The Journal of Neuroscience, May 1987, 7(5): 1447-1480
Growth Cone Morphology Varies with Position in the Developing Mouse Visual Pathway from Retina to First Targets
Paola Bovolentaa and Carol Mason Department of Pharmacology, New York University School of Medicine, New York, New York 10016
We have labeled the growth cones of retinal ganglion cell low well-defined common pathways, and more elaborate axons with HRP in intact mouse embryos. This has allowed filopodial forms appear when growth cones diverge, as they us (1) to visualize growth cone morphology during outgrowth turn or come to decision regions. Together with observations along an entire CNS pathway from origin to target; (2) to ask in vitro and in nonmammalian nervous systems in situ, these whether growth cone forms, and thus behaviors, differ at data serve as reference points for testing to what extent various points along the pathway; and (3) to study the re- growth cone form reflects intrinsic factors and interactions lationships of growth cones with the cellular environment. with the environment. During the major period of axon outgrowth between em- bryonic day (E) 12 and 15, growth cones in the optic nerve As the growing end ofa developing axon, the growth coneguides are highly elongated (up to 40 pm) and have lamellopodial the axon, choosesamong different substrates,leads the way into expansions, but the majority lack the microspikes or filo- target regions, and ultimately synapseswith certain target neu- podia characteristic of many growth cones. Within the optic rons (Rees et al., 1976; Johnston and Wessells, 1980; Letour- chiasm (El3-15), most growth cones shorten and spread, neau, 1982; Landis, 1983). Recent evidence from observations and project several short filpodia. In the optic tract, growth of neurons in vitro, in invertebrates, and in the PNS of lower cones become more slender and again lack filopodia, re- vertebrates in situ, suggeststhat the shapeof growth conescor- sembling sleeker versions of optic nerve growth cones. Near relates with their behavior (Raper et al., 1983; Argiro et al., the first target region (lateral geniculate nucleus), growth 1984; Tosney and Landmesser,1985a). One hypothesis derived cones with filopodia arise from individual axon lengths and from this recent work is that growth cone shapestransform turn medially toward the target. Within target regions, the during navigation along pathways, as a responseto environ- branches of immature axon arbors are tipped by minute mental cuesfor neurite extension and directionality (Caudy and swellings rather than by the enlarged growth cones prevalent Bentley, 1986a). during outgrowth toward targets. There is somereason to think that growth conesin the CNS, Electron-microscopic analysis of identified labeled growth especially in mammalian nervous systems,might not obey the cones in the optic nerve reveal intimate interactions between same rules. For example, it is unclear whether populations of growth cones and glia or other growth cones in the form of neuronshave a singleleading or “pioneer” neuron (Keshishian, invaginating contacts. In the optic nerve, growth cones con- 1980), which during some developmental stageshas a more tact immature glial (neuroepithelial) cells somewhere along complex shapethan the follower axons (Lopresti et al., 1973). their length, and also envelop bundles of neurites. In the Indeed, axon populations in CNS pathways are orders of mag- chiasm, single growth cones simultaneously relate to many nitude larger, perhapsobviating the need for axon “scouts.” different profiles. In this study, we were able to visualize growth conesduring These results demonstrate that in this single pathway from the outgrowth phase, in a major CNS pathway in mammalian origin to targets, growth cone morphology varies system- brain. We labeled retinal ganglion cell axons by injecting HRP atically with position along the visual pathway. During out- into the retina of isolated but intact mouse embryos. This ap- growth, simple growth cones are prominent when axons fol- proach hasproduced novel views of populations of growth cones along the entire visual pathway from retina to first targets in the thalamus and allowed us to observe whether the form of Received May 23, 1986; revised Nov. 3, 1986; accepted Dec. 3, 1986. retinal axon growth conesis invariant alonga pathway or changes This work was supported by NIH Grant NS- 1695 1 and a Research Career Develomnent award to C. M.. who also holds an Irma T. Hirsch1 Career Scientist as growth cones travel through different environments where Award.‘Elizabeth Gregory and I&sty Brown gave expert assistance in electron they display different behaviors. Axons grow relatively straight microscopy, Susan Catalan0 helped with illustration, and Yvel Calderon and Julia in the tubular pathways of optic nerve and tract, but in the optic Cohen typed the manuscript. We are grateful to Drs. Lloyd Greene and Mary Beth Hatten for their useful comments on the manuscript, and Drs. John Aletta, chiasm the majority cross over to the contralateral optic tract. Paul Letoumeau, Norm Wessells, and Rob Williams for their insightful discus- A turn is also required when axons enter thalamic targetsmedial sions. to the tract. Correspondence should be addressed to Carol Mason, Department of Phar- macology, New York University Medical Center, 550 First Avenue, New York, A secondquestion is whether growth cone forms are different NY 10016. during outgrowth, when axons must extend over great distances, z Present address: Department of Physiology and Cellular Biophysics, Columbia University, College ofphysicians and Surgeons, 630 West 168th Street, New York, as compared to synaptogenesiswithin target regions,where se- NY 10032. lection of target cells is made over shorter distances. Previous Copyright 0 1987 Society for Neuroscience 0270-6474/87/051447-14$02.00/O studies have characterized the form and cell-cell relations of 1448 Bovolenta and Mason - Morphology of Retinal Axon Growth Cones growth cones within targets of retinal axons in the kitten lateral cation, as well as photographed with phase-contrast optics. By placing geniculate nucleus and in the mouse cerebellum, during pre- the drawing of the 7 pm sections on the original drawing, pieces of the axons and growth cones of interest became evident. The selected 7 wrn and postnatal development of axon arbors and synaptogenesis sections were removed from slides and remounted onto faced-off block. (Mason and Robson, 1979; Mason and Gregory, 1984; Mason, Thin sections were cut and placed on single-holed Formvar-coated grids, 1985; Sretavan and Shatz, 1986). The tips of maturing axons stained with uranyl acetate and lead citrate and examined on a JEOL during this phase lack the typical forms of growth cones seen 100s EM. The drawings of the 50 and 7 pm sections, as well as the phase photographs, served as guides for relocating specific axons and during outgrowth in vitro, stereotypically, flattened flanks with growth cones. Over 170 preparations between E 12 and E 19 were ex- long filopodia (Bray and Bunge, 1973; Letoumeau, 1979). In- amined at the light microscope; of these, 15 were also analyzed with stead, most are minute tapered forms that lack microspikes, the EM. especially on axons that develop highly branched arbors. Definitions and quantitation ofgrowth conefeatures. Over 300 growth Another issue is how growth cones interact with the surround- cones were drawn with a camera lucida at 1000 x magnification. Growth cones were defined as the expansion of the distalmost constriction of ing cellular and extracellular environment in each portion of a the neurite. In many cases, growth cones appeared to consist of a series varied pathway during outgrowth and synaptogenesis. Because of expansions. If a thin segment of neurite was absent between these we have labeled individual growth cones with electron-dense expansions, the series was considered to be a single growth cone. HRP, we can begin to address this question and readily deter- There were 2 types of structures arising from the core or flanks of the mine how identified growth cones relate morphologically to body of the growth cone: (1) filopodia, thin (
Figure 1. Typical growth conemor- phologiesin the variousregions of the retinalaxon pathwayat El 3 (a, b) and El 5 (c). Photomicrographswere taken from osmicated50 rrn vibratomesec- tions. a, Growth cones in the optic nerve arelong and narrow, with concaveand convex lamellopodial expansions of the central core. b, Growth cones in the chiasm have shorter flanks and bear multiple filopodia (arrows). c, Fibers in the optic tract at the level of target re- gions have growth cone-like expansions that point medially (cuwed arrow) to- ward the target lateral geniculate nucle- us. Filopodia arise from these growth cones and extend both toward the target and into the tract (straight arrows). x 1070 (u-c).
niculate nucleus (LGN), both the main shaft of the axon and proportion bore short filopodia (Fig. 2, Table 1).Thus, the growth the branches were positioned in the more medial regions of the conesin the optic nerve differed strikingly from the stereotypic tract. In all portions of the pathway, fibers did not run straight growth conesin vitro, which have spreadforms with extensive but interwove to a considerable extent (Fig. 4). filopodia (Bray and Bunge, 1973). Variation in growth cone shapesappeared to be looselyrelated Growth cone morphology to the ageof the embryo. The earliest growing axons had more Growth cones had a broad spectrum of morphologies along the compact growth cones,while the “followers,” in the major phase pathway from the eye to the target, but their shape could be of outgrowth at E13-14, more commonly displayed lamello- grossly characterized by the region ofthe pathway through which podial expansions. they traversed (Fig. 1). Optic chiasm. Growth cone morphologieswere strikingly dif- Optic nerve. The silhouettes of optic nerve growth cones at ferent in the optic nerve and in the optic chiasm. The most various ages are illustrated with camera lucida drawings (Fig. obvious changewithin the chiasm was the presenceof several 2). At El 2 the growing tips of the few fibers labeled in the optic filopodia, 3-l 0 pm in length, arising from the main body of the stalk were simple, without many surface features or projections, growth cone (Figs. lb, 3 (closed arrowheads), and 4; Table 1). but relatively long (up to 20 wm). From El3 and E15, growth An additional changewas a reduction in length and slight broad- cones in the nerve had similar characteristics. Many optic nerve ening of the main body of the growth cone (Figs. 3, 4). At El 3 growth cones had a central core with lamellopodial extensions the averagelength of growth coneswas 14 pm, as compared to (asterisks in Fig. 2). Most growth cones were rather long, with 23 Km for optic nerve growth conesof the same age(Table 1). an average length of 23 pm, but some reached 40 pm (Fig. la Growth cones in the chiasm generally had more complex and Fig. 2). The most complex optic nerve growth cones con- 3-dimensional surface features than those in the optic nerve sisted of a series of concave and convex lamellopodial expan- (Fig. 3, asterisks).The only age-relatedfeature wasa slight short- sions (arrowheads in Fig. 2). The majority of growth cones with- ening of growth cone length by El 5 (Table 1). in the optic nerve lacked filopodia (Figs. la and 2; Table 1). By Most growth coneswithin the chiasm had filopodia and the E 15 the few growth cones that were visualized by HRP labeling majority of growth cones in optic nerve lacked filopodia, but were shorter and still had lamellopodial extensions, but a larger this feature was not the sole criterion for distinguishing them 1450 Bovolenta and Mason * Morphology of Retinal Axon Growth Cones
El2
El3
El4
El5
OPTIC NERVE
Figure 2. Cameralucida drawings of HRP-filledgrowth cones in the optic nerve duringthe major period of outgrowth.As in Figures3 and 5, the actualgrowth cone is considered to be the stippled expansionof the neurite,which is indicated in black. At E12 growth cones have more smooth surfacesthan at later ages.Many of the growthcones from E13-15are highly elongatedwith a seriesof thin convex and concavelamellopodial expansions(arrowheads). In somegrowth cones, a centralcore is visible (asterisks). Filopodiaare only occasionallyseen and rarely exceed2 in number(arrow). Scalebar, 10 pm.
(Figs. 3 (open arrowheads),and 4; Table 1). Growth cones in embryonic chiasm in isolatedbrains. Midway betweenthe optic the nerve were always much longer than they were wide, and if chiasm and LGN, growth cones were commonly located on they bore filopodia, never had more than 2 (Fig. 2, arrow; Table fibers that ran on the outer surface of the optic tract (Fig. 5a) 1). In the chiasm, the growth conesthat lacked filopodia, more and their shapesdid not seemto vary with age. Growth cones closely resembledoptic nerve growth cones, e.g., elongated la- on fibers in the optic tract were more slender versions of optic mellopodial forms (Fig. 3, open arrowheads,and Fig. 4, growth nerve growth cones (Fig. 5a, Table 1). They were relatively cones3 and 4). Filopodia on growth conesin the nerve or chiasm compact with tapered tips and were much shorter than optic were of similar length, but growth conesin the chiasm had, on nerve growth cones,with an averagelength of 9 km. Some tract the average, at least 2 more filopodia than did growth cones in growth cones had filopodia or small spines along their length the nerve. (Fig. 5a). A greater proportion of growth coneshad filopodia at An unusual feature of growing axons in both optic nerve and later ages(Table 1). chiasm was noted. In addition to the leading growth cone at the When fibers reached the border of target regions such as the end of the axon, velate or irregular expansions occurred en LGN, a further transformation occurred. Axons positioned more passant along the axon (Fig. 4, arrows on growth cones 2 and deeply in the tract gave rise to long smooth expansions that 5). By their shape, these expansions were interpreted as rem- bore several filopodia (Figs. lc and 5b, arrows). Some of these nants of growth cones rather than swellingsthat arose after expansionswere en passant but were always oriented medially extension. toward the target. Filopodia on these growth cones tended to Optic tract. In order to label growth cones in the optic tract, be more numerousand longer than growth conesin the chiasm HRP-coated glassmicropipettes were inserted directly into the (10-l 5 pm) and were oriented in all directions. Short filopodia The Journal of Neuroscience, May 1987, 7(5) 1451
El3
El4
El5
f
OPTIC CHIASM Figure 3. Camera lucida drawings of growth cones in the optic chiasm at the ages when growth cones are common in this region. Growth cones are shorter and wider than those in the optic nerve and have more complex 3-dimensional morphology (asterisks) with multiple filopodia (filled arrowheads). Some chiasm growth cones lack filopodia (open arrowheads) and resemble optic nerve growth cones, both in length and shape. As in the optic nerve, few age-related effects on morphology are evident (see Table 1). Scale bar, 10 pm. also arose along the length of these neurites. barely visible at the light-microscopic level. These minute swell- Targets. Once axons entered targets, dramatic changes took ings occurred on axons that projected to proper targets such as place in growth cone size and form. Rather than having the large the LGN (Fig. 6b), as well as on axons that projected to inap- growth cones seen on unbranched neurites, the branched arbors propriate regions (Fig. 6a). Some of the axons that were labeled were tipped in small swellings < 1 pm in diameter that were with HRP projected to nonvisual “inappropriate” targets. Fibers
Table 1. Position-related features of growth cones at different ages
Maximum Maximum Percentage Length of length width with Number of filopodia Tissue n (rm) (rm) filopodia filopodia (w@ Optic nerve El3 30 23.2 + 6.8 4.5 f 1.2 30 1.5 -t 0.1 3.4 + 1.6 El4 31 23.3 + 7.1 3.1 f 1.7 22 1.0 f 0.0 3.6 + 1.4 El5 17 15.1 * 1.1 2.9 e 1.1 41 1.4 + 0.5 3.1 + 1.8 Optic chiasm El3 35 15.1 k 2.8 6.7 f 2.0 71 2.4 f 1.4 3.8 + 1.7 El4 20 15.5 AI 4.1 6.4 f 2.3 70 2.4 k 1.2 2.8 t 1.0 El5 17 12.3 + 4.9 4.1 & 1.9 70 3.3 + 2.3 4.2 + 3.7 Optic tract El4 15 9.2 + 3.2 2.4 t- 0.8 40 2.5 T 1.8 3.5 + 2.3 El5 7 9.1 + 2.4 2.3 + 1.4 42 2.0 ck 0.0 2.5 + 1.0 El6 20 1.9 + 1.6 1.9 + 0.6 65 1.2 + 0.6 2.7 + 1.1 Dimensions of growth cones of the optic nerve, chiasm, and tract at different agesduring the major period of outgrowth in each region. Tract growth cones included only those found midway between chiasm and target entry zones. Average measurements arc given f SE. The average number and length of lilopodia (fourth and fifth columns) were calculated for only those growth cones having filopodia (third column). 1452 Bovolenta and Mason - Morphology of Retinal Axon Growth Cones
Figure 4. Camera lucida drawing of HRP-labeled retinal axons and growth cones at E14, in a single 50 pm osmicated vibratome section through the optic chiasm and diencephalon (inset), showing many of the labeled fibers in actual relationship to one another. Note that axons do not run parallel to one another but intertwine. The majority of fibers with growth cones run close to the pial surface (dotted lines). Growth cones I and 2 have filopodia and complex shapes, while growth cones 3 and 4 lack filopodia and resemble growth cones in the optic nerve (Fig. 2). Growth cone 5 has an intermediate shape. En passant swellings or expansions proximal to the growth cone occur on many fibers (arrows on axons2 and 5). Shaded structures are blood cells and vessels, marked with asterisks to refer to those in inset. Scale bar, 10 pm.
were seen within the zona incerta and formed branched arbors glial cell (Fig. 8~). The major enlargementof the labeledgrowth (Fig. 6~). Transient projections to such nonvisual structures cone grew apposed to glial cells on one aspect and contacted have been previously described in neonatal hamsters (Frost, other unlabeled neuritic elementson the other aspect. Sections 1984). through the concave expansion show that it was enveloping other neuritic profiles (Fig. 8~). This sort of section showsthat Cell-cell relationshipsof growth cones growth cones enfold other axons that run in a fasicle. The la- Several growth coneswere analyzed at the ultrastructural level, mellopodial expansions of the central core of the growth cone in a first attempt to characterize cell-cell relationships during could representthe amorphous profiles often observed in cross outgrowth along differen, parts of the pathway. All of the 10 sectionsof normally fixed optic nerve (Fig. 8b). These expan- growth cones analyzed in the optic nerve were either growing sionsusually lacked organellesand were positioned in between completely apposedto immature glial (neuroepithelial) cells or neuroepithelial cells and bundles of neurites. From simply ob- contacted them somewherealong their length. serving such unlabeledmaterial, it was formerly difficult to iden- One long but simple growth cone, located on the lateral por- tify these profiles as neuronal or glial (Williams et al., 1986; tion of the optic nerve close to the retina, was positioned ad- Carpenter and Guillery, personal communication). jacent to neuroepithelial cells that formed the border ofthe nerve In central portions of the nerve, growth cones contacted glial and closely followed their contours (Fig. 7a). An adjacent neu- cells at several points along their length but were not growing roepithelial cell made an intimate contact with the growth cone completely apposed to them (data not shown). In general, it by inserting a small protrusion of cytoplasm into an expanded appearedthat each labeledgrowth cone profile in the optic nerve portion of the growth cone (Fig. 7b). This insertion could be contacted only few other neighboring cells. followed for some distance in serial sectionsand later was seen In the chiasm,fibers regularly criss-crossand intertwine with to be completely engulfed by the growth cone (Fig. 7e). The tip one another and weave among radial glia. Growth coneswere of the same labeled growth cone also ran between a glial cell positioned among these crossings,and in thin sectionsit was and another unlabeled growth cone profile (Fig. 7, c, d, insert). apparent that each growth cone simultaneously touched many A small tongue of the HRP-filled growth cone insinuated into other profiles, either neurites, other growth cones, or glial pro- the unlabeled growth cone (Fig. 7c) and after few sectionswas cesses(Fig. 9). One growth conewith a highly convoluted surface enclosed in the cytoplasm of the unlabeled profile (Fig. 74. apposeda glial cell profile and several other neurites, some of Thus, the samegrowth cone was involved in glial-growth cone which were oriented perpendicular to the growth cone (Fig. 9a, insertions, as well as growth cone-growth cone insertions. arrows). Several profiles also contacted the base of a second Another sampled fiber (Fig. 8), tipped by a lamellopodial chiasm growth cone (Fig. 9b). At the light level, convoluted growth cone, at first ran in the central portion of the nerve and expansions were observed, while in the thin sectionsthrough then abruptly turned toward the lateral side. The tip of the this region, the labeledportions appearedin several pieces(Fig. growth cone was positioned between other neurites, close to a 9b), confirming the existence of the surface convolutions. One The Journal of Neuroscience, May 1987, 7(5) 1453
El4 E 15 E 16 El7
a.
b.
OPTIC TRACT Figure 5. Camera lucida drawings of retinal axons in the tract at various ages. Fibers in a are localized on the outer surface of the tract. The growth cones of these axons are shorter, more slender versions of optic nerve growth cones. Fibers in b are positioned in the inner part of the tract, adjacent to the lateral geniculate nucleus (lg, inset). Growth cone-like expansions (arrows) arise from the main shaft and project toward the target. Filopodia are prominent on these expansions and extend in all directions. Age of the embryo did not correlate with growth cone form in either position. Scale bar, 10 pm. of the filopodia on this growth cone insinuated between other filopodia in the optic nerve and tract, to shortenedspread forms neuritic profiles. Among the chiasm growth coneswith filopodia with filopodia in the optic chiasm,and filopodial expansionsat that were analyzed in the EM, no specialized contacts between the border of targets. Upon entering targets, including inappro- filopodia and other cells were noted. priate ones, retinal axon endings branch into arbors, but each branch is tipped by minute growing tips rather than the enlarged Discussion forms seenduring outgrowth. Shape changes of retinal axon growth cones are These data demonstrate that there are remarkable similarities position-specific in growth cone morphologies among a wide variety of inver- The major finding of this analysis,summarized in Figure 10, is tebratesand vertebrates, in regionswhere they follow a common that the growth conesof retinal ganglioncell axons have different pathway, in positionswhere they diverge, and in loci where they shapesaccording to their position alongthe visual pathway. The interact with target cells (Tosney and Landmesser,1985a; Caudy shapesrange from long contoured forms that generally lack and Bentley, 1986a). In the chick PNS, motor neuron growth 1454 Bovolenta and Mason - Morphology of Retinal Axon Growth Cones
a b
E 16 El7
Figure 6. Camera lucida drawings of retinal axon arbors within target regions. a, At E16, fibers within the optic tract branch into a nonvisual target, the zone incerta (ZI), which is thought to be a transient projection of immature retinal axons. b, At E17, labeled fibers arborize within the ventral lateral geniculate nucleus (LGN). Rather than ending with a single large growth cone, these axons have a finely divided branched terminal arbor, tipped with minute swellings (arrow). Note that the immature branching patterns and growing tips of retinal arbors are similar both within inappropriate and appropriate target regions. Scale bar, 10 pm. conesare larger and more elaborate in “decision regions,” such gated lamellopodial growth cones in mouse optic nerve (Jac- as the plexi or where musclenerves diverge from the main nerve kowski and Lieberman, 1979; Williams and Rakic, 1985). Short trunk (Tosney and Landmesser,1985a). In grasshopperneurons, filopodia were revealed on some chick retina and mouse optic growth cones are simple within connectives, but when growth nerve growth conesby the Golgi and HRP labeling techniques cones interact specifically with other identified neurons, their but were not detected on monkey optic nerve growth cones in morphology becomescomplex, with extremely long filopodia serial thin sections.Growth coneson amphibian sensoryaxons (Raper et al., 1983; Caudy and Bentley, 1986b). visualized with scanning electron microscopy display the flat- The forms observed in this study correspond to the few de- tened filopodial shapescommonly seenin vitro in certain cellular pictions of growth cones in vertebrate nervous systemsin situ environments such as myotomes (Roberts and Taylor, 1983). (e.g., Ramon y Cajal, 1960; Thanos and Bonhoeffer, 1983; In agreementwith the present study, growth cone form changes Nordlander, 1985; Tosney and Landmesser,1985a; Whitehead with the cellular surround and become more simple and blunt and Morest, 1985; Kuwada, 1986; Myers et al., 1986), regardless on skin basallamina. In regenerating frog optic nerve, growth of the technique used (Golgi impregnation, dye injection, and conesdisplay the range of forms seenin mouseoptic nerve with electron-microscopic methods). Retinal axon growth cones in HRP labeling (Scalia and Matsumoto, 1985). Position-specific chick retina and monkey optic nerve closely resemblethe elon- trends in morphology were not obvious, but regeneratingaxons The Journal of Neuroscience, May 1987, 7(5) 1455
Figure 7. Electron micrographs of an HRP-labeled growth cone in the optic nerve at El 3, demonstrating the invaginating contacts made by growth cones. Inset, Drawing of the light-microscopic image of this growth cone. a, Growth cone follows the contours of immature glial cells (G). b and e, Glial protrusion (G) inserts into the labeled growth cone (arrows). c, Toward the tip (c, d, inset), the growth cone inserts a small protrusion into the invaginated surface of a nearby growth cone (GC). d, As seen a few sections away, the expansion is completely enclosed by the membrane of the unlabeled growth cone (arrow, GC). Scale bar (inset), 10 pm. (a) x 7500; (b-e) x 20,000. 1456 Bovolenta and Mason * Morphology of Retinal Axon Growth Cones
Figure 8. Electron micrographs of an HRP-labeled optic nerve growth cone at El 3, showing that the convex and concave expansions seen at the light level (Fig. 2) wrap around other neurites. Inset, Drawing of the light-microscopic image of this growth cone. a, This expansion of the growth cone (a, inset) enfolds 2 other neurites and apposes glial cells (G). b, Cross section through an uninjected optic nerve at E13. Amorphous profiles (arrows) surround bundles of axons and appose glial cells (G). This sort of profile may represent the growth cone expansions that wrap around bundles of neurites shown in a in longitudinal section. c, The tip of the HRP-labeled growth cone is interposed among other neurites and contains large vesicles of the sort commonly seen in aldehyde-fixed growth cones. Scale bar (inset), 10 pm. (a, c) x 20,000; (b) x 12,500. The Journal of Neuroscience, May 1987, 7(5) 1457
Figure 9. Electronmicrographs of HRP-labeledgrowth cones in the chiasmat El 3. Insets showdrawings of the light-microscopicimages of these growthcones. a, Labeledportion of this highly convolutedgrowth cone contacts many profiles,some running perpendicular to others(arrows). Theseprofiles are most likely othergrowth cones, neurites, and glia. b, Onefilopodia of this growthcone is positionedbetween other neurites,but no specializedcontact was seen. Several nonlabeled profiles criss-cross and indent the labeledbody of the growthcone, causing it to appearin severalportions in this section, and possibly accounting for its complex shape. Scale bar (insets), 10 pm. (a, b) x 20,000.
may have different properties and responses(Grant and Tseng, cone movement and morphogenesisas axons extend through 1986). different environments are not understood.One popular concept Other studieson growth cones in situ support our observa- is that growth coneshave differential affinities to molecularcues tions that, just outside of targets, growth coneshave the elab- in the local environment and subsequentlychange form as they orate form of growth conesfound in decisionregions (Harris et register these cues. An example of such cues are gradients of al., 1985; Mason, 1985; Nordlander, 1985; Young and Rubel, adhesivemolecules (Nardi, 1983; Berlot and Goodman, 1984). 1986), but within targets, immature axon arbors have small The notion that growth cone form reflects the degreeof adhe- tapered tips or miniature growth cones (Mason, 1982, 1985; sivity wasderived from in vitro studies(Letourneau, 1979, 1982). Reh and Constantine-Paton, 1985; Sakaguchi and Murphey, On highly adhesive substrates,growth conesmove rapidly and 1985; Sachset al., 1986;Sretavan and Shatz, 1986).A surprising have a characteristic spread form with lamellopodia and filo- observation of the present study is that growth conesbecome podia, whereason lessadhesive substrates,growth conesmove smaller in both appropriate and inappropriate targets. slowly and have contracted forms. This model of hierarchical affinities hasbeen recently applied Factors responsible for changes in growth cone to growth cone behavior in developing grasshopperlimbs (Cau- shape and behavior dy and Bentley, 1986a). Filopodia and lamellopodia are most While the correlation between shapeand behavior is consistent numerousalong segmentalboundaries where growth conesre- even across phylogenetic lines, the factors controlling growth orient during navigation. The data at present do not distinguish 1458 Bovolenta and Mason - Morphology of Retinal Axon Growth Cones
neurite outgrowth with growth cone form in a single neuron population grown on a constant substrate. Growth cones that moved most rapidly had only lamellopodia, growth conesthat extended at moderate rates or pausedhad prominent filopodia, while the slowest moving growth cones were slender and ta- pered, with short filopodia. By extrapolation, the growth cones observed in the present study would extend fastest in the optic nerve, slow down in the chiasm, and move very slowly indeed in targets. The resemblanceof somegrowth conesin the chiasm to the lamellopodial growth cones in the optic nerve suggests that growth conesmove at different rates, or in a saltatory man- ner. The latter behavior has been observed in mouse cerebellar axon and PC 12 cell growth cones in vitro in real time (Aletta et al., 1986; Mason and Hatten, unpublished observations). In the present study, age-related differences in growth cone shapewere minimal, in agreementwith observations on motor neuron growth cones in chick peripheral nerve (Tosney and Landmesser,1985a). In other systems,the repertoire of growth cone form is related both to chronological age of the neuron (Argiro et al., 1984) and to order of outgrowth. In Xenopusspinal cord, growth cones both in younger animals and in the leading edgeof outgrowth are more complex than growth conesin older animals or in the follower population (Nordlander, 1985). The lead neurons in Daphnia eye during early development in the first part of the pathway have expanded specializationsat their tips, whereasthe followers do not (Lopresti et al., 1973). In cat Figure 10. Schematic representation of the changes in growth cone optic nerve, however, the earliestgrowth conesin the optic nerve morphology at different positions along the visual pathway. I, In the are simpler than later-growing ones(Williams et al., 1986), as optic nerve (ON), growth cones are long and indented and lack filopodia. 2, Within the optic chiasm (OCh), growth cones are shortened and have we observed in the present study. multiple filopodia. 3, In the optic tract (09, growth cones resemble optic nerve growth cones but are not as broad (1). 4, At the borders of Position and cellular relationshipsof growth target regions, such as the lateral geniculate nucleus (T2, LGN), filopodial conesin pathways growth cones arise from lengths of axon, and project toward the target. Retinal axon growth cones were differentially located in each 5, Within target regions, finely branched arbors have simple small tips. This arbor is depicted in the zona incerta (T,), which may be a transient portion of the visual pathway. In the optic nerve, growth cones target of retinal axons. are positioned at the edgesas well asinner portions of the nerve, agreeingwith the findings of Williams et al. (1986) in cat optic nerve. In contrast, growth conesgrow on the pial aspect of the whether affinity to segmentalborders is derived from adhesion chiasm and the optic tract, as they do in the ferret optic tract moleculeson cell membranesor in the extracellular matrix, or (Walsh and Guillery, 1985). In the latter system, new axons are from other more soluble factors that influence growth cone ex- added near the pial surface, displacing older axons toward the tension (Gunderson and Barrett, 1980; Haydon et al., 1984; deeper parts of the tract as development proceeds. Our obser- Connolly et al., 1985). Nonetheless,while the role of filopodia vations also showthat growth conesprojecting toward thalamic in neurite advance has been disputed (Marsh and Letourneau, targets arise from axons positioned away from the pia. This 1984; Bray and Chapman, 1985), these observations support suggeststhat there may be a lag time between outgrowth to, and the idea that filopodia mediate cell-cell interactions (Bastiani entry to, targets, as postulated by Schneider et al. (1985) for and Goodman, 1984). growing retinocollicular axons in the hamster. The transformation of retinal axons observed in the present The correlated light-microscopic and ultrastructural analysis study from predominantly nonfilopodial to more complex fil- of labeledgrowth coneshas shown that the curved lamellopodial opodial forms in regions where directionality changesis con- expansions on optic nerve growth cones envelop bundles of sistentwith this hypothesis. However, the precisemorphological other neurites. In this respect, fasiculatinggrowth conesin vitro transformations that occur in decision regionsor turning points closely resembleHRP-labeled retinal growth conesin the optic are not strictly the same in all animals. Motor neuron growth nerve. When extending on the culture substratum, growth cones conesin decisionregions in the chick PNS have more prominent have spreadfilopodial forms, but upon meeting other neurites, lamellopodia than at nondecision regions,and filopodia can be quickly elongateand embracethe other neurites with lamello- common on growth cones in both areas. The morphogenetic podia (Letoumeau, unpublished observations). changesmight reflect intrinsic properties of growth cones of The present observations demonstrate that some, but not all, different types of neuron (Haydon et al., 1985; Tosney and growing axons in the optic nerve contact glia, but the data do Landmesser,1985a; Nordlander, 1985). not necessarily implicate an obligatory relationship between There are at least 2 other factors that should be considered neurons and glia (Easter et al., 1984; Silver, 1984). The growth for any model of differential affinity and subsequentinduction cone-cell appositionsare, however, quite intimate, in the form of growth cone morphogenesis:rate of growth cone extension of protrusions of the growth cone into the invaginated mem- and age of the neuron. Argiro et al. (1984) correlated rate of brane of primitive glia, as well as into other growth cones. The The Journal of Neuroscience, May 1987, 7(5) 1459 earliest motor neuron growth cones in chick make similar in- Grant, P., and Y. Tseng (1986) Embryonic and regenerating Xenopus vaginating contacts into mesenchyme cells in the path of out- retinal fibers are intrinsically different. Dev. Biol. 114: 475-49 1. growth to the hindlimb (Tosney and Landmesser, 1985b). In Gundersen, R. W., and Barrett J. N. (1980) Characterization of the turning response of dorsal root neurites toward nerve growth factor. grasshopper embryos, filopodia of particular growth cones insert J. Cell Biol. 87: 546-554. into guidepost cells (Bastiani and Goodman, 1984), and coated Harris, W. A., C. E. Holt, T. A. Smith, and N. Gallenson (1985) pits and coated vesicles form near the filopodial insertion sites. Growth cones of developing retinal cells in vivo, on culture surfaces, In the grasshopper system, the invaginating contacts and as- and in collagen matrices. J. Neurosci. Res. 13: 101-122. sociated endocytosis are thought to provide information for Haydon, P. G., D. P. McCobb, and S. B. Kater (1984) Serotonin selectively inhibits growth cone motility and synaptogenesis ofspecific subsequent turning of the growth cone. Endocytotic events have identified neurons. Science 226: 561-564. also been observed in ependymal cells and in axons of the newt Haydon, P. G., C. S. Cohan, D. P. McCobb, H. R. Miller, and S. B. spinal cord during regeneration (Nordlander and Singer, 1978) Kater (1985) Neuron-specific growth cone properties as seen in iden- and at the base of filopodia in growth cones within developing tified neurons of Heliosoma. J. Neurosci. Res. 13: 135-147. chick optic tectum (Cheng and Reese, 1985). It was surprising Jackowski, A., and A. R. Lieberman (1979) Axonal growth cones of chick retinal aanalion cells. J. Anat. 129: 866-869. to find insertions between growth cones and other cells within Johnston, R. N, and N. K. Wessells (1980) Regulation of the elon- the main portion of the optic nerve, where, in contrast to the gating nerve fiber. Current Topics Dev. Biol. 16: 165-206. optic nerve head or the chiasm, turning or fiber resorting pre- Keshishian, H. (1980) The origin and morphogenesis of pioneer neu- sumably does not occur (Torrealba et al., 1982; Walsh et al., rons in the grasshopper metathoracic leg. Dev. Biol. 80: 388-397. 1985). Kuwada, J. 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