Report

Drosophila Synapse Formation: Regulation by Transmembrane Protein with Leu-Rich Repeats, CAPRICIOUS

See allHide authors and affiliations

Science  26 Jun 1998:
Vol. 280, Issue 5372, pp. 2118-2121
DOI: 10.1126/science.280.5372.2118

Abstract

Upon reaching the target region, neuronal growth cones transiently search through potential targets and form synaptic connections with only a subset of these. The capricious (caps) gene may regulate these processes in Drosophila. capsencodes a transmembrane protein with leucine-rich repeats (LRRs). During the formation of neuromuscular synapses, caps is expressed in a small number of synaptic partners, including muscle 12 and the motorneurons that innervate it. Loss-of-function and ectopic expression of caps alter the target specificity of muscle 12 motorneurons, indicating a role for caps in selective synapse formation.

The final step in formation of neural connectivity involves the recognition of target cells. Although earlier events of growth cone guidance greatly restrict the target region, neurons still have to choose a specific synaptic partner from among several potential targets (1). We studied synapse formation in the neuromuscular system of Drosophila melanogaster. In each abdominal hemisegment ofDrosophila larvae, ∼40 motorneurons innervate 30 muscle fibers in a specific manner (2). Once a motor axon enters its target region during late embryogenesis, its growth cone searches over the surface of many muscles but withdraws from most of these contacts, forming stable synapses only with its own target or targets (3–5). Here, we describe the caps gene that regulates the formation of some of the selective synaptic connections in this system.

We screened for enhancer trap lines that express a reporter gene in specific muscle fibers during the establishment of motorneuron innervation (6). caps was identified by analysis of one such line, E2-3-27 (7). In E2-3-27 embryos, the reporter (caps-LacZ) is expressed in four dorsal (1, 2, 9, and 10) and six ventral (12, 14 to 17, and 28) muscles (Fig. 1A). In muscle 12, caps-LacZ and caps RNA (see below) are expressed in a single nucleus of the syncytial muscle, near the contact site of the motorneuronal growth cone (Fig. 1, B, C, and F). caps-LacZ is also expressed in central nervous system (CNS) motorneurons aCC, RP2, RP5, and the most medial U, all of which innervatecaps-LacZ–positive muscles (Fig. 1, D and E) (8). caps-LacZ is not expressed in motorneurons that have been identified as innervating caps-negative muscles (for example, RP1, RP3, and RP4). Thus, the expression ofcaps-LacZ is correlated with neuromuscular specificity (Fig. 1H).

Figure 1

Expression of caps in motorneurons and muscles. (A to E) caps-LacZ expression. Stage 15 E2-3-27 embryos stained with an antibody to LacZ. (A) Expression in a single nucleus in muscle 12 (arrowheads) and in nuclei of other ventral muscles 14 to 17 and 28 (Cy3; red). Muscle morphology visualized with fluorescein isothiocyanate (FITC)-phalloidin (green). (B) Double staining with antibodies to LacZ (arrowhead; brown; horseradish peroxidase reaction) and to Fasciclin II (arrow; purple; alkaline phosphatase reaction), which visualizes the motor axons. (C) 4′,6′-diamidino-2-phenylindole staining to visualize all nuclei (blue). (D and E) Expression in a subset of motorneurons in the CNS (arrowheads; Cy3; red). Subset of CNS axons visualized with monoclonal antibody (mAb) 22C10 (FITC; green) (18). (F andG) caps RNA expression in a single nucleus in muscle 12 [(F), arrowhead] and in motorneurons aCC and RP2 [(G), arrowheads]. (H) Hemisegment structure.caps-positive muscles (yellow), caps-positive motorneurons in the intersegmental nerve (ISN; green) and in intersegmental nerve b (ISNb; blue) are shown. Still to be determined is whether motorneurons that innervate other caps-positive muscles (9, 14 to 17, 28) also express caps. (Ito L) CAPS protein localization. (I) CNS of a stage 15 embryo. (J to L) A first-instar larva. The same preparation is visualized with Nomarsky optics (J), or with antibodies to CAPS [(K) Cy3; red] or to Fasciclin II [(L) FITC; green]. Fasciclin II is expressed on all neuromuscular synapses (4). CAPS is detected at the synapses on muscle 12 (arrowheads) but not on muscle 13 (arrows). Bar: 30 μm in (A), (D), and (E); 20 μm in (B), (C), (F), (G), and (I) to (L).

We cloned the genomic DNA flanking the P-element insertion site (Fig. 2A) and identified a gene (caps) whose expression pattern was identical to that of the reporter gene (Fig. 1, F and G) (9). caps encodes a transmembrane protein with 14 Leu-rich repeats (LRRs) in its extracellular domain (Fig. 2, B and C). LRR, a ∼24 amino acid motif found in various proteins from sources as diverse as yeast to human, may mediate protein–protein interactions (10). Among the proteins with LRRs, CAPS protein was most closely related to the product of the tartan gene fromDrosophila (11), with amino acid similarity extending beyond the LRR region into the cytoplasmic region. Proteins with LRRs expressed on the cell surface may function in cell adhesion or recognition (6, 12). CAPS protein is expressed on the surface of developing motor axons (Fig. 1I) (13). In first-instar larvae, CAPS protein was detected in the mature synaptic sites of all caps-positive muscles (Fig. 1, J to L) (14, 15).

Figure 2

(A) Configuration of the caps gene in E2-3-27 enhancer trap line and in two caps alleles. The horizontal bar at the bottom of (A) indicates restriction mapping of the region. E, Eco RI; H, Hind III; K, Kpn I; P, Pst I; Sc, Sac I; Sl, Sal I; V, Eco RV; Xb, Xba I; and Xh, Xho I. (B) The deduced amino acid sequence of CAPS protein. The signal peptide is underlined, and the transmembrane domain is double underlined. Arrowheads indicate conserved Cys residues in the NH2-terminal and COOH-terminal flanking regions of LRRs. Single-letter codes for amino acids are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (C) Diagram showing the domain structure of Tartan (11), CAPS, and Connectin (6). Hatched rectangles indicate LRR, and dotted boxes denote NH2-terminal and COOH-terminal flanking regions. Conserved regions in the cytoplasmic domain of Tartan and CAPS are shaded with horizontal lines. SP, signal peptide; TM, transmembrane domain; GPI, GPI-anchor. The bar at the bottom of (C) shows the length of 100 amino acids.

To determine the function of caps in vivo, we first generated caps loss-of-function mutant alleles, which lack the first exon (Fig. 2A) and do not express CAPS protein detectable by our antibody (16). Most of the caps mutants die late in embryogenesis or soon after hatching, although a few survive to adulthood (17). Although no gross developmental defects were found in the CNS or musculature of caps mutant embryos and larvae (18), the target specificity of muscle 12 motorneurons was altered (19). In wild-type larvae, muscle 12 is innervated by the terminal branch of ISNb, including the RP5 axon, which projects to the boundary between muscles 12 and 13 and forms synaptic endings exclusively on muscle 12 (Fig. 3A) (20). In contrast, incaps mutant larvae, the terminal branch is often accompanied by additional varicosities on muscle 13, a neighboringcaps-negative muscle (Fig. 3B) (21, 22). Thus,caps restricts arborization of the nerve terminal to muscle 12.

Figure 3

The effects of loss-of-function and ectopic expression of caps. Body-wall fillet preparations of third-instar larvae were stained with mAb 1D4 to visualize all motor endings (A to C) and with antiserum GC1 (39) to visualize only type III endings (D and E). (A) Wild-type pattern of innervation of muscles 12 and 13. (B) In a caps mutant larva [caps 65.2/Df(3L)Ly], the muscle 12 nerve terminal sends a collateral back to form a few type Ib boutons on muscle 13 (arrow, enlarged in the inset). (C) In aG14-GAL4/+; UAS-caps-Ib/+ individual, the muscle 12 nerve terminal projects toward and forms several type Ib boutons on muscle 13 (arrows). (D) Wild type showing exclusively type III motorneuron innervation of muscle 12. (E) In aG14-GAL4/+; UAS-caps-Ib/+ larva, the type III motorneuron turns back and innervates muscle 13. Bar: 200 μm.

Ectopic overexpression of caps in all embryonic muscles by G14-GAL4 driver caused formation of more ectopic synapses (23, 24). In ∼70% of the hemisegments, the ISNb terminal formed one or more additional collaterals that formed more robust synaptic endings on muscle 13 (Fig. 3C) (25, 26). The ectopic nerve endings contained type III boutons, which are typical of muscle 12 but not muscle 13 neuromuscular synapses (Fig. 3, D and E) (22, 27, 28). Since the ectopic synapses were present in the first-instar larvae, caps may function while the connections are being formed (29). This possibility is further supported by the absence of such ectopic endings when caps expression was induced after completion of synaptogenesis byMhc 82-GAL4 (30).

We propose that caps mediates selective synapse formation. The loss-of-function phenotype may result from improper recognition of the target muscle, whereas the extra synapses on muscle 13 could reflect retention of inappropriate synaptic contacts. In contrast, the gain-of-function phenotype could indicate that the nerve terminal is attracted to muscle 13 and other muscles by ectopic caps(31). In both cases, however, muscle 12 motorneurons reach their target region normally (32) and extend along muscle 12 before making ectopic synapses on muscle 13 (33). Thus,caps may stabilize specific motorneuronal contacts during a late phase of target selection.

The expression of caps on both sides of the synaptic partners suggests that caps functions homophilically, as has been proposed for the candidate target recognition molecules, Connectin and Fasciclin III (6, 34, 35). However, expression ofcaps in S2 cells did not promote cell aggregation (36,37). Thus, caps may mediate synaptic target recognition through cell-cell signaling rather than adhesion.

Other molecules implicated in Drosophila neuromuscular target recognition (35, 38) include another LRR protein, Connectin, which is expressed both pre- and postsynaptically on a different subset of motorneurons and muscles (6, 34). Thus, neuromuscular connections may be specified in part by a combination of this group of genes in Drosophila.

  • * To whom correspondence should be addressed. E-mail: nose{at}bio.phys.s.u-tokyo.ac.jp

  • Present address: Department of Physics, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113–8654, Japan.

REFERENCES AND NOTES

View Abstract

Navigate This Article