Proc. Natl. Acad. Sci. USA Vol. 81, pp. 1849-1853, March 1984 Neurobiology

Neuronal growth cones: Specific interactions mediated by filopodial insertion and induction of coated vesicles (neuronal development/growth cones/grasshopper embryo/coated vesicles/cell interactions) MICHAEL J. BASTIANI AND COREY S. GOODMAN Department of Biological Sciences, Stanford University, Stanford, CA 94305 Communicated by Donald Kennedy, November 14, 1983

ABSTRACT We are interested in the factors that guide growth cones turn posteriorly and pioneer a second pathway individual neuronal growth cones during embryonic develop- (Fig. 1B). ment. Here we report on the discovery of a highly specific in- The MP1, dMP2, and vMP2 neurons arise from midline teraction between developing growth cones in the grasshopper precursors 1 (MP1) and 2 (MP2). There are two MP2s (one embryo as revealed by transmission electron microscope seri- on each side) and one MP1 (at the midline) in each segment. al-section reconstructions. Numerous filopodia from an identi- Each MP2 divides once to give rise to a ventral (vMP2) and fied growth cone (MP1) insert deep within another identified dorsal (dMP2) daughter. The single MP1 gives rise to a pair growth cone (pCC), inducing the formation of coated pits and of bilaterally symmetric daughters, each of which comes to vesicles. This interaction is highly specific, since filopodia lie dorsal to the two MP2 progeny, thus forming a trio of cells from other nearby growth cones that contact the surface of the on each side (Fig. 1B). All three cells send growth cones to two interacting neurons neither penetrate them nor induce the dorsal basement membrane. The growth cones reach the coated vesicles. These specific filopodial interactions may play basement membrane (4) at about the same time and in a vari- an important role in the subsequent development of these neu- ety of orientations relative to each other and to the body axis rons. (7, 9). Within several hours, and irrespective of their initial orientation along the basement membrane, the growth cones One ofthe most striking features about the nervous system is make divergent choices: the vMP2 growth cone turns anteri- the enormous diversity and remarkable specificity of neuro- orly, and the dMP2 and MP1 growth cones turn posteriorly nal morphology. The complex morphology of individual neu- (Fig. 1B). rons unfolds during the period of axonal outgrowth. A Our previous analysis using a light microscope suggested growth cone arises from the cell body and extends along ste- that the filopodia of the MP1 and dMP2 growth cones selec- reotyped routes that involve a series of cell-specific choices tively adhere to a small number of identified cells along their and turns, leaving behind an axon whose shape records the pathway as they make their posterior turn, including in par- growth cone's history. Thus one key to understanding the ticular the posterior corner cell (pCC) (7, 9) (Fig. 1C). A dis- development of neuronal diversity and specificity is likely to proportionate number of the MP1 filopodia adhere to the reside in the growth cone and its interactions with its embry- surface of the pCC cell body and its growth cone (which has onic environment. just been initiated), and the MP1 and pCC become strongly Growth cone motility involves three phases: extension, dye coupled (9). The aCC and pCC neurons are sibling prog- adhesion, and contraction (1, 2). Growth cones extend nu- eny from the first division of NB 1-1 from the next posterior merous long finger-like filopodia, -0.1 ,m in diameter and segment. up to 50 Am or more in length. Some of the filopodia contact These results on the MP1 growth cone in the central ner- other cell surfaces or extracellular basement membranes; vous system, and similar results on pioneer growth cones in to some of these surfaces they strongly adhere, and to the periphery, suggested to us the "landmark cell" hypothe- others their adhesion is much weaker. If adhesion is weak, sis for growth cone guidance (refs. 7-9; see also ref. 12). In the filopodium is retracted; if, however, its adhesion is the case of the MP1 growth cone, we proposed that the pCC strong, then tension in that direction is increased during the cell body and its growth cone serve as an important land- contractile cycle and the leading tip of the growth cone ad- mark cell for the MP1 by having a differentially labeled sur- vances toward the point of attachment (1, 2). face that the MP1 filopodia can distinguish from the other We are interested in the guidance of neuronal growth cell surfaces within filopodial grasp. cones during embryonic development and have focused our We have now examined the specific interactions of the fi- attention on the relatively simple and highly accessible ner- lopodia from the MP1 growth cone with the growth cone and vous system of the grasshopper embryo (3-12). What guides cell body of the pCC landmark cell by transmission electron the very first neuronal growth cones within the central ner- microscope serial section reconstructions. We report here vous system of the grasshopper embryo? Based on the pio- on a highly specific interaction between these cells (Figs. 2- neering work of Bate and Grunewald (4), we know the identi- 4). ties of these cells: the first growth cones extend from the MP1, dMP2, and vMP2 neurons (Fig. 1). The growth cones MATERIALS AND METHODS of these three individually identified neurons pioneer the Single clutches of synchronized eggs were collected from a very first longitudinal axonal pathways in each segment of laboratory colony of the grasshopper Schistocerca ameri- the grasshopper central nervous system (4, 7, 9). The growth cana and maintained at a constant temperature (330C) and cones are confronted with the same environment and yet humidity in an incubator. Under these conditions, the em- make divergent choices: the vMP2 growth cone turns anteri- bryo develops 5% per day and hatches on day 20 (13). The orly and pioneers one pathway, and the dMP2 and MP1 developmental stage of the embryos was determined by dis- secting the embryo from its egg case in saline and viewing The publication costs of this article were defrayed in part by page charge the MP1 cell body and growth cone in the living embryo with payment. This article must therefore be hereby marked "advertisement" a Zeiss compound microscope using a Leitz 50x water im- in accordance with 18 U.S.C. §1734 solely to indicate this fact. mersion lens and Zeiss Nomarski interference contrast op- 1849 Downloaded by guest on September 24, 2021 1850 Neurobiology: Bastiani and Goodman Proc. NatL Acad Sci. USA 81 (1984) A B C FIG. 1. Diagram of grasshop- per embryo showing the location and interaction of the MP1 and pCC neurons. (A) Schematic dia- gram of a 32% grasshopper em- ji bryo, showing metameric ar- rangement of cephalic, 3 thoracic, and 11 abdominal segments. (B) Cell bodies and growth cones of the identified neurons involved in pioneering the first longitudinal axonal pathways. The vMP2 growth cone turns anteriorly, pointing toward and contacting the medial (mLC) and lateral (ILC) landmark cells. The MP1 aCC and dMP2 growth cones turn pos- teriorly, pointing toward and con- tacting the aCC and pCC land- pCC mark cells. (C) Apparent selective filopodial adhesion from the MP1 growth cone to the pCC growth 20um cone and cell body (9).

tics. We reconstructed the MP1 growth cone and its filopo- growth cone and were found inserted anywhere from 0.1 to 7 dia from transmission electron microscope serial sections at gm into the cell. Coated pits and vesicles were present in the a developmental stage just after the MP1 growth cone turns membrane of the pCC at the filopodial tips of these inser- posteriorly and before it comes into direct contact with the tions in all but one case. pCC growth cone (Fig. 1C). Our proposed time course for this interaction (Fig. 3) is Embryos for electron microscopy were dissected directly based on two assumptions. First, filopodia originating far- into 2% paraformaldehyde/2.5% glutaraldehyde fixative ther back from the leading edge of the MP1 growth cone are made up in Millonig's buffer (pH 7.2) with 0.1% tannic generally older; and second, filopodia inserting deeper into acid/0.25% dimethyl sulfoxide. Embryos were left in the pri- the pCC growth cone are older than those with more shallow mary fixative for 1 hr at 0-5°C, briefly washed in primary insertions. These two assumptions are strongly correlated; fixative without the tannic acid, and then transferred directly the filopodia making deeper insertions in the pCC growth to a 2% osmium tetraoxide solution made up in Millonig's cone originate farther back on the MP1 growth cone. This is buffer for 2 hr at 0-50C. (This fixation schedule gave good just the opposite of what is seen for most other filopodia; the membrane preservation, but the high concentration of osmi- longer filopodia are in general those originating from the um caused fragmentation of the microfilaments.) Embryos leading tip of the growth cone. This implies a different time were then washed and stained en bloc with 2% uranyl ace- course for the retraction phase of those filopodia inserted tate (aq.) for 2 hr followed by 1% tannic acid for 1 hr. The into other cells vs. those exploring their surfaces. embryos were dehydrated in an ascending ethanol series and Our proposed time course for this interaction is shown in embedded in plastic. Thin sections were collected on form- Fig. 3. A filopodium from the MP1 growth cone initially con- var-coated slot grids and stained with uranyl acetate and lead tacts the surface of the growth cone of the pCC and forms a citrate. specialized contact with dense material apposed to both Approximately 60 ,um of serial thin sections (=600 sec- membranes (Fig. 3A). This quickly gives way to the forma- tions) were taken from the anterior edge of MP1 cell body to tion of a coated pit in the pCC just opposite the tip of the the posterior edge of the pCC cell body in the mesothoracic filopodium; junctions between the pCC and the filopodium (T2) segment. The cell bodies and axons of MP1, dMP2, now form along the sides of the filopodium (Fig. 2A) as it vMP2, aCC, and pCC were identified in thin sections by moves deeper into the growth cone (Fig. 3B). The space be- their characteristic positions and shapes. The reconstruction tween the filopodium and tube-like sleeve into which it in- of selected cells and their processes from one embryo was serts is 10-30 nm. Coated pit and vesicle formation contin- made from photographs taken of each section on a Hitachi ues at the tip and along the sides of the insertion, within 0.1 HU liE. The reconstruction of each cell began with its cell Am of the tip. At the same time, the filopodium moves deep- body; processes were followed and identified in sequential er into the growth cone (Fig. 3C) and punctate junctions sections until they terminated. form along the sides. Finally, coated pit formation ceases and the space between the sleeve and the filopodium dramat- ically increases, both along the sides and at the tip (Fig. 3D). RESULTS AND DISCUSSION After the junctions along the sides of the filopodium are lost, The MP1 growth cone had 28 filopodia extending from its the filopodium is probably retracted out of the sleeve, and leading 4 ,um; all 28 were reconstructed. Some of these filo- the sleeve collapses. podia contacted the dorsal basement membrane, the end feet It is unlikely that the filopodium is passively engulfed by of epidermal cells, the surfaces of unidentified cells, and the growth cone as it extends forward; the process of filopo- many unidentified filopodia. However, the majority of filo- dial insertion is likely to be an active process on the part of podia from MP1 (19 out of 28) contacted the pCC. Six filopo- both cells. First, filopodia have been observed that insert at dia came into intermittent and short (<1 gm) contact with the growth cone and penetrate all the way to the nucleus of the pCC cell body, growth cone, or filopodia. Six others con- the cell (Fig. 3D). Second, filopodia have been observed to tacted the pCC at its cell body and stayed in close contact for insert into cell bodies that have not yet extended growth several gm. The remaining 7 filopodia from MP1 were the cones and penetrate all the way to their nucleus (Fig. 2A). most interesting. They all first contacted the pCC at its The filopodial insertion and induction of coated pits is a Downloaded by guest on September 24, 2021 Neurobiology: Bastiani and Goodman Proc. NatL Acad. Sci USA 81 (1984) 1851

FIG. 2. Filopodia from neuro- nal growth cones insert deep into specific cell bodies and growth cones and induce the formation of coated pits and vesicles. (A) A fi- lopodium (f) is cut in longitudinal section to show its insertion into a neuronal cell body almost to the nucleus (N) of the cell. A coated pit is seen at the tip of the inser- tion (arrow) and punctate junc- tions are seen along the sides of the filopodium (arrowhead). (B) Five filopodial insertions are seen in this cross-section of a portion of the pCC growth cone. Four of the insertions are from the MP1 (small arrows) and one is from dMP2 (the large arrow). The filo- podium on the surface of the growth cone (curved arrow) is from MP1. This filopodium is in very close apposition to the growth cone membrane and a few micrometers distally inserts into the pCC growth cone and induces coated pits and vesicles. The larg- er profile (MP1) is the terminal fi- lum of the MP1 growth cone. (Bar = 0.5 Am.)

remarkably specific cell interaction. Coated pits and vesicles contact the pCC growth cone, yet do not insert and induce are induced only at the tips ofthe penetrating filopodia (Figs. coated pits. Moreover, the MP1 growth cone is closer to the 3 and 4). Filopodia from the MP1 growth cone that contacted aCC cell (the sibling of the pCC) than the pCC, and yet the the surface of the pCC cell body did not penetrate or induce interaction shows remarkable specificity as the MP1 filopo- coated pits; it thus appears that they must contact the sur- dia course around the aCC to contact and penetrate the pCC face of the growth cone to penetrate and induce coated pits. growth cone and cell body. Although we have focused on the The results suggest that after a cell has begun axonogenesis interaction of the MP1 filopodia with the pCC growth cone, filopodia only penetrate and induce the formation of coated the same phenomenon occurs between the pCC and the MP1 pits and vesicles if they contact the growth cone; before ax- growth cone and reciprocally between the MP1 and dMP2 onogenesis, coated pits can be formed by filopodia penetrat- growth cones. ing the cell body itself (Fig. 2A). The specificity of the filopodial interactions we describe Although coated pits form in other places on the pCC here has several interesting implications. First, only filopo- growth cone, there is no obvious relationship between their dia from particular identified growth cones (in this case MP1 position and membrane invaginations, surface filopodia, or and dMP2) are observed inserting into their other identified contact with other cells. It is quite striking that the pCC growth cones (in this case pCC), even though the filopodia of growth cone is riddled with 10 filopodial insertions; 7 of several other growth cones contact their surfaces. This sug- these are from MP1, 2 from dMP2, and 1 from an unidenti- gests that the filopodia from different identified neurons fied source. There are 3 growth cones closer to the pCC have different cell surfaces that growth cones can distin- growth cone than either the MP1 or dMP2; their filopodia guish. Downloaded by guest on September 24, 2021 1852 Neurobiology: Bastiani and Goodman Proc. NatL Acad Sci. USA 81 (1984) A

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dDt ;;gF' U card;.' .A *; FIG. 3. Proposed time course of filopodial insertion and induction of coated pits and vesicles. A filopodium from a specific identified growth cone (gc) contacts the growth cone ofanother specific cell (a). (A) The filopodium (f) initially makes a specialized contact with the growth cone. Soon a coated pit is seen opposite the tip ofthe filopodium and coated vesicles begin forming (arrow). (B) The filopodium inserts deeper into the growth cone (b), punctated junctions form along the sides of the filopodium, and a coated pit (arrow) region is very prominent at the tip of the insertion. Coated vesicles are seen in all stages of formation. (C) The filopodium penetrates further into the growth cone (c) and multiple sites of coated vesicle formation (arrows) are often seen. A coated vesicle has almost completely pinched off from the side of the insertion (open arrow). Coated vesicle formation occurs only within 0.2 Aum of the tip of the insertion. (D) The filopodium (d) has inserted to within a few tenths of a micron of the nucleus (N). Most of the punctate junctions along the sides of the filopodium are lost beginning near the tip. Concomitantly, the sleeve surrounding the filopodium enlarges and a region of coated membrane is no longer seen at the tip. At this time we speculate that the filopodium retracts out of the growth cone and the sleeve collapses. Few filopodia penetrate all the way to the nucleus; most complete the whole cycle after penetrating only a short distance (0.5-2.0 Am) into a growth cone. Notice that filopodia (asterisk) from the same growth cone (gc) that contacts the cell body do not insert or induce coated pits even though they are in close apposition to the cell body for several microns. The interaction is specific; filopodia from other nearby cells contact the growth cone, but do not insert. A, B, and C are longitudinal sections; D is a cross-section within 0.1 Am of the tip of the filopodium. (Bar = 0.2 ,m.) Second, coated pits are induced only at the tips of these induce the formation of coated pits and the subsequent re- inserting filopodia. This suggests that the tips of filopodia ceptor-mediated endocytosis as an inductive interaction. may be different from their shanks, a notion further support- Growth cones typically make a series of cell-specific path- ed by the different profiles of filopodial tips vs. shanks seen way choices on the way to their appropriate target. Growth in our serial transmission electron microscope analysis. The cones appear to be guided through each of these choice filopodial tips of free filopodia that are not contacting other points by the selective adhesion of their filopodia to specific cells appear quite different from the filopodial shanks. The surfaces (7-11). The adhesive properties of growth cones tips have more extensive fuzziness on the outer surface, and and axons may change during the course of these growth more extensive densities on the inner surface. Quite possibly cone navigations. The interactions involved in navigating specific informational molecules used to trigger this inser- through one choice point might induce the cell to change its tion and induce coated pits (and perhaps also involved in the expression of cell surface molecules involved in filopodial initial surface adhesion) may be concentrated at the tips of adhesion at that choice point or at a subsequent choice point, the filopodia relative to their shanks. Thus when the filopo- either by the disappearance of old receptors or the appear- dium comes into contact with the correct cell that has the ance of new receptors. The specific interactions described appropriate receptor, it may interact with the receptors to here might mediate such inductive changes. Downloaded by guest on September 24, 2021 Neurobiology: Bastiani and Goodman Proc. NatL. Acad. Sci. USA 81 (1984) 1853 tures or observe whether coated pit formation occurred at the tips. Slavkin and Bringas (18) describe long filopodia-like processes from mesenchymal cells protruding into epithelial cells of the developing tooth germ during the suggested in- ductive interaction between these two tissues. Thus, the spe- cific interaction of filopodial insertion and induction of coat- ed vesicles may be a general mechanism underlying many inductive events during development.

We thank Francis Thomas for valuable assistance with the elec- FIG. 4. Drawing showing important features of filopodial inser- tron microscopy. This work was supported by a National Institutes tion and the induction of coated vesicles. The induction of coated of Health postdoctoral fellowship to M.J.B. and by a National Sci- vesicle formation is localized to the membrane just opposite the tip ence Foundation grant and McKnight Foundation Scholars Award of the filopodium. The tip of the filopodium is morphologically dis- to C.S.G. tinct from the sides, with a denser matrix on both the extracellular and cytoplasmic side of the membrane. Punctate junctions are seen cone along the sides of the inser- between the filopodium and growth 1. Bray, D. (1982) in Cell Behavior, eds. Bellairs, R., Curtis, A., process filopodium grows tion. The insertion is probably active; the & Dunn, G. (Cambridge Univ. Press, London) pp. 299-318. forward at the same time thatjunctions form along the sides and the contact the 2. Letourneau, P. (1982) in Neuronal Development, ed. Spitzer, growth cone is advancing. Filopodia from other cells N. C. (Plenum, New York). cone not suggesting that insertion is not growth but do insert, Again Bate, C. M. (1976) J. Embryol. Exp. Morphol. 35, 107-123. a by the advancing growth 3. simply passive engulfment of filopodia 4. Bate, C. M. & Grunewald, E. B. (1981) J. Embryol. Exp. Mor- cone. phol. 61, 317-330. 5. Goodman, C. S. & Spitzer, N. C. (1979) Nature (London) 280, The observation of filopodial insertions is not limited to 208-214. the very first growth cones in the grasshopper central ner- 6. Goodman, C. S. & Bate, C. M. (1981) Trends Neurosci. 4, vous system; we have observed this phenomenon between 163-169. many different identified growth cones at different stages of 7. Goodman, C. S., Raper, J. A., Ho, R. K. & Chang, S. (1982) development. For example, we have found that filopodial Symp. Soc. Dev. Biol. 40, 275-316. insertion and the induction of coated vesicles occurs selec- 8. Ho, R. K. & Goodman, C. S. (1982) Nature (London) 296, P and the G neuron cone. We 404-406. tively between the cells growth 9. Taghert, P. H., Bastiani, M. J., Ho, R. K. & Goodman, C. S. have recently shown that the G growth cone selectively fas- (1982) Dev. Biol. 94, 391-399. ciculates on the P axons, and moreover, that the P axons are 10. Raper, J. A., Bastiani, M. J. & Goodman, C. S. (1983) J. necessary for the guidance of the G growth cone (ref. 14; Neurosci. 3, 20-30. unpublished observations). 11. Raper, J. A., Bastiani, M. J. & Goodman, C. S. (1983) 1. The presence of filopodial insertions is not limited to Neurosci. 3, 31-41. grasshopper growth cones. Vaughn and Sims (15) describe 12. Bentley, D. & Keshishian, H. (1982) Science 218, 1082-1088. filopodia-like processes from developing axonal collaterals 13. Bentley, D., Keshishian, H., Shankland, M. & Toroian-Ray- in mouse spinal cord that are associated with coated pits in mond, A. (1979) J. Embryol. Exp. Morphol. 61, 317-330. postsynaptic cells. They also suggest that "molecular infor- 14. Raper, J. A., Bastiani, M. J. & Goodman, C. S. (1983) Cold interactions Spring Harbor Symp. Quant. Biol. 48, 587-598. mation" may be passed between cells via these 15. Vaughn, J. E. & Sims, T. J. (1978) J. Neurocytol. 7, 337-363. and in their case induce changes in cell surface membranes 16. Rees, R. S., Bunge, M. B. & Bunge, R. P. (1976) J. Cell Biol. appropriate for specific synaptogenesis (see also ref. 16). 68, 240-263. Nordlander and Singer (17) have seen profiles in the growth 17. Nordlander, R. H. & Singer, M. (1978) J. Comp. Neurol. 180, cones of amphibian embryos that are similar to those seen in 349-373. Fig. 2B, but they did not identify the source of these struc- 18. Slavkin, H. C. & Bringas, P. (1976) Dev. Biol. 50, 428-442. Downloaded by guest on September 24, 2021