Overexpression of Agrin Isoforms inXenopusEmbryos Alters the Distribution of Synaptic Acetylcholine Receptors during Development of the Neuromuscular Junction

Developmental Biology 205, 22–32 (1999) Article ID dbio.1998.9104, available online at http://www.idealibrary.com on

Overexpression of Agrin Isoforms in Xenopus Embryos Alters the Distribution of Synaptic Acetylcholine Receptors during Development of the Neuromuscular Junction Earl W. Godfrey,* Jeremy Roe,* and R. David Heathcote† *Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; and †Department of Biological Sciences, University of Wisconsin—Milwaukee, Milwaukee, Wisconsin 53201

Synapse formation involves a large number of macromolecules found in both presynaptic nerve terminals and postsynaptic cells. Many of the molecules involved in synaptogenesis of the neuromuscular junction have been discovered through morphological localization to the synapse and functional cell culture assays, but their role in embryonic development has been more difficult to study. One of the best understood of these molecules is agrin, a synaptic extracellular matrix protein secreted by both motor neurons and muscle cells, that organizes the postsynaptic apparatus, including high-density aggregates of acetylcholine receptors (AChRs), at the neuromuscular junction. We tested the specific hypothesis that different agrin isoforms made by neurons and muscle cells contribute to agrin’s synapse organizing activity in the embryo. Agrin isoforms were overexpressed by injecting synthetic RNA into Xenopus laevis embryos at the one- or two-cell stage. To mark cells containing agrin RNA, green fluorescent protein (GFP) RNA was coinjected. The relative area of muscle AChR aggregates was measured by confocal microscopy and image analysis in GFP-positive segments of injected embryos. Innervated regions of myotomal muscles were compared in animals injected with a mixture of agrin and GFP RNAs or with GFP RNA alone. Overexpression of COOH-terminal 95-kDa fragments of a rat agrin isoform made only by neurons (4,8) and the major isoform (0,0) made by muscle cells both increased AChR cluster area by 100 –200%. Rat agrin protein was colocalized with AChR aggregates in innervated regions of muscles in injected embryos. These results show that agrin derived from both the nerve terminal and the muscle cell could contribute to synaptic differentiation at the embryonic neuromuscular junction. They further demonstrate the usefulness of overexpression by RNA injection as an assay for molecular function in embryonic synapse formation. © 1999 Academic Press Key Words: agrin; synaptogenesis; neuromuscular junction; acetylcholine receptors; Xenopus embryos.

INTRODUCTION Several molecules play a role in the formation and differentiation of neuromuscular synapses. Agrin is a widely distributed basal lamina protein that is concentrated at the neuromuscular junction (Reist et al., 1987; Godfrey et al., 1988a,b), where it is localized to the synaptic basal lamina (Reist et al., 1987). Agrin is made and secreted by both motor neurons and muscle cells (Magill-Solc and McMahan, 1988; Cohen and Godfrey, 1992; Ruegg et al., 1992; Lieth and Fallon, 1993; Ma et al., 1994, 1995). Agrin directs the formation of the postsynaptic apparatus, including a high-density aggregation of acetylcholine receptors (AChRs), and consists of several isoforms derived from alternative

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splicing of its mRNA (Ferns et al., 1992; Tsim et al., 1992). Among these, isoforms with inserts at both of two positions near the C terminus of the protein (“A” and “B,” Ruegg et al., 1992; “y” and “z;” Hoch et al. 1993) are exclusively neuronal (Smith and O’Dowd, 1994). Compared to isoforms (e.g., 0,0 and 4,0) made by nonneural tissues, including muscle (Ruegg et al., 1992; Hoch et al., 1993), the neuronal isoforms (e.g., 4,8) have much greater AChR aggregating activity on cultured muscle cells, when they are added to the culture medium as soluble proteins (Hoch et al., 1994; Gesemann et al., 1995). However, when the 0,0 and 4,0 isoforms are expressed on the surface of transfected cells, their ability to aggregate AChRs upon contact with cocultured myotubes is comparable to that of neuronal isoforms 0012-1606/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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Agrin Isoforms Alter Embryonic Synapse Formation

(Ferns et al., 1992, 1993). Here we show that embryonic overexpression of agrin isoforms normally secreted by both neurons (4,8) and muscle cells (0,0) increases the differentiation of postsynaptic structures, in contrast to some results from cell culture assays and transfection of adult muscles. These results suggest that both neuronal and muscle agrin isoforms play a role in postsynaptic differentiation of the embryonic neuromuscular junction. The function of proteins in synapse formation has largely been extrapolated from results of cell culture assays. For example, motor neuron–muscle cell cultures have been very useful for studying early stages of synapse formation (e.g., Cohen and Godfrey, 1992), but later stages of synaptogenesis have not been well characterized in these cultures. Another valuable tool is the elimination of candidate molecules by altering their genes in transgenic “knockout” mice. Use of knockout mice has shown that agrin (Gautam et al., 1996), muscle-specific kinase (DeChiara et al., 1996), and rapsyn (Gautam et al., 1995) are essential for the formation of embryonic neuromuscular junctions. However, since mice lacking expression of any of these proteins die at birth, the study of synaptic development is difficult in these animals. We have developed a novel approach to test the role of proteins in the formation of the neuromuscular synapse in the developing embryo. The test involves injecting RNA into early blastomeres to overexpress candidate molecules during development of the frog, Xenopus laevis. Injection of RNAs into early Xenopus embryos is frequently used to study the role of proteins in early development (Vize et al., 1991). Some studies have also utilized this method to evaluate the role of molecules involved in organogenesis. For example, Gove et al. (1997) used overexpression of the transcription factor GATA-6 to examine its effect on heart development in Xenopus, which they assayed in embryos at stages 26 –35. In the present study we used RNA injection to overexpress both neuronal and nonneuronal isoforms of agrin. We assayed the effects of overexpression at stages 28 –33/34 (1.3–1.8 days of development), during the period when synapses form in myotomal muscles. We show that RNA injection is a reliable method for overexpression of proteins in the developing neuromuscular system. We used confocal microscopy to image segmental groups of neuromuscular synapses labeled with fluorescent probes, thus enabling us to assay the effect of any protein on the development of the embryonic neuromuscular junction.

METHODS cDNAs and Subcloning To synthesize mRNAs that could be translated readily in Xenopus embryos, cDNAs were subcloned into the multiple cloning site of pCS21 (Rupp et al., 1994; Turner and Weintraub, 1994). Rat agrin cDNAs [0,0 “muscle” and 4,8 “neuronal” isoforms at positions y and z (Hoch et al., 1993); gift of Dr. Michael Ferns, Montreal General Hospital] encoding the COOH-terminal 95-kDa of the

agrin protein were used as templates for RNA synthesis after subcloning into pCS21 as ClaI/XbaI fragments. A cDNA encoding a red-shifted variant (F64L S65T) of the jellyfish green fluorescent protein (GFP-LT; gift of Dr. Richard Dorsky, University of California, San Diego) in pCS21 was used to prepare GFP RNA.

RNA Synthesis The pCS21 constructs were linearized with XbaI (for rat agrins) or NotI (for GFP). RNAs were synthesized with SP6 RNA polymerase using the mMessage mMachine kit (Ambion) and analyzed and quantitated after electrophoresis on agarose gels. Agrin RNAs were about 3.6 kb and GFP RNA was 1.2 kb, as expected. RNA concentrations of 0.5– 4 mg/ml (agrin) and 0.1 mg/ml (GFP) were used for injection.

RNA Injection Embryos of X. laevis were obtained by in vitro fertilization (Moon and Christian, 1987) and injected with synthetic RNAs at the one- or two-cell stages using a Nanoject (Drummond) or Picospritzer II (General Valve) through glass pipets with a tip diameter of 10 –20 mm. Embryos were injected (Moon and Christian, 1987) with 5 nl per blastomere containing GFP RNA (0.5 ng) or agrin (2.5–20 ng) plus GFP (0.5 ng) RNA.

Screening, Fixation, and Labeling Acetylcholine Receptors Following injection of RNAs, embryos were transferred into 0.13 modified Barth’s saline (Gurdon and Wickens, 1983) and allowed to develop at room temperature. The next day, they were screened for normal development and incubated at 10 –18°C overnight. Embryos were staged according to Nieuwkoop and Faber (1994). After neuromuscular synapse formation (usually stage 31), embryos were screened for GFP fluorescence. Normal embryos with fluorescence in the trunk myotomes and/or neural tube were fixed in 4% paraformaldehyde for 1 h at 4°C and rinsed in Ringer, and the skin was removed with fine forceps. Embryos were incubated in rhodamine–a-bungarotoxin (0.5–2 mg/ml, Molecular Probes) in 1% goat serum in Ringer overnight at 4°C, washed, incubated 1 h in mounting medium (Valnes and Brandtzaeg, 1985), and mounted on slides.

Confocal Microscopy AChR aggregates were imaged by confocal microscopy. Images were taken from the fifth through the seventh myotomes in embryos with GFP fluorescence in this region. These myotomes were chosen because the synaptic bands of AChR aggregates were more regular and continuous than in more rostral or caudal myotomes. To compare the extent of AChR clustering in synaptic regions of control and experimental animals, six different Z-series of four optical sections (at 1-mm intervals) were acquired from four to six animals in each group. Microscope settings remained constant during imaging of all groups of animals within an experiment to ensure comparability of the images.

Image Analysis The area of AChR aggregates in myotomal muscles was measured with Metamorph image analysis software (Universal Imag-

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Godfrey, Roe, and Heathcote

ing). Threshold was set to mark the aggregates in each series of confocal images, and aggregate area was converted to a percentage of the total area of the entire series of images.

Statistics Mean values and SE were calculated for each group of images. To create a more normal distribution of the data (percentages of area containing AChR aggregates), a one-tailed t test was performed after an arcsin conversion (Zar, 1974), comparing values of mean percentage area from each injected group of embryos with values from the GFP control group.

Immunofluorescence Rat agrin protein was detected in RNA-injected embryos with a rabbit anti-rat agrin antiserum (1:1000) made against the COOHterminal 95-kDa portion of isoform 4,8 (gift from Drs. Janice Sugiyama and Zach Hall, National Institute of Mental Health). This antibody did not cross-react with endogenous Xenopus agrin. Antibody-binding sites were revealed with FITC-labeled goat antirabbit antibodies (Cappel) and imaged by confocal microscopy.

Reverse Transcription–Polymerase Chain Reaction (RT-PCR) To determine how long injected RNA persisted in the embryo, RT-PCR was performed. Groups of five embryos, uninjected or injected with 10 ng of rat agrin (4,8) RNA, were frozen at stages 2, 9, 32, and 35/36. Total RNA was isolated using the acid guanidinium/phenol method of Chomzcynski and Sacchi (1987). cDNA synthesis and PCR amplification were performed using RT-PCR beads (Pharmacia) with 350 ng total RNA. Rat agrin forward and reverse primers were 59-AAG GTC AAC ACC AAC-39 (bases 5722–5736) and 59-GGT CGA TAA TAC GAC TCA CTA TAG GGA GGG AGT GGG GCA GGG-39 (T7 promoter sequence and bases 6007– 6021; Rupp et al., 1991). Primers for Xenopus ornithine decarboxylase (ODC; Bassez et al., 1990) were used as a control for RNA purification. Amplification was performed in a PTC-100 thermal cycler (M.J. Research) using 23 cycles of 95, 55, and 72°C (each 1 min). A control reaction without template RNA was included. The relative intensity of DNA bands was determined by laser scanning of ethidium bromide-stained agarose gels using a FluoroImager (Molecular Dynamics) and ImageQuant software.

RESULTS Innervation of Myotomal Muscles We chose the Xenopus embryo neuromuscular system because it allowed us to perform a convenient, rapid, and quantitative assay of synapse formation in the embryo. Synapses in the myotomal muscles of Xenopus tadpoles form in regular rows at both ends of each myotome, resulting in synaptic “stripes” bordering the septa between myotomes (arrows, Fig. 1b). The regular nature of the myotomes and the synapses within them resulted in small enough variability in the relative area of synaptic AChR aggregates that differences between control and experimental groups could readily be detected. Typically, this rela-

FIG. 1. Innervation of myotomal muscles in the Xenopus embryo, stage 31. (a) Diagram of stage 31 embryo showing myotomal muscles (shaded). (b) Innervation of both ends of muscle cells by motor axons is indicated by arrows. Bands of synapses from adjacent myotomes are closer together than indicated and cannot always be distinguished by light microscopy.

tively small variability resulted in SE of 10 –20% of the mean in uninjected and GFP RNA-injected embryos (e.g., Table 1). The organization of Xenopus embryo neuromuscular junctions allowed us to image large segmental ensembles of synapses by confocal microscopy (e.g., Fig. 5), thus averaging variation between synapses in a myotome. Development of these synapses occurs rapidly, and the free-living embryos are readily accessible. The first functional neuromuscular synapses form 1 day after fertilization, as evidenced by the onset of muscle movement (Nieuwkoop and Faber, 1994), the appearance of synaptic potentials and nerve–muscle contacts (Kullberg et al., 1977), and the aggregation of AChRs at these early synapses (Chow and Cohen, 1983; Cohen and Godfrey, 1992). Since some injected RNAs persist past this stage (see below; Fig. 4), altering expression of proteins during neuromuscular synaptogenesis by RNA injection into one- or two-cell embryos was possible.

GFP Is a Marker for RNA Injection and Translation To visualize the successful injection, diffusion, and translation of synthetic mRNA in the embryo, we injected a mixture of RNAs encoding agrin and GFP. Embryos injected with GFP RNA alone served as a control. In Fig. 2, an embryo is shown in which one blastomere had been injected with GFP RNA at

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Agrin Isoforms Alter Embryonic Synapse Formation

FIG. 2. GFP is a marker for successful RNA injection and indicates the extent of diffusion and translation of injected RNA and the persistence of the protein product. One blastomere of a two-cell stage embryo was injected with GFP RNA. GFP fluorescence is seen in most cells on the right (upper) half of this tadpole (stage 35/36, 2.1 days of development). FIG. 3. Rat agrin is colocalized with AChR aggregates in confocal images of the innervated region of myotomal muscles. Embryos were injected at the one-cell stage with rat agrin RNA (neuronal isoform 4,8) in the absence of GFP RNA. Whole mounts of fixed stage 31 (1.6 day) embryos were stained as described under Methods. (A) Rat agrin immunofluorescence (FITC-labeled antibody). (B) AChR aggregates labeled with rhodamine–a-bungarotoxin. (C) Superimposed images from A and B showing colocalized rat agrin and AChR aggregates (yellow). Arrows indicate synaptic regions adjacent to the myotomal septum. Bar, 20 mm.

the two-cell stage. At 2 days of development (stage 35/36), bright GFP fluorescence was seen in most cells derived from the injected blastomere, indicating that GFP RNA diffused widely and evenly within this blastomere before it divided. Thus, GFP is a reliable marker that enables us to screen large numbers of living embryos for successful injection and translation of RNA and to identify regions of injected embryos that are also likely to express the experimental gene product.

Overexpressed Rat Agrin Is Colocalized with AChR Aggregates in Embryonic Muscles Overexpressed rat agrin (neuronal isoform 4,8; Fig. 3A) was colocalized with AChR aggregates (Fig. 3B) by labeling agrin RNA-injected embryos with a species-specific anti-rat agrin antibody. Rat agrin protein was found in a punctate pattern in the innervated region of 1.6-day (stage 31) embryonic muscles (myotomal septum; arrows, Fig. 3), suggesting that it was

deposited at neuromuscular junctions in the injected embryos and that it was biologically active. Superimposing these two images shows considerable colocalization of rat agrin with AChR aggregates (yellow, Fig. 3C), particularly in the innervated region. In other embryos, punctate deposits of rat agrin immunoreactivity were also seen in other parts of myotomes (data not shown). The identity and AChR-aggregating activity of rat agrin encoded by the synthetic RNA were confirmed by in vitro translation, followed by agrin bioassay (AChR aggregation in primary chick muscle cultures) and immunoblotting of the translation product with the species-specific antibody (data not shown).

Rat Agrin RNA Persists during Neuromuscular Synaptogenesis Although the capped synthetic RNA that we injected into embryos should be stable, even stable RNAs are eventually

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FIG. 4. Injected agrin RNA persists throughout embryogenesis. Rat agrin RNA was detected in RNA-injected embryos by RT-PCR. (a) Rat agrin sequence. (b) Ornithine decarboxylase sequence from Xenopus as an internal control. Lanes: 1, no RNA control; 2–5, embryos injected with agrin RNA (10 ng) at the one-cell stage and analyzed at stages 2, 9, 32, and 35/36 (2 h–2.1 days); 6 and 7, uninjected control embryos (stages 9 and 32).

degraded. To determine whether rat agrin RNA persisted through the initial period of synaptogenesis, we used RTPCR and RNase protection assays. Rat agrin RNA was detected in injected tadpoles as late as 1.7 days (stage 32) by RNase protection (data not shown) and 2.1 days (stage 35/36) by RT-PCR (Fig. 4). The amount of rat agrin cDNA amplified from RNA in hatched tadpoles (2.1 days, stage 35/36) was 25% of that amplified from embryos '1 h after injection (stage 2), as determined by laser scanning and quantitation of fluorescence intensity of the bands shown photographically in Fig. 4. Thus, significant amounts of injected rat agrin RNA (Fig. 4) and protein (Fig. 3) persisted well after neuromuscular synaptogenesis began.

Overexpression of Neuronal and Muscle Agrin Isoforms Increases AChR Aggregation in Synaptic Regions of Embryonic Muscle To assay the effects of overexpression of agrin isoforms on synapse formation, we compared AChR aggregation at the neuromuscular junctions of animals injected with rat agrin RNAs plus GFP marker RNA, those injected with GFP RNA alone, and uninjected animals. Recombinant rat agrin aggregated AChRs on cultured Xenopus muscle cells (M. W. Cohen, personal communication). Overexpression of both neuronal (4, 8) and muscle (0, 0) isoforms of rat agrin

FIG. 5. Overexpression of neuronal and muscle agrin isoforms increases AChR aggregate area in innervated regions of myotomal muscles. Xenopus embryos were injected at the one-cell stage with RNAs encoding GFP (A), rat neuronal agrin (4,8) and GFP (B), or rat muscle agrin (0,0) and GFP (C). AChRs were labeled with rhodamine–a-bungarotoxin and images were obtained by confocal microscopy at stage 31 (1.6 days). Overlaid series of four images, taken at 1-mm intervals, are shown. Bands of AChR aggregates (bright clusters) running horizontally across the images represent innervated regions at the ends of two adjacent myotomes; muscle cells run vertically, parallel to the anterior–posterior axis of the embryo. The images shown were taken from the same experiment; AChR aggregate areas were 0.61% (A), 2.94% (B), and 2.16% (C) of area imaged. Bar, 20 mm.

Godfrey, Roe, and Heathcote

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Agrin Isoforms Alter Embryonic Synapse Formation

TABLE 1 Overexpression of Neuronal and Muscle Agrin Isoforms in Xenopus Embryos Increases Acetylcholine Receptor Aggregation in Myotomal Muscles Expt 1

2

RNA injected (ng) None GFP (0.5) Agrin 4,8 Agrin 0,0 GFP (0.5) Agrin 4,8 Agrin 4,8 Agrin 4,8

(10) 1 GFP (0.5) (10) 1 GFP (0.5) (2.5) 1 GFP (0.5) (5) 1 GFP (0.5) (10) 1 GFP (0.5)

Stage fixed

AChR aggregate area (%) 6 SE

Area ratio agrin:GFP

P value (vs GFP)

33/34 33/34 33/34 33/34 31 31 31 31

0.32 6 0.03 0.26 6 0.03 0.58 6 0.07 0.78 6 0.14 0.28 6 0.07 0.64 6 0.17 0.74 6 0.03 a 1.05 6 0.30

— — 2.23 3.00 — 2.29 2.64 3.75

1.00 — 0.001 0.006 — 0.049 0.003 0.007

Note. Agrin was overexpressed in Xenopus embryos by injection of RNA encoding either 4,8 (neuronal) or 0,0 (muscle) isoforms. Area ratio refers to the ratio of AChR aggregate area in agrin RNA-injected groups compared to corresponding GFP RNA-injected control groups. a Average of data from three microscopic fields. All other groups consists of data from six different fields from four to six embryos.

also resulted in a significant increase in AChR aggregation in embryonic muscle cells (Figs. 5B and 5C). Most of the increase in aggregation was in normally innervated regions of muscle (horizontal stripes of clusters, Fig. 5), but in some experiments there was also an increase in extrajunctional aggregation (above and below stripes of clusters). AChR aggregation was strikingly different in muscles of control (GFP RNA alone, Fig. 5A; uninjected, not shown) and experimental (agrin plus GFP RNA, Figs. 5B and 5C) embryos. There were more and larger AChR aggregates in muscles of embryos overexpressing agrin than in those expressing GFP alone. Overexpression of either neuronal or muscle agrin isoforms resulted in AChR aggregate areas two- to fourfold those of control embryos (Table 1). The increase in AChR aggregation was similar in regions of injected embryos in which GFP was expressed in the neural tube and regions with GFP in myotomes (data not shown). Although the AChR aggregate area in both control and experimental animals varied between experiments (not

shown), the relative increase due to agrin overexpression was similar (Table 2). Injection of increasing amounts of neuronal agrin RNA (4,8 isoform) led to increasing AChR aggregation in the embryos (Experiment 2, Table 1). Due to conflicting reports of AChR-aggregating activity of agrin isoforms in culture, it was unclear whether the 0,0 isoform, which is the major form made by muscle cells, would display activity in the embryo. However, overexpression of both 4,8 and 0,0 isoforms caused significant increases in AChR aggregation in embryonic muscles (Table 1). No obvious difference in the pattern of AChR aggregates induced by the two isoforms was noticed. The frequency of experiments showing significant increases in embryonic AChR aggregation (Table 2) was similar for groups of embryos injected with RNA encoding the 4,8 neuronal isoform (65%) and the 0,0 muscle isoform (50%). The potency of these isoforms in the embryo was also similar. In groups of embryos showing a significant increase, AChR aggregate area averaged 2.33- (4,8) and 2.68(0,0) fold that of controls, respectively. Thus, agrin isoforms

TABLE 2 Summary of Agrin RNA Injection Experiments

RNA injected Neuronal agrin 4,8 ($10 ng) Muscle agrin 0,0 ($10 ng)

Significantly increased groups*

Area ratio agrin:GFP (avg 6 SE)

No significant difference groups*

% of groups significantly increased

11

2.33 6 0.22

6

65

3

2.68 6 0.30

3

50

Note. Each group consisted of four to six imaged embryos which had been injected at the one- or two-cell stage with the same RNA (type and concentration) in the same experiment, and which contained GFP in the areas analyzed. * “Significantly increased groups” had an average AChR aggregate area significantly (P , 0.05) greater than that of control animals injected with GFP RNA alone, while “no significant difference groups” did not (P . 0.05). Area ratios shown were calculated from groups of embryos showing significant increases over control.

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Godfrey, Roe, and Heathcote

produced by both the nerve terminal and the muscle cell may contribute to the differentiation of the postsynaptic apparatus in the embryo.

DISCUSSION Overexpression of Synaptic Proteins during Embryonic Synapse Formation While many synaptic macromolecules have been discovered, knowledge of molecular mechanisms by which synapses are organized remains sketchy. Considerable progress has been made, however, in understanding some of the molecular interactions involved in the organization of the postsynaptic apparatus by agrin, different isoforms of which are externalized at the neuromuscular junction by motor nerve terminals and muscle cells (Ruegg et al., 1992; Ferns et al., 1993; Ma et al., 1994). Many of these interactions have been established by examining protein–protein interactions in cell-free systems (e.g., Campanelli et al., 1996; Gesemann et al., 1996; Hopf and Hoch, 1996; O’Toole et al., 1996) or in transfected cells (Apel et al., 1995, 1997; Denzer et al., 1997; Fuhrer et al., 1997). Corresponding studies of embryonic synapse formation have recently been focused on null mutant knockout mice, which have confirmed a requirement for agrin, MuSK, and rapsyn for the formation of the postsynaptic specialization of the neuromuscular junction in vivo (Gautam et al., 1995; DeChiara et al., 1996; Gautam et al., 1996). Because the developmental role of molecules in the living embryo can only be established by in vivo studies, additional embryonic assay systems are required to gain a more complete understanding of mechanisms of embryonic synaptogenesis. To address this need, we have utilized overexpression of synaptic molecules by RNA injection in Xenopus embryos to study their function in the formation of neuromuscular synapses. During development of myotomal muscles in the Xenopus embryo, innervation of muscle cells by motor axons from spinal cord begins at about 1 day of development (stage 22; Kullberg et al., 1977). One of the first events seen at nascent neuromuscular synapses is the aggregation of muscle cell AChRs at nerve–muscle contacts (Chow and Cohen, 1983; Cohen and Godfrey, 1992). Appearance of AChR aggregates can therefore reflect the establishment of new synapses in the embryonic muscles. We have measured the area of AChR aggregates as a reflection of the ability of the overexpressed molecules to affect the differentiation of the postsynaptic membrane.

Injected RNA and Encoded Protein Persist during Synaptogenesis in the Embryo Injected agrin RNA and protein translated from it were detectable in injected embryos up to the stage when they were fixed for analysis of synaptic differentiation. Although

the amount of RNA remaining at this stage was significantly lower than originally injected (25% or less of the original amount), the protein expressed was abundant enough that immunofluorescence revealed rat agrin colocalized at AChR aggregates in muscle. While these results show that protein translated from RNAs injected into embryos at the one-cell stage could be detected as late as stage 31, we found that injection of at least 10 ng of agrin RNA per embryo was required to stimulate increased AChR aggregation consistently. Thus, the RNA injection method is suitable for overexpression of synaptic molecules and observation of the effects on neuromuscular synapse formation.

Overexpression of Both Neuronal and Muscle Agrin Isoforms Increased AChR Aggregation in Embryonic Muscles Overexpression of agrin in the Xenopus embryo produced 100 –200% increases in AChR aggregate area in myotomal muscles. The extent of the increase in AChR aggregation depended on the amount of agrin RNA injected, consistent with the dose dependence of AChR aggregation in agrintreated muscle cell cultures (Godfrey et al., 1984). Thus, agrin levels can be manipulated in the developing embryo, resulting in an appropriate biological response. It appears that agrin receptors in these embryonic muscles were not saturated by endogenous agrin and that the area of AChR aggregates reflected the extent of agrin receptor activation in the embryo. An excess of agrin receptors at the neuromuscular junction might facilitate the growth of the synapse during development and its remodeling in the adult. The results were variable in two ways. First, the relative area of AChR aggregates in muscle varied between experiments in both controls and agrin RNA-injected embryos (Table 1 and data not shown). Second, some of the groups of agrin RNA-injected embryos did not show a significant increase in AChR aggregate area when compared to controls (Table 2). The variability in average area between groups of embryos in different experiments could reflect biological variability between groups of embryos with different parents, variability in labeling of AChRs, and differences between experiments in settings on the confocal microscope and resulting intensity of aggregates in images. The lack of a significant increase in AChR aggregate area in some groups of agrin RNA-injected embryos could be caused by variability in the stability or translation of agrin RNA, differences in the extent of diffusion of injected RNA (which would affect its concentration), and/or variability of AChR aggregate area within a group of embryos. All imaged regions of agrin RNA-injected embryos contained visible GFP; however, agrin RNA or protein may be degraded more rapidly than GFP. The doses of agrin RNA we used could be close to the minimum needed to consistently cause the observed effect. Injecting smaller amounts of RNA resulted in a larger proportion of experiments with no significant increase in area. However, we did not increase the amount

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Agrin Isoforms Alter Embryonic Synapse Formation

of RNA injected to more than 10 –15 ng/embryo because of increased toxicity seen after injecting larger amounts of RNA. Both an agrin isoform found only in neurons (4,8) and one found in many cell types that is also the major isoform in muscle cells (0,0) produced increased clustering in our experiments. The frequency and extent of the increase were similar for the two isoforms, indicating that their potency in embryonic muscle was similar. Some investigators have referred to the 0,0 isoform as “inactive,” since little or no AChR-aggregating activity is seen when recombinant agrin 0,0 is added to the culture medium of muscle cells (Ruegg et al., 1992; Ferns et al., 1993). However, even when assayed in this way, agrin extracted from adult skeletal muscle had a small but measurable activity (Godfrey, 1991). When agrin cDNA constructs were used to transfect adult rat muscle fibers, neuronal isoforms of agrin induced complete postsynaptic specializations extrajunctionally (Cohen et al., 1997; Jones et al., 1997), while other isoforms did not (Cohen et al., 1997). In contrast to these results, other studies have shown that both sets of isoforms can display AChR-aggregating activity. When the 0,0 and 4,0 isoforms were expressed on the surface of transfected cells which were cocultured with muscle cells (Ferns et al., 1993), these isoforms were about 80% as active as the neuronal isoforms. In another study, NG108-15 neuroblastoma– glioma hybrid cells induced AChR aggregation on muscle cells, but expressed only the 0,0 isoform of agrin (Pun and Tsim, 1997). Taken together, these results suggest that the agrin isoforms found in muscle cells have the ability to organize the postsynaptic apparatus, but this activity may not be apparent unless agrin is presented to the muscle cell on the surface of a cell or in the matrix. Such solid-phase presentation of agrin would allow agrin isoforms (such as 0,0), which may have a low affinity for the agrin receptors when presented in solution, to bind to these receptors with an effectively higher affinity, stimulating AChR aggregation. Thus, the method of assaying agrin’s synapse-organizing activity is critical. Assays using cell-attached agrin may more closely resemble the situation at the developing neuromuscular junction than assays in which agrin is added to the medium of cultured muscle cells. In Xenopus embryos, neuronal agrin is likely to be presented to muscle cells on the surface of motor axons rather than being released into the extracellular space. This hypothesis is suggested by previous results from Xenopus spinal cord–muscle cell cocultures (Cohen and Godfrey, 1992), in which most AChR aggregates at nerve–muscle contacts were colocalized with punctate aggregates of neuronal agrin on the surface of the axons. The punctate distribution of rat agrin in injected embryos (Fig. 3) is consistent with the findings in culture; it was seen in both innervated regions at the ends of myotomes (e.g., Fig. 3) and also scattered throughout the myotome in some embryos (not shown). Puncta containing agrin could result from the assembly of extracellular matrix molecules into particulate complexes, similar to those seen in early embryonic chick

heart (Mjaatvedt et al., 1987) and in chick and mouse embryo premuscle masses in the limb bud (Godfrey et al., 1988b; Godfrey and Gradall, 1998). Perhaps most of the agrin overexpressed by embryonic muscle cells in our experiments was more diffusely distributed on the surface of the cells, where it caused less AChR aggregation than agrin which was overexpressed by neurons and concentrated in the synaptic region by nerve terminals. Our results showing the AChR-aggregating activity of a muscle agrin isoform in the embryo raise the question of the role of agrin secreted by muscle cells at the normal embryonic synapse. Some neurons in the ciliary ganglion of chick embryos express the agrin 4,0 isoform, which is also expressed by muscle cells (Smith and O’Dowd, 1994; Hoch et al., 1993), but it is not known if this isoform is also expressed by motor neurons. However, the 0,0 isoform is the predominant one expressed in embryonic chick and rat muscles (Ruegg et al., 1992; Hoch et al., 1993; Ma et al., 1994). Unfortunately, the normal expression of agrin in muscle cells of Xenopus embryos has not been characterized, since the available anti-agrin antibodies do not label these cells (Cohen and Godfrey, 1992), and Xenopus agrin cDNA has not yet been cloned. Muscle-derived agrin is concentrated at AChR clusters induced by contact with motor axons (Lieth and Fallon, 1993), but agrin immunofluorescence surrounding embryonic muscle fibers is much less intense than at the neuromuscular junction (Godfrey et al., 1988b; Hoch et al., 1993). Thus, muscle agrin is likely to be more concentrated at the synapse than extrasynaptically, which could explain why isoforms expressed in muscle cells do not normally induce extrasynaptic AChR aggregation. Since muscle agrin is colocalized with AChR aggregates induced on muscle cells by neuronal agrin (Lieth et al., 1992), it is likely that muscle agrin isoforms also become concentrated at the embryonic synapse, where they could contribute to postsynaptic differentiation.

Distribution of AChR Aggregates Induced by Overexpressed Agrin In most of the embryos overexpressing agrin, AChR aggregation increased primarily in the regions of innervation at the ends of myotomes. This may have resulted from greater agrin overexpression by neurons than by muscle cells or it could reflect the cellular geometry of the synapse, at which nerve terminals concentrate neuronal agrin. Concentration of agrin receptors at the ends of the muscle cells could also contribute to preferential aggregation of AChRs at those sites. In our experiments, agrin RNA was injected at the one-cell stage, and regions of embryos expressing GFP in myotomes or in the spinal cord were selected for analysis. No apparent differences in the pattern or area of AChR aggregates were seen when GFP was expressed primarily in spinal cord or in muscle. Since injected agrin RNA was present in muscle cells and in the spinal cord, the synaptic localization of induced AChR aggregates may reflect greater stability or translation efficiency of rat agrin RNA in

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neurons than in muscle cells, and/or targeting or clustering of muscle agrin in synaptic regions of these cells, as discussed above. However, these parameters were not addressed in our experiments. In some experiments, overexpression of agrin resulted in an increase in AChR aggregation away from the ends of the myotomes, in regions of muscle cells that are not normally innervated. We assume that these ectopic aggregates resulted from increased secretion of agrin by muscle cells to levels normally found only at the synapse. While we saw rat agrin immunofluorescence concentrated in the innervated septal regions of myotomes in embryos overexpressing agrin (Fig. 3), we also observed punctate deposits of rat agrin throughout the myotomes of some injected embryos. However, we did not determine whether this “extrasynaptic” agrin was colocalized with AChR aggregates. While we cannot rule out the possibility that agrin overexpression stimulated the growth of motor axons into the myotomes, and that the extrasynaptic aggregates were induced by agrin presented by ectopic nerve terminals, we consider this possibility unlikely. Since eliminating agrin expression stimulates axonal overgrowth in muscle (Gautam et al., 1996), we would expect agrin overexpression to inhibit the growth of motor axons, consistent with agrin’s inhibitory effect on neurite outgrowth in culture (Chang et al., 1997).

General Applicability of RNA Injection for Overexpression of Synaptic Proteins Overexpression in Xenopus embryos has been used to test the role of specific proteins in function and development of cellular mechanisms involved in synaptic transmission at the neuromuscular junction. Synaptic proteins which were overexpressed by injection of either DNA or RNA into early embryos include acetylcholinesterase (AChE; Ben AzizAloya et al., 1993; Seidman et al., 1994; Shapira et al., 1994), acetylcholine receptors (Shapira et al., 1994), synapsin I (Lu et al., 1992), synapsin IIa (Schaeffer et al., 1994), and synaptophysin (Alder et al., 1995). These studies suggested that some of these proteins may be rate-limiting in the formation of functional embryonic synapses. Overexpression has not previously been used in Xenopus embryos to evaluate molecules believed to be involved in directing the differentiation of the neuromuscular junction. In this study, we have overexpressed agrin, a protein essential for organization of this synapse. The approach we have outlined can be applied to testing additional hypotheses about the role of specific proteins, their isoforms, and domains in embryonic synaptogenesis.

ACKNOWLEDGMENTS We thank Haig Tcheurekdjian, Daniel Byrne, Peter Schmidt, Sheila Baker, Jon Ekman, and Sarah Rickert for their assistance in developing the methods and performing experiments in this study. Drs. Richard Dorsky, Janice Sugiyama, Paul Krieg, Monroe Cohen, and Linda Gont and Mr. Brett Schroeder provided

valuable reagents, technical suggestions, and encouragement. We thank Drs. Monroe Cohen and Wesley Thompson for carefully and critically reading the manuscript, Carolyn Snyder for preparing Fig. 1, and Deb Generotzky for photography. This work was funded by the National Institute of Mental Health, NIH (1R01MH57545, E.W.G. and R.D.H.) and the Muscular Dystrophy Association (E.W.G.).

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