Expression of fasciclin I and II glycoproteins on subsets of axon pathways during neuronal development in the grasshopper

Cell, Vol. 48, 745-755,

March

13, 1987, Copyright

0 1987 by Cell Press

Expression of Fasciclin I and II Glycoproteins on Subsets of Axon Pathways during Neuronal Development in the Grasshopper Michael J. Bastiani, Allan L. Harm&n, Peter M. Snow, and Corey S. Goodman Department of Biological Sciences Stanford University Stanford, California 94305

Summary The “labeled pathways” hypothesis predicts that axon fascicles in the embryonic neutopil are differentially labeled by surface recognition molecules used for growth cone guidance. To identify candidates for such recognition molecules, we generated monoclonal antibodies (MAbs) that recognize surface antigens expressed on subsets of axon fascicles in the grasshopper embryo. The 3811 and 8C8 MAbs immunopmcipitate 70- and 95kd membrane glycoproteins called fasciclin I and II, respectively, which are expressed on different subsets of axon fascicles during development. These two glycoproteins are expressed regionally on particular portions of embryonic axons in correlation with their patterns of fasclculation, dynamically during the period of axon outgrowth in a manner consistent with a role in growth cone guidance, and at other times and places during embryogenesis, suggesting multiple developmental roles. Introduction How neurons find their appropriate targets during development is a central issue in developmental neurobiology. Neurons are often born far from their synaptic partners, thus their growth cones must traverse long distances along stereotyped and sometimes circuitous pathways to find their targets. A common observation from a variety of phyla is that different growth cones, confronted with the same environment, often make divergent and stereotyped pathway choices; these observations suggest both heterogeneous extrinsic cues and specificity in the ability of growth cones to respond to these cues (e.g., Bate and Grunewald, 1981; Goodman et al., 1982; Raper et al., 1983a, 1983b; Tosney and Landmesser, 1985a, 1985b; Eisen et al., 1986; Kuwada, 1986). Many studies devised to understand the mechanisms that mediate growth cone guidance have focused on the relatively simple and highly accessible developing nervous systems of insects such as the grasshopper and fruit fly. Many of the earliest differentiating neurons in insects extend growth cones that pioneer particular axon pathways; these pioneering growth cones often extend along a variety of substrates, including basement membranes, epithelial cells, primitive glial cells, other neurons, and mesodermal cells (e.g., Bate and Grunewald, 1981; Ho et al., 1983; Jan et al., 1985; Caudy and Bentley, 1986a, 1986b; Blair and Palka, 1985a, 1985b; Bastiani and Goodman, 1986).

In contrast, most of the later differentiating neurons in insects extend their growth cones along these preexisting axon pathways, using as their guidance cues the surfaces of specific axons (Raper et al., 1983a, 1983b, 1983c, 1984; Bastiani et al., 1984, 1986; du Lac et al., 1986). In the developing central nervous system (CNS), growth cones are confronted with an orthogonal scaffold of longitudinal and commissural axon bundles (or fascicles) in each segmental ganglion (Figure 1A). In the grasshopper embryo, the long filopodial extensions from individual growth cones can sometimes contact as many as 25 different fascicles (containing in total about 100 different axons), yet, invariably, these growth cones choose to extend along (i.e., fasciculate with) one particular bundle of axons. The specific affinities that growth cones display for particular axon surfaces give rise to the stereotyped patterns of selective fasciculation. Selective fasciculation is likely to play an important role during neuronal development in higher organisms as well (e.g., Bonhoeffer and Huf, 1985; Kapfhammer et al., 1986). For example, recent studies have revealed selective patterns of fasciculation during the development of the fish spinal cord (Kuwada, 1986). Studies in our laboratory and others on the behavior of individual growth cones in the developing CNS of insect embryos led to the proposal and subsequent refinement of the “labeled pathways” hypothesis (Ghysen and Janson, 1980; Goodman et al., 1982; Raper et al., 1983a, 1983b; Bastiani et al., 1984). The labeled pathways hypothesis predicts that axon fascicles in the embryonic neuropil are differentially labeled by surface recognition molecules used by growth cones for their selective fasciculation choices. This model has been experimentally tested and supported using specific cell ablations in grasshopper (Raper et al., 1983c, 1984; Bastiani et al., 1986; du Lac et al., 1986; Doe et al., 1986) and fish (Kuwada, 1986) embryos. To identify candidates for such axonal recognition molecules, we generated monoclonal antibodies (MAbs) that recognize surface antigens expressed on subsets of axon fascicles in the grasshopper embryo. Here we report on the 3811 and 8C6 MAbs, which are used to identify and study the expression of two different membrane glycoproteins. These two glycoproteins, called fasciclin I and II, are expressed on different subsets of axon fascicles during development in a spatiotemporal pattern that is consistent with the predictions of the hypothesis. Results The 3811 and 8C8 MAbs Recognize Surface Antigens Expressed on Different Subsets of Axon Pathways The antigens recognized by the 3Bll and 8C6 MAbs are localized on different subsets of axon bundles in the grasshopper embryo (Figure 1). Within the developing segmental ganglia, the 3811 MAb stains a small subset

Figure 1. 3811 and 8C6 MAbs Recognize Specific Subsets of Axon Pathways in the Grasshopper Embryo (A, B, C) Dorsal views with Nomarski optics of single focal planes of the whole-mount neuroepithelium of single segments of 43% grasshopper embryos stained with particular monoclonal antibodies (MAbs) and visualized with an HRP-conjugated second antibody and HRP immunocytochemistry. (A) The embryonic axon scaffold in a single segment of the grasshopper embryo showing the entire orthogonal array of longitudinal, commissural. and lateral axon fascicles in the neuropil of the T2 segment, as revealed with the l-5 MAb (Chang et al., 1983). (8) The 3811 MAb stains a specific subset of axon fascicles (open arrow) in the anterior and posterior commissures (it also strains a longitudinal fascicle in the connectives and the intersegmental nerve, both out of the plane of focus; see(E). (C) In contrast, the 8C6 MAb stains most of the longitudinal axon fascicles in the neuropil and in the connective (solid arrow). (D, E, P) Dorsal views with epifluorescence of single focal planes of the whole-mount neuroepithelium of pairs of segments of 40%-45% grasshopper embryos stained with particular antibodies and visualized with an FlTCconjugated second antibody. (D) Staining of all axon fascicles and cell bodies in two segments of the CNS using anti-HRP serum antibody (Jan and Jan, 1962). (E) By comparison, the 3811 MAb stains only a small subset of commissural (large open arrow) and longitudinal (solid arrow) axon fascicles, and the intersegmental nerve (small open arrow). (F) The 6C6 MAb stains all the major longitudinal axon fascicles in the connective (solid arrow) and the intersegmental nerve (small open arrow), but few of the commissural axons (large open arrow). The 8C6 MAb also binds to the neuroepithelial cells at the segment border and to some along the midline. A corn: anterior commissure: P corn: posterior commissure; SN: segmental nerve; ISN: intersegmental nerve; con: connective. Scale bar: (A, 8, C), 30 urn; (D, E, P), 50 pm.

of commissural 1E). In addition, of the longitudinal

pathways at this 8xon

in a 43% embryo (Figures 16, stage, the 3611 MAb stains one bundles between the developing

segmental segmental segmental

ganglia (Figure lE), all of the axons in the internerve (Figure lE), and two small bundles in the nerve root (data not shown). In contrast, in 43%

Expression 747

of Fasciclin

I and II Glycoproteins

Figure 2. Analysis by SDSPAGE of the Proteins Immunoprecipitated by the 3811 and 8C6 MAbs Membranes were prepared from adult CNS tissue and were labeled with 1251(see Experimental Procedures). Membrane proteins were solubilized in NWOtontaining buffer and subjected to immunoprecipitation using preformed antibody complexes made with either the 3811 or 8C6 MAbs. Immunoprecipitates were analyzed by SDS-PAGE on an 8.5% gel under reducing conditions. Lane 1: Target antigen immunoprecipitated by the 3811 MAb, called fasciclin I. Lane 2: Target antigen immunoprecipitated by the 8C6 MAb, called fasciclin II. Molecular masses are indicated on the left in kilodaltons.

and older embryos, the 8C6 MAb primarily stains longitudinal pathways (Figures lC, 1F). At early stages, the 8C6 MAb transiently stains several commissural bundles, but not the same subset as is stained by the 3811 MAb. In addition, the 8C6 MAb stains the intersegmental nerve (although 3811 and 8C6 stain different peripheral branches of this nerve; e.g., Figure 4B), and transiently stains two bundles in the segmental nerve root (data not shown). 3811 and 8C6 MAbs lmmunoprecipitate Different Membrane Glycoproteins Called Fasciclin I and II Membranes were prepared from both adult CNS and from 40%-50% grasshopper embryos. The membrane proteins were iodinated, and subsequently solubilized with Nonidet-P40. Following immunoprecipitation, the specific proteins recognized by the 3811 and 8C6 MAbs were analyzed by SDS-PAGE. The results (Figure 2) indicate that the 3811 and 8C6 MAbs recognize single proteins of 70 kd and 95 kd, respectively. Both membrane proteins are glycosylated, as indicated by their binding to the lectin Concanavalin A (data not shown). Because these two glycoproteins are expressed on different subsets of axons, we call them fasciclin I and fasciclin II.

Antisera Against Fasciclin I and II Recognize the Same Subsets of Axon Pathways as Do the MAbs We wondered whether the restricted spatiotemporal expression of the two antigens recognized by the 3811 and 8C6 MAbs (as described in further detail below) actually represents the restricted expression cjf the two core proteins, or, alternatively, whether it simply reflects the restricted expression of particular epitopes on otherwise more ubiquitously expressed proteins. Antisera against the purified proteins might help resolve this dilemma because they would recognize multiple epitopes on the core protein. To this end, large quantities of solubilized embryos were used to purify microgram quantities of each glycoprotein, using affinity chromatography followed by preparative gel electrophoresis. Antisera were generated in rats against each of the gel-purified glycoproteins. lmmunocytochemical studies revealed that the two antisera recognize the same subsets of axon pathways, as do the two MAbs (data not shown; identical to Figures lE, 1F). These results indicate that the fasciclin I and II glycoproteins are indeed expressed on restricted subsets of axon pathways, on restricted regions of particular axons, and at restricted times of development. These features are described below, with particular emphasis on the expression of fasciclin I; further details of fasciclin II expression will be presented later (Harrelson et al., unpublished data). Fasciclin I Is Positionally Expressed Transiently during Neurogenesis In addition to being expressed on a subset of axon pathways during axonogenesis, the fasciclin I glycoprotein is also transiently expressed in a segmentally repeated pattern on the surface of neuroepithelial cells during neurogenesis (Figure 3D) (for a review of grasshopper neurogenesis, see Doe et al., 1985). As segmentation occurs, fasciclin I expression begins in a graded fashion on approximately the posterior onethird and anterior one-third of the neurogenic ectodermal cells in each segment, with the greatest intensity at the segment border (Figure 3D). The staining becomes more localized and intense on a group of ectodermal cells that appear to give rise to the more medial neuroblasts (NB) in rows 1,2,3, and 7, and posterior midline cells that give rise to the median neuroblast (MNB) and midline precursors (MP) 4, 5, and 6 and their support cells. As the NBS form, most of the ectodermal cells stop staining, except for those surrounding NB l-l, 1-2, the MNB, and the ectodermal cells at the segment border. As development proceeds, most of the progeny of NB 1-1, 1-2, and the MNB do not express the antigen; the expression of fasciclin I during neurogenesis appears to be separate from its expression during axon outgrowth. Fasciclln I Is Positionally Expressed by Nonneuronal Cells in the Body Wall and Limb Buds Both glycoproteins are expressed not only on subsets of axon pathways and on neuroepithelial cells during neurogenesis, but also outside the developing CNS on the surface of nonneuronal cells during embryogenesis. Fas-

Cell 740

Figure

3. Fasciclin

I Is Positionally

Expressed

at Other

Times

and Places

throughout

Development

HRP immunocytochemistry using the 3811 MAb to reveal the expression of fasciclin I. (A) Fasciclin I expression on the cell bodies and axons of the pair of pioneer sensory neurons (open arrow) in the limb bud of a 32% embryo. Fasciclin I is also expressed on a large patch of epithelial cells at the base of the limb bud (solid arrow) and on another small patch of epithelial cells farther distal (asterisk). (B) In the adult connective between the Tl and T2 ganglia, only the ventromedial sensory axon pathway expresses fasciclin I. There is no labeling of a dorsolateral fascicle or of the nerve 6 (n6) branch of the intersegmental nerve. (C) Two patches (arrows) of body wall ectoderm (E) expressing fasciclin I (35% embryo). (D) The first expression of fasciclin I is in the neurogenic region, initially on the neuroepithelial cells, and then on both the neuroectodermal cells and neuro-

Expression 749

Figure

of Fasciclin

4. Expression

I and II Glycoproteins

of Fasciclin

I Correlates

with Axon

Fasciculation

HRP immunocytochemistry using the 3811 MAb to reveal the expression of fasciclin I in a single segment (A, B) or between two segments (C). (A) Fasciclin I is expressed on a small subset of neurons at about 32% of development. The solid arrows point to the growth cones of two neurons that turn medially and pioneer an axon fascicle in the anterior commissure. The arrowheads point to the growth cones of the Ul and U2 neurons that turn posteriorly and pioneer the longitudinal U fascicle. (B) At 38% of development, the major axon pathways expressing fasciclin I have formed in the anterior (A corn) and posterior (P corn) commissure, the longitudinal connective (con), and the segmental (SN) and intersegmental (ISN) nerves. This focal plane shows that the aCC neuron, which grows along the lf fascicle (Uf), expresses fasciclin I on its surface, while its sibling, the pCC neuron, which grows along the MPl fascicle (MPlf in (C)), does not express fasciclin I. The arrowhead shows the position of the EM section in Figure 5. (C) Photomicrograph of the longitudinal axon fascicles in a single connective (con) between two embryonic ganglia at 38% of development. Of the first three longitudinal axon fascicles (the vMP2, MPl, and U fascicles), only the U fascicle (Uf) expresses fasciclin I. The stained axon (arrow) from the aCC cell body can be seen crossing the unstained axon pathways to reach the U fascicle. The intersegmental nerve (ISN) turns laterally from the longitudinal pathways at the level of the segment boundary. MNB: median neuroblast; Scale bar: (A, B), 50 urn; (C), 25 pm.

I is localized on the surface of ectodermal cells in restricted and segmentally repeated regions of the body wall (Figure 3C) and limb bud (Figure 3A).

ciclin

Temporal and Regional Expression of Fasciclin I on Specific Axonal Processes During the early stages of axonal outgrowth (at about 32% of development), a small subset of neurons begins to express fasciclin I on their cell bodies and growth cones, including two well-characterized pairs of neurons (Figure 4A). The major observation is that the pattern of neurons that express fasciclin I on their growth cones and axons during axonal outgrowth are not correlated with the pat-

tern of epithelial cells and precursors that transiently express fasciclin I earlier during neurogenesis. Of the many axon pathways forming at this stage, these first neurons to express fasciclin I pioneer two particular axon pathways. The more anterior pair of neurons pioneers a single commissural pathway in the anterior commissure (Figures 5A, 58). The more posterior pair of neurons (consisting of the identified neurons Ul and U2) pioneers a single longitudinal pathway (the U fascicle, Figure 4C; Bastiani et al., 1986; du Lac et al., 1986), which turns laterally to establish the intersegmental nerve. The anterior group of fasciclin l-positive neurons appears

blasts (neuronal precursor cells) of approximately the anterior one-third and posterior open arrow, unstained neuroblasts. The arrows along the side demarcate the segment mental nerve; ISN: intersegmental nerve. Scale bar: (A, C), 30 urn; (B, D), 50 urn.

to express

the

glycoprotein

regionally

in that

it is

one-third of each segment. Solid arrow, stained neuroblasts; border (28% embryo). M: mesoderm; E: ectoderm; SN: seg-

Figure

5. lmmunoelectron

Microscopy

Showing

Localization

of Fasciclin

I on Specific

Axons

in the Anterior

Commissure

HRP (A, C) and gold (6) immuno-EM labeling using the 3811 MAb to reveal the expression of fasciclin I. (A, C) (A) is an enlargement of the axon pathway labeled A in (C); (C) is a lower magnification view showing most of the axon pathways in the anterior commissure of a 42% embryo (the level of this section is shown by the arrowhead in Figure 48). The fasciclin l-positive pathway in the anterior commissure consists of three tightly associated bundles of axons: other pathways in the anterior commissure do not express the protein (e.g., unlabeled arrow in (C)). The protein appears to be more abundant on axons in one of these associated bundles (dark arrow in A) and less abundant, although present, on axons in the other two associated bundles (open arrows in (A)). In the other two bundles, the protein appears to be more abundant on the surfaces of the axons that are closest to the axons that express higher amounts of the protein (A). (B) At a very early age (31%) before axons first begin to express fasciclin I, some filopodia have the protein on their surface, as shown with 5 nm gold bound to goat anti-mouse IgG. This suggests that these filopodia directly express the protein rather than acquiring it from other surfaces. Scale bar: (A, B), 5 pm; (C), IO urn.

localized only on portions of their surfaces. While pioneering their commissural pathway by looping up to the dorsal basement membrane and then extending across the mid-

line, they express the protein on their growth cones and axons. However, when they turn onto longitudinal axon pathways at the lateral edges of the commissure, they stop

Expression 751

of Fasciclin

I and II Glycoproteins

expressing the protein on their growth cones, and expression only persists where their axons fasciculate together in the commissure (Figure 16). Expression of Fasciclin I Corr6lates with Axon Fasciculation An interesting correlation between axon fasciculation and fasciclin I expression is observed for the wellcharacterized aCC and pCC neurons, sibling cells from the first ganglion mother cell of NB l-l (Goodman et al., 1982, 1984; Bastiani et al., 1985; Bastiani et al., 1986; du Lac et al., 1986). These sibling neurons migrate anteriorly from the next posterior segment to their final position just posterior to the posterior commissure; neither neuron expresses fasciclin I at this time (34%; Figure 4A). The growth cones from these two sibling neurons are then confronted with three different longitudinal bundles: the MPlWMP2 fascicle, the vMP2 fascicle, and the U fascicle. The pCC growth cone extends anteriorly along the MPll dMP2 fascicle, which does not express fasciclin I; the aCC growth cone extends posteriorly along the U fascicle, which does express fasciclin I (Figure 4C). After the aCC growth cone begins to extend posteriorly along the U fascicle, the aCC also begins to express fasciclin I on its cell body and axonal surface (Figure 4B). In contrast, its sibling, the pCC, never expresses fasciclin I on its surface. Ultrastructural Localization of Fasciclin I Expression The initial evidence for the surface expression of fasciclin I came from the binding of the MAb to the surface of neurons in living embryos. The ultrastructural localization of fasciclin I confirms that it indeed occurs on the surface of axons that fasciculate together (Figure 5). Moreover, at very early ages, before axons first begin to express the protein, some filopodia already express fasciclin I (Figure 58). At the light microscope level, the protein is seen on the surface of axons in only one of the ten or so major pathways in the anterior commissure (Figures lB, 1E). The electron microscope analysis reveals that the fasciclin l-positive pathway in the anterior commissure consists of three tightly associated bundles of axons (Figures 5A, 5C). The protein appears to be more abundant on axons in one of these associated bundles (dark arrow in Figure 5A) and less abundant, although present, on axons in the other two associated bundles (open arrows). Interestingly, in these other two bundles, the protein appears to be more abundant on the surfaces of the axons that are in closest proximity to the bundle that expresses higher amounts of the protein (Figure 5A). The different levels of expression of fasciclin I on axons within these three associated bundles has been consistently observed in analysis of several different embryos and several different segments within each embryo. The differential expression of fasciclin I on these three associated bundles may be due to differences in intrinsic expression and/or to subsequent stabilization of the protein on particular axon surfaces. Alternatively, the protein might be intrinsically expressed on the surface of axons in only one of the three bundles, and extrinsically acquired by the associated axons that they contact. Further studies

will be required to distinguish between these two alternatives. Whatever the mechanism, this differential expression may suggest that fasciclin I is partly responsible for the pattern of selective affinities both within and between associated bundles. Fasciclin I Is Expressed by a Subset of Sensory Neurons and Sensory Neuron Pathways In the peripheral nervous system (PNS), many sensory neurons express fasciclin I, including the initial pair of pioneer neurons in the limb bud (Figure 3A) (Bate, 1976; Bentley and Keshishian, 1982; Ho and Goodman, 1982; Caudy and Bentley, 1986a, 1986b). In the longitudinal connectives of the CNS at 45% of development, a small ventromedial fascicle, in addition to the dorsolateral U fascicle, begins to express fasciclin I. This fascicle continues to increase in size as it is joined by other fasciclin I-positive sensory axons that grow into the CNS along the segmental nerve (SN) and turn anteriorly along this pathway. Adult Expression of Fasciclin I In the adult segmental nervous system (Tl through A5), only the ventromedial longitudinal pathway expresses fasciclin I; this is the same sensory pathway that expressed fasciclin I in the embryo. Apparently all the interneurons and motoneurons in the CNS and some of the sensory neurons in the PNS (particularly those entering the intersegmental nerve) have stopped expressing fasciclin I by this time. A large number of fasciclin l-positive sensory neurons enter the CNS via the segmental nerve; some of these axons turn anterior along the ventromedial longitudinal sensory tract in the connective (Figure 3B). Thus in the adult CNS, none of the commissural, dorsolateral longitudinal (Figure 3B), or intersegmental nerve pathways express fasciclin I, as they did in the 45% embryo. Moreover, much of the fasciclin I expression disappears by the end of embryogenesis (data not shown). Discussion The primary objective of the present study was to identify molecular candidates for axonal recognition molecules. We generated MAbs that recognize surface antigens expressed on subsets of axon fascicles in the grasshopper embryo. We used two MAbs, 3811 and 8C6, to identify and study the expression of two different membrane glycoproteins (70 kd and 95 kd, respectively), and to purify these proteins for the generation of specific antisera. These two glycoproteins, fasciclin I and II, are localized on different subsets of axon fascicles during development in a spatiotemporal pattern which suggests that they play a role in the events of selective fasciculation. Parallel experiments in our laboratory have used MAbs to reveal a different surface glycoprotein, called fasciclin Ill, that is expressed on a different subset of axons in the Drosophila embryo (Pate1 et al., unpublished data). Moreover, similar experiments on other developing nervous systems have used MAbs to reveal surface antigens that distinguish, for example, subsets of axons in the leech embryo (McKay et al., 1983; Macagno et al., 1983) or different

Cell 752

subsets of dorsal root ganglion (e.g., Dodd et al., 1984).

neurons in the rat embryo

Expression of Fasciclin I and II Is Regional and Correlates with Axon Fasciculation The best evidence supporting the notion that the fasciclin I and II glycoproteins play a role as axonal recognition molecules during selective fasciculation is their regional expression. Both glycoproteins are expressed on particular axon pathways. They are not necessarily expressed over the entirety of an individual neuron, but rather their regional expression correlates with the patterns of axon fascicutation, and suggests that they may play a role as pathway labels. For example, fasciclin I is expressed on the surface of axons in a particular commissural pathway. At the point where the distal axons of these neurons leave this commissural pathway to extend onto other longitudinal pathways, they stop expressing fasciclin I. Similarly, fasciclin II is localized on most longitudinal pathways but not on most commissural pathways, even though most of the neurons whose axons run in these longitudinal pathways are interneurons whose axons first extend across one of the commissural pathways before turning into a longitudinal pathway. Thus the regional expression of these glycoproteins on particular portions of an axon often correlates with the identity of the axon pathway, and not the identity of the neuron. The expression of fasciclin I on groups of neurons appears to be correlated not with their lineage, but rather with their patterns of fasciculation. For example, the aCC and U neurons arise from different neuroblast lineages, share a common axon pathway (the U fascicle), and both express fasciclin I. In contrast, the aCC and pCC neurons arise as siblings from the same neuroblast lineage, choose different axon pathways (e.g., the U fascicle and MPl fascicle, respectively), and differ in their expression of fasciclin I: the aCC expresses it and the pCC does not (Figure 4). Expression of Fasciclin I Is Dynamic and Correlates with Time of Axon Outgrowth From the onset of axonal outgrowth, fasciclin I is expressed on the surface of specific commissural, longitudinal, and sensory axon pathways (Figures lB, lE, 4). In contrast, by the end of embryogenesis, the commissural and longitudinal pathways containing the axons of interneurons and motoneurons in the CNS, and some sensory axon pathways, no longer express fasciclin I. This correlates with the observation that all interneurons and motoneurons are born and differentiate during grasshopper embryogenesis. Thus fasciclin I is not expressed on the surface of mature CNS neurons, but rather is only expressed on their axonal surfaces during the period of axonal outgrowth. Moreover, many sensory pathways (including one ventromedial longitudinal pathway in the CNS) continue expressing fasciclin I throughout life (Figure 3B), in correlation with the continued addition of new sensory neurons. Thus fasciclin I expression persists

when such a putative pathway label is still needed as a guidance cue, whereas it disappears when it is no longer needed. Fasciclin I Is Positionally Expressed at Other Times and Places throughout Development Fasciclin I and II are both expressed on the surface of a variety of neuronal and nonneuronal cells at different times and places throughout embryonic development. For example, fasciclin I, in addition to being expressed on a subset of axon pathways, is transiently expressed in a segmentally repeated pattern during neurogenesis, and is also expressed in a segmentally repeated pattern on the ectoderm of the body wall and limb buds (Figure 4). Thus fasciclin I appears to be used at several different times and places during embryonic development, suggesting that it may serve different but perhaps related functions in different tissues. In each case, the expression of fasciclin I defines a particular subset of cells and/or regions of cells. If these conclusions were based solely on staining with the MA4 one might argue that different proteins sharing the same epitope were being expressed at these different times and places. However, this pattern of staining is seen with both the MAbs and the antiserum against each purified protein; the antisera would be expected to recognize multiple epitopes on each protein. In addition, each MAb immunoprecipitates only a single glycoprotein throughout embryogenesis (Figure 2). Fasciclin I and II Are Candidates for Pathway Labels The fasciclin I and II glycoproteins are good candidates for axon recognition molecules involved in the selective fasciculation choices of embryonic growth cones. However, whereas these molecules are good candidates for pathway labels, neither one alone is sufficient to account for the behavior of the growth cones we have previously observed (e.g., Raper et al., 1984; Bastiani et al., 1984; Bastiani et al., 1986; du Lac et al., 1986). The complexity of fascicles and specificity of growth cone choices suggests that many labels may be involved, and that the behavior of individual growth cones may often be based on the expression of several different molecules. In the future, we hope to determine the structure and function of fasciclin I and II, and to search for other related molecules. Fasciclin I and II have both been purified and characterized, and portions of them have been sequenced (Snow et al., unpublished data); thus it should now be possible to clone the genes encoding them. Because the patterns of selective fasciculation in the early Drosophila embryo are nearly identical to those of the grasshopper embryo (Thomas et al., 1984; Goodman et al., 1984), there may be a concomitant molecular conservation as well. If this is the case, then perhaps the molecular probes for fasciclin I and II isolated from the grasshopper will be useful in finding the homologous genes and proteins in Drosophila and will thus allow a detailed genetic analysis of this problem.

Expression 753

Experimental

of Fasciclin

I and II Glycoproteins

Procedures

Generation of MAbs The connectives (axon bundles connecting the segmental ganglia) from approximately 25 adult grasshoppers (Schistocerca americana) were dissected in Ringer solution, homogenized in PBS with 5 mM EDTA and 20 uglml PMSF, and centrifuged at 1000 x g (4OC) for 10 min. The supernatant was centrifuged at 200,000 x g (4’C) for 1 hr. The membrane pellet was resuspended by sonication in 106 nl of PBS and an equal volume of Freund’s adjuvant, and injected intraperitoneally into a BALB/c mouse (approximately 1 mg of protein per injection). The first injection was with Freund’s complete adjuvant; the remainder were with Freund’s incomplete adjuvant, except for the final injection without adjuvant 3 days before the fusion. Mice (BALBlc) were injected at 3-week or longer intervals, and received at least three injections before fusing their spleen cells with NS-1 myeloma cells (Kohler and Milstein, 1975; Oi and Herzenberg, 1980). Screening of MAbs Hybridoma supernatants were screened histochemically on wholemount 40% grasshopper embryos. Embryos were fixed in 2% paraformaldehyde in Millonig’s buffer for 15-30 min. washed in PBS with 0.1 mglml glycine for 10 min, washed in PBS with 2% Bovine serum albumin and 0.4% saponin (PBS+BSA+S) for 10 min, and incubated with equal volumes of the hybridoma supernatant and PBS+BSA+S overnight at 4OC. Embryos were then washed in PBS for 2 hr, and were finally incubated in a blocking solution of PBS+BSA+S and 5% normal goat serum for another 15 min before being incubated for 2 hr at 22% (or overnight at 4OC) in a l/t00 dilution of FITC-labeled goat anti-mouse IgMAG antibody (Cappel) in PBS+BS+S. Embryos were washed in PBS for at least 2 hr and then viewed at 400x magnification with a Zeiss compound microscope. FITC and Horseradish Peroxldase lmmunocytochemistry for Light Microscopy Further characterization of the MAb binding pattern was done using secondary antibodies labeled with either FITC, RITC, or horseradishperoxidase (HRP). In addition, a bridge system consisting of a biotinylated secondary antibody and a tertiary avidin-HRP complex (Cappel) was sometimes used. HRP was visualized using diaminobenzidine (0.5 mg/ml) and hydrogen peroxide (0.003%). For light-level histology using the fluorochromes FITC and RITC, the embryos were mounted either in PBS containing 0.2% ascorbic acid (pH 8.5) or in 90% glycerol containing 0.2 M n-propyl gallate (Giloh and Sedat, 1982). Embryos reacted for HRP histochemistry were mounted in 100% glycerol and viewed with Nomarski (DIC) optics. Adult CNS (Tl through A5) was fixed for 2 hr with 2% paraformaldehyde in Millonig’s buffer, rinsed in PBS for 10 min, briefly immersed in O.C.T. (Miles), and quick-frozen with carbon dioxide. Sections 10 sm thick were cut on a Slee cryostat and mounted on gelatin-coated slides. Sections were incubated for the same times and in the same solutions as described above for whole-mount embryos. Antigen was visualized with indirect immunofluorescence using FITC-labeled goat anti-mouse IgMAG. Slides were mounted in PBS with 0.2% ascorbic acid (pH 8.5). lmmunoelectron Microscopy Embryos were cultured in an RPMI-based medium (Raper et al.. 1984) and this medium was used in all incubation and washing steps. All steps were at 33°C. Embryos were incubated in a 1:lO dilution of the MAb hybridoma supernatant for 3 hr. washed for 1 hr, incubated for 2 hr in a 1 :lOO dilution of HRP-conjugated goat anti-mouse IgG (Cappel), and then washed for 2 hr. Embryos were fixed in a solution of 1% paraformaldehyde, 2.5% glutaraldehyde, and 0.5% DMSO in Millonig’s buffer (pH 7.2) for 1 hr at 4OC, and then washed in Millonig’s buffer with 1 mglml glycine for 15 min. The HRP was visualized using DAB (0.5 mglml), Beta-D-glucose (1 mglml), ammonium chloride (0.3 mglml), and glucose oxidase (2 U/ml) (Sigma) in PBS (pH 7.3). Embryos were reacted approximately 1 hr, washed in PBS for 15 min, fixed in 2% osmium tetraoxide for 2 hr. washed in PBS, dehydrated in an ethanol senes, and embedded in an Epon and Araldite mixture. Pale-gold sections were mounted on Formvar coated slot grids and viewed without further staining. Labeling with the 5 nm gold-bound goat anti-mouse

IgG (Janssen) was identical to that described above, embryos were not reacted for HRP histochemistry.

except that the

Membrane Preparation Dissected adult connectives were homogenized in a glass homogenizer with a Teflon pestle in 10 mM triethanolamine ((TEA) (pH 8.2) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 rig/ml of the following protease inhibitors: antipain, chymostatin, leupeptin, pepstatin, Nap-Tosyl-L-lysine Chloromethyl Ketone (TLCK), and N-To~yl-~phenylalanine Chloromethyl Ketone (TPCK). The disrupted connectives were centrifuged at 100,000 x g for 1 hr at 4’C. The membrane pellet was stored at -7OOC before being used for immunoprecipitation, at which time it was homogenized in PBS containing 1 mM PMSF. Membrane Protein Labeling and lmmunoprecipitatlon The membrane proteins were isotopically labeled with tz51 and lactoperoxidase as described in Haustein et al. (1975). After centrifugation at 13,000 x g for 15 min, the labeled membranes were resuspended in 10 mM TEA, 0.15 M NaCI, 1% Nonidet-WO (NP40), 0.5% sodium Deoxycholate (DOC), pH 8.2, containing the protease inhibitors described above, for 1 hr at OOC. The lysate was then centrifuged at 100,000 x g for 30 min at 4OC. The supernatant was subjected to immunoprecipitation using antibody preformed complexes as described in van Agthoven et al. (1981). The immunoprecipitated proteins were analyzed on a 7.5% SDS-polyacrylamide gel according to Laemmli (1970). Autoradiography was performed using Kodak XAR-5 film in combination with intensifying screens. Plotein Purification Grasshopper embryos (40%-50% of development) were dechorionated in 50% Chlorox for 5 min. The embryos were extruded from their egg cases and collected by centrifugation at 300 x g for 10 min, followed by three washes in PBS, all performed at 4’C. Membrane proteins were solubilized by the addition of IO mM TEA, 0.15 M NaCI, 2% NP40,0.5% DOC (pH 8.2) containing the protease inhibitors described above. Lysis was achieved by ten strokes in a glass dounce, followed by stirring at 4°C for 1 hr. The lysate was subsequently centrifuged at 13,000 x g for 30 min. Prior to application to affinity columns, the supernatant was clarified by centrifugation at 100,000 x g for 1 hr at 4OC. Affinity columns were prepared using the 3811 and 8C6 MAbs according to Schneider et al. (1982). The lysates were applied to the columns (1 ml) at a flow rate of 5-10 column volumes per hr. The columns were subsequently washed with lo-20 column volumes of the following four solutions: 10 mM TEA, 0.15 M NaCI, 1% NP40 (pH 8.2); 10 mMTEA, 0.15 M NaCl (pH 8.2); 10 mMTEA, 0.15 M NaCI, 0.5% DOC (pH 8.2); 10 mM TEA, 1.0 M NaCI, 1% NP40 (pH 8.2). Application of the lysate and subsequent washes were performed at 4OC, and all solutions contained protease inhibitors as described. The bound antigens were eluted with 10 column volumes of 50 mM triethylamine, 0.15 M NaCI, 1% NP40 (pH 11.5) containing protease inhibitors. Eluted proteins were precipitated by the addition of trichloroacetic acid (TCA) to 20%, followed by incubation with mixing for 30 min at 4OC. The precipitated proteins were collected by centrifugation at 1200 x g for 15 min, washed three times with ice-cold acetone, and dried under vacuum. Preparative SDS-PAGE was performed using a modification of the Laemmli procedure (1970). Generation of Antisera Coomassie blue-stained gel slices containing 5-10 pg of protein were soaked in water for 1 hr and homogenized in a glass dounce (5-10 strokes) using a Teflon pestle. The gel slices were subsequently mixed with an equal volume of complete Freund’s adjuvant and injected into the peritonea of 2- to 4-month-old male rats. Injections were repeated at intervals of 2-4 weeks, using incomplete Freund’s adjuvant for the boosts. Serum was collected 7-10 days following the boosts. Acknowledgments We thank Violette Paragas, Frances Thomas, and Lisa Belluui for technical assistance. This work was supported by grants from the National Institutes of Health, McKnight Foundation, and March-of-Dimes

Cdl 754

Birth Defects Foundation to C. S. G., and postdoctoral fellowships from the FESN Foundation (M. J. 6.) and ACS (A. L. H. and t? M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received

November

IO, 1986; revised

December

Bastiani, M. J., Raper, J. A., and Goodman, C. S. (1984). Pathfinding by neuronal growth cones in grasshopper embryos. III. Selective affinity of the G growth cone for the P cells within the A/P fascicle. J. Neurosci. 4, 2311-2328. J., du Lac, S., and Goodman, C. G. (1985). The first neucones in insect embryos: model system for studying the of neuronal specificity. In Model Neural Networks and BeSelverston, ed. (New York: Plenum Press), pp. 149-174.

Bastiani, M. J., and Goodman, C. G. (1986). Guidance growth cones in the grasshopper embryo. Ill. Recognition glial pathways. J. Neurosci. 6, 3542-3551.

of neuronal of specific

Bastiani, M. J., du Lac, S., and Goodman, C. G. (1986). Guidance neuronal growth cones in the grasshopper embryo. I. Recognition a specific axonal pathway by the pCC neuron, J. Neurosci. 3518-3531. Bate, C. M. (1976). Pioneer 54-55.

neurons

in an insect

embryo.

Nature

of of 6, 260,

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. Exp. Morph. 61, 317-330. Bentley, D. H., and Keshishian, oneer neurons in grasshoppers.

H. (1982). Pathfinding by peripheral Science 218, 1082-1088.

pi-

Blair, S. S., and Palka, J. (1985a). Axon guidance in cultured wing discs and disc fragments of Drosophila. Dev. Biol. 108, 411-419. Blair, S. S., and Palka, J. (1985b). Axon guidance sophila. Trends Neurosci. 8, 284-288.

of Drosophila, Siddiqi, Press), pp. 247-265.

Babu, Hall,

Goodman, C. S., Raper, J. A., Ho, R. K.. and Chang, S. (1982). Pathfinding of neuronal growth cones in grasshopper embryos. In Develop mental Order: Its Origin and Regulation, S. Subtelny and P B. Green, eds. (New York: Alan R. Liss), pp. 275-316. Goodman, C. S., Bastiani, M. J., Doe, C. Cf., du Lac, S., Helfand, S. L., Kuwada, J. Y., and Thomas, J. B. (1984). Cell recognition during neuronal development. Science 225, 1271-1279.

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