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.