Derailed Regulates Development of the ... - Semantic Scholar

J_ID: DNEU Wiley Ed. Ref. No.: 00049-2007.R1 Customer A_ID: DNEU20562 Date: 28-AUGUST-07

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 AQ4 44 45 46 47 48 49 50 51 52 53 54

Stage: I

Page: 1

Derailed Regulates Development of the Drosophila Neuromuscular Junction Faith L.W. Liebl,1 Yuping Wu,1 David E. Featherstone,2 Jasprina N. Noordermeer,3 Lee Fradkin,3 Huey Hing1,4 1

Cell and Developmental Biology Program, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

2

Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607

3

Laboratory of Developmental Neurobiology, Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands

4

Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received 9 April 2007; revised 20 June 2007; accepted 18 July 2007

ABSTRACT: Neural function is dependent upon the proper formation and development of synapses. We show here that Wnt5 regulates the growth of the Drosophila neuromuscular junction (NMJ) by signaling through the Derailed receptor. Mutations in both wnt5 and drl result in a significant reduction in the number of synaptic boutons. Cell-type specific rescue experiments show that wnt5 functions in the presynaptic motor neuron while drl likely functions in the postsynaptic muscle cell. Epistatic analy-

ses indicate that drl acts downstream of wnt5 to promote synaptic growth. Structure–function analyses of the Drl protein indicate that normal synaptic growth requires the extracellular Wnt inhibitory factor domain and the intracellular domain, which includes an atypical kinase. Our findings reveal a novel signaling mechanism that regulates morphology of the Drosophila NMJ. ' 2007 Wiley Periodicals, Inc. Develop Neurobiol 00: 000–000, 2007

Keywords: Wnt; Drl; Ryk; NMJ; synapse; boutons

INTRODUCTION This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1932-8451/ suppmat Correspondence to: H. Hing ([email protected]). Contract grant sponsor: NIH Institutional Training Grant (University of Illinois); contract grant number: HD07333. Contract grant sponsor: NIH Individual NRSA (NIH/NIDCD); contract grant number: 1 F32 DC08443-01. Contract grant sponsor: NIH-NINDS Grant; contract grant number: R01NS045628. Contract grant sponsor: Pionier Grant from the NOW. Contract grant sponsor: NIH/NIDCD; contract grant number: DC5408-01. Contract grant sponsor: Roy J. Carver Charitable Trust; contract grant number: 03-27. ' 2007 Wiley Periodicals, Inc. Published online 00 Month 2007 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/dneu.20562

The development and maintenance of synapses is of critical importance for nervous system development and higher order processes such as learning and memory. Once established, synapses undergo continuous remodeling in response to genetic and environmental cues. The molecular machinery that regulates synaptic growth and synaptic remodeling is not well understood. The Drosophila larval neuromuscular junction (NMJ) provides an excellent system for dissecting the molecular basis of synapse formation, growth, and remodeling. These synapses are similar to mammalian central synapses in that they are glutamatergic and remodel in response to activity 1

ID: sangeethag

Date: 28/8/07

Time: 13:45

Path: J:/Production/DNEU/VOL00000/070113/3B2/C2DNEU070113

55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108

J_ID: DNEU Wiley Ed. Ref. No.: 00049-2007.R1 Customer A_ID: DNEU20562 Date: 28-AUGUST-07

2

109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 AQ1 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162

Stage: I

Page: 2

Liebl et al.

(Gramates and Budnik, 1999; Collins and Diantonio, in press). The terminals of the presynaptic motor neuron arrive at its target muscle midway through embryogenesis. After the initial contact, the motor neuron growth cone collapses and forms nascent presynaptic processes, termed prevaricosities. Seventeen to 22 h after egg laying (AEL), sections of the prevaricosities begin to swell and/or constrict such that presynaptic terminals (known as synaptic boutons) are formed (Johansen et al., 1989; Halpern et al., 1991; Sink and Whitington, 1991; Yoshihara et al., 1997; Rose and Chiba, 1999). During larval development (24–120 h AEL), the presynaptic arborization grows dramatically to accommodate the rapidly growing larval muscles. This growth includes increasing the number of boutons, branches, and active zones per bouton (Schuster et al., 1996a,b; Zito et al., 1999). The coordinated growth of the presynaptic motor neuron and postsynaptic muscle requires transsynaptic signaling between cells (for reviews see Koh et al., 2000; Budnik and Ruiz-Canada, 2006). A number of molecular regulators of synaptic growth and plasticity have been identified. Mutations in ion channels proteins (Cacophony, GluRIIA, and Shaker), cytoplasmic signaling proteins (Nervous Wreck), or a cell adhesion molecule (FasII) lead to perturbations in bouton numbers (Budnik et al., 1990; Schuster et al., 1996a,b; Rieckhof et al., 2003; Schmid et al., 2006). Of particular interest are the discoveries that the secreted signaling proteins, Wingless (Wg) and Glass Bottom Boat (Gbb), reduces the size of the NMJ with mutations in Wg, reducing the number of synaptic boutons by *25% (Packard et al., 2002; McCabe et al., 2003). Wg and Gbb are evolutionary conserved proteins involved in a number of developmental processes including cell fate specification, axis patterning, and recently, neural development (for reviews see Logan and Nusse, 2004; Zou, 2004; Ille and Sommer, 2005; Marques, 2005). We now report a novel anterograde signaling pathway that regulates the development of the Drosophila NMJ. We show that Wnt5 regulates growth of the NMJ by signaling through Derailed (Drl), a transmembrane receptor of the atypical receptor tyrosine kinase (RYK) family. Mutations in either wnt5 or drl result in a significant reduction in the number of synaptic boutons. Epistatic analyses indicate that wnt5, which is expressed by the presynaptic motor neuron, signals through drl, which is expressed by the postsynaptic muscle, to regulate synaptic growth. Our study is the first to demonstrate that a RYK protein regulates synapse development.

METHODS Antibodies and Immunocytochemistry For staining and microscopy, animals were dissected and fixed for 30–60 min in either Bouin’s fixative (when GluRIIA or nc82 antibodies were used) or 4% paraformaldehyde (for all other staining). First and second instar larvae were dissected and fillet preparations were glued down using Sylgard-coated coverslips. Third instar larvae were dissected and fillet preparations were pinned down in Sylgard-lined Petri dishes. All dissections were done in Drosophila standard saline (135 mM NaCl, 5 mM KCl, 4 mM MgCl, 1.8 mM CaCl, 5 mM TES, 72 mM sucrose) with 2 mM glutamate to preserve neuronal morphology (Augustin et al., 2007) at RT. Mouse monoclonal anti-GluRIIA and nc82 (Iowa Developmental Studies Hybridoma Bank, Iowa City, IA) were used at 1:100 and 1:50, respectively. Rabbit polyclonal anti-Drl and anti-Wnt5 (Fradkin et al., 2004) were used at 1:100. Fluorescently conjugated anti-HRP (Jackson Immunoresearch Labs, West Grove, PA) was used at 1:100. Goat anti-rabbit or goat anti-mouse fluorescent (FITC or TRITC) secondary antibodies (Jackson Immunoresearch Labs) were used at 1:400. The 6/7 NMJ of abdominal hemisegments A3 or A4 were used for all studies. Confocal images were obtained using a Zeiss LSM 510 laser-scanning confocal microscope. Image analysis and quantification was performed using ImageJ and Adobe Photoshop software. The anti-Drl antiserum was raised in rabbits against a GST fusion protein, including Drl amino acids 123–222, and was affinity-purified against the same protein coupled to a column.

Molecular Biology Full-length drl cDNA was obtained from Open Biosystems (Huntville, AL) and subcloned into the pUAST vector to generate the UAS-drl transgene. To generate the UASdrlDWIF construct, nucleotides 472–1830 were amplified by PCR from drl cDNA and ligated to the first 60 nucleotides of drl cDNA. This construct was then subcloned into pUAST. All constructs were subcloned into pUAST using the EcoRI and XbaI sites. The sequence was verified for all constructs. Fly transformation was performed by DNA microinjection of embryos using standard methods. To verify transcription of these constructs, total RNA was isolated using trizol extraction, and reverse-transcribed using cDNA-specific primers.

Electrophysiology All electrophysiology was performed on the ventral body wall muscle 6. Larval recordings were performed on third instar larvae 110–120 h AEL. Muscle 6 was voltageclamped at
60 mV. Standard two-electrode voltage clamp techniques were used, as previously described (Liebl et al., 2005). Data were acquired and analyzed using an Axopatch amplifier and pClamp9 (Axon Instruments, Union City,

Developmental Neurobiology. DOI 10.1002/dneu

ID: sangeethag

Date: 28/8/07

Time: 13:45

Path: J:/Production/DNEU/VOL00000/070113/3B2/C2DNEU070113

163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216

J_ID: DNEU Wiley Ed. Ref. No.: 00049-2007.R1 Customer A_ID: DNEU20562 Date: 28-AUGUST-07

Stage: I

Page: 3

Derailed Signaling in NMJ Development

217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 F1 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270

CA). All dissections and recordings were done in standard Drosophila saline at 198C.

Data Acquisition and Statistics All animals were raised at 258C in standard fly vials with corn meal molasses medium. The total number of boutons was acquired from 6/7 NMJs of hemisegments A3 or A4. The density of nc82 labeling was quantified by counting the total number of nc82 puncta and dividing by the total NMJ area using ImageJ (NIH) software. Statistics were performed using GraphPad Prism (v. 4.01). All statistical comparisons were made using unpaired students t-tests. Control animals used were WT-Berlin, elav-Gal4, 24B-Gal4, ActinGal4, UAS-wnt5, and UAS-drl. There was no significant difference in bouton numbers between groups (WT-Berlin ¼ 56.36 6 3.609 boutons, n ¼ 11; elav-Gal4 ¼ 57.50 6 4.586 boutons, n ¼ 8, p ¼ 0.8459; 24B-Gal4 ¼ 52.11 6 3.080 boutons, n ¼ 9, p ¼ 0.4157; actin-Gal4 ¼ 57.38 6 4.496 boutons, n ¼ 8, p ¼ 0.8614; UAS-wnt5 ¼ 55.11 6 3.438 boutons, n ¼ 9, p ¼ 0.8263; UAS-drl ¼ 55.20 6 3.286 boutons, n ¼ 10, p ¼ 0.8178). Therefore, the data were combined into one control group. Statistical significance in figures is represented as follows: *p < 0.05, **p < 0.01, and ***p < 0.001. All error bars represent SEM.

RESULTS Wnt5 and Drl Are Expressed at the 6/7 NMJ We first investigated whether Drl and Wnt5 were expressed at the NMJ using immunolocalization. Drl [Fig. 1(B,D)] and Wnt5 [Fig. 1(A,C)] are each localized to the area of boutons in third instar larvae (Fig. 1). Wnt5 and Drl antibody immunoreactivity was almost undetectable in homozygous wnt5400 null mutants and dramatically reduced in drl2 null mutants (Fig. 1, right panels), indicating that these antibodies are specific. We were unable to distinguish whether the Wnt5 and Drl immunoreactivity were pre- or postsynaptic by colocalization experiments with the presynaptic marker CSP and the postsynaptic marker DLG (data not shown). The pre- and postsynaptic membranes of the NMJ are closely apposed (Atwood et al., 1993; Prokop et al., 1996) making it difficult to determine the cellular locations of proteins using confocal microscopy. The Drl labeling is consistent with previous reports, which showed that Drl is expressed in subsets of CNS axons (Yoshikawa et al., 2003) and ventral body wall muscles (Callahan et al., 1996). Wnt5 is expressed in posterior commissural axons of the CNS (Fradkin et al., 2004) but has not been localized to the NMJ. Our immunlocalization experiments suggest that Drl and Wnt5 may function at the NMJ.

3

Wnt5 Is a Positive Regulator of NMJ Size and Active Zone Formation To investigate the role of wnt5 at the NMJ, we examined the NMJs of the null mutant, wnt5400. Homozygous wnt5400 mutants are adult-viable as previously described (Fradkin et al., 2004). Mutant larvae exhibit no visible defects in muscle patterning or muscle size (muscle 6: WT ¼ 26,490 6 1526 lm2, n ¼ 6; wnt5400 ¼ 26,430 6 1077 lm2, n ¼ 7, p ¼ 0.9750; muscle 7: WT ¼ 20,020 6 764.6 lm2, n ¼ 6; wnt5400 ¼ 20,370 6 1313 lm2, n ¼ 7, p ¼ 0.8214). To examine NMJ morphology, we visualized presynaptic motor neurons with HRP to label the neuronal membranes. Our analysis revealed that wnt5 mutants have significantly fewer boutons compared with control animals [Fig. 2(A,C), control ¼ 55.85 6 3.059 boutons, n ¼ 13; wnt5400 ¼ 45.36 6 3.459 boutons, n ¼ 14, p ¼ 0.0329]. This reduction correlated with a reduction in NMJ size (WT ¼ 526.2 6 42.66 lm2, n ¼ 10; wnt5400 ¼ 382.0 6 37.80 lm2, n ¼ 10, p ¼ 0.0209). There was no significant difference in the number of branches at the 6/7 NMJ (control ¼ 4.938 6 0.4784 branches, n ¼ 16; wnt5400 ¼ 4.688 6 0.6240 branches, n ¼ 16, p ¼ 0.7527). We examined the synaptic expression and localization of the presynaptic protein, Bruchpilot/nc82 (Brp), and the postsynaptic glutamate receptor subunit, GluRIIA, by immunolabeling. Expression of Brp, which promotes the assembly of presynaptic active zones (Kittel et al., 2006; Wagh et al., 2006), is significantly reduced in wnt5 mutants compared with controls [Fig. 2(B,C); WT ¼ 0.5585 6 0.03462 puncta/lm2, n ¼ 10; wnt5400 ¼ 0.3904 6 0.02506 puncta/lm2, n ¼ 7, p ¼ 0.0026]. The expression and localization of GluRIIA appeared normal in wnt5 mutant animals (data not shown, normalized fluorescence: WT ¼ 1.000 6 0.1006, n ¼ 11; wnt5400 ¼ 0.8977 6 0.06632, n ¼ 9, p ¼ 0.4288). We placed wnt5400 in trans with the deficiency, Df (1) N19, which uncovers the wnt5 gene to exclude the possibility that the NMJ defects are caused by extraneous mutations in the wnt5 mutant background. wnt5400/Df (1) N19 animals exhibited a significant reduction in both the number of boutons and density of Brp (boutons: WT ¼ 55.85 6 3.059, n ¼ 13; wnt5400/Df (1) N19 ¼ 42.50 6 2.141, n ¼ 10, p ¼ 0.003; Brp: WT ¼ 0.5585 6 0.0346 puncta/lm2, n ¼ 10; wnt5400/Df (1) N19 ¼ 0.3766 6 0.0246 puncta/lm2, n ¼ 9, p ¼ 0.0006). These data indicate that wnt5 mutant animals exhibit a significant reduction in the number of boutons and suggest that the density of active zones in the presynaptic terminals is also significantly reduced. Developmental Neurobiology. DOI 10.1002/dneu

ID: sangeethag

Date: 28/8/07

Time: 13:45

Path: J:/Production/DNEU/VOL00000/070113/3B2/C2DNEU070113

271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 F2 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324

J_ID: DNEU Wiley Ed. Ref. No.: 00049-2007.R1 Customer A_ID: DNEU20562 Date: 28-AUGUST-07

4

C O L O R

325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358AQ6 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378

Stage: I

Page: 4

Liebl et al.

Figure 1 Wnt5 and Drl are localized to the NMJ. (A) Confocal fluorescent images showing NMJs on muscles 6 and 7 in third instar larvae. Animals were labeled with antibodies against HRP (magenta), which recognizes presynaptic membranes and Wnt5 (green). (B) Confocal fluorescent images showing NMJs labeled with antibodies against HRP (magenta) and Drl (green). Scale bar: 20 lm. (C) High magnification view of an area from A. (D) High magnification view of an area from B.

We performed electrophysiology to determine whether the reduction in the number of active zones affected synaptic function. Muscle 6 was voltage clamped at
60 mV and the presynaptic segmental nerve was stimulated (1 Hz, 5 V) to induce synaptic activity. The amplitude of evoked excitatory junctional currents (EJCs) was reduced 40% in wnt5 mutant animals [Fig. 2(D), WT ¼ 207.2 6 18.51 nA, n ¼ 8; wnt5400 ¼ 114.7 6 17.94 nA, n ¼ 6, p ¼ 0.0044]. Similarly, the frequency of spontaneous miniature EJCs (mEJCs) was significantly reduced in wnt5 mutants [Fig. 2(E), WT ¼ 2.030 6 0.3035 s, n ¼ 9; wnt5400 ¼

1.231 6 0.1985 s, n ¼ 8, p ¼ 0.0491]. These data suggest that presynaptic function is impaired in wnt5 mutants and is consistent with the reduction in the number of boutons and active zones observed immunocytochemically. Postsynaptic function, however, appeared normal, as there was no significant difference in mEJC amplitudes [Fig. 2(E), WT ¼ 0.8677 6 0.05136 nA, n ¼ 9; wnt5400 ¼ 0.8034 6 0.04789 nA, n ¼ 8, p ¼ 0.3781], an indicator of postsynaptic glutamate receptor function. Taken together, our results suggest that wnt5 promotes synaptic growth and the assembly of presynaptic active zones.

Developmental Neurobiology. DOI 10.1002/dneu

ID: sangeethag

Date: 28/8/07

Time: 13:45

Path: J:/Production/DNEU/VOL00000/070113/3B2/C2DNEU070113

379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432

J_ID: DNEU Wiley Ed. Ref. No.: 00049-2007.R1 Customer A_ID: DNEU20562 Date: 28-AUGUST-07

Stage: I

Page: 5

Derailed Signaling in NMJ Development

433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 F3 474 475 476 477 478 479 480 481 482 483 484 485 486

The Reduction in NMJ Size Is the Result of Underdevelopment Growth of the NMJ involves the addition of new boutons during larval development to correspond with the increase in muscle size (Schuster et al., 1996a,b; Zito et al., 1999). To ascertain whether the deficit in bouton number resulted from a failure to coordinate growth of the presynaptic motor neuron and postsynaptic muscle, we also examined wnt5 mutant NMJs during both first and second instar larval stages (24and 48-h AEL, respectively). There was no significant difference in the number of boutons between mutant and control first instar larvae [Supp. Fig. (A,B), WT ¼ 11.13 6 1.093 boutons, n ¼ 8; wnt5400 ¼ 9.429 6 1.232 boutons, n ¼ 7, p ¼ 0.3199]. However by second instar, there was a significant reduction in the number of boutons in wnt5 mutants compared with controls [Fig. Supp. Fig. (C,D), WT ¼ 19.44 6 0.9146 boutons, n ¼ 9; wnt5400 ¼ 15.13 6 1.217 boutons, n ¼ 8, p ¼ 0.0115]. There was no significant difference in the density of active zones during first or second instar larval stages (Fig. Supp. Fig., 1st instar: WT ¼ 0.6530 6 0.0534 puncta/lm2, n ¼ 8; wnt5400 ¼ 0.6084 6 0.0516 puncta/lm2, n ¼ 7, p ¼ 0.5613; 2nd instar: WT ¼ 0.5846 6 0.0716 puncta/lm2, n ¼ 7; wnt5400 ¼ 0.4614 6 0.0378 puncta/lm2, n ¼ 7, p ¼ 0.1383). These data suggest that mutant NMJs fail to keep pace with postsynaptic muscle development.

Restoration of wnt5 in Neurons But Not Muscle Rescues the Null Phenotype To identify the cell type in which wnt5 functions, we performed cell-type specific gene rescue experiments by expressing the UAS-wnt5 transgene specifically in neurons (using the elav-Gal4 driver) or muscle (using the 24B-Gal4 driver) in the wnt5400 mutant background. Expression of wnt5 in neurons but not in muscles restored the number of boutons to slightly greater than control levels [Fig. 3(A,B), wnt5400 ¼ 45.36 6 3.459 boutons, n ¼ 14; neuron expression ¼ 59.64 6 3.123 boutons, n ¼ 11, p ¼ 0.007, muscle expression ¼ 50.31 6 4.531 boutons, n ¼ 13, p ¼ 0.3893; control; compared with control animals the p values were 0.3978 and 0.3211 for neuronal and muscle expression, respectively]. Similarly, expression of wnt5 in neurons but not muscles restored the NMJ size to slightly greater than control levels (wnt5400 ¼ 382.0 6 37.80 lm2, n ¼ 10, neuron expression ¼ 595.8 6 29.72 lm2, n ¼ 10, p ¼ 0.0003; muscle expression ¼ 437.3 6 28.30 lm2, n ¼ 8, p ¼ 0.2794; compared with control animals the p values were 0.6837 and 0.0415 for neuronal and mus-

5

cle expression, respectively). Expression of the transgene in either neurons or muscle, however, restored the number of active zones/lm2 to near control levels [Fig. 3(A,C), wnt5400 ¼ 0.3904 6 0.02506, n ¼ 7; neuron expression ¼ 0.5140 6 0.03132, n ¼ 8, p ¼ 0.0099, muscle expression ¼ 0.5364 6 0.03560, n ¼ 8, p ¼ 0.0062]. These data indicate that wnt5 functions in the presynaptic motor neuron to promote synaptic growth, but may function in either the presynaptic neuron or the postsynaptic muscle cell to regulate active zone formation. The differential requirement for wnt5 suggests that synaptic growth and active zone development may be governed by distinct wnt5dependent signaling mechanisms.

Overexpression of wnt5 in Neurons Increases Synaptic Growth If wnt5 promotes NMJ development, we would expect that increasing the dose of wnt5 in wild-type animals would alter NMJ development. We therefore overexpressed wnt5 by driving the UAS-wnt5 transgene with cell-type specific Gal4 drivers in wild-type animals. Driving UAS-wnt5 using either a ubiquitous (ActinGal4) or neuron-specific drivers (elav-Gal4 and OK6Gal4) significantly increased the number of boutons of the 6/7 NMJ [Fig. 4(A,B), control ¼ 55.85 6 3.059 boutons, n ¼ 13; Act>wnt5 ¼ 78.07 6 5.274 boutons, n ¼ 14, p ¼ 0.0015; elav>wnt5 ¼ 77.75 6 4.254 boutons, n ¼ 8, p ¼ 0.0004; OK6>wnt5 ¼ 68.50 6 4.323 boutons, n ¼ 12, p ¼ 0.0239]. Driving UAS-wnt5 in postsynaptic muscle cells, using the 24B-Gal4 driver, significantly reduced the number of boutons (control ¼ 55.85 6 3.059 boutons, n ¼ 13; 24B>wnt5 ¼ 42.92 6 4.087 boutons, n ¼ 12, p ¼ 0.0176). Similar results were obtained when we overexpressed wnt5 using the GSV line, wnt51192, and used the Actin-Gal4, elavGal4, and 24B-Gal4 drivers (data not shown). The overexpression results support our rescue experiments, which show that wnt5 functions in the presynaptic neuron to positively regulate NMJ size. The inhibition of NMJ growth by postsynaptic wnt5 overexpression might be due to a deleterious effect of high levels of wnt5 on muscle physiology. Indeed, muscle defects are observed in wnt5-overexpressing muscle (R.W. Wouda, L.G.F. and J.N.N., in preparation). Our overexpression results support the idea that wnt5 functions in motor neurons to promote NMJ growth.

drl Mutant Animals Exhibit a Similar Morphology as wnt5 Mutants The observation that wnt5 acts presynaptically to regulate NMJ morphology raises the question as to Developmental Neurobiology. DOI 10.1002/dneu

ID: sangeethag

Date: 28/8/07

Time: 13:46

Path: J:/Production/DNEU/VOL00000/070113/3B2/C2DNEU070113

487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 F4 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540

J_ID: DNEU Wiley Ed. Ref. No.: 00049-2007.R1 Customer A_ID: DNEU20562 Date: 28-AUGUST-07

C O L O R

C O L O R

541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594

Stage: I

Page: 6

Figure 2 wnt5 mutant animals exhibit a reduction in the number of boutons and active zones. (A) Confocal fluorescent images showing NMJs labeled with antibodies against HRP (magenta) and the presynaptic protein Brp (nc82, green). Scale bar: 20 lm. (B) Magnification of a portion of the NMJ shown in A. (C) Quantification of active zones density (nc82 puncta/lm2, left) and bouton number (per NMJ, right). (D) Representative two-electrode voltage clamp recordings from muscle 6 of control and wnt5400 mutant third instar larvae, showing evoked endplate junctional currents (EJCs). (Right) Quantification of EJC amplitudes. (E) Representative voltage clamp recordings from control and wnt5400 third instar larvae, showing spontaneous excitatory junctional currents (mEJCs) in the muscle 6 NMJ. Quantification of mEJC frequency (middle) and amplitude (right).

Figure 3 Developmental Neurobiology. DOI 10.1002/dneu

ID: sangeethag

Date: 28/8/07

Time: 13:46

Path: J:/Production/DNEU/VOL00000/070113/3B2/C2DNEU070113

595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648

J_ID: DNEU Wiley Ed. Ref. No.: 00049-2007.R1 Customer A_ID: DNEU20562 Date: 28-AUGUST-07

Stage: I

Page: 7

Derailed Signaling in NMJ Development

649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 F5 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702

7

Figure 4 Overexpression of wnt5 in neurons produces an overgrowth of the NMJ. (A) Confocal fluorescent images showing NMJs labeled with antibodies against HRP (magenta) and the presynaptic protein Brp (nc82, green). For overexpression experiments, a UAS-wnt5 transgene was expressed ubiquitously (using Actin-Gal4, act>wnt5), or in neurons (elav>wnt5) or in muscle (24B>wnt5). Scale bar: 20 lm. (B) Quantification of the number of boutons per NMJ.

the downstream signaling mechanism involved. Our immnunolocalization experiments show that similar to Wnt5, the Drl receptor is also localized to the boutons of the NMJ [Fig. 1(B)]. To test whether Drl also functions in NMJ development, we examined the phenotype of drl2 mutant animals, which are protein nulls and homozygous-viable (Dura et al., 1995). Although the lateral ventral body wall muscles (specifically, muscles 21, 22, and 23) are abnormal and sometimes missing in mutant animals as previously described (Callahan et al., 1996), the medial ventral body wall muscles exhibited no visible defects in muscle size (muscle 6: WT ¼ 26,490 6 1526 lm2, n ¼ 6; drl2 ¼ 26,300 6 1392 lm2, n ¼ 6, p ¼ 0.9257; muscle 7: WT ¼ 20,020 6 764.6 lm2, n ¼ 6; drl2 ¼ 20,860 6 1020 lm2, n ¼ 6, p ¼ 0.5188). Similar to wnt5 mutants, drl mutant animals exhibited a significant reduction in the number of boutons [Fig. 5(A,B) and data not shown, control ¼ 55.85 6 3.059 boutons, n ¼ 13; drl2 ¼ 43.17 6 2.458 boutons, n ¼ 12, p ¼ 0.0040] and this correlated with a reduction in the size of the 6/7 NMJ (WT ¼ 526.2 6 42.66 lm2, n ¼ 10; drl2 ¼ 382.6 6 49.34 lm2, n ¼ 10, p ¼ 0.0410). Also similar to the wnt5 mutant phenotype, there was no significant

difference in the number of branches at the 6/7 NMJ (control ¼ 4.938 6 0.4784 branches, n ¼ 16; drl2 ¼ 4.167 6 0.8424, n ¼ 12 branches, n ¼ 12, p ¼ 0.4888) and the synaptic growth defect became apparent only during the second larval instar (Supp. Fig., first instar: WT ¼ 11.13 6 1.093 boutons, n ¼ 8; drl2 ¼ 9.000 6 0.9512 boutons, n ¼ 7, p ¼ 0.1720; second instar: WT ¼ 19.44 6 0.9146 boutons, n ¼ 9; drl2 ¼ 13.29 6 1.539 boutons, n ¼ 7, p ¼ 0.0028). We examined the synaptic expression and localization of Brp and the postsynaptic glutamate receptor subunit, GluRIIA by immunolabeling third instar mutant animals. Unlike the wnt5 mutant, the expression and localization of both Brp and GluRIIA were not significantly different than controls in drl mutants [Fig. 5(A,B), Brp: WT ¼ 0.5585 6 0.03462 puncta/ lm2, n ¼ 10; drl2 ¼ 0.4739 6 0.03352 puncta/lm2, n ¼ 8, p ¼ 0.1035; GluRIIA normalized fluorescence, WT ¼ 1.000 6 0.1006, n ¼ 11; drl2 ¼ 1.155 6 0.1140, n ¼ 12, p ¼ 0.3240]. In addition, drl mutant animals exhibited small statistically insignificant reductions in evoked amplitude and mini frequency [Fig. 5(C–F), evoked amplitude: WT ¼ 207.2 6 18.51 nA, n ¼ 8; drl2 ¼ 166.4 6 9.015 nA, n ¼ 7,

Figure 3 wnt5 expression in neurons rescues the mutant phenotype. (A) Confocal fluorescent images showing NMJs labeled with antibodies against HRP (magenta) and the presynaptic protein Brp (nc82, green). For rescue experiments, a UAS-wnt5 transgene was expressed in the wnt5 mutant background in neurons using the elav-Gal4 driver (neuron exp.) or in muscle using the 24B-Gal4 driver (muscle exp.). Scale bar: 20 lm. (B) Quantification of the number of boutons per NMJ. (C) Quantification of active zone density (nc82 puncta/lm2). Developmental Neurobiology. DOI 10.1002/dneu

ID: sangeethag

Date: 28/8/07

Time: 13:46

Path: J:/Production/DNEU/VOL00000/070113/3B2/C2DNEU070113

703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756

J_ID: DNEU Wiley Ed. Ref. No.: 00049-2007.R1 Customer A_ID: DNEU20562 Date: 28-AUGUST-07

8

C O L O R

757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810

Stage: I

Page: 8

Liebl et al.

Figure 5 drl mutant animals exhibit a reduction in the number of boutons but not active zones. (A) Confocal fluorescent images showing NMJs labeled with antibodies against HRP (magenta) and the presynaptic protein Brp (nc82, green). Scale bar: 20 lm. (B) Quantification of active zone density (nc82 puncta/lm2, left) and bouton numbers (per NMJ, right). (C) Representative two-electrode voltage clamp recordings from muscle 6 of control and drl2 third instar larvae, showing evoked endplate junctional currents (EJCs). (D) Quantification of EJC amplitudes. (E) Representative voltage clamp recordings from control and drl2 third instar larvae, showing spontaneous excitatory junctional currents (mEJCs) in the muscle 6 NMJ. (F) Quantification of mEJC frequency (left) and amplitude (right).

p ¼ 0.3080; mini amplitude: WT ¼ 0.8677 6 0.05136 nA, n ¼ 9; drl2 ¼ 0.8665 6 0.02951 nA, n ¼ 10, p ¼ 0.9832; mini frequency: WT ¼ 2.030 6 0.3035 Hz, n ¼ 9; drl2 ¼ 1.605 6 0.3264 Hz, n ¼ 11, p ¼ 0.2677]. To exclude the possibility of secondary mutations in the drl2 mutant background, we placed the drl2 mutation in trans to the deficiency, Df(2R) lio2, pigeon2, drl2. These animals also exhibited a significant reduction in the number of NMJ boutons, with no significant difference in the density of active zones (control ¼ 55.85 6 3.059 boutons, n ¼ 13; drl2/Df(2R) lio2, pigeon2, drl2 ¼ 43.78 6 4.390 boutons, n ¼ 9, p ¼ 0.030; Brp: WT ¼ 0.5585 6 0.03462 puncta/lm2, n ¼ 10; drl2/Df(2R) lio2, pigeon2, drl2 ¼

0.5240 6 0.0336 puncta/lm2, n ¼ 9, p ¼ 0.486). Taken together, these results indicate that drl regulates presynaptic neuronal morphology, but is not likely to regulate the formation of active zones.

drl Likely Functions in Postsynaptic Muscle To delineate the cell type in which drl functions, we sought to restore drl functions in specific cell types in the drl2 mutant background. Neither the presence of the UAS-drl transgene alone nor the expression of the transgene under the control of the pan-glial

Developmental Neurobiology. DOI 10.1002/dneu

ID: sangeethag

Date: 28/8/07

Time: 13:46

Path: J:/Production/DNEU/VOL00000/070113/3B2/C2DNEU070113

811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864

J_ID: DNEU Wiley Ed. Ref. No.: 00049-2007.R1 Customer A_ID: DNEU20562 Date: 28-AUGUST-07

Stage: I

Page: 9

Derailed Signaling in NMJ Development

865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 F6 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918

9

Figure 6 Expression of drl in muscle rescues the drl mutant phenotype. (A) Confocal images showing NMJs labeled with antibodies against HRP. For rescue experiments, a UAS-drl transgene was expressed in the drl mutant background in neurons using the elav-Gal4 driver or in muscle using the 24B-Gal4 driver. Scale bar: 20 lm. (B) For overexpression experiments, a UAS-drl transgene was expressed ubiquitously (act>drl), or in neurons (elav>drl) or muscle (24B>drl). Quantification of the number of boutons per NMJ for overexpression experiments. (C) Quantification of the number of boutons and branches for the rescue experiments.

driver, Repo-Gal4, rescued the growth phenotype [Fig. 6(A,C), drl2 ¼ 43.17 6 2.458 boutons, n ¼ 12, drl2; UAS-drl ¼ 40.80 6 2.476 boutons, n ¼ 10, p ¼ 0.509; glia expression ¼ 41.67 6 3.322 boutons, n ¼12, p ¼ 0.7201]. Expression of the transgene in muscle strongly rescued the drl2 mutant phenotype [Fig. 6(A,C), drl2 ¼ 43.17 6 2.458 boutons, n ¼ 12, muscle expression ¼ 56.83 6 3.303 boutons, n ¼ 12, p ¼ 0.0031] as the number of boutons is restored to slightly greater than control levels. Interestingly, drl expression in neurons produced an increase in the number of NMJ branches [Fig. 6(C), control ¼ 5.846 6 0.5644 branches, n ¼ 13; neuron expression ¼ 8.500 6 0.7638 branches, n ¼ 12, p ¼ 0.0096], a phenotype not seen in the wild type. The increased arborization produces an increase in bouton number [Fig. 6(A,C), drl2 ¼ 43.17 6 2.458 boutons, n ¼ 12, neuron expression

¼ 59.55 6 4.288 boutons, n ¼ 11, p ¼ 0.0028], leading to ambiguity about whether drl functions in the presynaptic motor neuron or postsynaptic muscle cell. Examination of NMJ size produced similar results (drl2 ¼ 382.6 6 49.34 lm2, n ¼ 10, neuron expression ¼ 564.8 6 49.61 lm2, n ¼ 9, p ¼ 0.019; muscle expression ¼ 559.6 6 37.99 lm2, n ¼ 9, p ¼ 0.012, glial expression ¼ 465.3 6 36.04 lm2, n ¼ 9, p ¼ 0.202). We also overexpressed drl in an otherwise wildtype background. Overexpressing drl using various Gal4 drivers did not significantly affect NMJ morphology [Fig. 6(B), control ¼ 55.85 6 3.059, n ¼ 13; Act>drl ¼ 60.50 6 6.425, n ¼ 12, p ¼ 0.5091; elav>drl ¼ 64.93 6 5.195, n ¼ 14, p ¼ 0.1521; 24B>drl ¼ 52.50 6 5.704, n ¼ 12, p ¼ 0.6023]. We conclude from the aforementioned rescue experiments that drl likely functions in the postsynaptic Developmental Neurobiology. DOI 10.1002/dneu

ID: sangeethag

Date: 28/8/07

Time: 13:47

Path: J:/Production/DNEU/VOL00000/070113/3B2/C2DNEU070113

919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972

J_ID: DNEU Wiley Ed. Ref. No.: 00049-2007.R1 Customer A_ID: DNEU20562 Date: 28-AUGUST-07

10

973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 F7 1022 1023 1024 1025 1026

Stage: I

Page: 10

Liebl et al.

Figure 7 Epistatic analyses of the relationship between wnt5 and drl. (A) Confocal images showing NMJs labeled with antibodies against HRP. Scale bar: 20 lm. (B) Quantification of the number of boutons per NMJ. (C) Confocal images showing NMJs labeled with antibodies against HRP. For the epistatic analysis, the UAS-wnt5 transgene was overexpressed in neurons in the drl mutant background (drl2; elav>wnt5 and drl2/Cyo; elav>wnt5). Scale bar: 20 lm. (D). Quantification of the number of boutons per NMJ.

muscle to regulate NMJ growth, but may also function in the presynaptic motor neuron for other developmental processes.

The NMJ Overgrowth Produced by Overexpression of wnt5 in Neurons Is Suppressed in the drl Mutant Background The reduction in bouton numbers in both wnt5 and drl mutants raised the possibility that the two genes may function in a signaling pathway to regulate synaptic growth. To test this possibility, we constructed animals bearing simultaneously the wnt5 and drl mutations. The NMJ morphology of wnt5400; drl2 double mutants is not statistically different than that of each of the single mutants [Fig. 7(A,B), drl2 ¼ 43.17 6 2.458 boutons, n ¼ 12; wnt5400 ¼ 45.36 6 3.459 boutons, n ¼ 14; wnt5400; drl2 ¼ 49.55 6 4.345, n ¼ 11, p ¼ 0.2056], supporting our hypothesis that wnt5 and drl act in the same signaling pathway to regulate synaptic growth.

We postulate that wnt5 acts upstream of drl. This hypothesis makes the prediction that mutations in the drl gene would block the effects of wnt5. To test this possibility, we overexpressed the UAS-wnt5 transgene in the drl mutant background. Neuronal overexpression of wnt5 in the wild-type background leads to overgrowth of the NMJ with a significant increase in the number of boutons [Figs. 4(A,B) and 7(C,D)]. Removal of a single copy of the drl gene (drl2/CyO) in the wnt5 overexpressing animal partially, but significantly suppressed the NMJ overgrowth phenotype [Fig. 7(C,D) elav>wnt5 ¼ 77.75 6 4.254 boutons, n ¼ 8; drl2/Cyo; elav>wnt5 ¼ 60.33 6 4.311 boutons, n ¼ 9, p ¼ 0.0119]. Removal of both copies of the drl gene in the wnt5 overexpressing animal results in a phenotype indistinguishable from that of the drl2 mutant [Fig. 7(C,D), drl2 ¼ 43.17 6 2.458 boutons, n ¼ 12; drl2; elav>wnt5 ¼ 47.55 6 2.855 boutons, n ¼ 11, p ¼ 0.2560]. These data indicate that the effect of neuronal overexpression of wnt5 is highly sensitive to the dose of the drl gene and that drl is epistatic to wnt5. This result supports the idea that wnt5 functions through drl to promote the growth of the NMJ.

Developmental Neurobiology. DOI 10.1002/dneu

ID: sangeethag

Date: 28/8/07

Time: 13:47

Path: J:/Production/DNEU/VOL00000/070113/3B2/C2DNEU070113

1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080

J_ID: DNEU Wiley Ed. Ref. No.: 00049-2007.R1 Customer A_ID: DNEU20562 Date: 28-AUGUST-07

Stage: I

Page: 11

Derailed Signaling in NMJ Development

1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 C 1093 O 1094 L O 1095 R 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 AQ5 1130 F8 1131 1132 1133 1134

11

Figure 8 Normal NMJ growth requires the WIF and intracellular domains of Drl. (A). Confocal fluorescent images showing NMJs labeled with antibodies against HRP (magenta). Scale bar: 20 lm. (B) Quantification of the number of boutons per NMJ. (C) Model of Wnt5-Drl signaling at the NMJ. (i) Wnt5 signals through Drl to regulate NMJ size. (ii, iii) In the absence of either Wnt5 (ii) or Drl (iii), the NMJ is reduced in size.

The Intracellular and WIF Domains of Drl Are Required for Normal Synaptic Growth The observation that Drl acts downstream of Wnt5 during NMJ development raises intriguing questions about the mechanisms by which Drl transduces the Wnt5 signal. The Drl protein is a member of the evolutionarily conserved RYK receptor tyrosine kinaserelated family. Members of this family possess a WIF motif in the extracellular domain and an intracellular domain that includes an \atypical" kinase. Our ability to rescue the drl mutant phenotype using transgenes afforded us the opportunity to probe the requirement of these domains of the Drl protein in synapse development. We expressed mutant drl constructs in the muscle of drl2 mutant animals to assess their ability to rescue the drl2 mutant phenotype. Deletion of the WIF motif completely abolished Drl’s capacity to rescue the mutant phenotype, indicating that the WIF motif is necessary for Drl function in NMJ development (Fig. 8, drl2 ¼ 43.17 6 2.458 boutons, n ¼ 12; drl2, 24B>
drlDWIF ¼ 49.85 6 3.123 boutons, n ¼ 13, p ¼ 0.1102; p-value when compared with control ¼ 0.280). Expression of the truncated DrlDcyt protein containing only the extracellular and transmembrane

domains failed to rescue the drl2 mutant phenotype, indicating that the cytoplasmic domain is necessary for Drl function in NMJ development (Fig. 8, drl2 ¼ 43.17 6 2.458 boutons, n ¼ 12, drl2, 24B>
drlDcyt ¼ 49.09 6 2.246 boutons, n ¼ 11, p ¼ 0.0915; p value when compared to control ¼ 0.171). Examination of NMJ size yielded similar results (drl2 ¼ 382.6 6 49.34 lm2, n ¼ 10, 24B>
drlDWIF ¼ 361.6 6 44.12 lm2, n ¼ 10, p ¼ 0.906; 24B>
drlDcyt ¼ 361.9 6 39.10 lm2, n ¼ 11, p ¼ 0.889). We conclude that the cytoplasmic and WIF domains of drl are required for normal growth of the presynaptic motor neuron.

DISCUSSION After its establishment, the Drosophila NMJ exhibits as much as a 10-fold increase in growth between the first and third instar larval stages by adding additional boutons and branches to correspond with the increase in surface area of the postsynaptic muscle (Schuster et al., 1996a,1996b; Zito et al., 1999). This growth requires coordination between the presynaptic motoDevelopmental Neurobiology. DOI 10.1002/dneu

ID: sangeethag

Date: 28/8/07

Time: 13:47

Path: J:/Production/DNEU/VOL00000/070113/3B2/C2DNEU070113

1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 AQ5 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188

J_ID: DNEU Wiley Ed. Ref. No.: 00049-2007.R1 Customer A_ID: DNEU20562 Date: 28-AUGUST-07

12

1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242

Stage: I

Page: 12

Liebl et al.

neuron and postsynaptic muscle. This process is likely mediated by a number of signaling mechanisms. Here we provide evidence that Wnt5 positively regulates NMJ growth by signaling through the Drl RYK receptor. This is the first study to demonstrate that a member of the RYK family plays a role in synapse formation.

Wnt5 Is an Anterograde Signal That Regulates NMJ Development Several pieces of evidence support our conclusion that the wnt5 gene acts as a positive regulator of NMJ growth and synaptic transmission. Mutation of wnt5 leads to stunted NMJs with decreased number of boutons. In addition to the structural defects, the NMJ shows reduced number of active zones. Electrophysiological recordings showed that the amplitudes of evoked releases and frequency of spontaneous releases are both significantly decreased. Examination of the mutant NMJs early in development showed that they are normal, indicating that wnt5 is needed for NMJ growth to maintain pace with muscle growth. Cell-type specific cDNA expression showed that wnt5 functions in the motoneurons. In contrast to the reduced bouton numbers in the loss-of-function wnt5 mutant, animals overexpressing wnt5 in the motoneurons exhibit significant overgrowth of the NMJ, indicating that NMJ development is highly sensitive to the dose of wnt5. Our results therefore indicate that Wnt5 is an anterograde signal that promotes the development of the NMJ. Our finding that Wnt5 apparently acts as a transsynaptic signal adds it to a growing list of Wnt proteins and Wnt signaling pathway components that play key roles in synaptogenesis (for review see Salinas, 2005). Most notably, Packard et al. (2002) observed that at the Drosophila NMJ, Wg is expressed by the presynaptic motor neuron where it governs the development of both pre- and postsynaptic structures (Packard et al., 2002; Mathew et al., 2005). In the mouse, Wnt3 and Wnt7a are necessary for the cessation of axon growth and the remodeling of the axons into presynaptic terminals, characterized by growth cone spreading and clustering of the synapsin I protein (Hall et al., 2000; Krylova et al., 2002). Dissection of the mechanisms by which these Wnt proteins regulate synapse development showed that they act through the Frizzled receptor (Packard et al., 2002; Mathew et al., 2005). However, our observations indicate that Wnt5 functions through the Drl RYK receptor to govern NMJ growth.

Drl Functions downstream of Wnt5 to Regulate Synapse Growth Several lines of evidence indicate drl is necessary for the growth of the Drosophila NMJ. Mutation of the drl gene results in a significant reduction in NMJ size, a phenotype that resembles that of the wnt5 mutant. As with the wnt5 mutant, the NMJs of the drl mutant appear normal during the first instar larval stage, indicating that drl regulates NMJ growth. Celltype specific gene rescue showed that drl likely functions in the postsynaptic muscle cell. Interestingly, despite the significant reduction in NMJ size in the drl mutant, this synapse displayed wild-type electrophysiology, and thus differs from wnt5 mutation in this respect. The density of active zones, the amplitudes of both evoked and spontaneous releases, and the frequency of miniature releases are normal compared with wild-type animals, suggesting that drl does not regulate the formation of active zones. Instead, drl appears to function specifically to regulate presynaptic growth. This observation raised the possibility that drl and wnt5 may function in the same pathway to regulate NMJ growth. That drl functions downstream to mediate wnt5 signaling is reinforced by the following observations. First, the wnt5; drl double mutants exhibit reduced NMJ sizes, a phenotype that resembles both the single wnt5 and drl mutants. Second, the overgrowth phenotype produced by neuronal overexpression of wnt5 is completely suppressed in the drl mutant background, indicating that drl acts downstream of wnt5. We conclude that wnt5 signals through the drl receptor to regulate NMJ growth. Collectively, our results suggest a novel anterograde signaling pathway [Fig. 8(C)] where Wnt5, expressed in the presynaptic cell, acts through Drl, likely in the postsynaptic cell, to promote synaptic growth.

The Drl Cytoplasmic and WIF Domains Are Necessary for NMJ Growth The capacity of the Drl RYK to mediate Wnt5 function in NMJ growth raises the question of the mechanism by which Drl transduces the Wnt5 signal. The Drl protein contains an extracellular WIF motif with homology to the Wnt-binding domain of the Wnt inhibitory factor-1 (WIF-1) protein (Yoshikawa et al., 2003) and an intracellular atypical kinase domain (Yoshikawa et al., 2001). To assess the requirements of these domains during NMJ development, we engineered Drl proteins lacking these domains and expressed them in muscle cells. Deletion of the extracellular WIF domain completely abolished NMJ

Developmental Neurobiology. DOI 10.1002/dneu

ID: sangeethag

Date: 28/8/07

Time: 13:47

Path: J:/Production/DNEU/VOL00000/070113/3B2/C2DNEU070113

1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296

J_ID: DNEU Wiley Ed. Ref. No.: 00049-2007.R1 Customer A_ID: DNEU20562 Date: 28-AUGUST-07

Stage: I

Page: 13

Derailed Signaling in NMJ Development

1297 1298 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350

function in NMJ development. Since the extracellular domain of Drl has been shown to bind to Wnt5 (Yoshikawa et al., 2003), our result suggests that Wnt5 may regulate growth of the NMJ by binding to the WIF motif of Drl. Deletion of the cytoplasmic domain of Drl also abolished its function in NMJ development. This result is consistent with observations in the embryonic ventral nerve cord, where the Drl cytoplasmic domain is needed for commissural axons to navigate properly in response to the Wnt5 signal (Yoshikawa et al., 2001). This domain is likely to play a role in signal transduction, although the mechanism by which it functions is unknown. Recently, the cytoplasmic tail of the mammalian RYK protein was shown to bind to the Dishevelled protein, raising the possibility that RYK proteins may act through the canonical pathway (Lu et al., 2004).

Wnt5 Signals Via Different Mechanisms to Regulate Structure and Function We found that the Wnt5-Drl signaling pathway mediates a signal that passes from motor neurons to muscle cells. This raises the question as to how the pathway regulates NMJ development. One intriguing possibility is that the signaling pathway stimulates the production of a signal in the muscle cell, which acts in a retrograde fashion to influence synaptic growth. One known retrograde signal is the BMP signaling pathway (for reviews see Marques, 2005). Intriguingly, mutations in components of this pathway (Gbb, Saxophone, Thick Veins, Wishful thinking, and Mad) lead to a reduction in NMJ size with fewer boutons (Aberle et al., 2002; Marques et al., 2002; McCabe et al., 2003, 2004; Rawson et al., 2003), a phenotype that resembles that of the wnt5 and drl mutants. We are currently exploring the possibility that BMP signaling is downstream of drl. Besides regulating NMJ structure, Wnt5 also regulates the function of the synapse. In this role, however, it does not appear to act via the Drl receptor. Wnts have been previously implicated in the development of presynaptic active zones (Hall et al., 2000; Krylova et al., 2002; Packard et al., 2002; AhmadAnnuar et al., 2006). We show that in the absence of wnt5, mutant NMJs have fewer active zones and impaired presynaptic function. Both the amplitude of evoked currents and the frequency of spontaneous currents are significantly reduced. It is unclear how Wnt5, which is produced by the presynaptic motor neuron, acts on the same cell that produces it. However, our data demonstrate that Wnt5 does not signal through Drl to regulate active zone formation. Drl

13

mutant animals are functionally normal with no detectable loss of Brp labeling. Further studies will hopefully identify the mechanisms by which Wnt5 and Drl govern NMJ structure and function. We thank the Iowa Developmental Hybridoma Bank for antibodies and the Bloomington Stock Center for fly stocks.

REFERENCES Aberle H, Haghighi AP, Fetter RD, McCabe BD, Magalhaes TR, Goodman CS. 2002. Wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila. Neuron 33:545–558. Ahmad-Annuar A, Ciani L, Simeonidis I, Herreros J, Fredj NB, Rosso SB, Hall A, et al. 2006. Signaling across the synapse: A role for Wnt and dishevelled in presynaptic assembly and neurotransmitter release. J Cell Biol 174:127–139. Atwood HL, Govind CK, Wu CF. 1993. Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae. J Neurobiol 24:1008–1024. Augustin H, Grosjean Y, Chen K, Sheng Q, Featherstone DE. 2007. Nonvesicular release of glutamate by glial xCT transporters suppresses glutamate receptor clustering in vivo. J Neurosci 27:111–123. Budnik V, Zhong Y, Wu CF. 1990. Morphological plasticity of motor axons in Drosophila mutants with altered excitability. J Neurosci 10:3754–3768. Callahan CA, Bonkovsky JL, Scully AL, Thomas JB. 1996. Derailed is required for muscle attachment site selection in Drosophila. Development 122:2761–2767. Collins CA, Diantonio A. Synaptic development: Insights from Drosophila. Curr Opin Neurobiol, in press. Dura JM, Taillebourg E, Preat T. 1995. The Drosophila learning and memory gene linotte encodes a putative receptor tyrosine kinase homologous to the human RYK gene product. FEBS Lett 370:250–254. Fradkin LG, van Schie M, Wouda RR, de Jong A, Kamphorst JT, Radjkoemar-Bansraj M, Noordermeer JN. 2004. The Drosophila Wnt5 protein mediates selective axon fasciculation in the embryonic central nervous system. Dev Biol 272:362–375. Gramates LS, Budnik V. 1999. Assembly and maturation of the Drosophila larval neuromuscular junction. Int Rev Neurobiol 43:93–117. Hall AC, Lucas FR, Salinas PC. 2000. Axonal remodeling and synaptic differentiation in the cerebellum is regulated by WNT-7a signaling. Cell 100:525–535. Halpern ME, Chiba A, Johansen J, Keshishian H. 1991. Growth cone behavior underlying the development of stereotypic synaptic connections in Drosophila embryos. J Neurosci 11:3227–3238. Ille F, Sommer L. 2005. Wnt signaling: Multiple functions in neural development. Cell Mol Life Sci 62:1100– 1108. Developmental Neurobiology. DOI 10.1002/dneu

ID: sangeethag

Date: 28/8/07

Time: 13:47

Path: J:/Production/DNEU/VOL00000/070113/3B2/C2DNEU070113

1351 1352 1353 1354 1355 1356 1357 1358 1359 1360 1361 1362 1363 1364 1365 1366 1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 AQ3 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 1403 1404

J_ID: DNEU Wiley Ed. Ref. No.: 00049-2007.R1 Customer A_ID: DNEU20562 Date: 28-AUGUST-07

14

1405 1406 1407 1408 1409 1410 1411 1412 1413 1414 1415 1416 1417 1418 1419 1420 1421 1422 1423 1424 1425 1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458

Stage: I

Page: 14

Liebl et al.

Johansen J, Halpern ME, Johansen KM, Keshishian H. 1989. Stereotypic morphology of glutamatergic synapses on identified muscle cells of Drosophila larvae. J Neurosci 9:710–725. Kittel RJ, Wichmann C, Rasse TM, Fouquet W, Schmidt M, Schmid A, Wagh DA, et al. 2006. Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release. Science 312:1051–1054. Koh YH, Gramates LS, Budnik V. 2000. Drosophila larval neuromuscular junction: Molecular components and mechanisms underlying synaptic plasticity. Microsc Res Tech 49:14–25. Krylova O, Herreros J, Cleverley KE, Ehler E, Henriquez JP, Hughes SM, Salinas PC. 2002. WNT-3, expressed by motoneurons, regulates terminal arborization of neurotrophin-3-responsive spinal sensory neurons. Neuron 35: 1043–1056. Liebl FL, Chen K, Karr J, Sheng Q, Featherstone DE. 2005. Increased synaptic microtubules and altered synapse development in Drosophila sec8 mutants. BMC Biol 3:27. Logan CY, Nusse R. 2004. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 20: 781–810. Lu W, Yamamoto V, Ortega B, Baltimore D. 2004. Mammalian Ryk is a Wnt coreceptor required for stimulation of neurite outgrowth. Cell 119:97–108. Marques G. 2005. Morphogens and synaptogenesis in Drosophila. J Neurobiol 64:417–434. Marques G, Bao H, Haerry TE, Shimell MJ, Duchek P, Zhang B, O’Connor MB. 2002. The Drosophila BMP type II receptor wishful thinking regulates neuromuscular synapse morphology and function. Neuron 33:529–543. Mathew D, Ataman B, Chen J, Zhang Y, Cumberledge S, Budnik V. 2005. Wingless signaling at synapses is through cleavage and nuclear import of receptor DFrizzled2. Science 310:1344–1347. McCabe BD, Hom S, Aberle H, Fetter RD, Marques G, Haerry TE, Wan H, et al. 2004. Highwire regulates presynaptic BMP signaling essential for synaptic growth. Neuron 41:891–905. McCabe BD, Marques G, Haghighi AP, Fetter RD, Crotty ML, Haerry TE, Goodman CS, et al. 2003. The BMP homolog Gbb provides a retrograde signal that regulates synaptic growth at the Drosophila neuromuscular junction. Neuron 39:241–254. Packard M, Koo ES, Gorczyca M, Sharpe J, Cumberledge S, Budnik V. 2002. The Drosophila Wnt, wingless, provides an essential signal for pre- and postsynaptic differentiation. Cell 111:319–330. Prokop A, Landgraf M, Rushton E, Broadie K, Bate M. 1996. Presynaptic development at the Drosophila neuro-

muscular junction: Assembly and localization of presynaptic active zones. Neuron 17:617–626. Rawson JM, Lee M, Kennedy EL, Selleck SB. 2003. Drosophila neuromuscular synapse assembly and function require the TGF-b type I receptor saxophone and the transcription factor Mad. J Neurobiol 55:134–150. Rieckhof GE, Yoshihara M, Guan Z, Littleton JT. 2003. Presynaptic N-type calcium channels regulate synaptic growth. J Biol Chem 278:41099–41108. Rose D, Chiba A. 1999. A single growth cone is capable of integrating simultaneously presented and functionally distinct molecular cues during target recognition. J Neurosci 19:4899–4906. Salinas PC. 2005. Retrograde signalling at the synapse: A role for Wnt proteins. Biochem Soc Trans 33:1295–1298. Schmid A, Qin G, Wichmann C, Kittel RJ, Mertel S, Fouquet W, Schmidt M, et al. 2006. Non-NMDA-type glutamate receptors are essential for maturation but not for initial assembly of synapses at Drosophila neuromuscular junctions. J Neurosci 26:11267–11277. Schuster CM, Davis GW, Fetter RD, Goodman CS. 1996a. Genetic dissection of structural and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilization and growth. Neuron 17:641–654. Schuster CM, Davis GW, Fetter RD, Goodman CS. 1996b. Genetic dissection of structural and functional components of synaptic plasticity. II. Fasciclin II controls presynaptic structural plasticity. Neuron 17:655–667. Sink H, Whitington PM. 1991. Pathfinding in the central nervous system and periphery by identified embryonic Drosophila motor axons. Development 112:307–316. Wagh DA, Rasse TM, Asan E, Hofbauer A, Schwenkert I, Durrbeck H, Buchner S, et al. 2006. Bruchpilot, a protein with homology to ELKS/CAST, is required for structural integrity and function of synaptic active zones in Drosophila. Neuron 49:833–844. Yoshihara M, Rheuben MB, Kidokoro Y. 1997. Transition from growth cone to functional motor nerve terminal in Drosophila embryos. J Neurosci 17:8408–8426. Yoshikawa S, Bonkowsky JL, Kokel M, Shyn S, Thomas JB. 2001. The Derailed guidance receptor does not require kinase activity in vivo. J Neurosci 21:RC119. Yoshikawa S, McKinnon RD, Kokel M, Thomas JB. 2003. Wnt-mediated axon guidance via the Drosophila Derailed receptor. Nature 422:583–588. Zito K, Parnas D, Fetter RD, Isacoff EY, Goodman CS. 1999. Watching a synapse grow: Noninvasive confocal imaging of synaptic growth in Drosophila. Neuron 22: 719–729. Zou Y. 2004. Wnt signaling in axon guidance. Trends Neurosci 27:528–532.

Developmental Neurobiology. DOI 10.1002/dneu

ID: sangeethag

Date: 28/8/07

Time: 13:47

Path: J:/Production/DNEU/VOL00000/070113/3B2/C2DNEU070113

1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472 1473 1474 1475 1476 1477 1478 1479 1480 1481 1482 1483 1484 1485 1486 1487 1488 1489 1490 1491 1492 1493 1494 1495 1496 1497 1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 1511 1512