Local transcriptional control of utrophin expression at the neuromuscular synapse.

Recently, the use of a transgenic mouse model system for Duchenne muscular dystrophy has demonstrated the ability of utrophin to functionally replace dystrophin and alleviate the muscle pathology (see Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349–353). However, there is currently a clear lack of information concerning the regulatory mechanisms presiding over utrophin expression during normal myogenesis and synaptogenesis. Using in situ hybridization, we show that utrophin mRNAs selectively accumulate within the postsynaptic sarcoplasm of adult muscle fibers. In addition, we demonstrate that a 1.3-kilobase fragment of the human utrophin promoter is sufficient to confer synapse-specific expression to a reporter gene. Deletion of 800 base pairs from this promoter fragment reduces the overall expression of the reporter gene and abolishes its synapse-specific expression. Finally, we also show that utrophin is present at the postsynaptic membrane of ectopic synapses induced to form at sites distant from the original neuromuscular junctions. Taken together, these results indicate that nerve-derived factors regulate locally the transcriptional activation of the utrophin gene in skeletal muscle fibers and that myonuclei located in extrasynaptic regions are capable of expressing utrophin upon receiving appropriate neuronal cues.

Duchenne muscular dystrophy (DMD) 1 is the most severe and prevalent primary myopathy. The genetic defect responsible for DMD is located on the short arm of the X chromosome and prevents the production of normal size dystrophin, a large cytoskeletal protein of 427 kDa (1,2). In 1989, Love and colleagues (3, see also Ref. 4) showed the existence of a gene on chromosome 6q24 that encodes a cytoskeletal protein, named utrophin, displaying a high degree of sequence identity with dystrophin.
In skeletal muscle, the level and localization of utrophin has been shown to vary markedly according to the state of differentiation and innervation of muscle fibers. In embryonic tissue, for instance, utrophin localizes to the sarcolemma along the entire length of developing fibers (5,6). As the muscle matures, the amount of utrophin decreases progressively, and utrophin becomes preferentially localized to the neuromuscular synapse (7,8). An exception to this occurs in muscle fibers from both DMD patients and mdx mice where utrophin persists at the sarcolemma in extrasynaptic regions (9 -11). Under specific conditions, therefore, utrophin presents a more homogeneous distribution along the sarcolemma of adult muscle fibers. Together, these studies therefore suggest that in addition to therapies based on dystrophin gene transfer, up-regulation of utrophin may be envisaged as an alternative strategy to prevent the relentless progression of DMD. In this context, we have recently shown that high expression of a truncated utrophin transgene markedly reduced the dystrophic muscle phenotype in mdx hind limb and diaphragm muscles indicating that systemic up-regulation of utrophin may indeed be an effective treatment for DMD (12). The next step is to decipher the regulatory mechanisms presiding over utrophin expression in attempts to ultimately induce expression of the endogenous gene product throughout skeletal muscle fibers. In the present study, we have thus initiated a series of experiments focusing on the molecular mechanisms involved in the restricted expression of utrophin at the neuromuscular synapse.

EXPERIMENTAL PROCEDURES
Surgery-Ectopic synapses were induced to form on soleus muscles from adult control and mdx mice. An incision was first made at the mid-calf region, and the common peroneal nerve was exposed by blunt dissection. Both branches of this nerve were isolated, cut, and transplanted onto the distal surface of the soleus (13). Fourteen days later, ϳ5 mm of the tibial nerve was cut and removed to denervate the muscle and to allow the foreign nerve to form synaptic contacts with soleus muscle fibers. Two weeks after sectioning the tibial nerve, the sciatic nerve was stimulated. Soleus muscles which demonstrated contractile activity in response to electrical stimulation were excised, mounted with Tissue Tek, and frozen.
Immunofluorescence-Immunofluorescence experiments were performed on longitudinal serial sections (12 m) of soleus muscles. The presence of synapsin was monitored using a rabbit anti-synapsin antibody (Alexis Corp., San Diego, CA). Utrophin immunoreactivity was detected using either a rabbit anti-utrophin antibody (from Dr. Tejvir Khurana, Harvard University) or a monoclonal anti-utrophin antibody (from Dr. Glen Morris, N.E. Wales Institute, UK). Synapsin and utrophin antibodies were applied onto separate serial muscle sections for 1 h.
In Situ Hybridization-Longitudinal serial cryostat sections (12 m) of hind limb muscles from control C57BL/6 and mdx mice were placed on alternate Superfrost Plus slides (Fisher Scientific Co., Pittsburgh, PA). Alternate slides were either processed for acetylcholinesterase (AChE) histochemistry (14) to visualize neuromuscular junctions or subjected to in situ hybridization using synthetic oligonucleotides for detection of utrophin transcripts. In situ hybridization was performed using two antisense oligonucleotides complementary to the mouse utrophin mRNA (oligonucleotide 1, 5Ј-TGTGCCCCTCAGCCACTCTTCCTTCTCCTTGATGG-TCTCCTC-3Ј, and oligonucleotide 2, 5Ј-TGCTGCCTGGTGGAACTGTG-GGCCTGGGTCAGTGTCAAGTG-3Ј) according to Schalling et al. (15). Analysis of in situ hybridization labeling was performed using an image analysis system equipped with Image 1.47 software (Wayne Rasband, NIMH) (16). The density of in situ hybridization labeling in synaptic versus extrasynaptic regions was determined by measuring the number of labeled pixels within a circular field of constant 100 m in diameter. To determine the difference in utrophin mRNA levels between control and mdx mouse muscles, 1-mm 2 areas of extrasynaptic regions were sampled. Optical density values were used as a measure of labeling with higher values indicating greater labeling (17). Twelve muscle sections were processed for each condition, and a minimum of four measurements were performed on each section. Three animals were used for each condition.
Expression of Utrophin Promoter-Reporter Gene Constructs-Four human utrophin promoter-reporter gene constructs were used in these experiments: 1.3-and 0.5-kb promoter fragments positioned in either the forward or reverse orientations (see Fig. 1 and Ref. 18). These promoter fragments were inserted upstream of the reporter gene lacZ and a nuclear localization signal (nlsLacZ). Plasmid DNA was prepared using the Qiagen Mega-prep procedure (Chatsworth, CA), and final pellets were resuspended in sterile phosphate-buffered saline to a final concentration of 2 g/l.
For direct gene transfer, 25 l of DNA solution was injected directly into the tibialis anterior (TA) muscle of 4-week-old mice (19 -21). At different time intervals thereafter (7-42 days), TA muscles were excised and quickly frozen for serial cryostat sectioning. Tissue sections were processed histochemically for the demonstration of ␤-galactosidase and AChE activity. The position of blue myonuclei indicative of utrophin promoter activity was determined and compared with the presence of neuromuscular synapses using the quantitative procedure established by Duclert et al (21).

RESULTS
In a first series of experiments, we examined by in situ hybridization the distribution of utrophin mRNAs along muscle fibers from both C57BL/6 and mdx mice. Our results disclosed a selective accumulation of utrophin transcripts within the postsynaptic sarcoplasm (Fig. 2, A and B). In these experiments, utrophin mRNAs were also detected in extrasynaptic regions of muscle fibers albeit at significantly lower levels in comparison to synaptic sites. As expected, utrophin transcripts were observed in blood vessels and capillaries (Fig. 2C). Control experiments performed with synthetic oligonucleotides corresponding to the sense strand of the mouse utrophin mRNA failed to label subcellular structures within these muscle sections (not shown).
Quantitative analyses revealed that of 375 neuromuscular junctions, 313 (ϳ83%) displayed an accumulation of silver grains corresponding to utrophin transcripts. Densitometric analysis further showed that the levels of utrophin mRNAs confined within the postsynaptic sarcoplasm were approximately 12-fold higher than those observed in extrasynaptic regions (Fig. 3A). In agreement with previous reports showing up-regulation of utrophin in mdx mouse muscle (see, for example, Ref. 22), we also noted that in comparison to control mice, levels of utrophin mRNA were significantly elevated (ϳ400%) in hind limb muscle fibers from mdx mice (Fig. 3B). However, the ratio of utrophin transcripts in synaptic versus extrasynaptic regions from mdx mouse muscle fibers was similar to that obtained with C57BL/6 mice.
To determine whether selective transcription of the utrophin gene accounts for the preferential accumulation of utrophin transcripts within the postsynaptic sarcoplasm, we performed  an additional set of experiments in which human utrophin promoter-reporter gene constructs were directly injected into skeletal muscle. Muscles injected with the 1.3-kb utrophin promoter-nlsLacZ construct demonstrated a strong level of expression (Fig. 4). In fact, quantitative analysis revealed that ϳ72% of muscles injected with this construct contained myonuclei expressing significant levels of ␤-galactosidase (Fig. 5A). By contrast, expression of the nlsLacZ construct driven by the 0.5-kb utrophin promoter fragment was markedly reduced since less than 30% of the injected muscles displayed blue myonuclei.
Injections of TA muscles with the construct containing the 1.3-kb human utrophin promoter fragment led to the preferential expression of ␤-galactosidase in myonuclei located in the vicinity of neuromuscular synapses (Fig. 4). Detailed quantitative analysis showed that in approximately 55% of the cases, the presence of blue myonuclei coincided with synaptic sites identified by AChE histochemistry (Figs. 4 and 5B). Similar patterns of expression were observed at different time intervals following DNA injection. Deletion of 800 bp 5Ј of this utrophin promoter fragment led to a marked reduction in the percentage of synaptic events (Fig. 5B). These results are nearly identical to those recently reported for the synapse-specific expression of AChR subunit gene promoters (50 -55%) and for the non-synapse-specific expression obtained with the muscle creatine ki-nase promoter (10 -12%; Refs. 19 -21). In our experiments, injections of constructs containing the utrophin promoter fragments cloned in the reverse orientation failed to induce nlsLacZ expression in TA muscles.
Finally, we induced the formation of ectopic synapses at sites distant from the original synaptic regions to: (i) examine the contribution of the nerve in the local accumulation of utrophin at the neuromuscular junction and (ii) determine whether utrophin could be expressed in extrasynaptic regions of adult muscle fibers. In these experiments, we observed numerous newly formed ectopic synapses in all soleus muscles that displayed a functional motor response. In fact, co-distribution between the presence of synapsin immunoreactivity and acetylcholine receptors (AChR) was routinely observed (Fig. 6, A and B). Immunofluorescence experiments performed on both control and mdx mouse soleus muscles using either one of the two utrophin antibodies revealed that utrophin was already present at the postsynaptic membrane of these ectopic synapses (Fig. 6, C and  D). DISCUSSION The postsynaptic sarcoplasm of the neuromuscular junction represents a highly differentiated domain within muscle fibers in which numerous organelles accumulate. These include morphologically distinct myonuclei referred to as fundamental by Ranvier (23), a synapse-specific Golgi apparatus (24,25), and a stable array of microtubules (26). Previous studies have also FIG. 5. Expression of utrophin promoter-reporter gene constructs in muscle fibers. A, percentage of TA muscles expressing the construct following injections with plasmids containing either the 1.3or 0.5-kb utrophin promoter fragment. Note that deletion of 800 bp from the 5Ј region of the 1.3-kb fragment reduced the percentage of muscles expressing the reporter gene. B, percentage of synaptic events (see "Experimental Procedures") following injections with the two different constructs. Note that the 1.3-kb utrophin promoter fragment confers preferential synaptic expression to the reporter gene nlsLacZ.

FIG. 4. Expression of utrophin promoter-reporter gene constructs in muscle fibers. A and B
show examples of TA muscles injected with plasmids containing the 1.3-kb utrophin promoter fragment and nlsLacZ. Brown precipitates correspond to AChE histochemistry indicating the presence of neuromuscular junctions. Note the co-localization between the presence of ␤-galactosidasepositive myonuclei and neuromuscular synapses following injections with this utrophin promoter fragment. C and D represent TA muscles injected with plasmids containing the 0.5-kb utrophin promoter fragment. Note that blue myonuclei are observed in extrasynaptic regions of muscle fibers. Bar ϭ 60 m.
shown the selective accumulation of transcripts encoding the various AChR subunits (27,28) as well as AChE (29,30) in the postsynaptic sarcoplasm of adult muscle fibers. In the present study, we show that accumulations of utrophin mRNAs are detectable at 83% of the neuromuscular junctions. This value is in fact similar to those reported recently for transcripts encoding other synapse-associated proteins (31).
In attempts to elucidate the mechanisms involved in the preferential accumulation of utrophin mRNAs in synaptic regions of muscle fibers (29,31) we injected various utrophin promoter-reporter gene constructs directly into muscle. Similar to the transcriptional activation of the various AChR subunit genes within the fundamental myonuclei (27,28), we observed that injection of constructs containing the 1.3-kb utrophin promoter resulted in synapse-specific expression of the reporter gene. Deletion of 800 bp 5Ј of this promoter fragment abolished synapse-specific expression indicating therefore that regulatory elements contained within this DNA fragment are necessary for conferring synapse-specific expression.
Sequence analysis of the deleted 800-bp fragment revealed the presence of an E box which is known to bind myogenic transcription factors. Interestingly, this site is the only consensus sequence that has been found common to all AChR promoters to date (28). Although myogenic factors contribute to the activity-dependent regulation of AChR subunit genes in muscle fibers, this binding site is not required for synapse-specific expression of the AChR ⑀-subunit gene (19). An N box motif constitutes another DNA element which may be involved in the local expression of the utrophin gene within nuclei located in the postsynaptic sarcoplasm (Ref. 20 and Fig. 1). Deletion and mutagenesis experiments have revealed that this DNA element is sufficient to confer synapse-specific expression to the mouse AChR ␦and ⑀-subunit genes, and that it binds a protein complex from muscle nuclear extracts in gel retardation assays (20,21). This DNA element may thus be responsible for the synapse-specific expression conferred by the 1.3-kb utrophin promoter fragment.
Ectopic nerve implants have been used successfully to study the development of the neuromuscular junction in vivo. Using this approach, we observed numerous ectopic synapses in "old" extrasynaptic regions of soleus muscle fibers. Immunofluorescence experiments further showed that utrophin appeared at these newly formed synaptic sites within 2 weeks following induction of ectopic synapses. These results are thus in agreement with previous studies which showed the presence of utrophin at agrin-induced AChR clusters in cultured myotubes (32). More importantly, our results indicate that the utrophin gene may be expressed in extrasynaptic regions of muscle fibers upon receiving appropriate neuronal cues. It appears therefore that nerve-derived factors play a crucial role in dictating the local expression of utrophin gene products.
Several nerve-derived factors are known to influence the localization and regulation of AChR. For example, ARIA/ heregulin has been shown to markedly influence expression of AChR and, in particular, the expression of the ⑀-subunit gene (33). Since the pattern of expression of the utrophin gene along muscle fibers is similar to that of the ⑀-subunit gene (Ref. 21 and this study) and since both genes appear largely insensitive to abolition of neuromuscular activity (34,35), ARIA/heregulin may thus be considered as a plausible candidate involved in the local regulation of utrophin at the synapse. Agrin represents another factor that may also contribute to the regulation of the utrophin gene within the postsynaptic sarcoplasm. A recent study has in fact shown that substrate-bound agrin induces a 2to 3-fold increase in the expression of the AChR ⑀-subunit gene in cultured myotubes (36) thereby providing support to the notion that agrin is also a transcriptional activator. Since utrophin may be involved in the early steps of synaptogenesis, it is thus possible that agrin stimulates expression of utrophin to ensure the presence of a cytoskeletal scaffold necessary for the assembly and stabilization of postsynaptic membrane domains. Preliminary results obtained in our laboratory indicate that, indeed, both Torpedo and recombinant agrin increase the levels of utrophin mRNA in cultured myotubes. 2 The identification of nerve-derived factors involved in modulating expression of the utrophin gene will provide key information essential for the up-regulation of utrophin as a therapeutic strategy for DMD.