Cell, Vol. 70, 553-567,

August 21, 1992, Copyright

0 1992 by Cell Press

Connectin: A Homophilic Cell Adhesion Molecule Expressed on a Subset of Muscles and the Motoneurons That Innervate Them in Drosophila Akinao Nose, Vinit B. Mahajan, and Corey S. Goodman Department of Molecular and Cell Biology Howard Hughes Medical Institute University of California, Berkeley Berkeley, California 94720

Summary Each abdominal hemisegment in the Drosophila embryo contains a stereotyped array of 30 muscles, each specifically innervated by one or a few motoneurons. We screened 11,000 enhancer trap lines, isolated several expressing j%gaiactosidase in small subsets of muscle fibers prior to innervation, and identified two of these as inserts in connectin and Toll, members of the ieucine-rich repeat gene family. Connectin contains a slgnai sequence, ten leucine-rich repeats, and a putative phosphatidyiinositol membrane linkage; in S2 ceils, connectin can mediate homophiiic cell adhesion. Connectin is expressed on the surface of eight muscles, the motoneurons that innervate them, and several gliai ceils along the pathways leading to them. During synapse formation, the protein localizes to synaptic sites; afterward, it largely disappears. Thus, connectin Is a novel cell adhesion molecule whose expression suggests a role in target recognition. introduction Neuronal growth cones follow specific pathways as they seek out and ultimately find and synapse with their appropriate targets. Monoclonai antibody screens have led to the identification of several cell adhesion molecules whose restricted patternsof expression in the developing nervous system suggest roles in growth cone guidance and pathway recognition in both vertebrates (e.g., Jessell, 1988; Furley et al., 1990; Burns et al., 1991) and invertebrates (e.g., Bastiani et al., 1987; Elkins et al., 1990; Grenningloh et al., 1991). However, little is known about the molecules or mechanisms involved in target recognition. Given the different kinds of responses, it is likely that different moiecuies, or at the very least different signal transduction mechanisms, are involved in pathway versus target recognition: during pathfinding, motility continues in a directed fashion, whereas during target recognition, motility shuts down as the growth cone transforms into a presynaptic terminal. The ability of motoneuron growth cones to find and recognize their correct muscles has been a model system for studies on the mechanisms of target recognition in both vertebrates and invertebrates. For example, in both chick (e.g., Tosney and Landmesser, 1985) and zebrafish (e.g., Eisen et al., 1988) embryos, motoneuron growth cones make stereotyped pathway choices as they extend toward the regions where their appropriate target muscles arise. Similarly, in both grasshopper (Ball et al., 1985b) and Dro-

sophiia (e.g., Sink and Whitington, 1990, 1991a; Halpern et al., 1991) embryos, identified motoneuron growth cones extend toward and innervate the appropriate target muscles in a highly stereotyped fashion. Target muscles or other nearby mesodermal cells appear to express attractive guidance cues. For example, the growth cones of chick motoneurons normally grow toward the regions of their appropriate target muscles, If displaced experimentally, these growth cones often take circuitous routes to reach their same targets (Landmesser, 1980; LanceJonesand Landmesser, 1981). identified motoneurons in zebrafish establish cell-specific terminal fields primarily by directed outgrowth of branches on appropriate muscle fibers (Eisen et al., 1989; Pike and Eisen, 1990; Liu and Westerfield, 1990). In the spf-7 mutation, which eliminates trunk muscles, the pattern of motoneuron axon branches is seriously disrupted (Eisen and Pike, 1991). In the grasshopper embryo, skeletal muscles develop from muscle pioneers (Ho et al., 1983; Ball et al., 1985a; Bali and Goodman, 1985a, 1985b), mesodermal cells that appear early in development, enlarge relative to the surrounding mesodermal cells from which they arise, and extend growth cone-like processes that find and establish the insertion sites of the muscles they prefigure. Smaller mesodermal cells cluster around and fuse with the muscle pioneers, which become multinucleate and later differentiate into muscle fibers. Motoneuron growth cones extend into the periphery before the differentiation of mature muscle fibers. They come in contact with several different muscle pioneers and display selective affinities for specific ones (Bail et al., 1985b). When a specific muscle pioneer is ablated prior to innervation, the motoneuron growth cone that would normally have innervated that muscle instead continues to extend past that location (Ball et al., 1985b). In Drosophila, homologs to the muscle pioneers have been described (Leisset al., 1988; Dohrmann et al., 1990; Bate, 1990). In Drosophila, these muscle precursors are thought to arise after fusion of surrounding myoblasts with smaller muscle founder ceils (Bate, 1990). As in the grasshopper, in Drosophilathese muscle pioneers prefigure the larval muscles by extending growth cone-like processes to the sites of muscle insertion, serve as the host for the fusion of surrounding mesodermai cells before differentiating into mature muscle fibers, and appear to provide guidance cues for motoneuron growth cones. Neuromuscular specificity in Drosophila is particularly well suited for studies on the molecular mechanisms of pathway and target recognition. The body wall muscuiature of Drosophila embryos and larvae consists of 30 individually identified muscle fibers in each abdominal hemisegment. Most, if not all, of these muscle fibers are innervated by either one or only a few motoneurons. For example, the RPl motoneuron innervates muscle fiber 13, and the RP3 motoneuron innervates muscle fibers 8 and 7(Sinkand Whitington, 1990,199la; Halpernetal., 1991).

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The RPl and RP3 growth cones initially contact and extend processes over a number of inappropriate muscles as well as contacting their correct target muscles. The processes to the inappropriate muscles are later withdrawn, generating the mature pattern of neuromuscular innervation (Sink and Whitington, 1991a). The synapses from these motoneurons onto their specific muscle fibers always form at defined locations; both the probing growth cones and the final synapses display characteristic shapes and morphologies (Johansen et al., 1989a, 1989b; Halpern et al., 1991). To test the specificity of motoneuron growth cones for specific muscle cell surfaces, a series of experiments was conducted that either duplicate or delete specific muscle fibers in Drosophila. For example, when muscle fibers 8 and 7 are surgically ablated, the RP3 growth cone arborizes over nontarget muscles in a variable and abnormal fashion (Sink and Whitington, 1991 b). Moreover, in numb mutant embryos, muscle fibers 12 and 13 are missing, and the RPl growth cone extends abnormally (Chiba and Keshishian, 1991, Sot. Neurosci., abstract). In specifically timed heat-shock experiments, muscle fiber 13 can be duplicated; in these embryos, RPl, which now has two fiber-13 targets, specifically innervates both muscle fibers 13 (Chibaand Keshishian, 1991, Sot. Neurosci., abstract). Taken together, theseobservations of normal and manipulated embryos in both grasshopper and Drosophila argue for a high degree of specificity in the ability of motoneuron growth cones to recognize particular muscle fibers (Ball et al., 1985b; Chiba and Keshishian, 1991, Sot. Neurosci., abstract; Sink and Whitington, 1991 b) and suggest that specific gene products active in target regions are responsible in part for determining the precise patterns of neuromuscular connectivity. To identify candidates for target recognition molecules in Drosophila, we performed a screen of -11,000 enhancer trap lines @‘Kane and Gehring, 1987; Bellen et al., 1990; Bier et al., 1990) in search of genes that are expressed by specific subsets of muscle fibers prior to and during the stage of motoneuron innervation. In this report, we describe a novel homophilic cell adhesion molecule, called connectin, which is expressed on the surface of a small subset of muscle fibers during the period of motoneuron outgrowth and innervation. Interestingly, connectin is also expressed on the surface of the very motoneuron growth cones that innervate these muscle fibers, as well as on the surface of a subset of peripheral glial cells along the pathways leading to these muscles. During synapse formation, the protein localizes to synaptic sites; afterward, it largely disappears. cDNA sequence analysis reveals that connectin belongs to a family of cell adhesion molecules and receptors with a repeating motif called the leucine-rich repeat (LRR) (Titani et al., 1987; Lopez et al., 1987, 1988; Krantz and Zipursky, 1990; Keith and Gay, 1990). In this paper we also show that another member of the LRR gene family, To// (known for its role in dorsoventral pattern formation; Hashimoto et al., 1988) is also expressed on the surface of a different subset of muscle fibers. The connectin gene has also been independently cloned

(A. P. Gould and R. A. H. White, submitted) based on a totally different screen in search of genes downstream of the homeotic gene Ultrebithorex (Gould et al., 1990). Results We performed a large-scale P element enhancer trap screen (the same as described in Kllmbt et al., 1991) to identify genes expressed by small su bsets of muscle fibers prior to or during innervation. Of the - 11,000 P element insertion lines screened, we isolated seven lines that express 8galactosidase (P-gal) in small subsets (l-8 out of 30) of muscle fibers prior to or during the stage of innervation. In this report, we describe the expression and molecular characterization of one of these lines, rF400, which is an insertion in a gene we call connectin. We also describe the expression of another line, AK80, which is likely to be an insertion in the Toll gene. We used antibodies against both the connectin and Toll proteins to describe their expression on subsets of muscle fibers during the period of motoneuron outgrowth and innervation. The P element insertion in enhancer trap line rF400 maps by polytene chromosome in situ hybridization (data not shown) to location 84C on the left arm of the third chromosome. This enhancer trap line expresses P-gal in the nuclei of 8 lateral (21-24, 18, and 5 [numbering of muscles according to Crossley, 1978; as modified by Bate, 1990)) and 2 ventral (27 and 29) muscle fibers (of the 30 in each abdominal hemisegment) and also in the nuclei of a small subset of central nervous system (CNS) neurons (data not shown). As described later, we identified a transcript near the P element insertion site whose expression pattern closely resembles the P-gal expression in the rF400 line. By using an antibody raised against the protein encoded by this transcript, we found that the protein is expressed on the surface of the same exact muscle fibers that express P-gal in the rF400 line; it is also expressed on the axons and growth cones of the motoneurons that innervate these muscles (see below). Based on this pattern of expression on pre- and postsynaptic cells, its transient localization to the sites of synapse formation, and its function in vitro as a homophilic cell adhesion molecule (described below), we call this molecule connectin. Connectin Is Expressed on a Subset of Muscle Fibers and Motoneurons In Drosophila embryos and larvae, the somatic muscle fibers are arranged in a stereotyped and segment-specific pattern. Each of the abdominal hemisegments 2-7 (A2A7) has an equivalent set of 30 muscles, with a slightly modified pattern in Al. Thoracic segments and the A8 segment have other segment-specific patterns. For simplicity, we describe in detail here only the pattern of connectin expression in Al-A7 (Figure 1). Connectin is expressed in a different but related pattern in the thoracic segments (Figure 1F). We also note that the overall expression level of connectin is somewhat higher in thoracic versus abdominal segments. The ability to visualize connectin expression is based on an antibody against a fusion protein.

Connectin Neuromuscular Development 555

Figure 1. Connectin Is Transiently Expressed on a Subset of Muscles and the Motoneurons That Innervate Them during Embryonic Development Dissected embryos were stained with anti-connectin antibodies and HRP immunocytochemistry. Two abdominal segments are shown in each photograph except for(F), in which one thoracic (Tl) and one abdominal (Al) segment are shown as an example of segmental differences. In this and the following figures (Figures 2-4) anterior is to the left and dorsal is up. Scale bar: 20 urn in (A)-(E) and (G); 25 urn in (F). (A) Early in muscle development (stage 12). connectin is expressed on a subset of muscle founder cells in the lateral (arrow) and ventral (arrowhead) region of the body wall. Several glial cells, including PGl and PG4, also begin expressing connectin among the ventral mesoderm. (B and C) At early stage 13, connectin is expressed on the pioneers for two ventral (27 and 29) and six lateral (21-24, 18, and 5) muscles and on three peripheral glial cells (PGl, PG3, and PG4). The set of muscle pioneers 21-24 are shown by the set of four arrowheads in (8) and (C), muscle pioneer 18 by the closed arrow, muscle pioneers 27 and 29 by the pair of arrowheads in (C). and the PGI and PG4 glia by the open arrows on the left and right, respectively. Muscle pioneer 5 and a peripheral glia PG3 are out of focus and are not shown here. (8) shows the dorsal-pointing growth cone-like extensions of muscle pioneers 21-24 as they grow toward their insertion sites in the epidermis. Connectin expression is also seen on the growth cone(s) of motoneurons exiting the CNS in the SN (long thin arrows) that extend toward and contact PGl (open arrows in [Cl). (0) Slightly later stage than (B) and (C), showing that some of the unfused myoblasts, which are about to fuse with connectin-positive pioneers, begin to express connectin prior to fusion (arrowheads). (E) At stages 14-15, the growth cones of the motoneurons in SNa (thin arrow) reach the lateral region and contact the ventral tip of lateral muscles 21-23. (F) Expression of connectin on muscles and neurons at stage 18 in the T3 and Al segments. Connectin is expressed in a different but related pattern in thoracic versus abdominal segments (note, for example, that the thoracic segments are missing muscle 18; short closed arrow). In each segment, the connectin expression is tightly linked between specific motoneurons (thin arrows) and the muscles they innervate. The open arrow marks the PG3 glial cell in (F) and (0); the asterisk in (F) marks connectin-positive motoneuron cell bodies in the CNS whose axons extend out the SN. (G) At stage 17, the muscle staining is now disappearing, and strong staining only persists at the sites of neuromuscular synapse formation by connectin-positive motoneurons onto connectin-positive muscles (thin arrows; the bottom right arrow marks a connectin-positive growth cone synapsing on muscle 5, which is mostly out of focus). Later in development, connectin expression disappears altogether from both muscles and motoneurons.

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Muscle fibers form by the fusion of myoblasts, beginning at the onset of germband shortening (late stage 11 to stage 12; staging according to Campos-Crtega and Hartenstein, 1985). By the completion of the germband shortening (stage 13) fused myoblasts form the precursor cells for each individual muscle fiber (homologous to grasshopper muscle pioneers; see Introduction). These muscle pioneers extend growth cone-like processes along the epidermis toward their appropriate attachment sites (Bate, 1990). While they are finding their attachment sites, neighboring myoblasts continue to fuse with these muscle pioneers and thus increase in size. By stages 15-18, muscle formation is essentially complete, with the final position and pattern of muscle fibers in the body wall, each attached to both of its appropriate insertion sites. Connectin staining is first seen in one to three myoblasts on the lateral side of the body wall at late stage 11 to early stage 12. At late stage 12, small numbers of myoblasts (7-10 ceils) in the ventral-lateral region of the body wall express connectin (Figure 1A). Some of these cells have the size of doublets or triplets of myoblasts, suggesting that cell fusion has already begun. By early stage 13, connectin is expressed on two groups of fused cells, one in the ventral and the other in the lateral region of the body wall, which can now be identified as the pioneers of individual muscle fibers (Figures 1 B and 1 C). The ventral group is comprised of two muscle pioneers, which form the external oblique muscles 27 and 29. The lateral group is comprised of the six muscle pioneers that form the pleural external muscles 21-24, which arise together; muscle 18, which migrates from a position more dorsal; and muscle 5. Figures 1 B-1G show the development of these muscle pioneers as they enlarge in size by the fusion of surrounding myoblasts and as they explore and establish their correct insertion sites in the epidermis. Connectin is expressed over the entire surface of these muscle pioneers and on their numerous filopodial extensions. Although filopodia are seen to extend in most directions, including areas that the muscles later will not occupy, more and longer filopodia are seen extending to the future insertion sites, suggesting that their selective contact and stabilization with appropriate epidermal sites may guide the extension of the muscle precursor. During this stage no connectin expression is seen in the majority of unfused myoblasts, some of which will later fuse with the connectin-positive muscle pioneers. However, some of the myoblasts that immediately surround and are about to fuse with connectin-positive pioneers do themselves begin to express connectin prior to fusion (Figure 1 D). At stage 18, connectin is expressed over the entire surface of those differentiated muscle fibers whose pioneers were connectin positive (Figure 1 F). Motoneurons extend growth cones out into the periphery toward their target muscles in five major peripheral nerve branches: the intersegmental nerve (ISN) and four branches of the segmental nerve (SNa, b, c, and d) (see Figure 4). In general, dorsal muscles are innervated via the ISN, while lateral and ventral muscles are innervated via one of the four branches of the SN. Motoneurons send their axons into the periphery at late stage 12 to early stage

13. At this stage, connectin is expressed on the axons and growth cones of a subset of motoneurons exiting the CNS via both the ISN and the SN (Figure 1C). Connectin is also expressed on two identified peripheral glial cells, PGl and PG3 (Fredieu and Mahowald, 1989; Klambt and Goodman, 1991), and another glial-like cell, PG4, which has not been previously described (Figures 1 C and 1 D). These glia may serve as guidepost cells for the motoneuron growth cones that express connectin. At this stage PGl sits lateral to the CNS and anterior to ventral muscle pioneers 27 and 29; this glial cell later elongates dorsoventrally to enwrap the SN. The motoneuron growth cones exiting the CNS in the SN grow toward and contact PGl as they leave the CNS. PG3 sits at the dorsal region of the body wall anterior to muscle precursor 18 and sends processes toward the CNS along the ISN; later in development, it enwraps the axons in the ISN. PG4 sits between the two ventral muscle pioneers 27 and 29, later elongates rostrocaudally, and appears at the light level to serve as a substrate for the SNc (see Figure 4). During late stage 13 to stage 15, connectin expression is observed first on a specific subset of motoneuron axons and growth cones in the SNa that grow dorsally along PGl and contact the ventral tip of lateral muscles 21-24 that express connectin (Figure 1 E), second on a subset of motoneuron axons and growth cones in the SNc that grow posteriorly from PGl and then contact PG4, and third in several of the ventral unpaired median (VUM) neurons in the ISN that grow dorsally and reach the dorsal region by stage 15. At stage 18, all or most motoneuron growth cones reach their target muscles and begin synaptogenesis. At stage 18, connectin is expressed on the axons and terminal branches of a specific subset of motoneurons in the SNa, in the SNc, on a subset of VUM axons in the ISN, and on the target muscles they innervate (Figure 1 F; see Figure 4). These motoneurons and muscles include, as identified by the target muscles, lateral muscles 21-24 and 5 innervated from the SNa, ventral muscles 27 and 29 innervated from the SNc, and dorsolateral muscle 18 innervated by a VUM axon from the ISN. At stage 16, no staining is seen on the axons in SNb and SNd; all of these connectin-negative axons innervate connectin-negative muscles. Moreover, with the exception of the one motoneuron that innervates connectinpositive muscle 18, the majority of the axons in the ISN are connectin negative and innervate connectin-negative muscles. Although we cannot exclude the possibility that a few other axons in the ISN express connectin, such expression would have to be highly regionalized (in that the staining stops precisely where the motoneuron that innervates muscle 18 leaves the ISN) and does not include any expression on the axon branches or growth cones that innervate connectin-negative muscles. Thus, connectin is expressed on the surface of a specific subset of motoneurons and their target muscles in a highly specific manner. However, we observe one exception to this rule: connectin staining is occasionally (but not always) seen on an axon innervating muscle 8 (see Discussion); this muscle does not express connectin. At stage 17, the overall expression level of connectin

b;nectin

Neuromuscular Development

both on muscles and the motoneuron axons decreases, andstrongstaining isseenonlyat thesitesof neuromuscular synapses onto connectin-positive muscles (Figure 1 G). In third instar larvae, little or no connectin expression is seen on muscles or in the peripheral nerves (data not shown). Connectin Is Also Expressed in the CNS and in Other Tissues A subset of axons and neuronal cell bodies within the CNS expresses connectin. The staining is first seen at late stage 11 in - 4 neurons per abdominal hemisegment. As axonogenesis begins around stage 12, connectin is expressed on a small number of the axons, including the identified interneuron SPl , several VUM motoneurons, and one of the RP neurons (probably RPl; connectin expression on RP neurons is transient and is not seen at later stages when they send their axons out into the periphery) (Figure 2A). Connectin is transiently expressed at high levels on many longitudinal glia and on some midline glia (Figures 2A and 1 B). During stage 13 to stage 15, axons and cell bodies of additional neurons begin to express connectin (Figure 28). Strong staining is seen on the axons of several VUM neurons that now exit the CNS and extend into the periphery via the ISN; connectin is also seen on the axons of some motoneurons exiting the CNS via the SN. At stage 16, when the entire axon scaffold is formed, connectin is expressed on many axons, many of which bundle together in specific longitudinal axon fascicles (Figure 2C). Stronger staining is seen on one or two fascicles each in the anterior and posterior commissure and in at least three axon bundles in the longitudinal connectives. Prominent staining is also seen on the axons and cell bodies of a group of motoneurons that sit lateral to the posterior commissure and send their axons into the periphery in the SN. Expression of connectin is also observed on subsets of visceral mesoderm and on at least one nerve that innervates the gut. It is also expressed on two to three cells in the dorsal cluster of the peripheral nervous system. Toll Is Expressed on a Small Subset of Ventral Muscles Another enhancer trap line, AK80, expresses P-gal strongly in a small subset of ventral muscles (7, 15, and 16) prior to innervation and more weakly in some other ventral muscles (including 28 and 17) and some lateral muscles. It also expresses P-gal along the midline of the CNS, as well as in the hindgut, the heart, and other tissues. This P element insert maps to chromosome location 97D, the known position of the To// gene (Hashimoto et al., 1988). The expression of Toll in muscles has not been previously described. However, Toll is known to be expressed in the midline of CNS, hindgut, heart, and other tissues in a similar pattern as that seen in AK80 (Gerttula et al., 1988; Hashimoto et al., 1991), raising the likelihood that AK80 is an insert in the To//gene. We therefore examined whether a subset of muscle fibers expresses Toll protein by using an antibody against Toll (Hashimoto et al., 1991). We found that Toll protein is indeed expressed

on the surface of the same muscle fibers that expressed P-gal in AK80. Toll starts to be expressed at late stage 11 to early stage 12 on two to three myoblasts (Figure 3A). During stages 13-14, Toll is expressed on two to four muscle pioneers (Figure 38). By stage 15, the overall expression of Toll on these muscles is decreasing, and strong staining is now seen only along the boundaries of the two pairs of differentiated muscles 6 and 7 and 15 and 16 (Figure 3C). It is also more weakly (and transiently) expressed at the boundary between muscles 28 and 15 and between 16 and 17. These regions correspond to the muscle innervation sites. Comparison of the pattern of P-gal expression in enhancer trap line AK80 with Toll antibody staining leads to the ambiguity of whether or not muscle 6 expresses Toll. In the CNS, Toll is expressed on a subset of midline glia as previously described (Figure 4; Hashimoto et al., 1991). No expression is seen on the axons or cell bodies of motoneurons or interneurons during embryogenesis. Toll was initially isolated as a maternal mutant that affects dorsoventral pattern formation (Anderson et al., 1985). Genetic analysis shows that Toll functions zygotically as well as maternally (Gerttula et al., 1988). To// encodes a transmembrane protein with numerous tandem copies of a 24 aa motif (LRR) in its extracellular domain (Hashimoto et al., 1988). The expression of both connectin and To// is summarized in Figure 4. Cloning of connect/n cDNA Genomic DNAsequences flanking the P element insertion site in line rF400 were recovered by the plasmid rescue method. These genomic sequences (approximately 1 kb of genomic sequence) were used as a probe to isolate overlapping genomic clones from a wild-type genomic library (Figure 5). Northern blot analysis shows that a 3.5 kb fragment, which includes the P element insertion site, hybridizes to a transcript of approximately 5 kb (data not shown). This fragment was used to isolate several overlapping cDNAs from a Igtll cDNA library made from 9-12 hr embryos (Zinn et al., 1988). To determine if the observed P-gal expression pattern in the rF400 line corresponds to the distribution of the transcript encoded by the cDNA, we performed in situ hybridization to whole-mount embryos, using one of the cDNA clones as a probe. The transcript was detected in the same muscle fibers that express @gal in rF400 (data not shown). The transcript was also detected in a similar manner to the P-gal expression in a small subset of CNS neurons and in visceral mesoderm. Connectin Is a Surface Molecule with LRRs The complete nucleotide sequence was determined for the longest cDNA of 3.2 kb (Figure 6). One open reading frame (ORF), extending from nucleotides 460-2505, was found, corresponding to a putative protein of 682 aa, with a calculated molecular size of 76 kd. The nucleotide sequence preceding the initiating methionine (TAAAATG) corresponds at three of four positions to the consensus sequence for Drosophila translational start sites (Cavener,

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Figure 2. Connectin

Is Also Expressed

on a Subset of Neuronal Cell Bodies, Axons, and Glia during the Embryonic

Development

of the CNS

(A) Just after the onset of axonogenesis (stage 12) connectin is expressed on a small number of axons, including the commissural interneuron SPI (arrowhead) and several of the commissural RP motoneurons (long thin arrows) within the CNS (it is not expressed on these motoneurons in the periphery); staining is also beginning on some of the longitudinal glia (closed arrows). (B) At early stage 13, axons and cell bodies of additional neurons begin to express connectin. Strong staining is seen on the growth cones and axons of the VUM neurons (short open arrows) and on two commissural axon bundles. Strong expression is also seen on many longitudinal glia (short closed arrows), particularly over the region within each neuromere where the neuropil forms. (C) At stage 16, when the entire axon scaffold has formed, stronger connectin staining is seen on one or two fascicles each in the anterior and posterior commissures and on at least three axon bundles in the longitudinal connectives. Prominent staining is also seen on a group of motoneurons (arrowhead) whose axons project out the CNS in the SN (long thin arrow). Scale bar: 20 urn in (A) and (B); 35 pm in (C). Figure 3. Toll Is Transiently

Expressed

on a Subset of Muscles during Embryonic

Development

Dissected embryos were stained with anti-Toll antibodies and HRP immunocytcchemistry. In each of these photographs, part of the CNS and ventral portion of the body wall of two abdominal segments are shown (note that Toll is expressed along the midline of the developing CNS; open arrow in [A] and [B]).

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Toll

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Figure 4. Summary of Connectin pression

and Toll Ex-

(Top) Schematic diagrams showing expression of connectin and Toll (yellow) on the surface of subsets of muscle fibers at stage 16 and expression of connectin of the subset of motoneurons that innervate the connectin-positive muscles (red). Left diagram is viewed from inside the embryo, while right diagram is viewed from outside in terms of layers of muscles. Blue muscles are the nonexpressing muscles. (Bottom) Schematic time line of connectin expression on mu6cle pioneers (yellow), motoneuron axons and growth cones (red lines and arrows), and glial cells (green). (For simplicity, muscle 5 is not shown). See text for details. ISN, intersegmental nerve; SN, segmental nerve; a, b, c, and d, branches of the SN.

stage 13

stage 14

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ISN

1987). This methionine is followed by a stretch of hydrophobic amino acids, a characteristic feature of a signal sequence (von Heijne, 1983). Hydropathy analysis reveals no other hydrophobic segment that could span the membrane. However, there is a stretch of some 17 mostly hydrophobic amino acids at the carboxyl end that are characteristic of proteins that are attached to the membrane via a phosphatidylinositol (PI) anchor(Ferguson and Williams, 1988).

These amino acids are preceded by the amino acids that fulfill the consensus for the cleavage and attachment site for a PI-lipid anchor. As described earlier, the antibodies directed against the protein stain the surface of cells in embryos. Furthermore, cell transfection experiments show that the protein is expressed on the cell surface and promotes cell aggregation (described below). These results suggest that the protein is attatched to the membrane, presumably via a PI anchor.

(A) At stage 12, Toll is expressed on two to three myoblasts per hemisegment (arrowheads) and on a subset of midline cells (open arrow) in CNS. (B) Stage 13 embryos. Toll is expressed on two to four muscle pioneers. (C) At stage 16, strong staining is seen along the boundaries of the differentiated muscles 15 and 16, which correspond to the muscle innervation sites (arrowheads). Scale bar: 20 urn.

I I I

I I -10 hFI

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m Figure 5. Structure

of the connecfin

Gene

A molecular map of the region surrounding the P[lacZ] enhancer trap element insertion in the rF400 line and the connectin transcription unit is shown. EcoAl and Xhol restriction sites in the genomic DNA are indicated on the line above the scale, which has its zero point located at the P element insertion site. The extent of the genomic )i phage and cosmid clones are shown under the scale. The approximate positions of the four major exons, mapped by comparison with cDNA and genomic clones, are indicated at the bottom. The ORF is indicated by the solid region; translation starts in the second exon (arrow).

To demonstrate further that connectin is PI linked, we incubated membranes from connectin-expressing S2cells (see below) either with or without PI-phospholipase C (PIPLC). The PLC treatment led to the release of over 50%1 of the membrane-bound connectin, whereas no connectin was released from membranes in the control (data not shown). These data further argue that connectin is PI linked. The connectin protein contains ten 24 aa LRRs (Figures 7A and 7C). LRRs have been identified in a variety of different proteins from a wide range of species, including, for example, human leucine-rich &-glycoprotein and a noncatalytic domain of yeast adenylate cyclase (Takahashi et al., 1985; Kataoka et al., 1985). These repeats are on average 24 aa in length and are characterized by a periodic distribution of hydrophobic amino acids, especially leucine residues, separated by more hydrophilic amino acids. Each repeat unit can potentially adopt an amphipathic structure. Several possible functions have been suggested for LRRs. They could play a role in protein-protein interactions(Titani et al., 1987; Lopezet al., 1987,1988) or in mediating interactions between the protein and cellular membranes (Takahashi et al., 1985; Kataoka et al., 1985). In Drosophila, prior to this report, LRRs were found in Toll, chaoptin, and slit (Figures 7A and 7C) (Hashimoto et al., 1988; Reinke et al., 1988; Rothberg et al., 1990). Toll is a transmembrane protein with two regions of some 16 and 3 LRRs (Figure 7C). The chaoptin protein is attached to the membrane via a PI anchor, and most of the protein is composed of some 41 LRRs (Figure 7C). Slit is a secreted protein with four tandem arrays of LRRs, each with five to six repeats. Some of the proteins with LRRs are known to share amino acid similarity extending to either amino-terminal

LRR-flanking sequences, carboxy-terminal LRR-flanking sequences, or both (Rothberg et al., 1990; Schneider et al., 1991). This sequence similarity is found in connectin in the carboxy-terminal LRR-flanking region but not in the amino-terminal flanking region (Figure 76). This similarity has been found in two other Drosophila LRR proteins, Toll and slit, and in some of the vertebrate LRR proteins, including human platelet glycoprotein lb (Titani et al., 1987; Lopez et al., 1987, 1988) and oligodendrocytemyelin glycoprotein (Mikol et al., 1990). One major characteristic of this region is the four cysteines that are highly conserved among this group of proteins. Connectin, however, lacks the fourth cysteine. A functional role for this region has been demonstrated in vivo; mutations in the cysteines contained within this region in Toll confer adominant phenotype. Aside from the LRRs and the carboxyflanking region, no significant amino acid similarity to any other protein in the data base was found, as determined by the BLAST program (Altschul et al., 1991). Connectln Cen Function as a Homophillc Cell Adhesion Molecule Many of the proteins with LRRs have been implicated in cell adhesion and/or cell recognition. In the cases of Drosophila chaoptin and Toll, there is evidence that these proteinscan mediate cell adhesion, chaoptin in a homophilit fashion (Krantz and Zipursky, 1990) and Toll in a heterophilic fashion (Keith and Gay, 1990). To study whether connectin can function as a cell adhesion molecule, we transfected nonadhesive Drosophila S2 cells (Snow et al., 1989) with a connectin cDNA under the control of the metallothionein promoter. The connectin cDNA (with the entire ORF) was cloned into the pRmHa3 vector (Bunch et al., 1988) downstream

Connectin 561

Neuromuscular

Figure 6. Nucleotide

Development

and Deduced

Amino Acid Sequence

of a connectin

The nucleotide sequence of the 3.2 kb cDNA clone and the translated in bold, and the LRR regions are underlined by a dashed line.

of the metalothionein promoter. This construct (pRmHacon) was cotransfected into the S2 cell line with the pPC4 plasmid, which encodes a Drosophila a-amanitin-resistant RNA polymerase II gene (Jokerst et al., 1989). S2 cells grow normally as single unclumped cells with low adhesivity and have been used to study the function of a variety of Drosophila cell adhesion molecules (for review see Hortsch and Goodman, 1991). After selection with a-amanitin, the resistant cells were plated in agarose gels at low density to isolate clonal cells. Aggregation experiments were conducted as both transient assays on mixed populations and as assays on clonal cell lines. One clonal line, connectin-expressing S2 (SPcon), which expresses connectin at high levels, was used for the following experi-

cDNA Clone

sequence of connectin

protein are presented. The signal peptide is underlined

ments, although the aggregation assays were replicated with mixed populations. Western blot analysis showed that S2-con cells express a 62 kd protein that is recognized by the anti-connectin antibodies and that is not present in the parent S2 cells (data not shown). The size of this protein is slightly smaller than that predicted from the cDNA sequence (76 kd, including signal peptide and PI-cleaved carboxy terminus). SP-con and control S2 cells were cultured overnight with gentle agitation in the presence of Cu2+, the inducer of the metallothionine promoter. As shown in Figure 8, Se-con cells form large aggregates consisting of hundreds to thousands of cells. No aggregation was observed in either control S2 cells or in SBcon cells without induction. To deter-

A

connectin

C

leucine rich repeats

V

K

ED:

I

!I;:

connectin

DRY HEG TSE GDT

Toll

K! consensus Connectin Toll Chaoptin Slit

B

sequences

P--LF-H--NL--L-L--N-L--L ----F--L--L--LDLS-N-L--I ----F--L--L--L-L--N-I--L

carboxy-flanking

regions

Connectin Toll-l Toll-2 Slit-1 SW-2 Slit-3 Slit-4

ELKNRTRHLQLRD----SLE QLV VHKPQYSRQFKLRTD k;T IFMERIG------DRN ---------LAP DYL KIPI ---------ETS

W

KI

consensus

L LSDNPF V Figure 7. Structure

CDC L WF L

of the Connectin

VWERI

kt: Ek

---------NGG ---------EPG

ZE

R

Protein and Comparison

NI

ARC with Other Drosophila

PE

I V

LP

FKCS

LRR Proteins

(A) The ten 24 aa LRRs of connectin are compared with the consensus sequences of other Drosophila LRR proteins: Toll, chaoptin, and slit. Amino acids representing greater than 40% identity at each position (for all repeats) are highlighted. (B) Carboxy-flanking region (flanking LRRs) from connectin is aligned with corresponding regions from Toll and slit. The four cysteine residues found in this region are marked (gray); the fourth one is missing in connectin. Other amino acids representing greater than 40% identity at each position are marked (black). (C)Schematic diagram showing the domain structure of connectin, Toll, and chaoptin. Each hatched rectangle represents one LRR. The horizontallined boxes denote carboxy-flanking regions and closed boxes indicate signal peptides and the transmembrane domain (in the case of Toll). The stippled region represents the plasma membrane.

mine whether connectin can bind in a homophilic fashion (or, alternatively, binds in a heterophilic fashion to a connectin receptor that is endogenously expressed by S2 cells), we examined whether S2 cells can bind to SP-con cells. S2 cells and SP-con cells were mixed together in a 1:l ratio and were allowed to aggregate as described above. To distinguish the cells, S2 cells were labeled with the lipophilic dye Dil prior to aggregation. Most or all of the S2 cells remained as single cells and were not found in the aggregates formed by SP-con cells (data not shown). In a control experiment, labeled and unlabeled SP-con cells were mixed. Both labeled and unlabeled cells were observed in the aggregates, showing that the labeling has no effect on the aggregating activity of the cells. These results indicate that, similar to chaoptin, connectin can also mediate cell adhesion in a homophilic fashion.

amount of mRNA; no null mutation yet exists. This is because the second exon, which contains the start of translation, is separated by an intron of over 20 kb from the first exon and the location of the P element (see Figure 5). This hypomorphic connectin allele shows no gross phenotype in that all eight normally connectin-positive muscles are still present and appear to be innervated, although the specificity of this innervation is impossible to assess. Even when a null mutation is available, a proper cellular analysis of phenotype awaits anatomical probes that, in the absence of normal connectin expression, will reveal the growth cones and axons of the specific motoneurons that normally express connectin. Only with such probes will it be possible to analyze the behavior of individual growth cones. Discussion

Prospects for a Genetic Analysis of connectin Function At present, it is difficult to do a proper cellular analysis of connectin function. Imprecise excisions of the P element inserted in the 5’ end of the connectin gene in line rF400 have generated a series of small deletions that lead to viable, hypomorphic mutant alleles, which by molecular analysis appear to be producing -1120 of the normal

Little is known about the molecular mechanisms that control the formation of specific synaptic connections. The goal of this study was to identify for future molecular genetic analysis candidate target recognition molecules that potentially control neuromuscular specificity in Drosophila. We used the enhancer trap method to identify a novel cell adhesion molecule, connectin, which is transiently ex-

Connectin 563

Neuromuscular

Figure 8. Aggregation nectin cDNA

Development

of Drosophila

52 Cells Transfected

with con-

The 3.2 kb connectin cDNA, under the control of the inducible metalothionein promoter, was cotransfected into S2 cells with a plasmid that confers a-amanitin resistance (SP-con cells). S2 cells (A) and S2-con cells (6) were allowed to aggregate with gentle agitation in the presence of Cu2+ for 18 hr. Scale bar: 100 pm.

pressed on a small subset of motoneuron growth cones and on the specific target muscles they innervate. Connectin belongs to the family of cell adhesion molecules with LRRs. Its specific expression on motoneuron growth cones and on target muscles, its transient localization to sites of synapses, its disappearance after synapse formation, and its function as a homophilic cell adhesion molecule in vitro all suggest that connectin is likely to play a role in neuromuscular specificity. Interestingly, Toll, a related member of the LRR gene family, is also transiently expressed on a different subset of muscle fibers, raising the possibility that these two related proteins may function as part of a broader recognition system. A remarkable aspect of connectin expression is its specificity both in presynaptic motoneurons and postsynaptic muscles (Figure 4). Connectin is expressed on two groups of muscle fibers (2 ventral [27 and 291 and 6 lateral muscles [21-24,16, and 51) of the 30 muscle fibers in each abdominal hemisegment. It appears to be expressed specifically by all of the motoneurons that innervate these eight mus-

cles, even though their axons extend in three different peripheral nerve branches. Connectin is not expressed on all of the axons in any one of these peripheral nerves; instead, it is only expressed on the specific subset of motoneurons that innervate connectin-positive muscle fibers. However, we have noticed one exception. Connectin is sometimes seen on at least one axon that innervates muscle 6 that does not express connectin. It is known, however, that muscle 6 is jointly innervated by a motoneuron that also innervates muscle fiber 5 (Cash et al., 1992) a connectin-positive muscle fiber. Moreover, since we do not see this connectin-positive axon to muscle 6 in all segments, it is also possible that this is a transient aberrant nerve branch from motoneurons targeted to other connectin-positive muscles. Transient axon processes over nontarget muscles have been reported in Drosophila (Sink and Whitington, 1991 a). In the peripheral nervous system, connectin is also expressed on at least two identified glial cells (PGl and PG3) and anotherglia-like cell (PG4) that sit along the peripheral pathways and that may serve as guidepost cells for motoneuron growth cones. In the CNS, connectin expression is revealed on a subset of axon pathways and some glia in a highly stereotyped fashion. These observations suggest that connectin may also play a role in other aspects of growth cone guidance in both the CNS and the peripheral nervous system. In this study, we also found that the Toll protein is strongly and transiently expressed on the surface of two other pairs of ventral muscles (6 and 7 and 15 and 16). Unlike connectin, the Toll protein is not observed on the motoneuron growth cones that innervate these muscles nor on any other neurons. However, it is possible that these growth cones express a receptor for Toll and specifically interact with their target muscles that express Toll. The function of Toll as a heterophilic adhesion molecule in vitro (Keith and Gay, 1990) is consistent with this idea. Both connectin and Toll start to be expressed at an early stage of muscle development (just at the onset of cell fusion) by a small number of myoblasts, thus sewing as good markers to study the formation of specific muscle fibers. The specific expression of connectin and Toll indicates that these founder cells (Bate, 1990) are already committed to particular muscle cell fates by this early stage of muscle development. These muscle founder cells fuse with surrounding cells to form the muscle pioneers (e.g., Ho et al., 1963; Bate, 1990) that extend numerous filopodia and appear to extend toward and attach to the appropriate muscle insertion sites in the epidermis. During this process, connectin or Toll expression is generally not obsewed on most of the surrounding myoblasts, some of which will later fuse with these pioneers. However, some of the unfused myoblasts that immediately surround the connectin-positive muscle pioneers do begin to express connectin prior to fusing with the muscle pioneers. This may suggest that the muscle pioneers induce the myoblasts immediately surrounding them, probably through direct cell-cell contact, to express muscle-specific molecules such as connectin and Toll and thus to entrain these cells to follow their specific developmental program.

Connectin and Toll both contain in their extracellular domains a series of tandem repeats of a 24 aa motif, the LRR. LRRs have been found in a variety of proteins in organisms from yeast to humans. Among the diverse LRR family of proteins, a subgroup can mediate cell adhesion, including human glycoprotein 1 b, Drosophila chaoptin, Toll, and, as reported here, connectin. Glycoprotein 1 b is a transmembrane heterodimeric protein and is involved in the von Willebrand factor (vWF)-dependent platelet to blood vessel adhesion (Titani et al., 1987; Lopez et al., 1987, 1988). The role of LRR domains in adhesion is suggested since a polypeptide fragment from the a chain containing LRR domains binds to vWF (Wicki and Clemetson, 1985; Handa et al., 1988). Chaoptin, a homophilic adhesion molecule, consists almost entirely of LRR domains, making it likely that LRR domains mediate its adhesive properties (Reinke et al., 1988; Krantz and Zipursky, 1990). By using the S2 cell transfection method (Snow et al., 1989; see review by Hortsch and Goodman, 1991) we have shown that connectin can mediate homophilic cell adhesion. This is consistent with the idea that connectin is involved in the specific interaction of motoneuron growth cones and their target muscles, both of which transiently express the protein on their surfaces during the period of synapse formation. Toll has also been shown to mediate cell adhesion in a heterophilic fashion by this same in vitro assay (Keith and Gay, 1990). Although these results point to a function of these proteins as cell adhesion molecules, it is possible that connectin and Toll may function as receptors mediating signal transduction during target recognition and synapse formation. Genetic evidence shows that Toll acts as a signal-transducing receptor in dorsoventral pattern formation; the cytoplasmic domain of Toll is similar to the interleukin 1 receptor (Schneider et al., 1991). We have no direct evidence for the role of connectin in signal transduction. Connectin lacks a cytoplasmic domain and appears to attach to the membrane via a PI linkage. However, connectin could transduce signals, as suggested for other PI-linked molecules (Stefanova et al., 1991). Because of the very different kinds of responses (continued growth cone motility versus transformation into a presynaptic terminal), it is possible that different signal transduction mechanisms might be involved in pathway versus target recognition. Nevertheless, the expression of connectin suggests that it could play a role in both events. Its expression in the CNS on a subset of axon pathways and its transient expression on a subset of longitudinal glia, as well as its expression in the periphery on a su bset of growth cones and the glia they follow, suggest a function in pathway recognition (similar, for example, to fasciclin II; see Grenningloh et al., 1991). However, its expression in the periphery on asubset of motoneuron growth cones and the very muscle fibers they innervate suggests an additional function in target recognition. If connectin does indeed help motoneuron growth cones recognize 8 (21-24, 18, 5, 27, and 29) of the 30 muscles in each abdominal hemisegment, it must be only part of the mechanism that generates the final pattern of specificity.

Although the connectin-expressing motoneurons innervate these muscles, they are able to distinguish among them. For example, certain motoneurons appear to innervate selectively 1 or 2 out of the cluster of 8 connectinpositive muscles (21-24, 18, and 5). It has been postulated for many years that both subsets of muscles and motoneurons must possess distinct molecular identities that allow the growth cones of motoneurons to find and synapse with their appropriate target muscles. In support of this notion, four homeodomain proteins (engrailed in zebrafish and S59, even-skipped, and apterous in Drosophila) have been shown to be expressed in specificsubsetsof musclefibers(Hattaet al., 1990; Dohrmann et al., 1990; Ball et al., 1991, Sot. Neurosci., abstract; C. Bourgouin et al., submitted). Moreover, Drosophila evenskipped isexpressed in asubset of identified motoneurons that innervate the even-skipped-expressing muscles (Ball et al., 1991, Sot. Neurosci., abstract), leading to the suggestion that this transcription factor might control the coordinated expression of recognition molecules both pre- and postsynaptically. But is there any evidence that different muscles express distinct surface labels during innervation? Halpern et al. (1991) have recently shown that Drosophila fasciclin III, a homophilic cell adhesion molecule of the immunoglobulin gene superfamily (Snow et al., 1989; Grenningloh et al., 1990), is transiently expressed on a subset of muscles. In the present study, we have shown that connectin and Toll, both members of the LRR gene family, are transiently expressed on different subsets of muscle fibers. These three examples provide strong evidence that individual muscle fibers are differentially labeled by specific cell adhesion molecules. Do motoneuron growth cones also express distinct surface labels during innervation? Are the same recognition molecules deployed on the motoneuron growth cones as on the muscles they innervate? Fasciclin Ill is transiently expressed on muscles 8,7,15, and 18 and on some motoneuron growth cones, at least one of which (RP3) innervates muscles 8 and 7 (Halpern et al., 1991). However, the correlation with motoneurons is not so precise, since RPl expresses fasciclin III but extends right over muscles 8 and 7 to innervate fasciclin Ill-negative muscle 13 and since muscles 15 and 18 appear to be innervated by fasciclin Ill-negative growth cones (Pate1 et al., 1987; Halpern et al., 1991; A. N. and D. Van Vactor, unpublished data). Moreover, fasciclin Ill is widely expressed on most epidermal cells and thus is not restricted to motoneurons, glia, and muscles during innervation. The most compelling case for pre- and postsynaptic specificity in the expression of a cell adhesion molecule is seen for connectin that is transiently expressed on the surface of eight muscles, the motoneurons that innervate them, and several glial cells along the pathways leading to them. During synapse formation, the protein localizes to synaptic sites; afterward, it largely disappears. This pattern of expression suggests a role for connectin in both pathway and target recognition during the generation of neuromuscular specificity.

glmectin

Neuromuscular

Development

Experimental Procedures Enhancer Trap Screen A large-scale enhancer trap screen was conducted as described before (Klambt et al., 1991). In brief, 5700 P[w’, IacZ] and 5000 P[ry+, IacZ] insertion lines were generated by crossing the transformant line to flies carrying the P[ry+; A2-31 insertion as a stable source of transposase activity. The embryos from the resultant 10,700 lines were stained with X-gal in custom-made 36well plexiglass dishes and were examined for B-gal expression under a dissecting microscope. Those that expressed B-gal in a subset of cells along the body wall were mounted under a coverslip and analyzed under a compound microscope to determine whether the expression was in muscles or in other tissues in the body wall. The lines that had muscle expression were then subjected to more detailed analysis by dissecting the stained embryos as described below.

p-Gal Staining and Horseradish Peroxidase lmmunocytochemiatry For a more detailed analysis of p-gal expression, embryos were dechorionated, fixed, devitellinized, and incubated in the staining solution overnight as described (Klambt et al., 1991). The stained embryos were cleared in 50% and 70% glycerol in phosphate-buffered saline (PBS) and filleted open on a glass slide by dorsal excision using tungsten needles. All viscera and yolk were removed to expose the nerve cord and body wall. Horseradish peroxidase (HAP) immunocytochemistry was used to study the expression pattern of connectin and Toll protein. Embryos were manually dechorionated and devitellinized on double-sided tape. The embryos were then attached to clean glass slides in PBS and dissected using tungsten needles to make fillet preparations. Glass slides were precoated with a thin layer of Silgard (Corning) for the dissection of stage 17 embryos. Preparations were fixed in 4% paraformaldehyde for 10 min, washed with PBT (PBS + 0.1% Triton X-100) for 30 min, blocked with PBT containing 5% normal goat serum for 30 min, and incubated with primary antibodies (anti-connectin mouse serum at a dilution of 1:300, and anti-Toll rabbit serum [Hashimoto et al., 19911 at a dilution of 1:50) overnight at 4OC. The embryos were washed for 1 hr with PBT and incubated in secondary antibodies (HRP-conjugated anti-mouse and anti-rabbit IgG, Jackson Immunoresearch labs, 1:300 dilution) for 2 hr at room temperature. After washing with PBT, they were reacted in 0.3 mglml diaminobenzidine in PBT for 7-10 min and cleared in 50% and 70% glycerol. The preparations stained by X-gal or HRP were examined and photographed using a Zeiss Axiophot microscope and Normarski optics.

Cloning and Sequencing The plasmid rescue method was used to recover DNA sequences flanking the rF400 Pelement insertion point asdescribed before (Mlodzik et al., 1990). The Drosophila genomic library (Maniatis et al., 1978) was screened using the rescued sequences (approximately 1 kb) as a probe. Three overlapping clones, including the first exon, were isolated. A cosmid library (J. Tamkun, unpublished data) was used to isolate more 3’genomic sequences, including second to fourth exons. cDNAs were isolated by screening a cDNA library made from 9-12 hr embryos (Zinn et al., 1988) with the genomic 3.8 kb EcoRI-Xhol fragment encompassing the P element insertion point. One of the cDNA clones was completely sequenced for both strands by the chain termination method (Sanger et al., 1977) using Sequenase (US Biochemical Company). The region including the stop codons was also sequenced in two other independent cDNAs, which gave identical sequences. The predicted protein sequences were analyzed using the FASTDP program (Intelligenetics). Other molecular biological techniques were performed using standard methods (Sambrook et al., 1989).

Production of Fusion Proteins and Generation of Antibodies A bacterial ffpE fusion gene was constructed by inserting a 845 bp Bglll-Xhol fragment of connectin cDNA, including LRR domains 5-l 0, into the BamHI-Sall site of the pATHI plasmid. Fusion protein was prepared by making an extract of purified inclusion bodies (Spindler et al., 1984). For immunization of mice, 100 ug of protein was emulsi-

fied in RIBI adjuvant (Immunochem Research). Mice were subsequently injected at 3-week intervals. Ten days after the third and fourth injection, the serum was collected and used for this analysis. The specificity of the antisera was confirmed by staining embryos derived from a genetic stock containing a large deficiency that removes the connectin gene as well as other neighboring genes.

Transfection of S2 Cells and Aggregation Assay For the expression of connectin in S2 cells, the complete 3.2 kb connecrin cDNA was cloned into the EcoRl site of pRmHa-3 (Bunch et al., 1988). The resultant plasmid, pRmHa-3-con, was cotransfected into S2 cells with the plasmid pPC4 (Jokerst et al., 1989) which confers a-amanitin resistance. For transfections, 50 PI of DNA solution containing 16 ng of pRmHa-con and 4 pg of pPC4 was mixed with an equal volume of lipofectin reagent (BRL) and added to S2 cells that wereplatedatadensityof 1 x 1OYml to2 x lOp/ml in Bmlof serum-free Schneider’s medium (GIBCO). After 18 hr, 3 ml of the medium with fetal calf serum (to 12%) was added. On the following day, a-amanitin (Sigma) at a final concentration of 5 fig/ml was added to the culture. After selection, the amanitin-resistant cells were cloned in 0.3% agar in the presence of an irradiated feeder layer of pPC4-transformed S2 cells. For cell aggregation assays, cells were collected by centrifugation and resuspended in the culture medium containing 0.7 m M CuSO, at a density of 1 x 1 OBcells/ml. Two to five milliliters of this suspension was added to a 50 ml conical tube and agitated on a rotary shaker at 150 rpm for 15-18 hr at 25OC. For mixing experiments, cells were labeled with the lipophilic dye Dil (Molecular Probes) prior to aggregation. After aggregation, Hoechst dye (Molecular Probes) was added to a final concentration of 250 pglml to allow all cells to be visualized. Aliquots were spotted onto slides for examination by fluorescence with a Zeiss epifluorescence compound microscope. Other details of cell culture and aggregation assays are as in Snow et al. (1989).

PLC Treatment The membranes from SP-con cells were incubated for 1 hr at 37OC either with (5 uglml in PBS)or without PI-PLC (Boehringer-Mannheim). After centrifugation at 100,000 x g for 1 hr in a table-top ultracentrifuge, the pellet (membrane fraction) versus supernatant were run on a Western blot. The PLC treatment led to the release of over 50% of the membrane-bound connectin, whereas no connectin was released from the membrane in the control.

Acknowledgments We thank Christian Klambt for his help with the enhancer trap screen, Karen Jepson-lnnes for technical assistance in the initial sequencing of the connectin cDNA, Ka Lai Chang for technical assistance with the genomic walk, Mark Seeger for help with cell transfections, Ursula Weber for help with chromosome in situs, and Nipam Pate1 for help with antibody staining. We also thank Carl Hashimoto and Kathryn Anderson for anti-Toll antibodies, John Tamkun for his cosmid library, and David Van Vactor and Mark Seeger for critical reading of the manuscript. We are grateful to Rob White for exchanging data on the connectin gene and revising our genomic map prior to publication. Research was supported by a Miller Institute Postdoctoral Fellowship and an American Cancer Society California Division Senior Postdoctoral Fellowship to A. N. and by National Institutes of Health grant 18366 to C. S. G., who is an Investigator with the Howard Hughes Medical Institute. 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

May I, 1992; revised June 17, 1992.

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Cdl 588

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Connectin 567

Neuromuscular

Development

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of neuromuscular

specific-

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GenBank

Accession

The accession M96647.

Number

number

for the sequence

reported

in this paper is