Synapse-specific gene expression

seminars in D E V E L O P M E N T A L B I O L O G Y , Vol 6, 1995: pp 175-183

Synapse-specific gene expression Gerald C. Chu, Mark A. Velleca and John P. Merlie

Inductive interactions between motor neuron and muscle result in the formation of synaptic structures at the neuromuscular junction. The localized appearance of synaptic proteins is due in part to the selective expression of specific genes in the muscle nuclei which lie beneath the motor endplate. For example, synapse-specific expression of the acetylcholine receptor subunit genes contributes to the restricted distribution of the'acetylcholine recepto'c. A transynaptic, motor neuron-derived signal is thought to induce changes in the transcriptional potential of synaptic nuclei. Although the precise mechanisms of gene activation have yet to be elucidated, a neuronally derived factor called ARIA is likely to play a central role in this process.

well as genes encoding other synaptically localized proteins, in a small population of nuclei lying directly beneath the neuromuscular endplate. In the multinucleate muscle fiber, these neuronally-induced nuclei are thus transcriptionally distinct from the vast numbers of extrasynaptic nuclei present within the same fiber. This review will describe mechanisms required to generate these differences in transcriptional potential. The gene encoding the AChR e subunit provides an excellent model for understanding synaptic gene regulation, and is used to illustrate mechanisms involved in this process. Finally, we will describe our current understanding of the signaling events involved in the transynaptic induction of gene expression, focusing on the role of ARIA, a putative neuromuscular inducing factor.

Key words: transynaptic gene regulation / synapdc expression / acetylcholine receptor genes / 7H4 / ARIA DURING DEVELOPMENT,inductive interactions between the growth cone of the elongating motor neuron and its target muscle fiber result in formation of the neuromuscular junction. Both pre- and post-synaptic structural components necessary for synaptic transmission arise as a result of these mutual and complementary interactions. 1 One of the most striking and best studied consequences of neuromuscular induction is the h i g h accumulation of nicotinic acetylcholine receptors (AChR) in the muscle postsynaptic membrane. Present at over 10,000 receptors/ ~m 2 at the synapse, the density of AChR falls off by three orders of magnitude within a few microns. A variety of mechanisms contribute to the formation and maintenance of these densely ordered AChR arrays. One mechanism involves the nerve-induced, post-translational reorganization of proteins already present on the muscle cell surface. 9"6 A second mechanism, the motor neuron-dependent induction of gene expression, is the focus of this review. A growing body of evidence indicates that the nerve supplies a stable, spatially restricted signal which preferentially stimulates expression of AChR genes, as

Synapse-specific e x p r e s s i o n o f A C h R subnnlt

genes The AChR is a pentameric channel comprised of four distinct yet structurally simila~ subunits. During embryogenesis, AChR are composed of a, [3, y and 6 subunits in the stoichiometry a213yS; in the postnatal period, AChR containing the e subunit replace those containing the 7 subunit, resulting in a physiological alteration of channel properties.'7 8 These five subunits are encoded by separate genes, and their expression is subject to multiple regulatory influences. Mechanisms of AChR subunit gene regulation are both nerve-dependent (regulated by local and global effects of the motor neuron),9 as well as nerveindependent (such as expression resulting from myogenic differentiation). A decade ago, Northern blot analysis of mouse diaphragm muscle first indicated that endplate-rich areas of muscle contain greater amounts of AChR mRNA compared to endplate-free regions. 1~ This observation suggested that the high concentration of AChR protein present at the postsynaptic membrane could in part result from the increased local expression of AChR subunit genes at the synapse. Since then, in-sitn hybridization studies of innervated muscle fibers have shown that a, [3, 6, and e mRNAs are

From the Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St Louis, 3/I0 63110, USA 9 Academic Press Ltd 1044-5 781/95/030175 + 09 $8.00/0 175

G. C. Chu et al

nuclei, a cDNA called 7H4 (due to its location on a gridded plate) was isolated. 2~ Based on Northern blot analysis of RNA from endplate-rich versus endplatefree muscle, 7H4 transcripts (1.6 kb and 5.4 kb in length) exhibit preferential accumulation at the synapse. In addition, 7H4 shares an expression pattern commonly associated with the AChR subunit genes; expression of 7H4 is muscle-specific, differentiation dependent, and is stimulated upon denervation. The onset of expression occurs postnatally, with a developmental time course quite similar to the AChR ~ subunit. Thus, based on its expression profile, one would predict that the 7H4 gene product plays a structural or functional role in formation of the neuromuscular synapse. Sequence analysis of 7H4 cDNA yielded a complete surprise: 7H4 does not seem to encode a protein. Stop codons are evenly distributed throughout each reading frame. Within the 5.4 kb transcript, the longest ATG-inifiated open reading frame is preceded by 30 ATG codons; it is in a poor context for translational initiation, and is only B12 bp in length. Other than some repetitive elements contained within the transcribed region, 7H4 shares no significant sequence homologies with other genes. The structure of the gene indicates that 7H4 contains no introns, and interestingly, that the 1.6 kb transcript originates completely from within the 5.4 kb RNA. This latter observation may indicate that regulatory elements which initiate transcription of the 1.6 kb RNA are themselves transcribed, or that this shorter species is a stable cleavage product of the longer 5.4 kb RNA. Despite these unusual characteristics, 7H4 RNA seems to be produced as a result of Pol II transcription: A TATA box and E box elements (binding sites for myogenic transcription factors) are found upstream of the 5.4 kb RNA transcriptional start site, and nontemplated polyadenylation occurs at the 3' end of the transcript. Most importantly, the sequence of 7H4 is highly conserved among rat, mouse .and human, suggesting functional evolutionary conservation. What might be the function of this non-coding synaptically associated RNA? A growing number of non-translated RNAs have been suggested to possess regulatory properties. 21"27 Dubbed 'riboregulators', these RNAs (either non-coding, or untranslated regions of coding mRNAs) have been implicated in mediating cellular growth a n d / o r differentiation. 26 Although the function of 7H4 has yet to be determined, one intriguing possibility is that 7H4 is a synapse-specific riboregulator involved in the development or maturation of the neuromuscular junction.

indeed highly enriched at the synapse. H'a4 Furthermore, these high resolution studies demonstrate that the greatest accumulations of these mRNAs are specifically associated with the synaptic, morphologically distinct nuclei, which are found direcdy beneath the motor endplate. The marked difference between synaptic and extrasynaptic nuclei in the expression of AChR genes has led to the hypothesis that localized nerve-induced assembly of the postsynaptic apparatus is partially mediated by changes in the transcriptional potential of synaptic nuclei.

Other genes preferentially expressed at the synapse It has been hypothesized that many of the postsynaptic proteins at the neuromuscular junction are encoded by genes which exhibit preferential expression at the synapse. Evidence for this hypothesis comes from studies of several proteins in addition to the AChR subunits. For example, the restricted distribution of acetylcholinesterase, an enzyme that hydrolyses acetylcholine and thereby limits the duration of synaptic transmission, may result from the increased levels of acetylcholinesterase mRNA found to be associated with synaptic nuclei. 15'16 Recently, mRNA for the 43 kDa AChR-associated cytoskeletal protein 43k rapsyn and the cellular adhesion molecule N-CAM have also been shown to exhibit preferential accumulation at synaptic nuclei; 17 however, some synaptically enriched proteins are encoded by mRNAs that are uniformly distributed) 7 Proteins encoded by genes which are synaptically expressed have diverse cellular localizations; extracellular (located in the basal lamina), transmembrane and intracellular, membrane-associated proteins are all represented. Some of the genes, such as AChR subunit genes, exhibit muscle-specific expression, while others do not: N-CAM and acetylcholinesterase are also found in the brain, and rapsyn is present in kidney and heart as well as in undifferentiated myocytes) sag Despite their complex regulation in muscle and other cell types, all of these genes appear to be positively induced by the motor neuron, resulting in synaptic enrichment of mRNA as well as the proteins they encode.

7H4, a synapse-associated, non-coding RNA In a subtractive hybridization designed to identify mRNAs selectively expressed in synaptic muscle 176

Synapse-specific gene expression Gain of function studies as well as mutational analyses in transgenic mice may help to elucidate the precise function of 7H4.

after innervation, a signaling pathway mediated by electrical activity may suppress this activation, a suppression overcome only by the presence of the motor neuron. Although the positive inducing influence of the motor neuron is a prerequisite for synapse-specific gene expression, it is equally important that extrasynaptic levels of expression remain low. This lack of extrasynaptic expression can be achieved in two ways: if activity-dependent down-regulation of expression sufficiently negates activation by the myogenic program, or if the gene is inherently less sensitive to myogenic activation. The e subunit gene exhibits low extrasynaptic expression due to this latter mechanism. In muscle cell tissue culture, in the absence of any neuronal influences, e subunit expression is 50-fold less than other AChR subunits. 4s Due to this low basal level of myogenic expression, the e subunit gene exhibits synaptically restricted expression under conditions where this property is lost for the other AChR subunits. For example, in denervated muscle, expression of the ct, 13, 3' and 6 subunits is stimulated 10-100 fold extrasynaptically, whereas e subunit expression remains endplate-specific. ~3'14 Due to its greater reliance on the inductive influence of the motor neuron for expression, the e subunit gene is a particularly good model for understanding transynaptic gene regulation.

Regulators of AChR gene expression Our current understanding of synapse-specific gene regulation has largely been obtained from analysis of the AChR subunit genes. In addition to their spatially restricted, motor neuron dependent activation in synaptic muscle nuclei, these genes are also regulated by two mechanisms which determine levels of gene expression throughout the muscle fiber. First, AChR genes are transcribed by fiuclei throughout the syncytial myofiber in a tissue-specific manner as part of the myogenic program. Second, these genes are down-regulated throughout the myofiber in response to electrical activity, a consequence of innervation. All three mechanisms - - synaptic, developmental, and activity d e p e n d e n t - - a c t in concert to determine levels ofAChR gene expression in the muscle fiber. In endplate-associated nuclei, positive, local induction by the motor neuron tends to be the predominant regulator. Outside the synapse, the two functionally antagonistic processes of developmental and activitydependent regulation compete to determine the levels of expression. Mechanisms involved in these two processes will be briefly reviewed below. Several potential mediators of developmental and activity-dependent AChR gene regulation have been characterized (Figure 2). The first is a group of muscle-specific transcriptional activators, known as the myogenic factors (or muscle differentiation factors, MDFs): MyoD, myogenin, MRF4 and myfS.2s These factors are activated during development and proceed to transactivate AChR and other musclespecific genes. Furthermore, some MDFs, most notably myogenin, are themselves regulated by electrical activity, suggesting that these factors also contribute to the activity-dependent expression of AChR genes. 29-a4 Another candidate regulator is protein kinase C (PKC). Electrical activity stimulates PKC, and stimulation of PKC can repress AChR genes, aS'a6 These regulatory pathways may converge: MDFs may be targets of PKC, aT'as and the potential of MDFs to stimulate transcription may depend on their phosphorylation state, sam A cAMP-dependent mechanism which couples electrical activity to gene regulation has also been suggested. 49 Thus, in the uninnervated muscle fiber, AChR gene expression is promoted by activation of the MDF-dependent myogenic program;

Transcriptional analysis of synapse-specific gene expression Although the selective accumulation of AChR mRNA at the synapse has been taken as evidence for preferential transcription of AChR genes by synaptic nuclei, other mechanisms can theoretically account for this effect. For example, the concentration of AChR mRNA at the synapse could result from selective stabilization of mRNA, or from selective transport of RNA to the neuromuscular junction. However, the results of AChR e subunit promoter analysis in transgenic mice support a major role for a transcriptional mechanism. A modified form of the E. coli lacZ gene possessing a nuclear localization signal sequence has been used as a reporter gene to investigate the expression pattern of AChR subunit transcriptional promoters. When the nuclear lacZ gene (nlacZ) is expressed, the [~alactosidase translation product is concentrated in nearby nuclei, permitting discrimination between transcriptionally active and inactive nuclei. Transgenic mice bearing this 177

G. C. Chu et al

reporter gene under the control of a 3500 bp AChR e subunit promoter (e3500nlacZ) exhibit a pattern of expression which reflects endogenous e subunit mRNA distribution; synaptic muscle nuclei exhibit strong expression of the nlacZ gene, yet ~alactosidase activity is not detectable short distances away in nuclei of the same myofiber (ref44, Figure 1). e3500nlacZ expression is muscle-specific, exhibits a postnatal onset, and does not appear extrasynapticaUy upon denervation. 44'4s Because the nlacZ transcript contains essentially no sequences in common with the endogenous transcript, differential RNA stability or transport are not likely mechanisms for synaptic mRNA accumulation. Similar studies have been performed with the chicken a subunit promoter. 44'46 In these studies, the synaptic localization is less robust, perhaps due to evolutionary divergence between the chicken and mouse promoter or the transacting factors needed to stimulate transcription. Using a different reporter gene, the 6-subunit promoter has also been shown to direct synapse specific expression, 47A8 and preliminary results indicate that the y subunit promoter can direct the synaptic expression of nlacZ during embryonic development (G.C.C. and J.P.M., unpublished). The observation that AChR subunit promoters can direct reporter gene activity at the motor endplate has

prompted a closer examination of these sequences to identify regulatory elements involved in synapsespecific gene expression. A 5' deletion series of the e promoter has been generated and tested in transgenic mice; these studies indicate that sequences within 280 bp upstream of the e transcriptional start site can direct the expression of nlacZ to synaptic nuclei. 45 In a separate study, a reporter construct bearing just 83 bp of the e promoter exhibited synaptically biased expression when injected into mouse muscle;49 however, the extrasynaptic expression observed in a significant percentage of fibers using this approach is somewhat puzzling. Similarly, transgenic analysis of the 6 subunit gene has defined the synaptically responsive promoter elements to be within 125 bp of the start of transcription. 48 Despite the observation that these proximal AChR subunit promoters can confer similar regulatory properties, sequence comparisons have revealed only limited homology between the promoters, giving little indication of the elements that might mediate synapse-specific gene activation. The analysis of AChR gene expression in muscle cell culture has helped characterize regulatory elements important for the myogenic activation of these genes. In the e subunit promoter, sequences within 150 bp of the transcriptional start site are sufficient to

letgm'e 1. The AChR e subunit promoter confers synapse-specificgene expression in transgenic mice. Transgenic mice bearing 3500 bp oft_he e subunit promoter fused to the nlacZ reporter gene were generated. The photograph above shows tibialis anterior muscle from a transgenic mouse which has been subject to [?%ralactosidase histochemistry. The dark staining indicates zones of transgene activity.Co-staining of this muscle for acetylcholinesterase, an independent marker for neuromuscular synapses (not shown) indicates that transgene activity occurs precisely in synaptic regions of the muscle. 178

Synapse-specific gvne expression confer differentiation-dependent, tissue-specific expression in muscle cell culture. 49-s2 This 150 bp sequence contains a single E box, the binding element for MDFs. The E box motif is present in the regulatory regions of all the AChR subunit genes and is involved in activation of these genes during the myogenic differentiation program. The E box within the e subunit promoter has been shown to be functional in tissue culture; deletion of this element impairs the ability of MDFs to transactivate the e promoter. 51 The ubiquitous presence of the E box motif in the AChR subunit genes makes it a plausible candidate to mediate synapse-specific transcription. However, transgenic constructs containing E box mutations do not result in" the loss of synaptic expression. 4s'49 Moreover, no MDF has been shown to be preferentially expressed at the synapse.53

growth factor (EGF) family, which serve as ligands for the Class I group of receptor tyrosine kinases (RTKs).68 The gene encoding ARIA is expressed prior to innervation in the ventral horn of the spinal cord, 64'67'69 where motor neuron cell bodies originate, and most recently, ARIA protein has been detected at the motor endplate. 7~ The cloning of ARIA has enabled in-vitro studies of AChR regulation using recombinant ARIA protein made in E. coli7~ A 68 amino-acid polypeptide corresponding to the EGF-like domain of ARIA was shown to be comparable to native ARIA protein in its ability to induce AChR e subunit mRNA levels in muscle culture. This recombinant ARIA polypeptide recapitulated in vitro some of the inducing activities associated with the motor neuron in vivo. For example, when muscle cells from transgenic mice bearing the e3500nlacZ transgene were dissociated and grown in tissue culture, the myotubes that formed exhibited only low levels of nlacZ expression m a n expected result given the absence of nerve. In the presence of ARIA, however, this transgene exhibited a remarkable degree of activation, whereas other factors tested yielded no induction at all. The shorter, synapsespecific e280nlacZ transgene also displayed an activation by ARIA as well. Transient transfection of reporter constructs containing the e promoter in a muscle cell line has delimited the sequences which confer ARIA-responsiveness to within 150 bp of the transcriptonal start site. Furthermore, responsiveness to ARIA was retained in the absence of functional E-boxes. Analysis of sequences which mediate ARIAdependent expression should thus provide a rapid means for the identification synaptic regulatory elements. The effects of ARIA on synaptically active genes other than the e subunit should also provide further insight into mechanisms of synapse-specific gene expression. Given the identity of ARIA as a ligand for receptor tyrosine kinases, we can now speculate about the regulatory factors and pathways that transduce the synaptic signal (see figure 2). Inductive interactions between ligands and their target RTKs, leading to the specification of cell fate, have been well characterized in other developmental systems. For example, in the nematode C. elegans, the product of lin-3, a ligand structurally related to ARIA, locally induces vulval structure formation by binding to let-23, a Class I RTK.71 Synaptic nuclei may represent a 'cell fate' that is distinct from extrasynaptic nuclei, a fate that is possibly specified by ARIA. The mammalian homologne of ARIA has been shown to interact directly or

Neurotrophic control of synapse-specific genes The ability of neuronal cells to influence the number and distribution of AChRs in co-culture, even in the absence of direct contact, led to the hypothesis that motorneurons secrete factors that are directly responsible for induction of AChR. 54 The basal lamina protein agrin can induce the clustering of extant AChRs but does not appear to stimulate new AChR synthesis or the transcription of AChR subunit genes. ~'s Calcitonin gene related peptide (CGRP) increases muscle surface AChR number, 5s as well as a subunit mRNA by a cAMP dependent mechanism, 56'57 and has been p r o p o s e d as a neuronal inducing agent. 58 However, CGRP may not be present early enough in embryonic development to account for synapse-specific gene induction. 59'6~ ARIA (acetylcholine receptor inducing activity) is the strongest candidate for the neuronal inducing factor which directs synaptic gene expression. This factor was initially identified as an activity present in chicken brain and spinal cord extract that could stimulate AChR synthesis in cultured chicken myotubes. 61 In addition to increasing AChR number, ARIA has also been shown to induce the expression of several AChR subunit mRNAs. 62'63 Most dramatically, the level of e subunit mRNA increases 10-fold when ARIA is added to myotube cultures. 6s Recently, the gene encoding ARIA has been cloned; 64 surprisingly, the sequence turns out to be the chicken homologne of human heregulin [also known as Neu differentiation factor (NDF) and glial growth factor (GGF)]. 6~67 This factor belongs to the epidermal 179

G. C. Chu et al

~

~

0

~

I= 0

u'~,,

~'~

~ ,~

.~

~

~

~C)~

~

~Nu~

~_~ 0~"--.-.~

.-J

~

'~ |

.

~

o

-~

o~

~

o :

~

~

~

/x.Z./ ~,

9-

0

~

~'0~

'~

~.~

~-

0

0

~..~

~.~

m'~ u

~

.~, ~

~

~

~

I~

C ~ ' ~

~ c ,. ~ . . 0 . ~ 0

-

0

I~"~

~ ' ~ "~

,~ ~

..0

o~

."I, *~ , ~ ' ~ I

~ .'~'~ 0~"

~

9 .~ o ~

o I~ ~ . ~ :~ ~0 -~

~,

'a

~_

o-~ ~ ~ ~ u ~'l~ ~ ..~ ~

~s~.~.

180

~

~'~

~ 1 7 6 1 7 6 1 7 6 1 7 6 I~. ~

0

o., ~

t~O

~c~

~. ~

._=..~

o

~.~'~ u ~..~.~ ~> o

ti~0.~ .o. ~

~

-

o~

~.~o ~ ~ .~

~ "o

~ .~ ~'~0 .~

Synapse-specific grae expression 10. Merlle JP, Sanes JR (1985) Concentration of acetylcholine receptor mRNA in synaptic regions of adult muscle fibres. Nature 317:66-68 11. Fontaine B, Sassoon D, Buckingham M, CbangeuxJP (1988) Detection of the nicotinic acetylcholine receptor alpha-subunit mRNA by in situ hybridization at neuromuscular junctions of 15-day-old chick striated muscles. EMBOJ 7:603-609 12. Fontaine B, Changeux JP (1989) Localization of nicotinic acetylcholine receptor alpha-subunit transcripts during myogenesis and motor endplate development in the chick.J Cell Biol 108:1025-1037 13. Goldman D, StapleJ (1989) Spatial and temporal expression of acetylcholine receptor RNAs in innervated and denervated rat soleus muscle. Neuron 3:219-228 14. Brenner HR, Witzemann V, Sakmann B (1990) Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses. Nature 344:544-547 15. Jasmin BJ, Lee RK, Rotundo RL (1993) Compartmentalization of acetylcholinesterase mRNA and enzyme at the vertebrate neuromuscular junction. Neuron 11:467-477 16. Michel RN, Vu CQ, Tetzlaff W, Jasmin BJ (1994) Neural regulation of acetylcholinesterase mRNAs at mammalian neuromuscular synapses. J Cell Biol 127:1061-1069 17. Moscoso LM, Merlie.JP, Sanes JR (1995) N-CAM, 43K-rapsyn, and S-Laminin mRNAs are concentrated at synaptic sites in muscle fibers. Mol Cell Neurosci 6:80-89 18. Frail DE, Musil LS, Buonanno A, MerlieJP (1989) Expression of RAPsyn (43K protein) and nicotinic acetylcholine receptor genes is not coordinately regulated in mouse muscle. Neuron 2:1077-1086 19. Musil LS, Frail DE, Merlle JP (1989) The mammalian 43-kD acetylcholine receptor-associated protein (RAPs)m) is expressed in some nonmuscle cells. J Cell Biol 108:1833-1840 20. Velleca MA, Wallace MC, Mealie .[P (1994) A novel synapseassociated noncoding RNA. Mol Cell Biol 14:7095-7104 21. Brannan CI, Dees EC, Ingram RS, Tilghman SM (1990) The product of the H19 gene may function as an RNA. Mol Cell Biol 10:28-36 22. Brockdorff N, Ashworth A, Kay GF, Cooper P, Smith S, McCabe VM, Norris DP, Penny GD, Patel D, P.Astan S (1991) Conservation of position and exclusive expression of mouse Xist from the inactive X chromosome. Nature 351:329-331 23. Brockdorff N, Ashworth A, Kay GF, McCabe VM, Norris DP, Cooper PJ, Swift S, Rastan S (1992) The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus. Ceil 71:515-526 24. Brown CJ, Hendrich BD, Rupert JL, Lafreniere RG, Xing Y, LawrenceJ, W'dlard I-IF (1992) The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell 71:527-542 25. Rastinejad E Blau HM (1993) Genetic compiementation reveals a novel regulatory role for 3' untranslated regions in growth and differentiation. Cell 72:903-917 26. Rastinejad F, Conboy MJ, Rando TA, Blau HM (1993) Tremor suppression by RNA from the 3' untranslated region of a-Tropomyosin. Cell 75:1107-1117 27. Askew DS, Li J, Ihle JN (1994) Retroviral insertions in the murine His-I locus activate the expression of a novel RNA that lacks an extensive open reading frame. Mol Cell Biol 14:1743-1751 28. Edmondson DG, Olson EN (1993) Helix-loop-helixproteins as regulators of muscle-specific transcription, j Biol Chem 268:755-758 29. Effimie R, Brenner ~ Buonanno A (1991) Myogenin and myoD join a family of skeletal muscle genes regulated by electrical activity. Proc Nail Acad Sci USA 88:1349-1353 30. Witzemann V, Sakmann B (1991) Differential regulation of

i n d i r e c t l y with e r b B 2 , e r b B 3 a n d e r b B 4 ( H E R 2 , H E R 3 a n d HER4),72 m e m b e r s o f t h e Class I f a m i l y o f RTKs. e r b B 2 a n d e r b B 3 a r e f o u n d in m u s c l e a n d p o t e n t i a l l y m e d i a t e t h e effects o f A R I A (L.M. M o s c o s o , G.C.C., J.P.M., J.R. Sanes, u n p u b l i s h e d ) however, t h e t r u e A R I A r e c e p t o r m a y b e a n o v e l RTK, y e t to b e discovered. Signal transduction pathways involving Class I RTKs h a v e b e e n c h a r a c t e r i z e d in o t h e r cell types. S H 2 d o m a i n - c o n t a i n i n g p r o t e i n s , w h i c h b i n d p h o s p h o r y l a t e d t y r o s i n e r e s i d u e s , a s s o c i a t e w i t h activ a t e d RTKs. 7s F o r e x a m p l e , G r b 2 a n d t h e p 8 5 s u b u n i t o f p h o s p h a t i d y l i n o s i t o l 3-kinase (PI3 k i n a s e ) a r e t h o u g h t to i n t e r a c t w i t h s p e c i f i c p h o s p h o t y r o s i n e r e s i d u e s in t h e c y t o p l a s m i c d o m a i n o f e r b B 2 a n d e r b B 3 , respectively. 72 T h u s , a c t i v a t i o n o f t h e G r b 2 m e d i a t e d Ras pathway, s t i m u l a t i o n o f PI3 k i n a s e , o r activation of some unique combination of signaling p a t h w a y s m a y t r a n s d u c e a s p a t i a l l y r e s t r i c t e d s i g n a l to s y n a p t i c n u c l e i . T h e b i n d i n g o f A R I A to a R T K m a y trigger a cascade involving the phosphorylation of s i g n a l i n g i n t e r m e d i a t e s , r e s u l t i n g in t h e a c t i v a t i o n o f a s p e c i f i c set o f g e n e s . G e n e s a c t i v a t e d in t h e ARIAdependent cascade may encode regulatory molecules w h i c h m a y in t u r n s t i m u l a t e t h e t r a n s c r i p t i o n o f structural genes needed for the formation of the neuromuscular junction ( F i g u r e 2). A d d i t i o n a l r e s e a r c h in this a r e a will b e n e e d e d to e l u c i d a t e t h e p r e c i s e m e c h a n i s m s i n v o l v e d in this t r a n s y n a p t i c signaling process.

References 1. Hall ZW, SanesJR (1993) Synaptic structure and development: the neuromuscular junction. Cell/Neuron 72 (Suppl.):99-121 2. McMahan UJ (1990) The agrin hypothesis. Cold Spring Harbor Symp Quant Biol 55:407-418 3. Nastuk MA, Fallon JR (1993) Agrin and the molecular choreography of synapse formation. Trends Neurosci 16:72-76 4. Hoch W, Campanelli JT, Scheller RH (1994) Agrin-induced clustering of acetylcholine receptors: a cytoskeletal link. J Cell Biol 126:1-4 5. Fallon JR, Hall ZW (1994) Building synapses - - agrin and dystroglycan stick together. Trends Neurosci 17:469-473 6. Apel ED, Merlie JP (1995) Assembly of the postsynaptic apparatus. Curr Opin Neurobiol 5:62-67 7. Mishina M, Takai T, Imoto K, Noda M, Takahashi T, Numa S, Methfessel C, Sakmann B (1986) Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature 321:406-411 8. Gu Y, Hall ZW (1988) Immunological evidence for a change in subunits of the acetylcholine receptor in developing and denervated rat muscle. Neuron 1:117-125 9. Laufer R, ChangeuxJP (1989) Activity-dependentregulation of gene expression in muscle and neuronal cells. Mol Neurobiol 3:1-53 181

G. C. Chu et al

31. 32.

33. 34.

35.

36. 37.

38. 39.

40. 41.

42.

43. 44.

45. 46.

47. 48. 49.

MyoD and myogenin mRNA levels by nerve induced muscle activity. FEBS Lett 282:259-264 Duclert A, Piette J, ChangeuxJP (1991) Influence of innervation of myogenic factors and acetylcholine receptor aiphasubunit mRNAs. Neuroreport 2:25-28 Buonnano A, Apone L, Morasso MI, Beers R, Brenner HR, Eftimie R (1992) The myoD family of myogenic factors is regulated by electrical activity:isolation and characterization of a mouse myf-5 cDNA. Nucl Acids Res 20:539-544 Neville CM, SchmidtJ (1992) Expression of myogenic factors in skeletal muscle and electric organ of Torpedo californica. FEBS Lett 305:23-26 MerlieJP, MuddJ, Cheng TC, Olson EN (1994) Myogenin and acetylcholine receptor alpha gene promoters mediate transcriptionai regulation in response to motor innervation.J Biol Chem 269:2461-2467 Klarsfeld A, Laufer R, Fontaine B, Devillers TA, Dubreuil C, ChangeuxJP (1989) Regulation of muscle AChR alpha subunit gene expression by electrical activity: involvement of protein kinase C and Ca2+. Neuron 2:1229-1236 Huang CF, Tong J, SchmidtJ (1992) Protein kinase C couples membrane excitation to acetylcholine receptor gene inactivation in chick skeletal muscle. Neuron 9:671-678 Laufer R, Klarsfeld A, Changeux JP (1991) Phorboi esters inhibit the activity of the chicken acetylcholine receptor aiphasubunit gene promoter. Role of myogenic regulators. Eur J Biochem 202:813-818 Huang CF, Neville CM, SchmidtJ (1993) Control of myogenic factor genes by the membrane depolarization/protein kinase C cascade in chick skeletal muscle. FEBS Lett 319:21-25 Li L, ZhouJ, James G, Heller HR, Czech MP, Olson EN (1992) FGF inactivates myogenic helix-loop-helix proteins through phosphorylation of a conserved protein kinase C site in their DNA-binding domains. Cell 71:1181-1194 Olson EN, Burgess R, StaudingerJ (1993) Protein kinase C as a transducer of nuclear signals. Cell Growth Diff 4:699-705 Mendelzon D, ChangeuxJP, Nghiem HO (1994) Phosphorylation of myogenin in chick myotubes - - regulation by electricalactivity and by protein kinase C - - implications for acetylcholine receptor gene expression. Biochemistry 33:2568-2575 Chahine KG, Baracchini E, Goldman D (1993) Coupling muscle electrical activity to gene expression via a cAMPdependent second messenger system. J B i o l Chem 268:2893-2898 MartinouJC, MerlieJP (1991) Nerve-dependent modulation of acetylcholine receptor epsilon-subunitgene expression. J Neurosci 11:1291-1299 Sanes JR, Johnson YR, Kotzbauer PT, Mudd J, Hanley T, Martinou JC, Merlie JP (1991) Selective expression of an acetylcholine receptor-lacZ transgene in synaptic nuclei of adult muscle fibers. Development 113:1181-1191 Gundersen K, SanesJR, MerlieJ~P (1993) Neural regulation of muscle acetycholine receptor epsilon and alpha subunit gene promoters in transgenic mice. J Cell Biol 123:1535-1544 Klarsfeld A, Bessereau JL, Salmon AM, Triller A, Babinet C, Changeux JP (1991) An acetylcholine receptor aipha-subunit promoter conferring preferential synaptic expression in muscle of transgenic mice. EMBOJ 10:625-632 Simon AM, Hoppe P, Burden SJ (1992) Spatial restriction of AChR gene expression to subsynaptic nuclei. Development 114:545-553 Tang JC, Jo SA, Burden SJ (1994) Separate pathways for synapse-specific and electrical activity-dependent gene expression in skeletal muscle. Development 120:1799-1804 Duclert A, Savatier N, Changeux JP (1993) An 83-nucleotide promoter of the acetylcholine receptor epsilon-subunit gene confers preferential synaptic expression in mouse muscle. Proc Nail Acad SCi USA 90:3043-3047 182

50. Numberger M, Durr I, Kues W, Koenen M, Witzemann V (1991) Different mechanisms regulate muscle-specific AChR gamma- and epsilon-subunit gene expression. EMBO J 10:2957-2964 51. Sunyer T, Merlie JP (1993) Cell type- and differentiationdependent expression from the mouse acetylcholine receptor epsilon-subunitpromoter. J Neurosci Res 36:224-234 52. Waike W, Staple J, Adams L, Gnegy M, Chahine IL Goldman D (1994) Calcium-dependent regulation of rat and chick muscle nicotinic acetylcholine receptor (nAChR) gene expression. J Biol Chem 269:19447-19456 53. Velleca MA, Subtractive cloning of a novel synapse-specific noncoding RNA. 1994, Ph.D. Thesis, Washington University 54. Fischbach GD, Cohen SA (1973) The distribution of acetylcholine sensitivity over uninnervated and innervated muscle fibers grown in cell culture. Dev Biol 31:147-162 55. New HV, Mudge AW (1986) Caicitonin gene-related peptide regulates muscle acetylcholine receptor synthesis. Nature 323:809-811 56. Laufer R, ChangeuxJP (1987) Caicitonin gene-related peptide elevates cyclic AMP levels in chick skeletal muscle: possible neurotrophic role for a coexisting neuronal messenger. EMBO J 6:901-906 57. Fontaine B, Klarsfeld A, ChangeuxJP (1997) Caicitonin generelated peptide and muscle activity regulate acetylcholine receptor aipha-subunit mRNA levels by distinct intracellular pathways. J Cell Biol 105:1337-1342 58. Changeux .]P, Duclert A, sekine S (1992) Caicitonin generelated peptides and neuromuscular interactions. Ann New York Acad Sci 657:361-378 59. Peng HB, Chen Q, DeBiasi S, Zhu D (1989) Development of calcitonin gene-related peptide (CGRP) immunoreactivity in relationship to the formation of neuromuscular junctions in Xenopus myotomal muscle. J Comp Neurol 290:533-543 60. Li JY, Dahlstrom AB (1992) Development of calcitonin generelated peptide, chromgranin A, and synaptic vesicle markers in rat motor endplates, studied using immunofluorescenceand confocai laser scanning. Muscle Nerve 15:984-992 61. Usdin TB, Fischbach GD (1986) Purification and characterization of a polypeptide from chick brain that promotes the accumulation of acetylcholine receptors in chick myotubes. J Cell Biol 103:493-507 62. Harris DA, Fails DL, Dill-Devor RM, Fischbach GD (1988) Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor alpha subunit. Proc Nail Acad Sci USA 85:1983-1987 63. Martinou JC, Fails DL, Fischbach GD, Merlie JP (1991) Acetylcholine receptor-inducing activity stimulates expression of the epsilon-subunit gene of the muscle acetylcholine receptor. Proc Nail Acad Sci USA 88:7669-7673 64. Fails DL, Rosen KM, Corfas G, Lane WS, Fischbach GD (1993) ARIA, a protein that stimulates acetylcholine receptor synthesis, is a member of the neu ligand family. Cell 72:801-815 65. Wen D, Peles E, Cupples R, Suggs SV, Bacus SS, Luo Y, Trail G, Hu S, Silbiger SM, Ben Levy R, Koski RA, Lu HS, Yarden Y (1992) Neu differentiation factor: a transmembrane glycoprotein containing an EGF domain and an immunoglobulin homology unit. Cell 69:559-572 66. Holmes WE, SliwkowskiMX, Akita RW, Henzel WJ, Lee J, Park JW, Yansura D, Abadi N, Raab H, Lewis GD, Shepard HM, Ku~mg W-J, Wood WI, Goeddel DV, Vandlen RL (1992) Identification of heregulin, a specific activator of p185erbB2. Science 256:1205-1210 67. Marchionni MA, Goodearl AD, Chen MS, Bermingham MO, Kirk C, Hendricks M, Danehy F, Misumi D, Sudhaiter J, Kobayashi IL Wroblewski D, Lynch C, Baidassare M, Hiles I, DavisJB, HsuanJJ, Totty NF, Otsu M, McBurney RN, Waterfield MD, Stroobandt P, Gwynne D (1993) Gliai growth factors are

Synapse-specific gene expression

68. 69. 70.

71.

alternatively spliced erbB2 ligands expressed in the nervous system. Nature 362:312-318 Prigent SA, Lemoine NR (1992) The type 1 (EGFR-related) family of growth factor receptors and their ligands. Prog Growth Factor Res 4:1-24 Meyer D, Birchmeier C (1994) Distinct isoforms of neuregulin are expressed in mesenchymai and neuronal cells during mouse development. Proc Nard Acad Sci USA 91:1064-1068 Chu GC, Moscoso LM, Sliwkowski MX, Merlie JP (1995) Regulation of the acetylcholine receptor ~ subunit gene by recombinant ARIA: an in vitro model for transynaptic gene regulation. Neuron 14:329-339 Hill RJ, Steinberg PW (1993) Cell fate patterning during C. elegans vulval development. Dev Suppl 1993:9-18

183

72. Cart-awayKI, Canfley LC (1994) A neu acquaintance for erbB3 and erbB4: a role for receptor heterodimerizafion in growth signaling. Cell 78:5-8 73. Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, Neel BG, Birge RB, Fajardo JE, Chou MM, Hanafusa H, Schaffhausen B, Canfley LC (1993) SH2 domains recognize specific phosphopeptide sequences. Cell 72:767-768 74. Jo SA, Zhu X, Marchioni MA, Burden SJ (1995) Neuregulins are concentrated at nerve-muscle synapses and activate AChreceptor gene expression. Nature 373:158-161