Cloning of cDNAs encoding Xenopus neuregulin: expression in myotomal muscle during embryo development

Molecular Brain Research 58 Ž1998. 59–73

Research report

Cloning of cDNAs encoding Xenopus neuregulin: expression in myotomal muscle during embryo development Jie F. Yang a , Hong Zhou b, San Pun a , Nancy Y. Ip a , H. Benjamin Peng b, Karl W.K. Tsim a,) a

b

Department of Biology and Biotechnology Research Institute, The Hong Kong UniÕersity of Science and Technology, Clear Water Bay Road, Hong Kong, China Department of Cell Biology and Anatomy and Curriculum in Neurobiology, UniÕersity of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Accepted 24 March 1998

Abstract Neuregulin has diverse functions in neural development, and one of them is the up regulation of acetylcholine receptors ŽAChRs. at the muscle fiber during the formation of neuromuscular junctions. Although the primary source of neuregulin is derived from motor neuron, the expression in muscle has also been demonstrated. The precise role of neuron-derived and muscle-derived neuregulin during the early stages of development is not known. In order to study the role of neuregulin during early embryo development, we isolated the cDNAs encoding Xenopus neuregulin by cross-hybridization with its chick homologue. The amino acid sequence of Xenopus protein is 50 to 70% identical to members of the neuregulin family. The cDNAs encoding different isoforms of Xenopus neuregulin were identified, and these isoforms have two variation sites: Ži. the spacer domain with either 0 or 43 amino acid insertion; and Žii. the C-terminus of EGF-like domain to derive either a or b isoform. When the EGF-like domain of Xenopus neuregulin was expressed in mammalian cells, the recombinant protein was able to induce the expression of AChR and the tyrosine phosphorylation of erbB receptors in cultured myotubes. An ; 6.5 kb transcript corresponding to neuregulin was detected in RNA isolated from brain and muscle. Various splicing variants were expressed in different Xenopus tissues. In situ hybridization showed a strong expression of neuregulin in developing brain and spinal cord of Xenopus embryo. In addition, it was also prominently expressed in the myotomal muscle. These data suggest that in addition to motor neurons, the postsynaptic muscle cells can also contribute neuregulin for synaptogenesis. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Acetylcholine receptor; Development; Neuromuscular junction; Synaptogenesis

1. Introduction During the development of neuromuscular junctions, motor neurons make contact with muscle fibers and direct the formation of postsynaptic specializations w15,40x. These specializations include the aggregation of acetylcholine receptors ŽAChRs., acetylcholinesterase ŽAChE. and other synaptic proteins w30x. The increase in postsynaptic AChR density, up to approximately 10,000 receptorsrm m2 , is primarily due to the aggregation of AChRs already present in the membrane at the time of nerve–muscle contact as well as an increase in local AChR synthesis w15,40x. In the local synthesis of post-synaptic molecules, RNAs encoding ) Corresponding [email protected]

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0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 0 8 5 - 0

AChR subunits, AChE and other synapse-specific proteins are highly concentrated in the synaptic regions w22,32,34,48x. The up regulation of AChR synthesis at the neuromuscular junction is induced by motor neurons. The motor nerve provides two distinct mechanisms to achieve this striking localization of AChRs: Ži. it releases factors, such as calcitonin gene-related peptide w6,14x, ascorbic acid w21x and acetylcholine receptor inducing activity ŽARIA. w10,18x, that stimulate the synaptic expression of AChR; and Žii. nerve-evoked electrical activity, on the other hand, represses the synthesis of AChR in the extrasynaptic regions w26,53x. ARIA, first isolated from chick brain, is the best candidate for the nerve-derived signal for postsynaptic gene regulation. ARIA could mimic several effects of motor axons on the muscle target that include: inducing the

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synthesis of AChR in aneural myotubes w10x, increasing the number of voltage-gated sodium channels w8x and the expression of the ´-subunit of AChR characteristic of the adult AChR w23,29,46x. In addition, ARIA was shown to stimulate the tyrosine phosphorylation of both erbB2 and erbB3, and the activation of mitogen-activated protein kinase indicating that ARIA’s signaling pathway in muscle might be mediated by erbB2 andror erbB3 receptors w2,46,47,50x. Based on these observations, Falls et al. w10x, Loeb and Fischbach w27x, and Sandrock et al. w43x proposed that ARIA released from developing motor nerve terminals, activates its receptor on the postsynaptic muscle membrane and induces the postsynaptic gene expression at the neuromuscular junction. The cDNA encoding ARIA was first isolated from a lgt 10 chick brain library. The cloned ARIA cDNA encodes a considerably larger transmembrane precursor designated pro-ARIA with a predicted protein of 602 amino acids. From N- to C-terminus, pro-ARIA has immunoglobulin ŽIg.-like, epidermal growth factor ŽEGF.like, hydrophobic and intracellular domains w11x. Mature ARIA, which is 42 kDa in size, is believed to be produced by proteolytic cleavage of pro-ARIA at the dibasic amino acid residues ŽK 205 and R 206 . adjacent to the hydrophobic domain w11,38x. Sequence analysis shows that ARIA belongs to a family of proteins that have been called neuregulins which have diverse functions in neural development w23,33x. Members of neuregulin include rat neu differentiation factor ŽNDF. w52x, human heregulin ŽHRG. w20x and bovine glial growth factor ŽGGF. w28x. Thus, the term neuregulin has been used to describe all splice variations observed for this family w28x. Through alternative RNA splicing, many isoforms are generated from this family. The most common splicing site is at the C-terminus of the EGF-like domain that determines two major classes of isoforms termed a and b . Within each class, there are subclasses that are classified according to the region downstream of the intracellular domain w20,52x. The Ig-like domain at N-terminus is able to bind the extracellular matrix at the synaptic clefts through charged interactions w27x. The EGF-like domain can be released by unknown proteases to act on the postsynaptic muscle membrane. This domain from several members of the neuregulin family causes the tyrosine phosphorylation of erbB receptors and the induction of AChR expression w27,46,54x. At vertebrate neuromuscular junctions, the predominant source of neuregulin is from motor neuron w9,11x. Immunohistochemical analysis on mouse muscle fibers revealed the expression of neuregulin at synaptic sites, and the immunoreactivity was also extended around the circumference of muscle fibers w7,23,34,42x. In comparison with the synaptic staining, the level of extrasynaptic staining is reduced during development from the first postnatal week to the adult stage w34x. In addition to neurons, the expression of neuregulin has also been reported in muscle and Schwann cell w36x. Neuregulin mRNA has been de-

tected in chick muscles w38x, rat muscles and in cultured sol 8 mouse muscle cells w34x. Various isoforms of neuregulin are found in chick muscle and their expression profile is changed during development, denervation and regeneration w36x. The relative role of neuron-derived and musclederived neuregulin in the formation of neuromuscular junctions is not known. In order to study the role of neuregulin in embryo development and the formation of neuromuscular junctions at early stage, we cloned the cDNAs encoding neuregulin from embryonic Xenopus cDNA libraries, and characterized the expression of its transcripts during development in different tissues.

2. Materials and methods 2.1. Screening cDNA libraries An embryonic stage 17 to 24 Xenopus cDNA library was screened by using procedures described in Sambrook et al. w41x. In brief, about 1 = 10 6 recombinant phages from the amplified lgt 11 library were screened with random-primed 32 P-labeled chick neuregulin cDNA Ž; 1.0 kb from nucleotide 24 to 953. which had a specific activity of about 1 = 10 9 cpmrm g of DNA w12,38x. Hybridization was carried out overnight at 428C in 30% formamide, 5 = SSC, 5 = Denhardt’s reagent, 0.1% SDS, 0.l mgrml salmon sperm DNA to which about 5 = 10 5 cpmrml of radioactive probe had been added. Filters were washed twice for 30 min in 2 = SSC, 0.2% SDS at room temperature and then twice for 30 min at 558C. Three rounds of screening were done to ensure the positive clones. A partial ; 2.4 kb cDNA insert, namely XA-1, was isolated. Using XA-1 as a probe to rescreen another stage 40–45 cDNA library in l ZAPII ŽStratagene, La Jolla, CA. by similar hybridization methods, six more positive clones were isolated. The identified l phages were isolated as described by the supplier ŽStratagene.. All cDNA clones were sequenced by the dideoxy chain termination method w45x using Sequenase ŽUSB, Cleveland, OH.. The internal sequences of these cDNAs were sequenced by using synthetic oligonucleotides. Both strands of the cDNA clones were sequenced at least twice and the DNA sequences were aligned by using MacVector ŽKodak, New Haven, CT. software package. 2.2. Functional expression of Xenopus neuregulin The cDNA encoding EGF-like domain of Xenopus neuregulin was tagged with human immunoglobulin Fc. Briefly, an artificial leader sequence was inserted into a mammalian expression vector pcDNA I ŽInvitrogen, San Diego, USA.. The downstream of the leader sequence was an EcoRI cloning site and then followed by a ; 0.6 kb cDNA fragment encoding Fc region of human immunoglobulin G1 w54x. The EGF-like domain of Xenopus

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neuregulin was constructed by PCR with a pair of primers flanking G187 to K 259 , with an artificial EcoRI site at both ends. The PCR was carried out for 30 cycles in standard reaction mixture containing Xenopus neuregulin cDNAs, 2.5 U of Vent DNA polymerase and 0.3 m g of forward primer: 5X CGG AAT TCC GGT CAC CTT ATT AAG 3X Žsense, 719–733., and backward primer: 5X TGG AAT TCT TTT TGG TAC AAC TCC 3X Žanti-sense, 922–937.. The DNA fragment was subcloned into EcoRI site of the modified pcDNA I vector. The modified pcDNA I containing Fc-tagged neuregulin cDNA was transfected into human embryonic kidney fibroblast ŽHEK. 293 cells by calcium phosphate precipitation w38,41x. The Fc-tagged neuregulin EGF-like fragment was purified by protein-G column as described previously w54x. 2.3. RNA isolation and RT-PCR Total RNA was prepared from the collected tissues by using LiCl method w5x. For the cultured myotubes, total RNA was isolated by using Micro RNA Isolation Kit ŽStratagene.. RNA concentration and purity were determined by ultraviolet absorbance at 260 nm. In RT-PCR analysis, 5 m g of total RNAs was reverse transcribed by Moloney Murine Leukemia Virus reverse transcriptase ŽGIBCO-BRL, Grand Island, NY. by random oligonucleotide priming in a 20 m l reaction. One-fifth of the reverse transcription product was used as a template in PCR analysis with primers described below. PCR was carried out for 30 cycles of 948C for 1 min, 608C for 2 min and 728C for 2 min in a 25 m l volume containing 0.8 mM dNTPs, 1 = PCR buffer and 0.625 U of Taq Polymerase ŽGIBCO-BRL.. PCR products were analyzed in a 12% polyacrylamide gel w41x and directly cloned into pCR II vector ŽInvitrogen, La Jolla, CA.. The identity of the cloned PCR products was confirmed by DNA sequencing. The PCR primers were designed according to Xenopus neuregulin cDNA sequence. Set of primers flanking the EGF-like domains, S-1: 5X-GGT CAC CTT ATT AAG TG-3X Žsense; from 719–735. and AS-1: 5X-TTT TTG GTA CAA CTC CT-3X Žantisense; from 937–921., and set of primers flanking the spacer domain S-2: 5X-AAC CAG CTT GGA AAT GA-3X Žsense; from 518–534. and AS-2: 5X-ACA CTC TCC TCC ATT GAC ACA-3X Žantisense; from 778–758. were used. 2.4. Northern blot analysis RNA samples were fractionated on a 1% formaldehyde gel. Ethidium bromide was used to assess the equivalency of loading of different samples. After the electrophoresis, samples were transferred to a charged nylon membrane ŽHybond-N, Amersham, UK. and were UV cross-linked. Blots were hybridized with a partial neuregulin cDNA fragment ŽXA-1., or an ; 1.2 kb chick AChR a-subunit cDNA w38,48x. Probes were labeled with w a-32 Px dCTP

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and the hybridization was performed at 428C overnight in 40% deionized formamide, 5 = Denhardt’s solution, 0.5% SDS, 5 = SSC, 10% dextran sulfate and 0.1 mgrml denatured salmon sperm DNA. After hybridization, the filters were washed twice with 2 = SSC with 0.1% SDS at room temperature for 30 min each, and then twice with 0.1 = SSC with 0.1% SDS at 558C for 30 min each. The washed filters were exposed to X-ray film with double intensifying screens at y808C. 2.5. Cell culture Primary chick myotube cultures were prepared from the hind–limb muscles dissected from embryonic day 11 chick embryos according to modified protocol w13,51x. Muscle cells were cultured in MEM supplement with 10% heat inactivated horse serum, 2% Žvrv. chick embryo extract, 1 mM L-glutamine, 100 Urml penicillin and 100 m grml streptomycin. The C2C12 myoblasts were maintained in DMEM supplemented with 10% heat inactivated horse serum, 1 mM L-glutamine, 100 Urml penicillin and 100 m grml streptomycin. Cultures were incubated at 378C in a 5% CO 2 humidified incubator and the medium was replaced every 3 to 4 days. The C2C12 myoblasts were induced to fuse at confluent stages by serum starvation to 2% in final w46x. In AChR a-subunit induction assay, the chick myotubes were treated with Fc-tagged neuregulin overnight, and total RNA was collected from the treated chick myotubes. In the phosphorylation studies, fused C2C12 myotubes were treated with Fc-tagged neuregulin for 30 min, and then cells were collected for immunoprecipitation assay. 2.6. Immunochemical analysis In the phosphorylation studies, fused C2C12 myotubes were treated with purified recombinant Fc-tagged neuregulin for 30 min. The treated cells were resuspended in RIPA buffer ŽPBS pH 7.4, 1% NP-40, 0.5% dexocoxylate, 0.1% SDS, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM aprotinin.. The erbB3 receptor was immunoprecipitated with an antibody against erbB3 ŽC17; Santa Cruz Bitotech. Santa Cruz, CA. at 1:1000 dilution. The immunoprecipitated proteins were collected on protein G agarose beads, fractionated by 7.5% SDS-PAGE w36,49x. Electrophoresed proteins were transferred onto nitrocellulose membrane. The membrane was blocked with 2.5% dry milk containing 20 mM Tris–HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20, for 1 h at 378C, followed by incubation with horseradish peroxidase-conjugated antityrosine phosphorylation antibodies RC 20 ŽTransduction Lab. Lexington, KY. diluted 1 in 1000. Immunoreactivity was detected by ECL Western Blot System ŽAmersham. followed the instructions from the supplier. In Western blot analysis, anti-neuregulin a isoform specific antibody ŽTransduction Lab.. was used. In immunohistochemical

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Fig. 1 Žcontinued..

studies, anti-neuregulin polyclonal antibody was used; this antibody was raised specifically for both a and b isoforms in our laboratory w36x. The immunohistochemical procedure was performed as described in ref. w49x. 2.7. Induction of AChR a-subunit promoter The pnlacZ plasmid containing 850 bp chick AChR a subunit promoter tagged with b-galactosidase gene was described in ref. w44x and provided by Dr. Joshua Sanes from Washington University School of Medicine. The cDNA was transfected into 2-day-old chick myotube cultures by using calcium phosphate precipitation w38,41x. Two days after transfection, the myotubes were treated with Fc-tagged neuregulin for 36–48 h. The neuregulin-induced b-galactosidase activity was determined according to ref. w41x. In b-galactosidase staining, cell cultures were fixed for 5 min in PBS containing 2% paraformaldehyde and 0.2% glutaraldehyde at room temperature, and then rinsed with PBS. The staining reaction was developed for 16–24 h at 378C in PBS, pH 7.4, containing 1 mgrml X-Gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM MgCl 2 . After the color development, the culture was fixed with ethanol and observed under a Zeiss Axiophot microscope. 2.8. In situ hybridization Whole-mount in situ hybridization was conducted as described w16,17x. Albino Xenopus embryos were collected at different stages and fixed in MEMFA Ž0.1 M MOPS, pH 7.4, 2 mM EGTA, 1 mM MgSO4 , 3.7% formaldehyde. for 2 h, then stored in absolute methanol at

y208C. Before hybridization, the embryos were rehydrated by 5 min washes in 75% methanol: 25% DEPC water, 50% methanol: 50% DEPC water and 25% methanol:75% PBS–Tween ŽPBS with 0.1% Tween., rinsed twice for 5 min each in 0.1 M triethanolamine ŽSigma. and rinsed in 0.1 M triethanolamine with 12 m l acetic anhydride ŽSigma. for 5 min, and then another 12.5 m l acetic anhydride was added. The embryos were re-fixed in 4% paraformaldehyde for 20 min, washed 5 = 5 min in PBS–Tween and replaced with hybridization buffer Ž50% formamide, 5 = SSC, 1 mgrml Torula RNA, 100 mgrml heparin, 1 = Denhardt’s, 0.1% Tween-20, 0.1% CHAPS, 10 mM EDTA.. This prehybridization step was carried overnight at 608C. The solution was then replaced with 0.5 ml probe solution Žhybridization buffer with 0.1 m grml probe. and the incubation was carried out at 608C overnight. Digoxigenin-labeled Xenopus neuregulin cRNA probe Ž; 3.1 kb XA-5. was synthesized in antisense direction using SP6 RNA polymerase with Riboprobe In Vitro Transcription kit according to manufacturer’s protocol ŽPromega, Madison, WI.. Digoxigenin-11-UTP was purchased from Boehringer Mannheim ŽIndianapolis, IN.. After hybridization, the embryos were placed into hybridization buffer without probe for 10 min at 608C, washed two times in 2 = SSC for 20 min each at 378C, once in 2 = SSC with RNaseA Ž20 m grml. and RNase T1 Ž10 Urml. for 30 min at 378C, rinsed in 2 = SSC and 0.2 = SSC for 30 min at 608C. They were then washed 4 times in maleic acid buffer ŽMAB: 100 mM maleic acid, pH 7.5, 150 mM NaCl. at room temperature and in MAB with 20% FBS at room temperature for 1 h. The solution was replaced with fresh MAB containing 20% FBS and a 1:2000 dilution of antidigoxigenin antibody coupled to alkaline phosphatase

Fig. 1. Nucleotide and deduced amino acid sequences of Xenopus neuregulin. ŽA. The sequence is derived from XA-7 that encodes for neuregulin SP43 a 1 . Ig-like domain Žunderlined., EGF-like domain Ždotted line. and hydrophobic domain Žboxed. are shown. Cysteines within Ig-like and EGF-like domains are circled. Potential N-glycosylation sites are boxed by a square. A potential proteolytic cleavage site within a pair of basic amino acids ŽK 259 –R 260 . is X X indicated by an arrow head. Part of the 5 and 3 untranslated sequences are not shown. ŽB. Hydrophilicity profile of deduced Xenopus neuregulin. The method of Kyte and Doolittle w25x was used with a window size of seven residues. Negative values indicate increasing hydrophobicity. Amino acid numbers are shown below the profile. The predicted structure of neuregulin is shown above the profile for reference.

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ŽBoehringer Mannheim., and the incubation was carried out overnight at 48C on a rocking platform. The embryos were washed at least 5 times for 1 h at room temperature each time with MAB to remove excess antibody. For chromgenic reaction, the embryos were washed twice with alkaline phosphatase buffer ŽAP buffer: 100 mM Tris HCl, pH 9.5, 50 mM MgCl 2 , 100 mM NaCl, 0.1% Tween-20, 5 mM levamisol. and then treated with AP buffer containing NBT and BCIP. The chromogenic reaction was stopped when satisfactory signal-to-background ratio was achieved by replacing the AP buffer with MEMFA. Before microscopic examination, the embryos were cleared with a 2:1 mixture of benzyl benzoate:benzyl alcohol in a glass dish. For the control, another set of embryos were hybridized with digoxigenin-labeled Xenopus neuregulin cRNA probe synthesized in the sense direction using T7 RNA polymerase and processed in the same fashion.

consensus sequence of an EGF-like domain ŽC 192 to C 222 .. Between Ig-like and EGF-like domains ŽP137 to R 179 . is the spacer domain. Two potential N-glycosylation sites

3. Results 3.1. Cloning of Xenopus neuregulin An oligoŽdT.-primed stage 17 to 24 Xenopus cDNA library, constructed in the phage lgt 11, was screened by cross-hybridization with ; 1 kb chick neuregulin cDNA. A single clone, namely XA-1, was isolated. This ; 2.4 kb cDNA was sequenced and found to share high homology with the neuregulin family. The very 5X- end of XA-1 is not full-length and corresponds to the transmembrane domain of neuregulin. By using XA-1 as a probe, we screened a stage 40–45 l ZAPII cDNA library and six more positive clones were isolated. These clones were XA-2 Ž; 2.6 kb., XA-3 Ž; 2.6 kb., XA-4 Ž; 2.8 kb., XA-5 Ž; 2.9 kb., XA-6 Ž; 3.1 kb., and XA-7 Ž; 3.1 kb.. The longest clone XA-7 was sequenced. XA-7 has 2031 bp of open reading frame that encodes a polypeptide of 677 amino acids with a predicted molecular weight Ž Mr . of 75,788 Da ŽFig. 1 A.. The first ATG of XA-7 is at nucleotide 161 and follows the empirical rules for translation initiation site w24x. Hydropathy analysis w25x on the predicted protein revealed that there is no nonpolar amino acid at the N-terminus that could function as a signal peptide, but it has one stretch of 24 nonpolar amino acids ŽV261 to C 284 . that is of sufficient length and hydrophobicity to transverse the lipid bilayer ŽFig. 1B.. A pair of basic amino acids ŽK 259 –R 260 . adjacent to the hydrophobic region was found. This is consistent with the nature of neuregulin as a transmembrane protein, and the K 259 –R 260 dibasic residues could serve as a cleavage site for the release of biologically active neuregulin. Sequence analysis shows that there is an Ig-like domain at the N-terminal portion of XA-7 encoded protein. The spacing of the cysteines and the residues around it fit the consensus for an Ig-like domain of C2 type. The spacing of the cysteines before the transmembrane region fits the

Fig. 2. Alignment of Xenopus neuregulin to members of the family. Xenopus neuregulin SP0 a 2 is chosen to compare with human ŽHu., chick ŽCh., rat ŽRat. and bovine ŽBov. proteins. Identical residues among all species are shaded. The amino acid sequence of Xenopus protein is ;64% identical overall to that of human, ;61% identical to chick, ; 70% identical to rat, ; 53% identical to bovine.

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exist at the C-terminus of the Ig-like domain. The C-terminus of the hydrophobic domain accounts for over 60% of the predicted protein. Similar to other members of neuregulin, this sequence contains a high percentage of proline, serine and threonine. However, its function is not known, and it has been proposed to be a site for regulating the proteolytic release of the N-terminal end. Thus, the deduced XA-7 sequence is believed to encode for Xenopus neuregulin and the EGF-like sequence of XA-7 protein shows that is an a 1 isoform of neuregulin. The coding sequence of individual clones were sequenced. XA-4, XA-5, XA-6 and XA-7 encode the fulllength sequence of Xenopus neuregulin with different extension at 5X untranslated region. In addition, these cDNAs encode various isoforms of neuregulin family with variation at two sites: Ži. the spacer domain with either 0 or 43 amino acid insertion, namely SP0 or SP43, respectively; Žii. the C-terminus of EGF-like domain to give rise to either a or b isoform. The identified l clones from our screening are: XA-2; neuregulin SP0 a 2 , XA-3;

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neuregulin SP43 a 2 , XA-4; neuregulin SP0 a 1 , XA-5; neuregulin SP0 b 2 , XA-6; neuregulin SP43 a 2 , XA-7; neuregulin SP43 a 1. 3.2. Sequence comparison with the neuregulin family The cloned Xenopus neuregulin is highly homologous to other members of the neuregulin family. Xenopus neuregulin SP0 a 2 was chosen to compare with its counterparts. The amino acid sequence of Xenopus neuregulin is ; 64% identical overall to that of human protein, ; 61% identical to chick protein, ; 70% identical to rat protein, and ; 53% identical to bovine protein ŽFig. 2.. The highest homology among all species is in the intracellular domain of neuregulin Žfrom 65% to 80% identity.. However, Xenopus neuregulin differs strikingly from other members of the neuregulin family at the N-terminal and the C-terminal ends of the protein ŽFig. 2.. Various isoforms of identified Xenopus neuregulin Ž a 1, a 2, b 2. share a high homology with its counterparts. Fig. 3 shows the sequence of neuregulin a 1, a 2 and b 2 isoforms and its comparison to chick and rat proteins. The

Fig. 3. Various isoforms of Xenopus neuregulin. ŽA. The splicing variants at the C-terminus of EGF-like domain to derive a 1, a 2 and b 2 isoforms are shown. ŽB. Sequence comparison of neuregulin a 1, a 2 and b 2 isoforms to different counterparts. ŽC. A spacer domain with either 0 or 43 amino acid insertion is shown. The spacer domain is rich in serine and threonine, and two potential N-linked glycosylation sites are just up stream of the spacer domain. Identical residues are boxed and the number of residues derived from Fig. 2 are indicated. Spacer domains of chick and rat proteins are given for reference.

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a 1 and a 2 isoforms of Xenopus neuregulin show over 60% in amino acid identity with its counterparts ŽFig. 3B.; the amino acid sequence of b 2 isoform is identical to the rat protein, and is just one residue different from the chick protein ŽFig. 3B.. However, the 43 amino acids insertion at the spacer domain shows no close homology to its counterparts of neuregulin family ŽFig. 3C.. Other cDNAs related to neuregulin were also identified. Some of the cDNAs share homology to the neuregulin isoform having a cysteine-rich domain at the N-terminal end w19x or a new neuregulin-like ligand, namely neuregulin-2 w3,4x. However, the exact identity of these cDNAs will be described elsewhere. 3.3. Functional expression of Xenopus neuregulin To determine the biological activities of identified Xenopus neuregulin, cDNA encoding the EGF-like domain of neuregulin was tagged with human immunoglobulin Fc cDNA and subcloned into mammalian expression vector. The chimeric construct was transfected into HEK 293 cells and the neuregulin-Fc fusion protein was purified by protein-G column w54x. The Fc-tagged neuregulin was recognized by anti-neuregulin and anti-human Fc antibodies at ; 60 kDa Ždata not shown.. This recombinant protein was used in biological assays. The pnlacZ plasmid containing AChR a-subunit promoter was transfected into cultured myotubes, and neuregulin-Fc recombinant protein was applied. Fig. 4 A shows the b-galactosidase staining on the transfected myotubes which indicates the neuregulin-induced expression of AChR a-subunit promoter. The AChR-inducing activity of a and b forms of neuregulin was relatively equal, and their induction effect was in dose

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dependent manner ŽFig. 4B.. Moreover, the purified Fctagged recombinant Xenopus neuregulin induced: Ži. the expression of AChR a-subunit mRNA by ; 5-fold in cultured chick myotubes ŽFig. 5A., and Žii. the tyrosine phosphorylation of erbB3 receptor in cultured C2C12 myotubes ŽFig. 5B.. Although we did not quantify the potency of different neuregulin isoforms, all isoforms of cloned Xenopus neuregulin exhibited AChR-inducing activity in cultured myotubes Ždata not shown.. 3.4. Expression of neuregulin mRNA and its isoforms Anti-neuregulin antibody was used to stain the neuromuscular junctions of adult Xenopus leg muscle. The antibody staining was co-localized with rhodamine-conjugated a-bungarotoxin indicating the restricted localization of neuregulin at the neuromuscular junctions ŽFig. 6.. This synaptic expression of neuregulin in Xenopus muscle is similar to other species w23,34,36x. Northern blot analysis using cDNA derived from XA-1 Ž; 2.4 kb. to probe for corresponding transcripts in different Xenopus tissues provided results comparable to those obtained from other species. Transcripts were detected in RNA isolated from brain and muscle tissues, while the level of expression was too low to be detected in the liver ŽFig. 7A.. The length of the corresponding transcripts was ; 6.5 kb that is smaller than homologous transcripts in chick Ž7.5 kb.; rat tissues have three transcripts Ž6.8 kb, 2.6 kb, 1.7 kb.. The intensity of the transcript revealed in brain RNA was ; 15-fold higher than the RNA isolated from muscle ŽFig. 7A.. By using primers ŽS-1 and AS-1. flanking the carboxyl-terminus of EGF-like domain of Xenopus

Fig. 4. Neuregulin induces the expression of AChR a-subunit promoter. The pnlacZ plasmid containing 850 bp chick AChR a-subunit promoter tagged with b-galactosidase gene was transfected into 2-day-old chick myotube cultures by using calcium phosphate precipitation. ŽA. Two days after transfection, 1 m g Fc-tagged Xenopus neuregulin a 1 was applied onto 1 ml cultured myotubes. The b-galactosidase staining was developed. Blue color indicates the induction of the promoter. The denotation are: the pnlacZ transfected but without neuregulin treatment Žcontrol., pnlacZ transfected and treated with neuregulin a 1 Ž a 1-Fc., a higher magnification of a stained myotube Ž a 1-Fc; the bottom figure.. The b isoform of neuregulin also showed similar results. Scale bar: Žthe top two figures. 120 m m; Žthe bottom figure. 20 m m. ŽB. Different amount of a 1 or b 2 forms of Xenopus neuregulin were applied onto 5 ml cultured myotubes. Two days later, the induced b-galactosidase activity was determined. Values are expressed in % of control Žno neuregulin treatment as 100%., and are in mean " S.E.M., n s 4. Fig. 5. Neuregulin induces the up regulation of AChR a-subunit and the tyrosine phosphorylation of erbB3 on cultured myotubes. ŽA. Five ml 4-day-old chick myotubes were treated with ; 3 m g of purified neuregulin-Fc Ž a 1-Fc or b 2-Fc. for overnight. Tetrodotoxin ŽTTX; 1 m M. serves as a positive control and the untreated myotube is the background control Žcontrol.. Total RNA was isolated from the cells and 10 m g of RNA was subjected to a 1% formaldehyde–agarose gel. The membrane was probed with AChR a-subunit cDNA Ž; 1.2 kb., and a transcript of ; 3.2 kb was detected. Lower panel shows the ribosomal RNA staining with 18S and 28S as markers. ŽB. The C2C12 myotubes were treated with purified neuregulin-Fc Ž; 3 m g per 5 ml cultures. for 30 min. Control is untreated myotubes. The treated cells were lysed in RIPA buffer. The erbB3 receptor was immunoprecipitated with 1:1000 of a rabbit antibody ŽC17. against erbB3. The immunoprecipated proteins were collected on protein G agarose beads and fractionated by 7.5% SDS-PAGE. Electrophoresed proteins were transferred onto nitrocellulose membrane, and detected by peroxidase-conjugated anti-tyrosine phosphorylation antibodies RC 20 Župper panel.. The same filter was washed and probed with anti-erbB3 receptor antibody as to confirm the identity of the recognized band Žlower panel.. A 200 kDa molecular marker is indicated. Fig. 6. Co-localization of neuregulin immunoreactivity with AChR aggregate at the neuromuscular junctions of Xenopus. Sixteen micrometer sections of adult Xenopus leg muscle was used. The muscle section was double stained with rhodamine-conjugated a-bungarotoxin and anti-neuregulin antibodies followed by fluorescein isothiocyanate-conjugated secondary antibody. ŽA. Rhodamine-conjugated a-bungarotoxin staining; ŽB. Same view as in ŽA. but stained with anti-neuregulin antibody specific for both a and b isoforms. Bar: 20 m m.

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Fig. 7. Northern blot and RT-PCR analysis of Xenopus neuregulin mRNA distribution. ŽA. RNAs Ž40 m g. isolated from adult Xenopus brain, muscle and liver were blotted onto a nitrocelluose paper. The blot was probed with the XA-1 fragment under the conditions of high stringency. A transcript with a size of ; 6.5 kb can be detected in both brain and muscle. Arrow heads indicate the ribosomal RNA markers. ŽB. Reverse transcription was used to prepare first strand cDNA from total RNAs. The cDNAs were amplified by PCR with S-1 and AS-1 primers flanking the EGF-like domain of neuregulin. The PCR products were revealed on a 12% acrylamide gel and stained with ethidium bromide. The a 1, a 2 and b 2 isoforms were detected in RNA isolated from adult Xenopus brain, spinal cord, liver and heart, while muscle expressed only a 1, a 2 isoforms. ŽC. The cDNAs were amplified by PCR with S-2 and AS-2 primers flanking the Ig-like domain of neuregulin. The PCR products were revealed as in ŽB.. Neuregulin SP43 are expressed in adult Xenopus brain, spinal cord and muscle.

neuregulin, RT-PCR was performed on various tissues including brain, spinal cord, muscle, liver and heart from adult Xenopus. All tissues, except muscle, gave three major PCR products with sizes of 218 bp and 194 bp, and 185 bp; they represented the partial sequence at the Cterminus of the EGF-like domain of Xenopus neuregulin a 1, a 2 and b 2 based on their sequence ŽFig. 7B.. However, muscle expresses only a 1 and a 2 isoforms of neuregulin. In order to determine the splicing variants at the spacer domain, S-2 and AS-2 primers were used for RT-PCR analysis. Splicing variants of neuregulin at the known spacer domain were identified in the Xenopus cDNA libraries. These neuregulin variants are: a zeroamino acid insertion, a 43-amino acid insertion, namely neuregulin SP0 and neuregulion SP43 , respectively Žsee Fig.

3C.. Neuregulin SP0 was detected in nerve tissue, muscle, liver and heart while the expression of neuregulin SP43 found only in brain, spinal cord and muscle ŽFig. 7C.. Thus, the neuromuscular junctions could contain a variety of neuregulin isoforms which could be derived from both muscle and spinal cord in Xenopus. 3.5. In situ hybridization of neuregulin To understand the spatial relationship of neuregulin expression to neural development, whole-mount in situ hybridization was conducted at different stages. Neuregulin mRNA was detected within blastomeres at the earliest stage of embryo development. A 4-cell embryo is shown in Fig. 8A, and a blastula stage embryo is shown in Fig. 8B.

Fig. 8. Expression of neuregulin mRNA during embryo development. Localization of neuregulin mRNA by whole-mount in-situ hybridization. ŽA. An embryo undergoing second cleavage to become four cells. The mRNA was located in the animal pole of these cells. ŽB,C. A blastula-stage embryo, a view of the animal hemisphere ŽB. and a lateral view ŽC.. The message was restricted to the animal cap ŽAn. and was absent from the vegetal side ŽVe.. The horizontal line in ŽC. is due to the edge of the glass used to support the embryo resting on its side. ŽD. A stage 20 embryo. The mRNA was localized to the neural tube and the somites. ŽE. A stage 22 embryo. Labeling was observed in the developing brain and somites. The anterior 8–9 somites were distinct at this stage. Neuregulin mRNA was observed in both the segregated and unsegregated somitic mesoderm. ŽF. A stage 25 embryo. Prominent labeling of the neural tube ŽNT. and myotomes ŽMT. was seen. The eye vesicle showed intense labelling Žinsert.. ŽG,H. A stage 28 embryo. Myotomal labeling was found both in the nuclear area Župper arrow. and at the intersomitic junctions Žlower arrow.. ŽG. lateral view; ŽH. dorsal view. ŽI. A stage 32 embryo. Messages were concentrated in the brain, eye, myotomes, branchial arches and the area surrounding the optic vesicle. ŽJ. Stage 30 embryo hybridized with the sense probe.

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Neuregulin mRNA was restricted to the animal cap of the embryo ŽFig. 8C.. This early expression suggests the existence of maternal neuregulin messages. During neurulation, its expression became restricted to the area of the neural plate and the somites ŽFig. 8D and E.. Within the central nervous system, neuregulin mRNA was abundant in the brain and the spinal cord ŽFig. 8F and H, ‘NT’..

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Within the developing brain, the eye vesicle also showed prominent expression ŽFig. 8F insert.. In addition, it was also concentrated in the branchial arches ŽFig. 8F–I.. The myotomal muscle, which is derived from the somite and is the earliest skeletal muscle to form in the embryo also showed prominent mRNA localization ŽFig. 8F–I, ‘MT’.. The message was initially seen within the entire

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myotome blocks ŽFig. 8D., but it then became restricted to bands corresponding to the area occupied by myo-nuclei Župper arrow in Fig. 8G.. Interestingly, it was also enriched in the area flanking the intersomitic junctions Žlower arrow in Fig. 8G.. This latter area is where the myotomal neuromuscular junctions are located. This suggests that neuregulin mRNA is localized near the neuromuscular junctions in these skeletal muscle cells. For the control, embryos at similar stages were hybridized with a probe synthesized in the sense direction and processed in identical fashion. As shown in the example in Fig. 8J, these control embryos showed no labeling.

4. Discussion Several lines of evidence suggest that the cDNAs that we cloned from Xenopus libraries encode for neuregulin. First, the deduced proteins contain Ig-like, EGF-like, hydrophobic and intracellular domains which are the characteristic of members of the neuregulin family. The Xenopus protein shares a great homology with all members of neuregulin: ; 64% identical in amino acid sequence to that of human, ; 61% identical to chick, ; 70% identical to rat, ; 53% identical to bovine neuregulin. Besides, the splicing variants of neuregulin found in Xenopus are also closely related to its counterparts. Second, the recombinant Fc-tagged Xenopus neuregulin was recognized by antineuregulin antibody, thus indicating the cloned cDNA encodes a protein having similar epitopes as neuregulin. Third, the recombinant neuregulin induced the tyrosine phosphorylation of erbB3 receptor, and the expression of AChR a-subunit mRNA and the b-galactosidase-tagged a-subunit promoter in cultured myotubes. 4.1. Structure of Xenopus neuregulin and its isoforms While sequence analysis indicates that the N-terminal ends of different members of neuregulin family contain no classical hydrophobic signal peptide, they are secretory protein in nature. The mechanism underlying the secretion of neuregulin, however, remains unknown. Several membrane-bound and secreted proteins are devoid of the hydrophobic signal peptide in their N-terminal ends. Accordingly, an alternate pathway for protein secretion has been proposed for those proteins lacking a signal peptide w31x. The transmembrane domain of neuregulin is highly conserved from frog to human, and is flanked at both ends with clusters of basic residues. N-terminal to the transmembrane domain, there is the dibasic residue ŽK 259 –R 260 . that is highly conserved among all neuregulin members. The dibasic residues have been proposed to be involved in the proteolytic cleavage of soluble active neuregulin fragment at the neuromuscular junction w27x. Although the intracellular domain that account for over 60% of the total protein is conserved among all members of neuregulin, the

exact function of this domain within neuregulin is yet unknown. Moreover, the intracellular domain is highly hydrophic in nature and rich in serine and threonine residues which are highly conserved. Some of these residues are located at potential sites for phosphorylation by protein kinase C and casein kinase II. However, the exact phosphorylation sites of the intracellular domain have not been reported. One of the proposed functions of this domain is to regulate the release of soluble active neuregulin fragment at the K 259 –R 260 dibasic residues w52x. Two splicing variants of Xenopus neuregulin at the spacer domain with either 0 or 43 amino acid insertion were found. The 43-amino acid stretch at the spacer domain is distinct and shares no close sequence homology with other members of neuregulin w36x. Similar to other homologues, the spacer domain of Xenopus neuregulin is rich in serine and threonine and that could be a potential attachment sites for O-linked glycosylation. The Nterminus of the spacer domain contains two potential N-linked glycosylation sites and are highly conserved in all species. Thus, the spacer domain of neuregulin may be rich in both O-linked and N-linked sugar. The functions of the heavy glycosylated spacer domain have been proposed: Ži. to control the susceptibility of neuregulin to protease action in the release of the active EGF-like fragment at the synaptic cleft w27x; and Žii. to keep the adjacent functional domains, Ig-like and EGF-like domains, in proper exposure and being accessible for molecular interactions w37x. Mutation analysis is being carried out in our laboratory to determine these possibilities. In chick neuregulin, there are three splicing variants at the spacer domain; they have either 0, 17, or 34 amino acids insertion. The expression of these isoforms in different chick tissues is distinct. Chick brain and spinal cord express only neuregulin SP0 but not neuregulin SP17 or neuregulin SP34 , while muscle expresses all variants at the spacer domain w36x. On the other hand, two splicing variants at the spacer domain with either 0 or 43 amino acids insertion were revealed in Xenopus neuregulin. The expression of neuregulin SP0 and neuregulin SP43 could be detected in Xenopus brain, spinal cord and muscle, and this profile of expression contrasts that observed in chick species. Indeed, the precise role of the splicing variation at the spacer domain of neuregulin in the formation of neuromuscular junction is not clear. The splicing variants at the C-terminus of the EGF-like domain of Xenopus neuregulin are a 1, a 2 and b 2 isoforms. These isoforms were identified in our library screening as well as in our RT-PCR analysis on RNAs isolated from different Xenopus tissues. Other isoforms having variation at the carboxyl-terminus of EGF-like domain, as identified in avian or mammalian tissues, could exist in Xenopus. However, they are not further investigated in this study. The two splicing sites, the carboxylterminus of EGF-like domain Ž a 1, a 2, b 2. and the spacer

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domain ŽSP0, SP43., were interchangeable. cDNAs encoding five different combinations of neuregulin isoforms, except neuregulin SP43 b 2 , were identified in our library screening. Two different splicing sites were also interchangeable in chick neuregulin w36x. Moreover, the expression profile of a 1, a 2, b 2 isoforms are very different to that in chick tissues. In chick spinal cords, b 1 is the predominant species being expressed throughout the development, while a 2 and b 2 isoforms are expressed only in embryonic muscles. In adult chicken muscle, b 1 isoform is the only species detected w36x. We do not know whether the expression of different neuregulin isoforms could be altered in Xenopus muscle during development and denervation. However, the expression profile of muscle-derived neuregulin in Xenopus is very different when compared to that in chicken. Adult Xenopus muscle expresses a 1 and a 2 isoforms, while the spinal cord expresses a 1, a 2 and b 2 isoforms. It will be interesting to determine the change of these neuregulin isoforms in spinal cord, muscle and Schwann cells during the formation of the neuromuscular junction in Xenopus. 4.2. Expression of neuregulin in muscle Neuregulin has diverse functions in neural development, but the major role at the neuromuscular junction is to induce the synthesis of AChR in the muscle fiber. Although the potency of neuregulin derived from different species in AChR up regulation has not been determined, the AChR-inducing activity of neuregulin has been observed for amphibian, avian and mammalian proteins. Besides, the localization of neuregulin, recognised by antichick neuregulin antibody, is restricted to the neuromuscular junction of Xenopus muscle. This synaptic expression pattern is similar to that observed in avian w36x and mammalian muscles w23,34x. These lines of evidence suggest that neuregulin induces the postsynaptic gene expression during the formation of vertebrate neuromuscular junctions. In addition to the central nervous system, neuregulin mRNA is also present in Xenopus skeletal muscle as shown by in situ hybridization and by northern blot analysis. This is consistent with results obtained in chick that several isoforms of neuregulin are expressed by the muscle w36x. Thus, both the motor neuron and the target muscle cell can contribute neuregulin to the developing neuromuscular junction. This redundancy in the secretion of synaptically related molecules by both pre and the postsynaptic cell seems to be a general principle for the neuromuscular junction. Another example is agrin, a heparan-sulfate proteoglycan involved in signaling the clustering of AChRs. Both neurons and muscle cells can secrete agrin, although the form secreted by muscle has much lower AChR-aggregating activity than the neuronal forms w1,39x. Our in situ hybridization study has also shown that the neuregulin mRNA appears very early during development.

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In fact, there exists a pool of maternal neuregulin message as shown by its expression in the animal hemisphere of embryos during the early blastula stage. Although the significance of this maternal mRNA is unknown, its early expression suggests that in addition to neural development, neuregulin may also play a role in embryonic development. Other functions of neuregulin are also suggested by the localization of its message in other parts of the embryo, e.g., the branchial arches, besides the central nervous system and muscle during later development. Within the central nervous system, neuregulin is strongly expressed in the eye vesicle and thus may participate in the development of the neural retina. Within myotomes, neuregulin message is localized to the region of the myonuclei as expected as well as at the intersomitic junctions where the neuromuscular junction is found. This raises the interesting question on the localization of messages of postsynaptic protein near the neuromuscular junction. Localization of mRNAs, such as Vg-1, is well documented in Xenopus oocytes w35x. At vertebrate neuromuscular junction, messages for synaptic proteins are known to be synthesized specifically by synaptic nuclei w15,22x. During the stages of Xenopus synaptic development examined in this study, the myotomal muscle fibers are mononucleated. Thus, the localization of neuregulin mRNA may result from a mechanism different from differential nuclear expression. The elucidation of this mechanism should shed more light on the formation of the molecular complex at the postsynaptic membrane. Acknowledgements We are grateful to Tina Dong, Lisa Yung, H.Y. Choi and Matthew Au from our laboratory for their expert technical assistance. We thank Dr. Joshua Sanes from Washington University School of Medicine for providing pnlacZ plasmid, Drs. Tom Sargant and Doug DeSimone for providing Xenopus cDNA libraries. The research was supported by grants from Research Grants Council of Hong Kong and the Biotechnology Research Institute at The Hong Kong University of Science and Technology Žto N.Y.I and K.W.K.T.. and by US NIH grant NS-23588 Žto H.B.P... References w1x M.A. Bowe, J.R. Fallon, The role of agrin in synapse formation, Annu. Rev. Neurosci. 18 Ž1995. 443–462. w2x K.L. Carraway, L.C. Cantlet, A neu acquaintance for erbB3 and erbB4: a role for receptor heterodimerization in growth signaling, Cell 78 Ž1994. 5–8. w3x K.L. Carraway, J.L. Weber, M.J. Unger, J. Ledesma, N. Yu, M. Gassmann, C. Lai, Neuregulin-2, a new ligand of erbB3rerbB4 receptor tyrosine kinase, Nature 387 Ž1997. 512–516. w4x H. Chang, D.J. Riese II, W. Gilbert, D.F. Stern, U.J. McMahan, Ligands for erb-family receptors encoded by a neuregulin-like gene, Nature 387 Ž1997. 509–512.

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