Proc. Natl. Acad. Sci. USA Vol. 90, pp. 3043-3047, April 1993 Neurobiology An 83- promoter of the acetylcholine receptor e-subunit gene confers preferential synaptic expression in mouse muscle (neuromuscular junction/promoter regulation) AYMERIC DUCLERT, NATHALIE SAVATIER, AND JEAN-PIERRE CHANGEUX Unite de Recherche Associ&e, Centre National de la Recherche Scientifique 0210, Neurobiologie Moleculaire, Departement des Biotechnologies, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France Contributed by Jean-Pierre Changeux, December 29, 1992

ABSTRACT The expression of the acetylcholine receptor purpose, we used a simple method of transient expression of e-subunit gene is restricted to the endplate of adult muscle DNA constructions in muscle tissue after direct DNA injec- fibers. We have started to study the regulatory elements of the tion (15). In parallel, we examined the expression of several E-subunit gene promoter that are important for its synaptic constructions ofthe E-subunit gene promoter by transfection expression. We used, for this purpose, a rapid method ofin vivo in different in vitro culture systems to compare the regulatory expression after DNA injection into the muscle tissue [Wolff, elements important for localized expression in vivo with J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., those sufficient for general expression in vitro. Jani, A. & Felgner, P. L. (1990) Science 247, 1465-14681. Our results show that a construction containing 83 upstream from the transcription start site is sufficient to obtain MATERIAL AND METHODS preferential endplate expression. Moreover, mutation of a Constructions. A 1.1-kb fragment of the E-subunit gene MyoD located around position -70 does not alter promoter was initially cloned by inverse PCR and used as a this synaptic expression. We also studied the expression of this probe to screen 106 clones of a DBA/2J mouse genomic promoter in vitro in muscle primary cultures and showed the library (Clontech). One clone was isolated that had 3.5 kb presence ofa positive element between positions -122 and -83. downstream and >10 kb upstream ofthe ATG. The different Comparison of in vivo and in vitro results reveals that the constructions of the --subunit gene promoter were obtained elements important for in vivo localization at the synapse and by III deletion and subcloning in a KS Bluescript in vitro expression in cultured muscle cells may differ. luciferase or KS nlsLacZ vector. The muscle creatine kinase (MCK) promoter construction contained 3.3 kb of promoter The motor endplate is a highly specialized structure devoted sequence. The SVP promoter construction was a 350-nt to signal transmission between motor nerve and skeletal HindIII promoter fragment containing the 72-bp viral en- muscle. A number of proteins such as the acetylcholine hancer. receptor (AcChoR) or collagenic forms of the acetylcholin- In Vivo Expression Studies. Plasmid DNA was prepared and (AcChoEase) accumulate at this level (1). An im- purified on cesium chloride gradients (16). It was resus- portant step in the comprehension ofAcChoR localization to pended to a final concentration of 3 ,Agl,ul with 20% (wt/vol) the endplate was accomplished by the discovery that the sucrose. Three-week-old C57/BL6 x SJL hybrid mice were mRNAs coding for the different subunits of this pentameric anesthetized and tibialis anterior muscles were injected a2(3y/68 receptor were themselves localized in the endplate slowly with -20 Al of DNA in sucrose solution as described region (2) at the level of "fundamental nuclei" (3-5) just (15). One week later, injected mice were killed and tibialis beneath the postsynaptic domain of the muscle fibers. Fur- anterior muscles were removed. The few damaged muscles ther, a fragment of the a-subunit gene promoter allowed a were discarded to avoid possible extrasynaptic expression of transiently higher expression of the reporter gene ,B-galacto- the E-subunit gene due to muscle regeneration (17). Whole sidase at junctional nuclei in transgenic mice (6, 7). Frag- muscles were fixed for 20 min, rinsed, and stained overnight ments of the promoters of the E-subunit and 8-subunit genes at 25°C for f-galactosidase using 5-bromo-4-chloro-3-indolyl were also shown to confer endplate expression to reporter ,B-D-galactoside (7). Blue fibers were removed, further fixed genes in transgenic mice (8, 9), suggesting that a higher for several hours, stained for AcChoEase (18), and analyzed transcription rate in junctional nuclei is responsible for at under the microscope. least some of the localized accumulation of these mRNAs. In Vitro Transfection Studies. Rat primary muscle cultures During early development in rat and calf, the AcChoR is were prepared by collagenase/hyaluronidase/trypsin diges- first expressed as an a2PYS pentamer along the entire length tion of muscle tissue from 1- to 2-day-old rats as described of the muscle fibers. Then around birth, the E-subunit re- (19) and transfected 1 day later with 1 ,ug of DNA per 35-mm places the y-subunit and causes changes in the properties of plate by the calcium phosphate precipitation technique (20). the AcChoR channel. The mRNA coding for this E-subunit is Luciferase activity was measured 3 days later (21). The 293 restricted from the outset ofits expression tojunctional areas cell line was transfected as primary muscle cultures. (5), suggesting that the regulation of the E-subunit gene should be easier to study. Several of the promoters of AcChoR subunit genes have RESULTS been cloned and their expression has been analyzed (10-14), In Vivo Expression of a 2.2-kb Fragment of the e-Subunit most often in cultured cells. The aim of the present work was Gene Promoter. We first injected into muscle tissue (15) two to analyze the promoter elements of the E gene responsible constructions containing either a 3.3-kb fragment ofthe MCK for the localized expression in the junctional area. For this gene that confers muscle-specific expression (22, 23) or the ubiquitously expressed simian virus 40 early promoter up- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: AcChoR, acetylcholine receptor; AcChoEase, in accordance with 18 U.S.C. §1734 solely to indicate this fact. ; MCK, muscle creatine kinase.

3043 Downloaded by guest on September 30, 2021 3044 Neurobiology: Duclert et al. Proc. Natl. Acad. Sci. USA 90 (1993) stream of the reporter gene nlsLacZ. This reporter gene of the s-subunit gene promoter in transgenic mice (8). More- contains the nuclear localization signal from the simian virus over, the intensity of blue staining for the most intensely 40 large tumor antigen (24), which limits diffusion of the stained nuclei of an event was, on average, higher for protein product along the muscle fiber by trapping it into synaptic events than for extrasynaptic ones (data not shown). nuclei (7, 8). Both constructions (MCKnlsLacZ and SVP- Fig. 1A presents an example of a synaptic event obtained nlsLacZ) gave similar expression patterns, which, using this with -2.2-kb nlsLacZ. Thus, -2.2 kb conferred preferential technique, appeared restricted to muscle fibers (15). A mi- synaptic expression compared with the ubiquitous promoters nority of muscle fibers expressed the lacZ product. In ex- MCK and SVP. pressing fibers, blue nuclei occurred in groups and extended In Vivo Expression of Deletion and Point Mutants of the to only a small portion of the total length of the fiber with a E-Subunit Gene Promoter. We next generated a number of 5' gradient of blue intensity at each end of the stained domain deletion mutants that shared the same 3' terminus (nucleotide (Fig. 1). This type of expression probably reflects localized +65) and placed them upstream of the nlsLacZ gene. Both entry and expression of plasmid DNA molecules within the constructions, -158-nt nlsLacZ and -83-nt nlsLacZ (see muscle fiber combined with limited diffusion of /3-galacto- Fig. 2), gave a percentage of synaptic events around or sidase. Blue fibers were then dissected and AcChoEase was >50%, much higher than the percentage obtained with revealed histochemically (18). The positions of the blue MCKnlsLacZ or SVPnlsLacZ (Table 1 and Fig. 3). The nuclei and of the brownish AcChoEase staining were com- intensity of the blue staining obtained with - 158-nt nlsLacZ pared. A blue fiber, referred to as an "event," was called and -83-nt nlsLacZ did not significantly differ between "synaptic" only when AcChoEase staining was located synaptic and extrasynaptic events. Thus, a construction approximately at the center of the set of blue nuclei. Other- containing 83 nt upstream of the transcription start site is wise, the event was called "extrasynaptic." With these sufficient to confer preferential synaptic expression. In con- definitions, about 10% of the events could be called synaptic trast, no expression of a -36-nt nlsLacZ construction could with each construction, MCKnlsLacZ or SVPnlsLacZ (see ever be detected in any of the 48 muscles injected (Table 1). Table 1 and Fig. 3). Fig. 1D illustrates an example of an Thus, this -36-nt nlsLacZ construction has lost positive extrasynaptic event obtained with MCKnlsLacZ. elements present in the -83-nt nlsLacZ construction and A similar experiment was then undertaken with a 2.2-kb important for its synaptic expression in vivo. fragment of the s-subunit gene promoter ending at position The myogenic proteins ofthe MyoD family oftranscription +65 (Fig. 2) relative to the transcription start site (8) inserted factors bind to a consensus CANNTG sequence referred to upstream of the nlsLacZ reporter (construction -2.2-kb as an E box (25). Such elements have been shown to regulate nlsLacZ). Fig. 3 shows that 75-80% of the events obtained the expression of AcChoR subunit genes (26) and other were of the synaptic type. The synaptic events were highly muscle-specific genes (27, 28). Numberger et al. (13) have reminiscent ofthe expression obtained with a 3.5-kb fragment shown the presence of a MyoD binding site in the rat A: B

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FIG. 1. Sample of expressing muscle fibers obtained by DNA injection in vivo. (A) Synaptic event obtained with -2.2-kb nlsLacZ (with two other adjacent nonexpressing fibers). (B) Synaptic event at high magnification obtained with -83-nt nlsLacZ. (C) Extrasynaptic event obtained with -83-nt nlsLacZ. (D) Extrasynaptic event obtained with MCKnlsLacZ. The brownish precipitate of the AcChoEase staining (arrows) contrasts with the blue 8-galactosidase staining of the nuclei. (Bars = 100 um.) Downloaded by guest on September 30, 2021 Neurobiology: Duciert et al. Proc. Natl. Acad. Sci. USA 90 (1993) 3045

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w >1 (o 50 -158 -122 4-4 r -150 -140 -130 -o20 -110 -100 0 ATGACAGGCCTTGTGGATTACAGAACTGGCTGAAACCCAGGCAAAGGATGCTGGGATGGG 07)u -83 CO -90 r -0 -70 -60 -50 -40 -4-i GTGGGGCAAGGGACACAGGATGGGGCAGCTGCCTCCCCCACCCCCACAGCAGGGGCAGAG C-

CA -36 _14TG 1 r -30 -20 r -10 +10 +20 01) GATTAGGTGACAGTCCCCAAACCTAGCCCGGAACTAACACCCTCCTCCCCTTCACACAGG f2+ 10

+30 +40 +50 +60 CACCTTGGCCTGTTCCCTCAAGCTTGTCAAAGCTCAGAATAAC 4-i~~~~~~~ yI C;b C~ luci <0) B -2.9kb # I. -Of / 0,-. -158 1 __ -122 '4 FIG. 3. Summary diagram of the in vivo expression studies. This -83 '4 __ diagram is a bar representation of the data from Table 1. When several experiments were done with one construction, the percent- -3 6 - age of synaptic events was directly averaged and the error bar -14 &t represents the SEM. -158 MyoD- -158A-112/-84 (29, 30). Also, fragments ofthe 6-subunit gene promoter may -158A-76/-37 confer expression to a reporter gene in cultured muscle cells (8, 13). Thus, an analysis of the E-subunit gene promoter by FIG. 2. (A) Restriction map and sequence of the --subunit gene transfection in different cell types could help to define promoter showing the different mutants used. A simplified restriction activating/repressing elements, the role of which could later map of the 2.9-kb proximal fragment of the E-subunit gene promoter is shown at the top with the position of the -2.2-kb construction be assessed in vivo. For this quantitative analysis, we used indicated. The sequence of the E-subunit gene promoter around the the luciferase gene instead ofthe lacZ gene because ofits high site of initiation of transcription (+ 1) determined by Sanes et al. (8) sensitivity. is also presented. The boundaries of 5' deletion mutants ( r) with their position number are indicated. The MyoD consensus site is Table 1. Summary of in vivo expression experiments underlined and point mutations ( I ) are shown just below. Putative AP2 consensus sites are doubly underlined. A SHUE box homology No. of Total no. of % of is underlined with a dashed line. The sequence ofthis proximal region muscles blue fibers synaptic of the s-subunit gene promoter was identical to that reported Promoter injected Obtained Dissected events previously (8) except for two point mutations and one point deletion at positions -100, -65, and -43, respectively. (B) Schematic -2.2 kb 14 42 40 75 diagram of the different constructions used for in vitro experiments. 16 49 40 80 x, MyoD mutation. Each arrow represents the site of initiation of -158 nt 14 37 35 48 transcription. 18 52 31 48 17 33 32 59 E-subunit gene promoter. An identical consensus sequence -158-nt MyoD- 6 30 30 40 (GGCAGCIGCC) is found at the same position in the mouse 14 23 20 60 E-subunit gene promoter (Fig. 2A; ref. 8). We thus addressed 18 50 32 50 the issue of the importance of this sequence for the synapse- -83 nt 36 21 21 71 specific expression of the s-subunit gene by mutating all 22 39 26 46 bases of the consensus; CAGCTG was thus replaced by 14 63 41 58 TGGCCA (Fig. 2A). This mutation was introduced in the -36 nt 26 0 -158-nt context to avoid the influence of other putative 22 0 upstream MyoD binding sites. The - 158-nt MyoD- nlsLacZ MCK 8 33 33 9 resulting construction gave a percentage of synaptic events 20 446 53 8 (50% on the average) that was not significantly different from SVP 8 85 55 11 that given by -158-nt nlsLacZ (Table 1 and Fig. 3). We 16 81 58 15 that therefore conclude this proximal MyoD binding site is For each experiment the number of muscles injected and blue not essential for the preferential synaptic expression of the fibers obtained are indicated. For some experiments only a random E-subunit gene promoter. sample of the blue fibers obtained was stained for AcChoEase and Deletion Analysis of the e-Subunit Gene Promoter by Trans- dissected, either because blue fibers were lost or because they were fection. Cultured mouse muscle cells express E-subunit too numerous. For each experiment the percentage of synaptic mRNA and adult AcChoR channels in the absence of nerve events is indicated. Downloaded by guest on September 30, 2021 3046 Neurobiology: Duclert et al. Proc. Natl. Acad. Sci. USA 90 (1993) Different 5' deletion mutants that all end up at nucleotide level of expression that did not significantly differ from that +65 (Fig. 2 A and B) were placed upstream of the luciferase of -158-nt luci (0.05 < P < 0.1). In conclusion, deletion gene and transfected either in rat muscle primary cultures or analysis reveals the presence of a positive element between in nonmuscle 293 cells (human embryonic kidney cells; ref. positions -122 and -83. 31). All results were normalized to the activity of the SVPluci plasmid, transfected in parallel, which was set at 100. DISCUSSION The -2.9-kb luci construction containing 2.9 kb of the E-subunit gene promoter was 20-fold more active than the We have combined an in vitro analysis by transfection with promoterless reporter plasmid called KS luci in primary an in vivo study by muscle injection to define elements muscle cultures (Fig. 4). It was also 5-fold more active in important for the expression ofthe e-subunit gene. We report muscle cultures than in 293 cells. Since -158-nt nlsLacZ still three main findings: (i) a construction containing 83 nt had a preferential synaptic expression in vivo, we then upstream from the transcription start site of the E-subunit measured the activity of - 158-nt luci in muscle culture; it was gene promoter suffices to obtain preferential synaptic ex- not significantly different from that of -2.9-kb luci. The pression, though it gives only low levels of expression in construction -122-nt luci still showed the same activity. primary cultures of rat myotubes; (ii) the MyoD binding site However, expression dramatically decreased when the pro- present in this construction is not required for synaptic moter was further reduced from -122 nt to -83 nt (around expression; (iii) a positive element important for expression 5-fold). The luciferase activity remained more or less con- in rat muscle primary cultures is localized between positions stant and was low for the following constructions: -83-nt, -122 and -83. - 36-nt, and - 14-nt luci. These experiments thus indicate the These results were obtained by using a simple method of presence of a positive element between positions -122 and transient expression after injection ofDNA into muscle tissue -83. This element displays some muscle specificity because (15). To our knowledge, this technique has not been used the fall between -122-nt and -83-nt was previously to investigate the compartmentalized expression luci much greater in of a promoter. It allows a rapid test for the expression of a muscle cells than in 293 cells. To examine the importance of given construction, though absolute levels of expression in the (-112, -84) region, a gap was introduced in the -158-nt synaptic and extrasynaptic areas cannot be quantified. In luci construction (Fig. 2B). The construction obtained, called particular, we cannot assess the significance of the 20% -158A-112/-84 luci, had a level of expression significantly fraction of extrasynaptic events obtained with -2.2-kb lower than that of the -158-nt luci construction (Fig. 4). nlsLacZ. This might stem either from a bonafide expression To assess the functional role of the MyoD binding site in in extrasynaptic areas or from the high number of plasmid vitro, we introduced into -158-nt luci the same mutation as molecules introduced in a small portion of the fiber. in -158-nt MyoD- nlsLacZ (Fig. 2). The resulting construc- The -83-nt nlsLacZ construction contains some, but prob- tion, -158-nt MyoD- luci, had a level of expression 40% ably not all, of the elements important for the compartmen- lower than that of -158-nt luci (Fig. 4) (P < 0.05 according talized expression of the s-subunit gene. Indeed, -2.2-kb to the Mann-Whitney U test). However, -158A-76/-37 nlsLacZ was found to give a higher percentage of synaptic luci, which has a gap between positions -76 and -37, had a events. Whether the difference between the two construc- tions depends upon activating or repressing elements remains lon to be investigated. The level ofexpression of -36-nt nlsLacZ was below the detection threshold of our method. Thus we cannot assess whether the expression ofthis construction has c myotlues a preferential synaptic localization or not. We can only note that an important positive element has been lost. This ele- ment might include the MyoD binding site and/or the puta- 20~~~~~~~~~ q tive adjacent AP2 consensus sequence CCCCACCCC. Yet, if the MyoD binding site is important for expression in vivo, it is not required for endplate expression. Transfection experiments with primary cultures of rat my- otubes have shown that a positive element is present between positions -122 and -83. However, this element is not re- quired for preferential synaptic expression (Table 1 and Fig. 3). We have noted two homologies with known elements in this fragment: a putative AP2 consensus site (GGGGTGGGG) and Thsbrdarmisteaeaeo four 293ue) rtre(23cls a SHUE box (32) (CCAGGCA) (Fig. 2). The approximate position of this element was confirmed by the gap mutant -158A-112/-84 luci (Fig. 4). A small effect could be ob- served after mutation of the MyoD binding site. However, a gap mutation suppressing this site did not produce a significant lbI>> Cbv> decrease in expression (see - 158A -76/-37 luci in Fig. 4). At variance with an earlier report from another group on rat E-subunit gene promoter (13), we found no increase in activity after MyoD binding site suppression in 293 cells (Fig. 4) nor in FIG. 4. Luciferase activities of 5' deletion mutants in rat primary primary cultures of rat fibroblasts (data not shown). cultures of myotubes and in 293 cells normalized to SVP luci activity. Our data show that a -83-nt construction that is expressed This bar diagram is the average offour (myotubes) or three (293 cells) at only low levels in vitro remains preferentially expressed at different experiments with the error bar representing the SEM. For sites in vivo. an each experiment, each transfection was repeated in duplicate and the synaptic Moreover, obvious difference of results were normalized to the activity of the simian virus 40 early expression between the -83-nt and -36-nt constructions was promoter that had been transfected in parallel in independent plates. observed in vivo but not in vitro. This suggests that the Shaded bars represent the values obtained for rat primary myotubes; elements important for the expression in vivo at the neuro- open bars represent those obtained for 293 cells. KS, vector without muscular junction and those important for expression in promoter. cultured muscle cells may be different. Also, the E-subunit Downloaded by guest on September 30, 2021 Neurobiology: Duclert et al. Proc. Natl. Acad. Sci. USA 90 (1993) 3047 gene promoter does not have a strong muscle specificity 12. Baldwin, T. J. & Burden, S. J. (1988) J. Cell Biol. 107, 2271- when compared with the MCK promoter (see Fig. 4). In fact, 2279. the levels ofmRNA coding for the E-subunit in muscle culture 13. Numberger, M., Durr, I., Kues, W., Koenen, M. & Witze- are much lower than those coding for the other AcChoR mann, V. (1991) EMBO J. 10, 2957-2964. subunits (30). This leads to the suggestion that E-subunit 14. Prody, C. A. & Merlie, J. P. (1991) J. Biol. Chem. 266, 22588- regulatory sequences contain few of the muscle-specific 225%. at 15. Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, elements active early stages of differentiation and this G., Jani, A. & Felgner, P. L. (1990) Science 247, 1465-1468. might explain the absence of high levels of expression of 16. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular E-subunit mRNA before birth (33). Further analysis of the Cloning: A Laboratory Manual (Cold Spring Harbor Lab., mechanisms governing compartmentalized expression of the Plainview, NY). E-subunit gene will require the detailed identification of the 17. Goldman, D., Carlson, B. M. & Staple, J. (1991) Neuron 7, DNA regulatory elements involved and might be facilitated 649-658. by the experimental method used in this work. 18. Koelle, G. B. & Friedenwald, J. S. (1949) Proc. Soc. Biol. Med. 70, 617-622. We thank Dr. S. D. Hauschka for the generous gift of an MCK 19. Cossu, G., Zani, B., Coletta, M., Bouche, M., Pacifici, M. & promoter construction, Dr. J. Piette for helpful discussions, and Drs. Molinaro, M. (1980) Cell Differ. 9, 357-368. M. Yaniv, J. Massoulie, J. L. Bessereau, A. Bessis, A. Devillers- 20. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. Thiery, and M. Picciotto for critical reading of the manuscript. This Cell. Biol. 2, 1044-1051. work was supported by the Centre National de la Recherche Scien- 21. Nguyen, V. T., Morange, M. & Bensaude, 0. (1988) Anal. tifique, the College de France, the Institut National de la Sante et de Biochem. 171, 404-408. la Recherche Medicale (Contract 872004), and the Direction des 22. Jaynes, B., Chamberlain, J. S., Buskin, J. N., Johnson, J. E. & Recherches et Etudes Techniques (Contract 87/211). Hauschka, S. D. (1986) Mol. Cell. Biol. 6, 2855-2864. 23. Jaynes, B., Johnson, J. E., Buskin, J. N., Gartside, C. L. & 1. Changeux, J.-P., Babinet, C., Bessereau, J. L., Bessis, A., Hauschka, S. D. (1988) Mol. Cell. Biol. 8, 62-70. Cartaud, J., Cartaud, A., Daubas, P., Devillers-Thiery, A., 24. Kalderon, D., Roberts, B. L., Richardson, W. D. & Smith, Duclert, A., Huchet, M., Hill, J., Jasmin, B., Laufer, R., A. E. (1984) Cell 39, 499-509. Nghiem, H. O., Piette, J., Roa, M. & Salmon, A. M. (1990) 25. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, Cold Spring Harbor Symp. Quant. Biol. 55, 381-406. M., Benezra, R., Blackwell, K., Turner, D., Rupp, R., Hol- 2. Merlie, J. P. & Sanes, J. R. (1985) Nature (London) 317, 66-68. lenberg, S., Zhuang, Y. & Lassar, A. (1991) Science 251, 3. Fontaine, B., Sassoon, D., Buckingham, M. & Changeux, J.-P. 761-766. (1988) EMBO J. 7, 603-609. 26. Piette, J., Bessereau, J. L., Huchet, M. & Changeux, J.-P. 4. Goldman, D. & Staple, J. (1989) Neuron 3, 219-228. (1990) Nature (London) 345, 353-355. 5. Brenner, H. R., Witzemann, V. & Sakmann, B. (1990) Nature 27. Lassar, A. B., Buskin, J. N., Lockshon, D., Davis, R. L., (London) 344, 544-547. Apone, S., Hauschka, S. D. & Weintraub, H. (1989) Cell 58, 6. Klarsfeld, A., Bessereau, J. L., Salmon, A. M., Triller, A., 823-831. Babinet, C. & Changeux, J.-P. (1991) EMBO J. 10, 625-632. 28. Wentworth, B. M., Donoghue, M., Engert, J. C. & Berglund, 7. Salmon, A.-M. & Changeux, J.-P. (1992) NeuroReport 3, E. B. (1991) Proc. Natl. Acad. Sci. USA 88, 1242-1246. 973-976. 29. Pinset, C., Mulle, C., Benoit, P., Changeux, J.-P., Cheily, J. & 8. Sanes, J. R., Johnson, Y. R., Kotzbauer, P. T., Mudd, J., Montarras, D. (1991) EMBO J. 10, 2411-2418. Hanley, T., Martinou, J. C. & Merlie, J. P. (1991) Development 30. Martinou, J. C. & Merlie, J. P. (1991) J. Neurosci. 11, 1291- 113, 1181-1191. 1299. 9. Simon, A. M., Hoppe, P. & Burden, S. J. (1992) Development 31. Harrison, T., Graham, F. & Williams, J. (1977) Virology 77, 114, 545-553. 319-329. 10. Klarsfeld, A., Daubas, P., Bourachot, B. & Changeux, J.-P. 32. Baldwin, T. J. & Burden, S. J. (1989) Nature (London) 341, (1987) Mol. Cell. Biol. 7, 951-955. 716-720. 11. Wang, Y., Xu, H. P., Wang, X. M., Ballivet, M. & Schmidt, J. 33. Witzemann, V., Barg, B., Criado, M., Stein, E. & Sakmann, B. (1988) Neuron 1, 527-534. (1989) FEBS Lett. 242, 419-424. Downloaded by guest on September 30, 2021