chick where motor axons destined for particular muscles sort out into their correct nerve roots. (B) Higher magnification of the plexus region showing fascicles breaking up and axons regrouping with other axons. (C) After treatment with Endo-N to remove sialic acid residues from N-CAM, the axons do not defasciculate properly and stay in large fascicles. As a result, innervation errors are made. © 2019 Elsevier Inc. All rights reserved. 26

Fig. 5. 26 The fas. II loss-of-function and (A) In the wild-type the posteriorly directed axons of d. MP 2 and MP 1 fasciculate with the anteriorly directed axons of p. CC and v. MP 2. These axons all express fas. II on their membranes. (B) Loss-of-function mutants of fas. II leads to a defasciculation phenotype. © 2019 Elsevier Inc. All rights reserved. 27

Fig. 5. 27 An experiment supporting the labeled pathway hypothesis. (A) In a control embryo, the G-growth cone, after crossing the midline, fasciculates with P-axons and not A-axons. (B) When the P-neuron is ablated, the G-growth cone stalls and does not fasciculate with the A-axons. © 2019 Elsevier Inc. All rights reserved. 28

Fig. 5. 28 CAM changing. (A) Two panels showing fas. I (top) and fas. II (bottom) distribution in the embryonic CNS of Drosophila as revealed with specific antibodies. (B) Axons express different CAMs on different segments. A commissural axon in an embryonic Drosophila CNS. This axon expresses fas. II in the longitudinal pathway to help it fasciculate with other fas. II-expressing axons in this pathway, switches to fas. I while it is in the commissure and fasciculating with other fas. I-expressing axons, and then switches back again to fas. II once it has reached the other side. © 2019 Elsevier Inc. All rights reserved. 29

Fig. 5. 29 Repulsive guidance. (A) The central projections of most DRG axons do not enter the ventral horn of the spinal cord, but rather make synapses in the dorsal horn. (B) When cultured together, DRG neurons avoid ventral spinal cord explants to grow to dorsal targets. (C) The telencephalon shows olfactory tract fibers originating from the olfactory bulb traveling in the lateral region, far away from the medial septum. (D) When cultured together, olfactory bulb axons travel away from the septum indicating the existence of a diffusible chemorepellent. (E) Surround repulsion. DRG axons outside the spinal cord elongate in a bipolar fashion between the dermomyotome and the ventral spinal cord and notochord. Many surrounding tissues, including the epidermis, the dermomyotome, the floorplate, and the notochord, secrete diffusible repellents. (F) When placed in a collagen gel between a piece of notochord and dermomyotome, DRG axons extend in a bipolar fashion, similar to their pattern in vivo. © 2019 Elsevier Inc. All rights reserved. 30

Fig. 5. 30 Growth-cone collapse. A time-lapse series of a growth cone from a retinal ganglion cell encountering an axon of a sympathetic axon in culture. Upon first contact, the growth cone retracts and collapses. © 2019 Elsevier Inc. All rights reserved. 31

Fig. 5. 31 Comparison of peripheral projections in wild type (+/+) and neuropilin knockout (−/−) mice. Top, trigeminal projections. Middle, intersomitic projections of spinal nerves, and bottom, projections into the limb, are all overgrown in mutant mice. © 2019 Elsevier Inc. All rights reserved. 32

Fig. 5. 32 Growth cones can rely on chemotaxis to orient their growth. (A) sensory neuron turns toward a pipette that is ejecting nerve growth factor (NGF) and thus producing a diffusible gradient. Each time the pipette is moved, the axon reorients its growth. © 2019 Elsevier Inc. All rights reserved. 33

Fig. 5. 33 Chemotactic agents from target tissues. Sensory axons from the trigeminal ganglion heavily innervate the maxillary pad of the mouse face, the site of the whisker field. When the trigeminal ganglion is placed into a three-dimensional collagen gel with the maxillary pad tissue and another piece of epithelium, the axons leaving the ganglion grow toward their appropriate target, suggesting that it is releasing a chemotropic agent. © 2019 Elsevier Inc. All rights reserved. 34

Fig. 5. 34 Dorsal commissural interneurons are attracted by a gradient of Netrin. (A) Dorsal commissural interneurons grow directly to the ventral midline of the spinal cord along a gradient of Netrin that is released by floorplate neurons. (B) In collagen gels, dorsal interneurons are attracted at a distance and orient to the floorplate. (C) They are also attracted to Netrin released from a pellet of COS cells which have been transfected with the netrin gene. © 2019 Elsevier Inc. All rights reserved. 35

Fig. 5. 35 Retinal axons follow local guidance cues in the neuroepithelium. (A) When the tectum is removed, the axons still grow correctly to the tectum, indicating that the tectum is not the source of a diffusible attractant. (B) A piece of neuroepithelium in front of the retinal axons is rotated 90 degrees (top). When the retinal axons enter the rotated piece, they are deflected in the direction of the rotation, but they correct their trajectories when they exit the rotated piece, showing that these axons pay attention to localized cues within the epithelium. © 2019 Elsevier Inc. All rights reserved. 36

Fig. 5. 36 Local gradients of morphogens can orient axons. (A) Commissural interneurons of the spinal cord, once they cross the ventral midline, grow anteriorly toward the brain, and up the Wnt 4 concentration gradient. (B) These axons can be seen well in a filleted preparation grown in culture. The neural tube is sliced open at the dorsal midline and flattened out. Label is applied to the commissural interneurons. (C) Commissural interneurons grow posteriorly if a ball of COS cells expressing Wnt 4 is placed on the posterior side of such an explant. (D) In an fz 3 knockout, lacking the Wnt 4 receptor, commissural interneurons do not grow either anteriorly or posteriorly once they cross the midline. © 2019 Elsevier Inc. All rights reserved. 37

Fig. 5. 37 Midline crossing mutants in Drosophila. (A) In normal flies, many neurons cross the midline once in a commissure and then travel in longitudinal fascicles on the other side. (B) In commissureless mutants, the axons do not cross but travel in longitudinal tracts on the same side. (C) In roundabout mutants, the longitudinal tracts do not form properly because the axons keep crossing back and forth. (After Seeger et al. , 1993. ) © 2019 Elsevier Inc. All rights reserved. 38

Fig. 5. 38 A schematic of a vertebrate commissural neuron before and after crossing the ventral midline which expresses Slit, Sema, Netrin, and Shh. Before the axons cross, their growth cones are attracted to Netrin and Shh and not repelled by Slit and Sema. After crossing, the same growth cones are repulsed by Slit, Sema, and Shh, and no longer attracted by Netrin. © 2019 Elsevier Inc. All rights reserved. 39

Fig. 5. 39 In a filleted explant of the neural tube in chicks, labeling the commissural axons on each side with crystals of Di. I shows that precrossing axons are attracted to either an ectopic floorplate or aggregate of Netrin-expressing COS cells, whereas postcrossing axons that have experienced the floorplate are no longer attracted to either the ectopic floorplate or Netrin. © 2019 Elsevier Inc. All rights reserved. 40

Fig. 5. 40 Repulsive guidance by Netrin through Unc-5. (A) In C. elegans, guidance of the Av. M neuron GC by the Unc-6/netrin guidance cue and the Unc-40 and Unc-5 receptor subtypes. Av. M neurons normally express the UNC-40 but not the Unc-5 receptor subtype. Av. M GCs migrate ventrally to the ventral nerve cord, then turn anteriorly and migrate within the ventral nerve cord to the nerve ring. The normally ventral migration phase depends on ventrally expressed Unc-6 and other, unknown guidance cues. Ectopic expression of the Unc-5 receptor in Av. M causes GCs to migrate in a dorsal direction to the dorsal nerve cord, and this is also dependent on Unc 6. (B) Trochlear motor neurons in the vertebrate embryo are repelled by netrin. Trochlear motor neurons arise in the ventral neural tube at the midbrain/hindbrain region. They grow away from the ventral midline to decussate and leave the brain dorsally. (C) Trochlear neurons in a collagen gel explant culture grow away from the floorplate, and away from COS cells expressing Netrin. © 2019 Elsevier Inc. All rights reserved. 41

Fig. 5. 41 c. AMP modulates growth cone turning. (A) When internal c. AMP is high, the growth cone of embryonic spinal neurons grows toward a source of Netrin ejected by a pipette. (B) When c. AMP is pharmacologically lowered, the same neurons are repelled by Netrin. © 2019 Elsevier Inc. All rights reserved. 42

Fig. 5. 42 Local translation in the growth cones of retinal ganglion cells (RGCs). (A) An RGC growth cone showing m. RNAs for beta-actin (in purple) and dynamic tubulin (in green). (B) Guidance cues act on their receptors to activate the translation of specific m. RNAs relevant to signaling and cytoskeletal dynamics. (C) A gradient of attractive Netrin leads to the upregulation of beta-actin on the near-side of the growth cone in accordance with the differential translation model. © 2019 Elsevier Inc. All rights reserved. 43

Fig. 5. 43 (A) The embryonic frog brain showing a cross-section at the level of the diencephalon. (B) A micrograph of such a cross-section showing a single retinal ganglion cell. (C) Four stages of retinal ganglion cell navigation with some of the relevant guidance systems shown for each stage. © 2019 Elsevier Inc. All rights reserved. 44