Chapter 5 - Elsevier...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)

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Chapter 5

Wiring Up the Brain: Axon Navigation

© 2019 Elsevier Inc. All rights reserved. 2

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.


Fig. 5.2 A Purkinje cell after 5 weeks of culture has sent out one long thin axon and a heavily branched set of dendrites reminiscent of its

morphology.


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.


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.


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.


Fig. 5.6 The growth of the Ti1 pioneers is aided by guidepost or stepping stone cells. (A) A grasshopper embryo showing the developing

legs. (B) The Ti1 pioneers in red reach from one of the guidepost neurons(blue) to the next, successively contacting fe1, Tr1, and Cx1 on

their way to the CNS. (C) When the Cx1 cells are ablated, the Ti1 cells lose their way and do not cross into the coxal segment of the

embryonic leg.


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.


Fig. 5.8 (A) An axon growing to its target is like B, a driver navigating through city streets. See text for details.


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.


Fig. 5.10 Early observations of growth cones. (A) In the late 1800s, Ramón y Cajal saw expansions of axons near the ventral midline of the

chick neural tube. (B) In the 1930s, Speidel observed growing nerve fibers tipped with axons in the frog tail. (C) In the early 1900s, Harrison

grew neural explants in culture and watched them extend axons, or leader axons of the opposite side, tipped with motile growth cones.


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.


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.


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).


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.


Fig. 5.15 Actin filaments are necessary to guide growth cones. (A) In the grasshopper limb, the Ti1 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).


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 mM 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.


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 Ti1 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.


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.


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 wild-

type 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 FP4-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.


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.


Fig. 5.21 Mechanosensitivity of RGC axons in vitro. (A, B) Cultures of Xenopus eye primordia (asterisks) on (A) soft (0.1 kPa) and (B) stiff

(1 kPa) substrates. Arrows indicate axons. (C) Eye primordium grown on a stiff substrate and treated with spider venom component

GsMTx4 which blocks stretch activated mechanical sensing channels in the growth cone. Scale bar: 200 μm.


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.


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.


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 IgG 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.


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.


Fig. 5.26 The fasII loss-of-function and (A) In the wild-type the posteriorly directed axons of dMP2 and MP1 fasciculate with the anteriorly

directed axons of pCC and vMP2. These axons all express fasII on their membranes. (B) Loss-of-function mutants of fasII leads to a

defasciculation phenotype.


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.


Fig. 5.28 CAM changing. (A) Two panels showing fasI (top) and fasII (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 fasII in the longitudinal pathway to help it fasciculate with other fasII-expressing axons in this

pathway, switches to fasI while it is in the commissure and fasciculating with other fasI-expressing axons, and then switches back again

to fasII once it has reached the other side.


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.


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.


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.


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.


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.


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.


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.


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 Wnt4 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 Wnt4 is placed on the posterior side of such

an explant. (D) In an fz3 knockout, lacking the Wnt4 receptor, commissural interneurons do not grow either anteriorly or posteriorly once

they cross the midline.


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.)


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.


Fig. 5.39 In a filleted explant of the neural tube in chicks, labeling the commissural axons on each side with crystals of DiI 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.


Fig. 5.40 Repulsive guidance by Netrin through Unc-5. (A) In C. elegans, guidance of the AvM neuron GC by the Unc-6/netrin guidance cue and the Unc-40

and Unc-5 receptor subtypes. AvM neurons normally express the UNC-40 but not the Unc-5 receptor subtype. AvM 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 AvM 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.


Fig. 5.41 cAMP modulates growth cone turning. (A) When internal cAMP is high, the growth cone of embryonic spinal neurons grows

toward a source of Netrin ejected by a pipette. (B) When cAMP is pharmacologically lowered, the same neurons are repelled by Netrin.


Fig. 5.42 Local translation in the growth cones of retinal ganglion cells (RGCs). (A) An RGC growth cone showing mRNAs for beta-actin

(in purple) and dynamic tubulin (in green). (B) Guidance cues act on their receptors to activate the translation of specific mRNAs 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.


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.

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Chapter 5 - Elsevier...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)