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Guidance of pioneer growth cones: Filopodial contacts and coupling revealed with an antibody to Lucifer Yellow
DEVELOPMENTAL
BIOLOGY
94,
391-399
(1982)
Guidance of Pioneer Growth Cones: Filopodial Contacts and Coupling Revealed with an Antibody to Lucifer Yellow PAUL H. TAGHERT, MICHAEL J. BASTIANI, ROBERT K. Ho, AND COREY S. GOODMAN Department
of Biological
Sciences, Stanford
Uniuersity,
Stanford,
California
94305
Received May 19, 1982; accepted in revised form July 12, 1982 We are interested in the factors that guide individual neuronal growth cones during embryonic development. We have developed an antibody to the fluorescent dye Lucifer Yellow. We use the antibody here to examine the specific filopodial contacts and dye coupling by the first growth cones in the grasshopper embryo that navigate in an axonless environment. We have studied the distribution and apparent selective adhesion of the filopodia from these pioneering growth cones in the central nervous system and periphery. Our results suggest that selective filopodial adhesion to specific “landmark” cells may play an important role in the guidance of pioneer growth cones. INTRODUCTION
The complex morphology of individual neurons is generated during the period of axonal outgrowth. An ameboid process, called the “growth cone,” arises from the cell body and extends; it leaves behind an axon whose shape records the growth cone’s history (Cajal, 1894; Harrison, 1910). During development, growth cones traverse long distances to their correct targets by an active process of precise pathfinding (e.g., LanceJones and Landmesser, 1981a,b; Raper et al., 1982a,b). What factors guide the advancing tip of individual growth cones? The key to answering this question may reside in the adhesive amd contractile properties of the numerous filopodia that extend from the growth cone (Bray, 1982). Many filopodia are randomly extended and then retracted in a contractile cycle (Nakai, 1960; Wessells et al., 1980). In cell culture, if a filopodium makes contact and its adhesion is weak, then it is retracted, if adhesion is strong, however, tension is then increased in that direction during the contractile cycle and the leading tip of the growth cone advances toward the point of attachment (Bray, 1982). Letourneau (1975) grew neurons on patterned substrata of different adhesivity in tissue culture. He observed that contact with an area of greater adhesiveness by a single filopodium was sufficient to guide the growth cone toward that surface. Can we demonstrate the role of specific filopodial adhesion in the precise pathfinding by neuronal growth cones in developing embryos? How long are the filopodia, which cells do they contact, which ones do they selectively adhere to, and to what extent does this selective adhesion account for the growth cone’s migratory behavior? We have begun to answer these questions by
the development and use of an antibody to Lucifer Yellow (anti-LY). The grasshopper embryo is an excellent preparation in which to study the question of filopodial adhesion because its nervous system is relatively simple, and its individual identified neurons are large and highly accesible from the time of axonal outgrowth (e.g., Goodman and Spitzer, 1979; Goodman and Bate, 1981). The cell bodies, axons, and growth cones of individual neurons are large and can be visualized with Nomarski interference contrast optics, penetrated with microelectrodes, and filled with a variety of markers, including the fluorescent dye Lucifer Yellow (Stewart, 1978). Many axons and growth cones in the grasshopper embryo can also be visualized using specific monoclonal antibodies (Chang et al., 1982; Kotrla and Goodman, in preparation). Since Harrison’s initial observations (1910), increasing evidence has supported the notion that axonal pathways are “pioneered” early in development when distances are short and the terrain relatively simple; “the fibers which develop later follow, in the main, the paths laid down by the pioneers” (Harrison, 1910). In the grasshopper embryo, the cells subserving this pioneering role are typically large and conspicuous in both the peripheral (Bate, 1976a,b; Keshishian, 1980) and central nervous systems (Bate and Grunewald, 1981). We have studied these peripheral (Ho and Goodman, 1982) and central pioneer neurons (Goodman et al., 1982) and in both cases our previous results implicated specific “landmark” cells in the guidance of these pioneering growth cones, pathfinders that establish axonal pathways by navigating in an axonless environment. In this paper we use the anti-LY antibody to suggest that the filopodia of pioneering growth cones in 391 OOlZ-1606/82/120391-09$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
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the CNS and periphery selectively adhere to (and become coupled to) these specific landmark cells in their environment as they make cell-specific turns toward these cells. In another series of papers (Raper et al., 1982a,b), the anti-LY antibody and other intracellular markers are used to implicate specific filopodial adhesion in the process of selective fasciculation by embryonic growth cones onto specific axonal pathways. MATERIALS
Anti-LY
Antibody
AND
METHODS
Production
Lucifer Yellow (LY) was covalently bound to bovine serum albumin (BSA) by two different methods: carbodiimide coupling (Moore et al., 1977) and coupling with 0.4% paraformaldehyde (4-hr incubation at room temperature). The conjugate solutions were emulsified 1:l with Freund’s complete or incomplete (for boosts) adjuvant. One and one-half milliliters was injected intradermally into multiple sites along the backs of New Zealand White rabbits at 2-week intervals for 6 weeks and bled 1 week after the final boost. The two different conjugates were each injected into two rabbits and the resultant sera from all four rabbits contained specific anti-LY antibodies. All immunocytochemical staining was blocked by preincubating a l/500 dilution of the primary antiserum with 1 pg/ml LY overnight at 4’C.
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dHzO. Constant negative current between 0.5 and 2 nA filled most cells in about 2 min. Following dye injections, embryos were fixed for 1 hr at room temperature in 2% paraformaldehyde in Millonig’s buffer solution (pH 7.3). Immunocytochemistry
Following fixation, preparations were rinsed in buffer and incubated overnight in primary antiserum at 4°C: anti-LY-a l/500 dilution in PBS containing 1% Triton X-100 and 1% BSA; I-5 monoclonal-a l/l dilution of the supernatant in PBS containing 2% BSA and 0.2% Saponin. Following the primary antibody incubation, preparations were rinsed in PBS for 1 hr then incubated overnight in a l/250 dilution of HRP-labeled goat antirabbit IgG (Miles, for the anti-LY preparations) or a l/400 dilution of HRP-labeled rabbit anti-mouse IgG (Miles, for the I-5 monoclonal). Secondary antibody dilutions were made in PBS and contained BSA and Triton or Saponin as above. Preparations were then rinsed for 1 hr in PBS, incubated for 1 hr in 0.5 mg/ml 3,3’-diaminobenzidine in PBS, then developed for 1/21 hr in the same solution containing 0.006% H202. Preparations were finally rinsed in buffer, cleared in a glycerin series, and mounted in glycerin. The tissues were viewed under Nomarski interference contrast optics, and photographed and drawn at 1250X magnification. RESULTS
of Central
AND
Pioneer
DISCUSSION
Dye Injection
Filopodia
Growth
Cones
We used grasshopper embryos from a laboratory colony of Schistocerca americana. Eggs were immersed in saline, cut open at their posterior end, and the embryos were gently squeezed out of their egg shells. After adhering yolk was removed, the ventral nerve cord was exposed by slitting the dorsal membrane from head to tail. The embryo was then transferred in a drop of saline onto a glass slide lightly coated with Sylgard 184 (Corning). For experiments in the CNS, the embryo was positioned dorsal side up in a small rectangular coffin cut out of the Sylgard, and held in place by wire pins projecting from underneath the Sylgard layer and over the embryo’s head and tail. For experiments in the limb buds, embryos were positioned ventral side up. To aid visibility, the saline used in the study was slight hypotonic and consisted of 150 mM NaCl, 3 mA4 KCl, 2 mM CaCl,, 1 mM MgS04, and 5 mA4 TES adjusted to pH 7.0. Neurons were visualized using a Zeiss compound microscope equipped with Nomarski interference contrast optics and a Leitz 50X water immersion lens. Cell bodies were impaled under direct visual control. Glass microelectrodes were drawn on a Sutter Instruments puller and filled with 10% LY (Stewart, 1978) dissolved in
The grasshopper central nervous system (CNS) is segmentally arranged and forms from the ectodermal epithelium that runs down the middle of the embryo from head to tail as a strip of contiguous plates of cells. There are two types of neuronal precursor ceils for the segmental ganglia: neuroblasts (NBS) (Bate, 1976b) and midline precursors (MPs) (Bate and Grunewald, 1981). Each segment has 61 NBS and 7 MPs. Whereas each NB divides repeatedly and generates a clone of progeny, each MP divides only once and gives rise to two progeny. The dorsal surface of the neural epithelium is covered by a conspicuous noncellular basement membrane which separates the neural epithelium from the mesoderm; this basement membrane is probably secreted by the ectoderm at an early stage (Bate and Grunewald, 1981). The first growth cones in the CNS arise from the progeny of midline precursors 1 (MPl) and 2 (MP2); there are two MP2’s (one on each side) and one MPl (at the midline) in each segment. Each MP2 divides once to give rise to a ventral (vMP2) and a dorsal (dMP2) daughter cell. The single MPl gives rise to a pair of bilaterally symmetric daughters, each of which comes to lie dorsal to the two MP2 progeny (forming a trio of cells on each side). All three cells (on each side)
TAGHERT ET AL.
Guidance
send growth cones up to the dorsal basement membrane. The growth cone of the vMP2 extends anteriorly, while the growth cones of the dMP2 (its sibling) and MPl extend posteriorly on the neural surface of the dorsal basement membrane (Fig. 1) (Bate and Grunewald, 1981). The cell bodies and often the axons and growth cones of the MPl and MP2 progeny are visible with Nomarksi interference contrast oyptics; their axons and growth cones can be clearly visualized using the I-5 monoclonal antibody (mab) which selectively stains a subset of neurons in the grasshopper embryo (Chang et al., 1982). Preparations using the I-5 mab show that the growth cones of the MPl, dMPf!, and vMP2 reach the basement membrane at about the same time and in a variety of orientations relative to each other and to the body axis (Fig. 2A) (Goodman et al., 1982). For example, of the two sibling cells (the vMP2 and dMP2), in some cases the vMP2 growth cone i:s the more anterior and in other cases the dMP2 growth cone is the more anterior; similarly, either growth cone can be the more lateral of the two. Within several hours, and irrespective of their initial orientation along thie membrane, their growth cones begin to make divergem choices; the vMP2 turns anteriorly and the dMP2 and MPl turn posteriorly (Fig. 2B). What guides the growth cones of the MPl, dMP2, and vMP2? Perhaps thle growth cones passively follow mechanical guidance cues: for example, anatomical studies in amphibian elmbryos have revealed channels between ependymal cells that are subsequently invaded by neuronal growth cones (e.g., Singer et al., 1979; Silver and Robb, 1979; Silver and Sidman, 1980). These observations have given rinse to the “blueprint” hypothesis whereby the germinal neuroepithelium is thought to contain the pattern for channels which in turn serve as mechanical guides for elongating axons. It is possible that clefts with similar functions might exist in the neuroepithelium of grasshopper embryos (Bate and Grunewald, 1981). However, the divergent and cell-specific choices made by the pioneering growth cones in the CNS of the grasshopper embryo suggest the independant existence of active guidance mechanisms. In this regard, it should be noted that the blueprint hypothesis also requires the presence of specific biochemical cues to direct the elongating axons during pathway selection. Studies of these pioneering growth cones in grasshopper embryos support the notion that there is “information” in their environment, and that they are differentially determined to respond to that information in cell-specific ways. Tlheir environment could provide polarity information, positional information, or both. This information could1 be in the form of a diffusible gradient, a substrate gradient (on either the basement
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membrane or cell surfaces), or specific positional information (on either the basement membrane or cell surfaces). In trying to define sources of guidance information in the growth cone’s environment, it is important to understand just how large that environment is. As will be described in detail below, each of these growth cones has many long, thin filpodia extending radially for more than 50 pm. Our results suggest that one source of guidance for these growth cones is likely to be positional information in the form of landmark cells. We define a landmark cell as an identified cell whose differentially labeled surface distinguishes it from the cells around it. In the posterior direction, likely candidates for landmark cells are the two conspicuous corner cells. The anterior (aCC) and posterior (pCC) corner cells are sibling progeny from the first cell division of NB l-l (Goodman et al., 1982). The aCC and pCC cells migrate anteriorly at about 5 pm per hour on the neural surface of the dorsal basement membrane; their total migration is only about 50 pm and from birth they are never more than 60 pm from the MPl and dMP2 cell bodies. As the corner cells migrate anteriorly, they and the growth cones of the dMP2 and MPl appear to point and grow directly toward one another (Fig. 2B). In time, the corner cells finish migrating and the growth cones of the dMP2 and MPl then pass between them and the Ql cell body [one of the two progeny from the first cell division of NB 74 (Raper et al., 1982a)] (Fig. 2C). Interestingly, the dMP2 and MPl growth cones first become selectively dye coupled to the corner cells and later to Ql (Goodman et al., 1982; Raper and Goodman, 1982). [Dye coupling refers to the spread of the fluorescent dye Lucifer Yellow (450 MW) from the interior of one cell to another.] Do the filopodia from the dMP2 and MPl growth cones in the CNS selectively adhere to the corner cells, and use them for guidance (Figs. 2A-C)? We could not answer these questions using Lucifer Yellow injections alone. Although the dye quickly spreads into the filopodia (when the living preparation is viewed in fluorescence) the fragile filopodia lose structural integrity within seconds due to the intense fluorescence. When the fixed preparation is viewed with fluorescence, the filopodia (0.1 pm in diameter) are often faint and fade rapidly; furthermore, halos from more intensely fluorescent growth cones, axons, or cell bodies often obscure the filopodia. Finally even if the filopodia are visible with fluorescence, it is difficult to distinguish which unstained cells they are touching. We wanted a technique that would routinely show the filopodia and their contacts in whole-mount preparations in the light microscope without the need for serial reconstruction from electron micrographs. Although we can fill these embryonic cells with horseradish peroxidase (HRP) for
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TAGHERT ET AL.
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electron microscopy (Raper et al., 1982b; Bastiani, Raper, and Goodman, in preparation) in only a small percentage of preparations can we see the filopodia at the light level. Thus we made rabblit serum antibodies to Lucifer Yellow. After Lucifer Yellow is injected into a cell, the embryo is fixed, incubated with the anti-LY antibody, then incubated with a HRP-labeled second antibody. The embryo is processed for the HRP reaction, mounted in glycerine, and viewed at up to 1250X with Nomarski optics (Fig. 1). Given the ability of Lucifer Yellow (450 MW) to travel long distances within a cell and to cross gap junctions, there is every rea.son to suspect that all filopodia are filled by the dye. The antibody technique appears to be at least as sensitive (perhaps more) in detecting the number and extent of the dye-filled filopodia as is direct observation of the fluorescent dye in living preparations. Photographs of whole-mount preparations using the Lucifer Yellow antibody are shown in Fig. 1; parts A-C show fills of the MPl cell in sequentially older embryos. Figures 2 (D, E), 3, and 4 (D, E) are camera lucida drawings of representative preparations showing the filopodial ‘contacts and coupling from the MPl cell in the CNS and the 1B cells in the periphery. As the MPl makes its cell-specific turn posteriorly, one or several of its filopodia contact the corner cells (Fig. 3A); the longest lilopodia from any growth cone often reach for more than 50 pm and the corner cells are about 50 pm away. Although there are many other cells in its environment and near the neural surface of the basement membrane (and thus within filopodial grasp), a disproportionate number of the MPl’s filopodia soon contact the corner cells (Fig. 3B). Furthermore, those that do contact the corner cells tend to continue running along them (Figs. 2D, E). We interpret this to imply selective adhesion, although we have not directly measured adhesive forces. It may be that just one or a few filopodia contacting the corner cells are sufficient to mediate the cell-specific turn (compare
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D
MPl
FIG. 2. Apparent selective filopodial adhesion from the MPl growth cone to the anterior and posterior corner cells (aCC, pCC) in the developing CNS of the grasshopper embryo. (A-C) Camera lucida drawings of the MPl, dMP2, vMP2, aCC, pCC, and Ql cell bodies based on whole-mount preparations using the I-5 monoclonal antibody. The MPl growth cone turns posteriorly and points at the aCC and pCC cell bodies which are migrating anteriorly. In time, and MPl (and dMP2) growth cones grow between these two cell bodies and the Ql cell. (D,E) Two examples of the MPl growth cone and its filopodia at the same stage of development: LY was injected into the cell body and the embryo was processed with the anti-LY antibody. Note the extensive filopodial contact with the aCC and pCC cell bodies. Selective dye coupling was observed to both these cells; the only sites of cellular contact that could mediate this coupling are the filopodia. Note several long filopodia in (E) that are not contacting the aCC or pCC cell bodies.
Figs. 3A and B). Unfortunately, it has been very difficult to obtain preparations before the stage shown in Fig. 3A in which we are confident that we see the full extent of the filopodia. When the growth cone is first initiated, the dye-filled filopodia often appear as broken strings
FIG. 1. Filopodia and dye coupling from midline precursor 1 (MPl) at three stages of development, as visualized with an antibody to Lucifer Yellow, in the central nervous system of the grasshopper embryo. Only a very thin plane is in focus with Nomarski optics; thus only some of the filopodia are visible in the photographs. (A) The filopodia from the MPl growth cone extensively cover the posterior corner cell (pCC); note dye coupling to pCC mesdiated via the filopodia. The anteriorly extending axon is the dye-coupled vMP2, and the cell bodies out of focus around the MPl are those of the dye-coupled dMP2 and vMP2. (B) The MPl growth cone has just grown between the pCC and the Ql; note the MPl’s long leading filopodia that extend posteriorly beyond the pCC and Ql cells, and the relative amounts of dye coupling to the pCC and Ql cell bodies. (C) Dye coupling is observed from the MPl to the pCC, Ql, Q2 (Ql’s sibling, the cell body just lateral to Ql), and to two other identified cell bodies further posterior and out of the plane of focus (open arrow). The dye coupled vMP2 axon has extended anteriorly and is dye coupled to a conspicuous identified cell via that cell’s axon. The MPl growth cone has entered the next posterior segment (arrow heads show segmental boundaries); the dMP2 growth cone has extended about the same distance. The dye coupled vMP2 axon has extended anteriorly into the next anterior segment. The growth cones of the dye-filled, posteriorly directed MPl and dMP2, and the unfilled, anteriorly directed vMP2 of the next posterior segment, pass each other near the segmental boundary and continue extending within a few microns of each other. Yet, while their filopodia overlap extensively, they do not become dye coupled to one another. Similarly, the dye-filled, anteriorly directed vMP2 and the unfilled, posteriorly directed MPl and dMP2 of the next anterior segment do not become dye coupled to one another, although their filopodia overlap extensively.
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A M Pl
aCC
PCC
\,,“,,,;
,
“\ ‘/)/
‘1,
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20 urn
FIG. 3. Filopodia from the MPl growth cone at successively later stages of development. (A) Radiation of MPl filopodia just after the growth cone turns posteriorly. A few filopodia contact the aCC cell. (B) With time, the aCC and pCC (corner cells) migrate further anteriorly (and thus closer to the MPl). The MPl growth cone broadens and has a greater proportion of its filopodia in contact with the corner cells (the MPl is dye coupled to the corner cells at this stage). Note also a smaller growth cone extension in the anterior direction. This anterior extension is along the vMP2 axon (see Fig. 2B) and is retracted in time. (C) At a later stage of development, the MPl growth cone has extended further posteriorly and has extensive filopodial contact (and dye coupling) with the corner cells and an additional identified cell, Ql. Furthermore, many long filopodia extend posteriorly from its tip and contact one of a pair of identified cells just on the other side of the segment boundary.
of beads rather than smooth threads. These observations may indicate that at the earliest stages of growth cone initiation (i.e., before the growth cone makes its turn) the filopodia are more sensitive to perturbations by the intracellular penetration and dye injection than at later stages. However, at these early times, we do see a large expanse of dye-filled beads in many directions, suggesting an initially broad radiation of filopodia. When we examined 11 preparations at the stage of development just after the MPl growth cone turned posteriorly, we consistently found extensive filopodial contacts from the MPl growth cone to the corner cells, strongly suggesting selective filopodial adhesion with the surface of these cells (two examples are shown in Figs. 2D, E). In three of these preparations, we counted about 20 cell bodies within a one cell diameter radius of the posteriorly directed growth cone. This is likely to be a very conservative estimate of the number of cells within filopodial grasp: a cell body diameter is about 15 pm and the filopodia can reach for over 50 pm. In 6 of the 11 preparations, we counted the total number of filopodia that extended from the posteriorly directed growth cone and the number contacting the corner cells (Table 1). On average, 45% of the filopodia contacted the surface of the corner cells, although these cells constitute less than 10% of the available cell surfaces. Thus, the MPl growth cone at this stage demonstrates a
strong tendency for filopodial contact with the corner cells, and no tendency for selective contacts with any of the other cells in its immediate environment. Within a short period, however, some of the MPl filopodia begin to selectively adhere to the Ql cell body (Fig. 3C), and shortly thereafter to two other, identified cells further posteriorly along its route and just on the other side of the segment boundary (Figs lB, lC, 3C). Filopodia
of Peripheral
Pioneer Growth Cones
The first growth cones in the limb bud arise from the 1B cells (Bate, 1976a,b; Keshishian, 1980; Ho and Goodman, 1982). The 1B growth cones extend proximally toward the CNS along the basement membrane on the inside of the ectodermal epithelium of the limb bud. However, they make a characteristic turn about halfway down the limb bud (Figs. 3A-C) (Bate, 1976a,b; Keshishian, 1980). This turn in direction occurs about the time they reach the 3B cell (although the growth cones need not reach this cell); they always, however, turn toward the 1A cells (Ho and Goodman, 1982). The pair of 1A cells migrate out of the ectodermal epithelium and come to lie on its inside surface. Depending upon the segment, the 1A cells either have or have not sent growth cones into the CNS by the time the 1B growth cones arrive at their cell bodies. In the prothoracic (Tl)
TAGHERT ET AL.
Guidance
segment, they have extended growth cones, whereas in the metathoracic (T3) segment, they have not (Ho and Goodman, 1982). Upon reaching the 1A cell bodies, the 1B growth cones then continue extending toward the CNS. Our results suggest that one possible source of guidance for the 1B growth cones is likely to be positional information in the form of landmark cells; the likely candidates are the 1A cells. Having seen that the filopodia of the MPl growth cone selectively adhere to the corner cells in the CNS, we wanted to know if the filopodia from the 1B growth cones in the periphery iselectively adhere to the 1A cells, and use them for guidance (Figs. 4A-C). The 1B growth cones are easily within filopodial reach of the 1A cells when they make their cell-specific turn toward them (Fig. 4): the 1A cells are less than 50 pm away. Although they are growing on the inside of an epithelial tube, a disproportionate number of the 1B filopodia contact the 1A cells rather than the other ectodermal cells, as can be seen in Figs. 4D, E. Even before the 1B growth cones make their turn toward the 1A cells, they already have
FIG. 4. Apparent selective filopodial adhesion from the 1B growth cones to the 1A cell bodies in the metathoracic limb bud of the grasshopper embryo. (A-C) ICamera lucida drawings of the lB, lA, 3B, and 2B cells based on whole-mount preparations using the I-5 monoclonal antibody. The 1B growth cones make a characteristic turn toward the 1A cell bodies on tlneir way to the CNS. (D,E) Two examples of the 1B growth cones and their filopodia at the same stage of development: LY was injected into the cell body of one of the two 1B cells and the embryo was processed with the anti-LY antibody. Note the extensive filopodial contact with the 1A cell bodies and the 3B cell body. In both examples, one of the growth cones has not yet made the turn, and yet filopodia already span the distance and contact the 1A cell bodies (arrowheads). Selective dye coupling was observed to all of the cell bodies drawn (in D, 1A cells, and 3B cell; in E, 1A cells, 3B cell, and 2B cell).
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TABLE 1 FILOPODIAL CONTACTSBYMP~GROWTHCONE
Preparation 1 2 3 4 5 6
Number of filopodia 32 31 39 34 39 38
Number of filopodia contacting corner cells (%) 14 (44) 14 (45)
19 (49) 11 (32) 20 (51)
18 (47)
long filopodia in contact with the surface of the 1A cells (e.g., arrows in Figs. 4D, E). Selective Dye Coupling from Pioneer Growth Cones The Lucifer Yellow antibody also reveals patterns of selective dye coupling (Raper and Goodman, 1982; Goodman et al., 1982) from the MPl growth cone in the CNS to the corner cells (with the strongest coupling typically to the pCC) (Figs. 1, 2D, E), then Ql (Figs. lB, 3C), and then to two other cell bodies more posterior (Figs. lC, 3D). Dye coupling is also seen from the 1B growth cones in the periphery to the 3B cell and the 1A cells (Figs. 4D, E). The antibody allows us to visualize the relative amount of dye coupling to different cells (e.g., Figure 1B). The amount of reaction product following the antibody technique accurately reflects the graded extent of Lucifer Yellow dye coupling that is often seen with fluorescence. The antibody technique appears to be at least as sensitive in detecting the dye as is direct observation following blue irradiation. It is evident that in several cases this dye coupling must be mediated via the filopodia since they are the only sites of cellular contact (e.g., Figs. lA, 2D, E, and 4D). However, filopodia and growth cones do not become dye coupled to every cell they contact. One striking example is observed when the posteriorly directed growth cones of the MPl and dMP2 meet the anteriorly extending growth cone of the vMP2 of the next posterior segment at the segment border. Despite their close proximity (~5 pm) and extensive filopodial overlap, the vMP2 does not become dye coupled with the MPl or dMP2. Landmark
Cell Hypothesis
Our results suggest a landmark cell hypothesis whereby (i) the filopodia of pioneering growth cones selectively adhere to the surfaces of specifically labeled landmark cells, and (ii) this specific filopodial adhesion serves to guide the pioneering growth cones toward the landmark cells as they make cell-specific turns or pathway alterations. In the CNS, the filopodia of the MPl growth cone
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contact and appear to adhere to the surfaces of the corner cells just as they make their cell-specific turn toward these cells. The landmark cell hypothesis predicts that the filopodia from the dMP2 growth cone also selectively adhere to the surface of the corner cells in the posterior direction, whereas the filopodia from the vMP2 growth cone selectively adhere to the surface of a landmark cell(s) in the anterior direction. It is possible that contact by only one or a few filopodia by the initial MPl radiation may be sufficient to direct the course of its growth cone toward the corner cells (e.g., Figs. 2E, 3A). While there may in addition be some form of anterior/posterior polarity information in their environment, nevertheless the behavior of the MPl filopodia suggests that selective filopodial adhesion to specific landmark cells plays an important role in the guidance of pioneering growth cones. Likewise, in the periphery, the filopodia of the 1B growth cones contact and appear to adhere to the surfaces of the 1A cells just as they make their cell-specific turn toward these cells. It appears as if the filopodia that contact the landmark cells are on average longer than the filopodia that contact other cells (e.g., Figs. 2D, E). Why is this? There are two possible explanations. First, our Lucifer Yellow dye injections present a single static picture of a very dynamic process. According to Bray’s model (1982), filopodia are actively extended and retracted over a several-minute cycle. In tissue culture, filopodia that attach to axons can be seen to pull on the axon at intervals of about 2 min (Nakai, 1960; Wessells et al., 1980). At any one moment, only a small percentage of the filopodia (on nonadhesive surfaces) will be at their maximum length. We occasionally see very long filopodia that are not touching specific landmark cells (e.g., Figs. 2E, 3A). However, those filopodia that contact a particularly adhesive surface will retain their full length as their contractile cycle produces tension rather than retraction. Thus, at any one moment, one might expect to find the filopodia on the landmark cells to be on average longer than those filopodia touching other cells. The second alternative, however, is that the apparent difference in the average length of the filopodia may reflect an artifact produced by penetrating the cell body with a microelectrode and filling the cell with dye for up to several minutes. A change in membrane potential or ion fluxes might cause a partial retraction of some filopodia, leaving those filopodia that are touching adhesive surfaces at their full length. In the CNS, the filopodia from the MPl and dMP2 growth cones first appear to selectively adhere to the corner cells, then Ql (Fig. 3C), and then to two cell bodies about 50 pm further posterior (Figs. lB, C, 3D). These next landmark cells are just on the other side of the segment border. Furthermore, the first sign of seg-
VOLUME 94, 1982
mentation in the limb bud appears proximal to the 1B cells and is located just distal to the IA cells (this border is the division between the femur and the proximal leg). Thus, in both the CNS and the periphery, specific landmark cells are located just across the segment boundaries in the direction of growth cone extension. These observations are particularly interesting in light of theories concerning iterated segmental gradients in insects and the inhibition of axon growth across segment boundaries (e.g., Lawrence, 1981). It may be that the pioneering growth cones require particularly adhesive cell surfaces on the other side to get across segment boundaries, and that later cells can simply follow the pioneer axons. One final question is why the MPl growth cone continues to extend from landmark cell to landmark cell in a stepping stone fashion (Bate, 1976a) rather than simply staying on the first adhesive cell surface it finds. One need not suppose that each successive landmark has a different and more adhesive label. Rather, the cells may have a proclivity to grow and simply follow those surfaces that are most adhesive. Alternatively, the next landmark may have the same label but may, with time, be more adhesive than the first, simply because the extensive filopodial contacts with the first landmark changed that cell’s surface (e.g., by binding or altering the adhesive component). The Lucifer Yellow antibody can thus be a powerful histological tool, particularly when used to resolve the intricate details of cell shape, contacts, and coupling during development. It has already helped us to answer one important question heretofore difficult to approach in a developing embryo. Our results suggest that selective filopodial adhesion to specific landmark cells may play an important role in the guidance of pioneer growth cones. We now plan to test this landmark cell hypothesis by examining how these pioneer growth cones navigate in the absence of their normal landmarks. We thank Susannah Chang for the I-5 monoclonal antibody and John Kuwada for careful reading of the manuscript. Support was provided by a Muscular Dystrophy fellowship to P.H.T., N.I.H. fellowship to M.J.B., and grants from the NSF and McKnight Foundation to C.S.G. who is a Sloan Fellow. REFERENCES
BATE, C. M. (1976a). Pioneer neurones in an insect embryo. Nature (London) 260, 54-56. BATE, C. M. (1976b). Embryogenesis of an insect nervous system. I. A map of the thoracic and abdominal neuroblasts in Locusta migratoria. J. Embryol. Exp. Morphol. 35, 107-123. BATE, C. M., and GRUNEWALD, E. B. (1981). Embryogenesis of an insect nervous system. II. A second class of precursor cells and the origin of the intersegmental connectives. J. Embryol. Erp. Morphol.
61,317-330. BRAY, D. (1982). Filopodial
contraction
and growth cone guidance. In
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“Cell Behavior” (R. Bellairs, A. Curtis, and G. Dunn, eds.), Cambridge Univ. Press, London/New York. CAJAL, S. R. Y. (1894). “Die retina der Wirbelthiere.” Bergmann Verlag. CHANG, S., Ho, R. K., and GOODMAN, C. S. (1982). Monoclonal antibodies that reveal specific patterns of neurons and mesodermal cells early in grasshopper elmbryogenesis. Dev. Biol. in press. GOODMAN, C. S., and SPITZEI~:, N. C. (1979). Embryonic development of identified neurons: differentiation from neuroblast to neuron. Nature (London) 280, 208--214. GOODMAN, C. S., and BATE, C. M. (1981). Neuronal development in the grasshopper. Trends Neurosci. 4, 163-169. GOODMAN, C. S., RAPER, J. A., Ho, R. K., and CHANG, S. (1982). Pathfinding by neuronal growth cones during grasshopper embryogenesis. In “40th Symposium, Society for Developmental Biology” (S. Subtelny and P. Green, eds.), pp. 275-316. Alan R. Liss, New York. HARRISON, R. G. (1910). The outgrowth of the nerve fiber as a mode of protoplasmic growth. J. Bxp. 2001. 9, 787-845. Ho, R. K., and GOODMAN, C. S. (1982). Perhipheral pathways are pioneered by an array of central and peripheral neurons. Nature (London) 297, 404-406. KESHISHIAN, H. (1980). The origins and morphogenesis of pioneer neurons in the metathoracic leg of the grasshopper. Dev. Biol. 80, 388-397. LANCE-JONES, C., and LANDMESSER, L. (1981a). Pathway selection by chick lumbrosacral motoneurones during normal development. Proc. Roy. Sot. Land. B 214,1-18. LANCE-JONES, C., and LANDMESSER, L. (1981b). Pathway selection by embryonic chick motoneurones in an experimentally altered environment. Proc. Roy. Sot. Lond. B 214. 19-52. LAWRENCE, P. A. (1981). The cellular basis of segmentation in insects.
Cell 26, 3-10. LETOURNEAU, P. C. (1975). Cell-to-cell substratum adhesion and guidance of axonal elongation. Deu. Biol. 66, 183-196. MOORE, G., LUTTERODT, A., BURFORD, G., and LEDERIS, K. (1977)
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A highly specific antibody 101, 1421-1435.
for arginine
vasopressin.
Endocrinology
NAKAI, J. (1960). Studies on the mechanism determining the course of nerve fibers in tissue culture. II. The mechanism of fasciculation. Z. Zellforsch. 51, 427-449. RAPER, J. A., and GOODMAN, C. S. (1982). Transient dye coupling between developing neurons reveals patterns of intracellular communication during embryogenesis.; 6th Symp. “Ocular Visual Development,” in press. RAPER, J. A., BASTIANI, M. J., and GOODMAN, C. S. (1982a). Pathfinding by neuronal growth cones in grasshopper embryos. I. Divergent choices made by the growth cones of sibling neurons. J. Neurosci., in press. RAPER, J. A., BASTIANI, M. J., and GOODMAN, C. S. (1982b). Pathfinding by neuronal growth cones in grasshopper embryos. II. Selective fasciculation onto specific axonal pathways. J. Neurosci., in press. SILVER, J., and ROBB, R. M. (1979). Studies on the development of the eye cup and optic nerve in normal mice and in mutants with congenital optic nerve aplasia. Deu. Biol. 68, 175-190. SILVER, J., and SIDMAN, R. L. (1980). A mechanism for the guidance and topographic patterning of retinal ganglion cells axons. J. Comp. Neural. 189, 101-111. SINGER, M., NORDLANDER, R. H., and EGAR, M. (1979). Axonal guidance during embryogenesis and regeneration in the spinal cord of the newt: The blueprint hypothesis of neuronal pathway patterning. J. Comp. Neurol. 185, l-22. STEWART, W. W. (1978). Functional connection between cells as revealed by dye coupling with a highly fluorescent naphthalamide tracer. Cell 14, 741-759. WESSELLS, N. K., LETOURNEAU, P. C., NUTTALL, R. P., LUDUENAANDERSON, M., and GUIDUSCHEK, J. M. (1980). Responses to cell contacts between growth cones neurites and ganglionic nonneuronal cells. J. Neurocytol. 9, 647-664.
BIOLOGY
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Guidance of Pioneer Growth Cones: Filopodial Contacts and Coupling Revealed with an Antibody to Lucifer Yellow PAUL H. TAGHERT, MICHAEL J. BASTIANI, ROBERT K. Ho, AND COREY S. GOODMAN Department
of Biological
Sciences, Stanford
Uniuersity,
Stanford,
California
94305
Received May 19, 1982; accepted in revised form July 12, 1982 We are interested in the factors that guide individual neuronal growth cones during embryonic development. We have developed an antibody to the fluorescent dye Lucifer Yellow. We use the antibody here to examine the specific filopodial contacts and dye coupling by the first growth cones in the grasshopper embryo that navigate in an axonless environment. We have studied the distribution and apparent selective adhesion of the filopodia from these pioneering growth cones in the central nervous system and periphery. Our results suggest that selective filopodial adhesion to specific “landmark” cells may play an important role in the guidance of pioneer growth cones. INTRODUCTION
The complex morphology of individual neurons is generated during the period of axonal outgrowth. An ameboid process, called the “growth cone,” arises from the cell body and extends; it leaves behind an axon whose shape records the growth cone’s history (Cajal, 1894; Harrison, 1910). During development, growth cones traverse long distances to their correct targets by an active process of precise pathfinding (e.g., LanceJones and Landmesser, 1981a,b; Raper et al., 1982a,b). What factors guide the advancing tip of individual growth cones? The key to answering this question may reside in the adhesive amd contractile properties of the numerous filopodia that extend from the growth cone (Bray, 1982). Many filopodia are randomly extended and then retracted in a contractile cycle (Nakai, 1960; Wessells et al., 1980). In cell culture, if a filopodium makes contact and its adhesion is weak, then it is retracted, if adhesion is strong, however, tension is then increased in that direction during the contractile cycle and the leading tip of the growth cone advances toward the point of attachment (Bray, 1982). Letourneau (1975) grew neurons on patterned substrata of different adhesivity in tissue culture. He observed that contact with an area of greater adhesiveness by a single filopodium was sufficient to guide the growth cone toward that surface. Can we demonstrate the role of specific filopodial adhesion in the precise pathfinding by neuronal growth cones in developing embryos? How long are the filopodia, which cells do they contact, which ones do they selectively adhere to, and to what extent does this selective adhesion account for the growth cone’s migratory behavior? We have begun to answer these questions by
the development and use of an antibody to Lucifer Yellow (anti-LY). The grasshopper embryo is an excellent preparation in which to study the question of filopodial adhesion because its nervous system is relatively simple, and its individual identified neurons are large and highly accesible from the time of axonal outgrowth (e.g., Goodman and Spitzer, 1979; Goodman and Bate, 1981). The cell bodies, axons, and growth cones of individual neurons are large and can be visualized with Nomarski interference contrast optics, penetrated with microelectrodes, and filled with a variety of markers, including the fluorescent dye Lucifer Yellow (Stewart, 1978). Many axons and growth cones in the grasshopper embryo can also be visualized using specific monoclonal antibodies (Chang et al., 1982; Kotrla and Goodman, in preparation). Since Harrison’s initial observations (1910), increasing evidence has supported the notion that axonal pathways are “pioneered” early in development when distances are short and the terrain relatively simple; “the fibers which develop later follow, in the main, the paths laid down by the pioneers” (Harrison, 1910). In the grasshopper embryo, the cells subserving this pioneering role are typically large and conspicuous in both the peripheral (Bate, 1976a,b; Keshishian, 1980) and central nervous systems (Bate and Grunewald, 1981). We have studied these peripheral (Ho and Goodman, 1982) and central pioneer neurons (Goodman et al., 1982) and in both cases our previous results implicated specific “landmark” cells in the guidance of these pioneering growth cones, pathfinders that establish axonal pathways by navigating in an axonless environment. In this paper we use the anti-LY antibody to suggest that the filopodia of pioneering growth cones in 391 OOlZ-1606/82/120391-09$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
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the CNS and periphery selectively adhere to (and become coupled to) these specific landmark cells in their environment as they make cell-specific turns toward these cells. In another series of papers (Raper et al., 1982a,b), the anti-LY antibody and other intracellular markers are used to implicate specific filopodial adhesion in the process of selective fasciculation by embryonic growth cones onto specific axonal pathways. MATERIALS
Anti-LY
Antibody
AND
METHODS
Production
Lucifer Yellow (LY) was covalently bound to bovine serum albumin (BSA) by two different methods: carbodiimide coupling (Moore et al., 1977) and coupling with 0.4% paraformaldehyde (4-hr incubation at room temperature). The conjugate solutions were emulsified 1:l with Freund’s complete or incomplete (for boosts) adjuvant. One and one-half milliliters was injected intradermally into multiple sites along the backs of New Zealand White rabbits at 2-week intervals for 6 weeks and bled 1 week after the final boost. The two different conjugates were each injected into two rabbits and the resultant sera from all four rabbits contained specific anti-LY antibodies. All immunocytochemical staining was blocked by preincubating a l/500 dilution of the primary antiserum with 1 pg/ml LY overnight at 4’C.
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dHzO. Constant negative current between 0.5 and 2 nA filled most cells in about 2 min. Following dye injections, embryos were fixed for 1 hr at room temperature in 2% paraformaldehyde in Millonig’s buffer solution (pH 7.3). Immunocytochemistry
Following fixation, preparations were rinsed in buffer and incubated overnight in primary antiserum at 4°C: anti-LY-a l/500 dilution in PBS containing 1% Triton X-100 and 1% BSA; I-5 monoclonal-a l/l dilution of the supernatant in PBS containing 2% BSA and 0.2% Saponin. Following the primary antibody incubation, preparations were rinsed in PBS for 1 hr then incubated overnight in a l/250 dilution of HRP-labeled goat antirabbit IgG (Miles, for the anti-LY preparations) or a l/400 dilution of HRP-labeled rabbit anti-mouse IgG (Miles, for the I-5 monoclonal). Secondary antibody dilutions were made in PBS and contained BSA and Triton or Saponin as above. Preparations were then rinsed for 1 hr in PBS, incubated for 1 hr in 0.5 mg/ml 3,3’-diaminobenzidine in PBS, then developed for 1/21 hr in the same solution containing 0.006% H202. Preparations were finally rinsed in buffer, cleared in a glycerin series, and mounted in glycerin. The tissues were viewed under Nomarski interference contrast optics, and photographed and drawn at 1250X magnification. RESULTS
of Central
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Dye Injection
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We used grasshopper embryos from a laboratory colony of Schistocerca americana. Eggs were immersed in saline, cut open at their posterior end, and the embryos were gently squeezed out of their egg shells. After adhering yolk was removed, the ventral nerve cord was exposed by slitting the dorsal membrane from head to tail. The embryo was then transferred in a drop of saline onto a glass slide lightly coated with Sylgard 184 (Corning). For experiments in the CNS, the embryo was positioned dorsal side up in a small rectangular coffin cut out of the Sylgard, and held in place by wire pins projecting from underneath the Sylgard layer and over the embryo’s head and tail. For experiments in the limb buds, embryos were positioned ventral side up. To aid visibility, the saline used in the study was slight hypotonic and consisted of 150 mM NaCl, 3 mA4 KCl, 2 mM CaCl,, 1 mM MgS04, and 5 mA4 TES adjusted to pH 7.0. Neurons were visualized using a Zeiss compound microscope equipped with Nomarski interference contrast optics and a Leitz 50X water immersion lens. Cell bodies were impaled under direct visual control. Glass microelectrodes were drawn on a Sutter Instruments puller and filled with 10% LY (Stewart, 1978) dissolved in
The grasshopper central nervous system (CNS) is segmentally arranged and forms from the ectodermal epithelium that runs down the middle of the embryo from head to tail as a strip of contiguous plates of cells. There are two types of neuronal precursor ceils for the segmental ganglia: neuroblasts (NBS) (Bate, 1976b) and midline precursors (MPs) (Bate and Grunewald, 1981). Each segment has 61 NBS and 7 MPs. Whereas each NB divides repeatedly and generates a clone of progeny, each MP divides only once and gives rise to two progeny. The dorsal surface of the neural epithelium is covered by a conspicuous noncellular basement membrane which separates the neural epithelium from the mesoderm; this basement membrane is probably secreted by the ectoderm at an early stage (Bate and Grunewald, 1981). The first growth cones in the CNS arise from the progeny of midline precursors 1 (MPl) and 2 (MP2); there are two MP2’s (one on each side) and one MPl (at the midline) in each segment. Each MP2 divides once to give rise to a ventral (vMP2) and a dorsal (dMP2) daughter cell. The single MPl gives rise to a pair of bilaterally symmetric daughters, each of which comes to lie dorsal to the two MP2 progeny (forming a trio of cells on each side). All three cells (on each side)
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send growth cones up to the dorsal basement membrane. The growth cone of the vMP2 extends anteriorly, while the growth cones of the dMP2 (its sibling) and MPl extend posteriorly on the neural surface of the dorsal basement membrane (Fig. 1) (Bate and Grunewald, 1981). The cell bodies and often the axons and growth cones of the MPl and MP2 progeny are visible with Nomarksi interference contrast oyptics; their axons and growth cones can be clearly visualized using the I-5 monoclonal antibody (mab) which selectively stains a subset of neurons in the grasshopper embryo (Chang et al., 1982). Preparations using the I-5 mab show that the growth cones of the MPl, dMPf!, and vMP2 reach the basement membrane at about the same time and in a variety of orientations relative to each other and to the body axis (Fig. 2A) (Goodman et al., 1982). For example, of the two sibling cells (the vMP2 and dMP2), in some cases the vMP2 growth cone i:s the more anterior and in other cases the dMP2 growth cone is the more anterior; similarly, either growth cone can be the more lateral of the two. Within several hours, and irrespective of their initial orientation along thie membrane, their growth cones begin to make divergem choices; the vMP2 turns anteriorly and the dMP2 and MPl turn posteriorly (Fig. 2B). What guides the growth cones of the MPl, dMP2, and vMP2? Perhaps thle growth cones passively follow mechanical guidance cues: for example, anatomical studies in amphibian elmbryos have revealed channels between ependymal cells that are subsequently invaded by neuronal growth cones (e.g., Singer et al., 1979; Silver and Robb, 1979; Silver and Sidman, 1980). These observations have given rinse to the “blueprint” hypothesis whereby the germinal neuroepithelium is thought to contain the pattern for channels which in turn serve as mechanical guides for elongating axons. It is possible that clefts with similar functions might exist in the neuroepithelium of grasshopper embryos (Bate and Grunewald, 1981). However, the divergent and cell-specific choices made by the pioneering growth cones in the CNS of the grasshopper embryo suggest the independant existence of active guidance mechanisms. In this regard, it should be noted that the blueprint hypothesis also requires the presence of specific biochemical cues to direct the elongating axons during pathway selection. Studies of these pioneering growth cones in grasshopper embryos support the notion that there is “information” in their environment, and that they are differentially determined to respond to that information in cell-specific ways. Tlheir environment could provide polarity information, positional information, or both. This information could1 be in the form of a diffusible gradient, a substrate gradient (on either the basement
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membrane or cell surfaces), or specific positional information (on either the basement membrane or cell surfaces). In trying to define sources of guidance information in the growth cone’s environment, it is important to understand just how large that environment is. As will be described in detail below, each of these growth cones has many long, thin filpodia extending radially for more than 50 pm. Our results suggest that one source of guidance for these growth cones is likely to be positional information in the form of landmark cells. We define a landmark cell as an identified cell whose differentially labeled surface distinguishes it from the cells around it. In the posterior direction, likely candidates for landmark cells are the two conspicuous corner cells. The anterior (aCC) and posterior (pCC) corner cells are sibling progeny from the first cell division of NB l-l (Goodman et al., 1982). The aCC and pCC cells migrate anteriorly at about 5 pm per hour on the neural surface of the dorsal basement membrane; their total migration is only about 50 pm and from birth they are never more than 60 pm from the MPl and dMP2 cell bodies. As the corner cells migrate anteriorly, they and the growth cones of the dMP2 and MPl appear to point and grow directly toward one another (Fig. 2B). In time, the corner cells finish migrating and the growth cones of the dMP2 and MPl then pass between them and the Ql cell body [one of the two progeny from the first cell division of NB 74 (Raper et al., 1982a)] (Fig. 2C). Interestingly, the dMP2 and MPl growth cones first become selectively dye coupled to the corner cells and later to Ql (Goodman et al., 1982; Raper and Goodman, 1982). [Dye coupling refers to the spread of the fluorescent dye Lucifer Yellow (450 MW) from the interior of one cell to another.] Do the filopodia from the dMP2 and MPl growth cones in the CNS selectively adhere to the corner cells, and use them for guidance (Figs. 2A-C)? We could not answer these questions using Lucifer Yellow injections alone. Although the dye quickly spreads into the filopodia (when the living preparation is viewed in fluorescence) the fragile filopodia lose structural integrity within seconds due to the intense fluorescence. When the fixed preparation is viewed with fluorescence, the filopodia (0.1 pm in diameter) are often faint and fade rapidly; furthermore, halos from more intensely fluorescent growth cones, axons, or cell bodies often obscure the filopodia. Finally even if the filopodia are visible with fluorescence, it is difficult to distinguish which unstained cells they are touching. We wanted a technique that would routinely show the filopodia and their contacts in whole-mount preparations in the light microscope without the need for serial reconstruction from electron micrographs. Although we can fill these embryonic cells with horseradish peroxidase (HRP) for
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electron microscopy (Raper et al., 1982b; Bastiani, Raper, and Goodman, in preparation) in only a small percentage of preparations can we see the filopodia at the light level. Thus we made rabblit serum antibodies to Lucifer Yellow. After Lucifer Yellow is injected into a cell, the embryo is fixed, incubated with the anti-LY antibody, then incubated with a HRP-labeled second antibody. The embryo is processed for the HRP reaction, mounted in glycerine, and viewed at up to 1250X with Nomarski optics (Fig. 1). Given the ability of Lucifer Yellow (450 MW) to travel long distances within a cell and to cross gap junctions, there is every rea.son to suspect that all filopodia are filled by the dye. The antibody technique appears to be at least as sensitive (perhaps more) in detecting the number and extent of the dye-filled filopodia as is direct observation of the fluorescent dye in living preparations. Photographs of whole-mount preparations using the Lucifer Yellow antibody are shown in Fig. 1; parts A-C show fills of the MPl cell in sequentially older embryos. Figures 2 (D, E), 3, and 4 (D, E) are camera lucida drawings of representative preparations showing the filopodial ‘contacts and coupling from the MPl cell in the CNS and the 1B cells in the periphery. As the MPl makes its cell-specific turn posteriorly, one or several of its filopodia contact the corner cells (Fig. 3A); the longest lilopodia from any growth cone often reach for more than 50 pm and the corner cells are about 50 pm away. Although there are many other cells in its environment and near the neural surface of the basement membrane (and thus within filopodial grasp), a disproportionate number of the MPl’s filopodia soon contact the corner cells (Fig. 3B). Furthermore, those that do contact the corner cells tend to continue running along them (Figs. 2D, E). We interpret this to imply selective adhesion, although we have not directly measured adhesive forces. It may be that just one or a few filopodia contacting the corner cells are sufficient to mediate the cell-specific turn (compare
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D
MPl
FIG. 2. Apparent selective filopodial adhesion from the MPl growth cone to the anterior and posterior corner cells (aCC, pCC) in the developing CNS of the grasshopper embryo. (A-C) Camera lucida drawings of the MPl, dMP2, vMP2, aCC, pCC, and Ql cell bodies based on whole-mount preparations using the I-5 monoclonal antibody. The MPl growth cone turns posteriorly and points at the aCC and pCC cell bodies which are migrating anteriorly. In time, and MPl (and dMP2) growth cones grow between these two cell bodies and the Ql cell. (D,E) Two examples of the MPl growth cone and its filopodia at the same stage of development: LY was injected into the cell body and the embryo was processed with the anti-LY antibody. Note the extensive filopodial contact with the aCC and pCC cell bodies. Selective dye coupling was observed to both these cells; the only sites of cellular contact that could mediate this coupling are the filopodia. Note several long filopodia in (E) that are not contacting the aCC or pCC cell bodies.
Figs. 3A and B). Unfortunately, it has been very difficult to obtain preparations before the stage shown in Fig. 3A in which we are confident that we see the full extent of the filopodia. When the growth cone is first initiated, the dye-filled filopodia often appear as broken strings
FIG. 1. Filopodia and dye coupling from midline precursor 1 (MPl) at three stages of development, as visualized with an antibody to Lucifer Yellow, in the central nervous system of the grasshopper embryo. Only a very thin plane is in focus with Nomarski optics; thus only some of the filopodia are visible in the photographs. (A) The filopodia from the MPl growth cone extensively cover the posterior corner cell (pCC); note dye coupling to pCC mesdiated via the filopodia. The anteriorly extending axon is the dye-coupled vMP2, and the cell bodies out of focus around the MPl are those of the dye-coupled dMP2 and vMP2. (B) The MPl growth cone has just grown between the pCC and the Ql; note the MPl’s long leading filopodia that extend posteriorly beyond the pCC and Ql cells, and the relative amounts of dye coupling to the pCC and Ql cell bodies. (C) Dye coupling is observed from the MPl to the pCC, Ql, Q2 (Ql’s sibling, the cell body just lateral to Ql), and to two other identified cell bodies further posterior and out of the plane of focus (open arrow). The dye coupled vMP2 axon has extended anteriorly and is dye coupled to a conspicuous identified cell via that cell’s axon. The MPl growth cone has entered the next posterior segment (arrow heads show segmental boundaries); the dMP2 growth cone has extended about the same distance. The dye coupled vMP2 axon has extended anteriorly into the next anterior segment. The growth cones of the dye-filled, posteriorly directed MPl and dMP2, and the unfilled, anteriorly directed vMP2 of the next posterior segment, pass each other near the segmental boundary and continue extending within a few microns of each other. Yet, while their filopodia overlap extensively, they do not become dye coupled to one another. Similarly, the dye-filled, anteriorly directed vMP2 and the unfilled, posteriorly directed MPl and dMP2 of the next anterior segment do not become dye coupled to one another, although their filopodia overlap extensively.
396
DEVELOPMENTAL BIOLOGY
A M Pl
aCC
PCC
\,,“,,,;
,
“\ ‘/)/
‘1,
/ a
VOLUME 94. 1982
20 urn
FIG. 3. Filopodia from the MPl growth cone at successively later stages of development. (A) Radiation of MPl filopodia just after the growth cone turns posteriorly. A few filopodia contact the aCC cell. (B) With time, the aCC and pCC (corner cells) migrate further anteriorly (and thus closer to the MPl). The MPl growth cone broadens and has a greater proportion of its filopodia in contact with the corner cells (the MPl is dye coupled to the corner cells at this stage). Note also a smaller growth cone extension in the anterior direction. This anterior extension is along the vMP2 axon (see Fig. 2B) and is retracted in time. (C) At a later stage of development, the MPl growth cone has extended further posteriorly and has extensive filopodial contact (and dye coupling) with the corner cells and an additional identified cell, Ql. Furthermore, many long filopodia extend posteriorly from its tip and contact one of a pair of identified cells just on the other side of the segment boundary.
of beads rather than smooth threads. These observations may indicate that at the earliest stages of growth cone initiation (i.e., before the growth cone makes its turn) the filopodia are more sensitive to perturbations by the intracellular penetration and dye injection than at later stages. However, at these early times, we do see a large expanse of dye-filled beads in many directions, suggesting an initially broad radiation of filopodia. When we examined 11 preparations at the stage of development just after the MPl growth cone turned posteriorly, we consistently found extensive filopodial contacts from the MPl growth cone to the corner cells, strongly suggesting selective filopodial adhesion with the surface of these cells (two examples are shown in Figs. 2D, E). In three of these preparations, we counted about 20 cell bodies within a one cell diameter radius of the posteriorly directed growth cone. This is likely to be a very conservative estimate of the number of cells within filopodial grasp: a cell body diameter is about 15 pm and the filopodia can reach for over 50 pm. In 6 of the 11 preparations, we counted the total number of filopodia that extended from the posteriorly directed growth cone and the number contacting the corner cells (Table 1). On average, 45% of the filopodia contacted the surface of the corner cells, although these cells constitute less than 10% of the available cell surfaces. Thus, the MPl growth cone at this stage demonstrates a
strong tendency for filopodial contact with the corner cells, and no tendency for selective contacts with any of the other cells in its immediate environment. Within a short period, however, some of the MPl filopodia begin to selectively adhere to the Ql cell body (Fig. 3C), and shortly thereafter to two other, identified cells further posteriorly along its route and just on the other side of the segment boundary (Figs lB, lC, 3C). Filopodia
of Peripheral
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The first growth cones in the limb bud arise from the 1B cells (Bate, 1976a,b; Keshishian, 1980; Ho and Goodman, 1982). The 1B growth cones extend proximally toward the CNS along the basement membrane on the inside of the ectodermal epithelium of the limb bud. However, they make a characteristic turn about halfway down the limb bud (Figs. 3A-C) (Bate, 1976a,b; Keshishian, 1980). This turn in direction occurs about the time they reach the 3B cell (although the growth cones need not reach this cell); they always, however, turn toward the 1A cells (Ho and Goodman, 1982). The pair of 1A cells migrate out of the ectodermal epithelium and come to lie on its inside surface. Depending upon the segment, the 1A cells either have or have not sent growth cones into the CNS by the time the 1B growth cones arrive at their cell bodies. In the prothoracic (Tl)
TAGHERT ET AL.
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segment, they have extended growth cones, whereas in the metathoracic (T3) segment, they have not (Ho and Goodman, 1982). Upon reaching the 1A cell bodies, the 1B growth cones then continue extending toward the CNS. Our results suggest that one possible source of guidance for the 1B growth cones is likely to be positional information in the form of landmark cells; the likely candidates are the 1A cells. Having seen that the filopodia of the MPl growth cone selectively adhere to the corner cells in the CNS, we wanted to know if the filopodia from the 1B growth cones in the periphery iselectively adhere to the 1A cells, and use them for guidance (Figs. 4A-C). The 1B growth cones are easily within filopodial reach of the 1A cells when they make their cell-specific turn toward them (Fig. 4): the 1A cells are less than 50 pm away. Although they are growing on the inside of an epithelial tube, a disproportionate number of the 1B filopodia contact the 1A cells rather than the other ectodermal cells, as can be seen in Figs. 4D, E. Even before the 1B growth cones make their turn toward the 1A cells, they already have
FIG. 4. Apparent selective filopodial adhesion from the 1B growth cones to the 1A cell bodies in the metathoracic limb bud of the grasshopper embryo. (A-C) ICamera lucida drawings of the lB, lA, 3B, and 2B cells based on whole-mount preparations using the I-5 monoclonal antibody. The 1B growth cones make a characteristic turn toward the 1A cell bodies on tlneir way to the CNS. (D,E) Two examples of the 1B growth cones and their filopodia at the same stage of development: LY was injected into the cell body of one of the two 1B cells and the embryo was processed with the anti-LY antibody. Note the extensive filopodial contact with the 1A cell bodies and the 3B cell body. In both examples, one of the growth cones has not yet made the turn, and yet filopodia already span the distance and contact the 1A cell bodies (arrowheads). Selective dye coupling was observed to all of the cell bodies drawn (in D, 1A cells, and 3B cell; in E, 1A cells, 3B cell, and 2B cell).
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TABLE 1 FILOPODIAL CONTACTSBYMP~GROWTHCONE
Preparation 1 2 3 4 5 6
Number of filopodia 32 31 39 34 39 38
Number of filopodia contacting corner cells (%) 14 (44) 14 (45)
19 (49) 11 (32) 20 (51)
18 (47)
long filopodia in contact with the surface of the 1A cells (e.g., arrows in Figs. 4D, E). Selective Dye Coupling from Pioneer Growth Cones The Lucifer Yellow antibody also reveals patterns of selective dye coupling (Raper and Goodman, 1982; Goodman et al., 1982) from the MPl growth cone in the CNS to the corner cells (with the strongest coupling typically to the pCC) (Figs. 1, 2D, E), then Ql (Figs. lB, 3C), and then to two other cell bodies more posterior (Figs. lC, 3D). Dye coupling is also seen from the 1B growth cones in the periphery to the 3B cell and the 1A cells (Figs. 4D, E). The antibody allows us to visualize the relative amount of dye coupling to different cells (e.g., Figure 1B). The amount of reaction product following the antibody technique accurately reflects the graded extent of Lucifer Yellow dye coupling that is often seen with fluorescence. The antibody technique appears to be at least as sensitive in detecting the dye as is direct observation following blue irradiation. It is evident that in several cases this dye coupling must be mediated via the filopodia since they are the only sites of cellular contact (e.g., Figs. lA, 2D, E, and 4D). However, filopodia and growth cones do not become dye coupled to every cell they contact. One striking example is observed when the posteriorly directed growth cones of the MPl and dMP2 meet the anteriorly extending growth cone of the vMP2 of the next posterior segment at the segment border. Despite their close proximity (~5 pm) and extensive filopodial overlap, the vMP2 does not become dye coupled with the MPl or dMP2. Landmark
Cell Hypothesis
Our results suggest a landmark cell hypothesis whereby (i) the filopodia of pioneering growth cones selectively adhere to the surfaces of specifically labeled landmark cells, and (ii) this specific filopodial adhesion serves to guide the pioneering growth cones toward the landmark cells as they make cell-specific turns or pathway alterations. In the CNS, the filopodia of the MPl growth cone
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contact and appear to adhere to the surfaces of the corner cells just as they make their cell-specific turn toward these cells. The landmark cell hypothesis predicts that the filopodia from the dMP2 growth cone also selectively adhere to the surface of the corner cells in the posterior direction, whereas the filopodia from the vMP2 growth cone selectively adhere to the surface of a landmark cell(s) in the anterior direction. It is possible that contact by only one or a few filopodia by the initial MPl radiation may be sufficient to direct the course of its growth cone toward the corner cells (e.g., Figs. 2E, 3A). While there may in addition be some form of anterior/posterior polarity information in their environment, nevertheless the behavior of the MPl filopodia suggests that selective filopodial adhesion to specific landmark cells plays an important role in the guidance of pioneering growth cones. Likewise, in the periphery, the filopodia of the 1B growth cones contact and appear to adhere to the surfaces of the 1A cells just as they make their cell-specific turn toward these cells. It appears as if the filopodia that contact the landmark cells are on average longer than the filopodia that contact other cells (e.g., Figs. 2D, E). Why is this? There are two possible explanations. First, our Lucifer Yellow dye injections present a single static picture of a very dynamic process. According to Bray’s model (1982), filopodia are actively extended and retracted over a several-minute cycle. In tissue culture, filopodia that attach to axons can be seen to pull on the axon at intervals of about 2 min (Nakai, 1960; Wessells et al., 1980). At any one moment, only a small percentage of the filopodia (on nonadhesive surfaces) will be at their maximum length. We occasionally see very long filopodia that are not touching specific landmark cells (e.g., Figs. 2E, 3A). However, those filopodia that contact a particularly adhesive surface will retain their full length as their contractile cycle produces tension rather than retraction. Thus, at any one moment, one might expect to find the filopodia on the landmark cells to be on average longer than those filopodia touching other cells. The second alternative, however, is that the apparent difference in the average length of the filopodia may reflect an artifact produced by penetrating the cell body with a microelectrode and filling the cell with dye for up to several minutes. A change in membrane potential or ion fluxes might cause a partial retraction of some filopodia, leaving those filopodia that are touching adhesive surfaces at their full length. In the CNS, the filopodia from the MPl and dMP2 growth cones first appear to selectively adhere to the corner cells, then Ql (Fig. 3C), and then to two cell bodies about 50 pm further posterior (Figs. lB, C, 3D). These next landmark cells are just on the other side of the segment border. Furthermore, the first sign of seg-
VOLUME 94, 1982
mentation in the limb bud appears proximal to the 1B cells and is located just distal to the IA cells (this border is the division between the femur and the proximal leg). Thus, in both the CNS and the periphery, specific landmark cells are located just across the segment boundaries in the direction of growth cone extension. These observations are particularly interesting in light of theories concerning iterated segmental gradients in insects and the inhibition of axon growth across segment boundaries (e.g., Lawrence, 1981). It may be that the pioneering growth cones require particularly adhesive cell surfaces on the other side to get across segment boundaries, and that later cells can simply follow the pioneer axons. One final question is why the MPl growth cone continues to extend from landmark cell to landmark cell in a stepping stone fashion (Bate, 1976a) rather than simply staying on the first adhesive cell surface it finds. One need not suppose that each successive landmark has a different and more adhesive label. Rather, the cells may have a proclivity to grow and simply follow those surfaces that are most adhesive. Alternatively, the next landmark may have the same label but may, with time, be more adhesive than the first, simply because the extensive filopodial contacts with the first landmark changed that cell’s surface (e.g., by binding or altering the adhesive component). The Lucifer Yellow antibody can thus be a powerful histological tool, particularly when used to resolve the intricate details of cell shape, contacts, and coupling during development. It has already helped us to answer one important question heretofore difficult to approach in a developing embryo. Our results suggest that selective filopodial adhesion to specific landmark cells may play an important role in the guidance of pioneer growth cones. We now plan to test this landmark cell hypothesis by examining how these pioneer growth cones navigate in the absence of their normal landmarks. We thank Susannah Chang for the I-5 monoclonal antibody and John Kuwada for careful reading of the manuscript. Support was provided by a Muscular Dystrophy fellowship to P.H.T., N.I.H. fellowship to M.J.B., and grants from the NSF and McKnight Foundation to C.S.G. who is a Sloan Fellow. REFERENCES
BATE, C. M. (1976a). Pioneer neurones in an insect embryo. Nature (London) 260, 54-56. BATE, C. M. (1976b). Embryogenesis of an insect nervous system. I. A map of the thoracic and abdominal neuroblasts in Locusta migratoria. J. Embryol. Exp. Morphol. 35, 107-123. BATE, C. M., and GRUNEWALD, E. B. (1981). Embryogenesis of an insect nervous system. II. A second class of precursor cells and the origin of the intersegmental connectives. J. Embryol. Erp. Morphol.
61,317-330. BRAY, D. (1982). Filopodial
contraction
and growth cone guidance. In
TAGHERT ET AL.
Guidance
“Cell Behavior” (R. Bellairs, A. Curtis, and G. Dunn, eds.), Cambridge Univ. Press, London/New York. CAJAL, S. R. Y. (1894). “Die retina der Wirbelthiere.” Bergmann Verlag. CHANG, S., Ho, R. K., and GOODMAN, C. S. (1982). Monoclonal antibodies that reveal specific patterns of neurons and mesodermal cells early in grasshopper elmbryogenesis. Dev. Biol. in press. GOODMAN, C. S., and SPITZEI~:, N. C. (1979). Embryonic development of identified neurons: differentiation from neuroblast to neuron. Nature (London) 280, 208--214. GOODMAN, C. S., and BATE, C. M. (1981). Neuronal development in the grasshopper. Trends Neurosci. 4, 163-169. GOODMAN, C. S., RAPER, J. A., Ho, R. K., and CHANG, S. (1982). Pathfinding by neuronal growth cones during grasshopper embryogenesis. In “40th Symposium, Society for Developmental Biology” (S. Subtelny and P. Green, eds.), pp. 275-316. Alan R. Liss, New York. HARRISON, R. G. (1910). The outgrowth of the nerve fiber as a mode of protoplasmic growth. J. Bxp. 2001. 9, 787-845. Ho, R. K., and GOODMAN, C. S. (1982). Perhipheral pathways are pioneered by an array of central and peripheral neurons. Nature (London) 297, 404-406. KESHISHIAN, H. (1980). The origins and morphogenesis of pioneer neurons in the metathoracic leg of the grasshopper. Dev. Biol. 80, 388-397. LANCE-JONES, C., and LANDMESSER, L. (1981a). Pathway selection by chick lumbrosacral motoneurones during normal development. Proc. Roy. Sot. Land. B 214,1-18. LANCE-JONES, C., and LANDMESSER, L. (1981b). Pathway selection by embryonic chick motoneurones in an experimentally altered environment. Proc. Roy. Sot. Lond. B 214. 19-52. LAWRENCE, P. A. (1981). The cellular basis of segmentation in insects.
Cell 26, 3-10. LETOURNEAU, P. C. (1975). Cell-to-cell substratum adhesion and guidance of axonal elongation. Deu. Biol. 66, 183-196. MOORE, G., LUTTERODT, A., BURFORD, G., and LEDERIS, K. (1977)
of Growth
399
Cones
A highly specific antibody 101, 1421-1435.
for arginine
vasopressin.
Endocrinology
NAKAI, J. (1960). Studies on the mechanism determining the course of nerve fibers in tissue culture. II. The mechanism of fasciculation. Z. Zellforsch. 51, 427-449. RAPER, J. A., and GOODMAN, C. S. (1982). Transient dye coupling between developing neurons reveals patterns of intracellular communication during embryogenesis.; 6th Symp. “Ocular Visual Development,” in press. RAPER, J. A., BASTIANI, M. J., and GOODMAN, C. S. (1982a). Pathfinding by neuronal growth cones in grasshopper embryos. I. Divergent choices made by the growth cones of sibling neurons. J. Neurosci., in press. RAPER, J. A., BASTIANI, M. J., and GOODMAN, C. S. (1982b). Pathfinding by neuronal growth cones in grasshopper embryos. II. Selective fasciculation onto specific axonal pathways. J. Neurosci., in press. SILVER, J., and ROBB, R. M. (1979). Studies on the development of the eye cup and optic nerve in normal mice and in mutants with congenital optic nerve aplasia. Deu. Biol. 68, 175-190. SILVER, J., and SIDMAN, R. L. (1980). A mechanism for the guidance and topographic patterning of retinal ganglion cells axons. J. Comp. Neural. 189, 101-111. SINGER, M., NORDLANDER, R. H., and EGAR, M. (1979). Axonal guidance during embryogenesis and regeneration in the spinal cord of the newt: The blueprint hypothesis of neuronal pathway patterning. J. Comp. Neurol. 185, l-22. STEWART, W. W. (1978). Functional connection between cells as revealed by dye coupling with a highly fluorescent naphthalamide tracer. Cell 14, 741-759. WESSELLS, N. K., LETOURNEAU, P. C., NUTTALL, R. P., LUDUENAANDERSON, M., and GUIDUSCHEK, J. M. (1980). Responses to cell contacts between growth cones neurites and ganglionic nonneuronal cells. J. Neurocytol. 9, 647-664.