A role for , a renal , in vertebrate skeletal muscle cell fusion

Regina Lee Sohna,1,2, Ping Huanga,1, Genri Kawaharaa, Matthew Mitchella, Jeffrey Guyona,3, Raghu Kallurib, Louis M. Kunkela,c, and Emanuela Gussonia,4

aDivision of Genetics and Program in Genomics, Children’s Hospital Boston, 300 Longwood Avenue, Boston, MA 02115; cThe Manton Center for Orphan Disease Research, Children’s Hospital Boston and the Howard Hughes Medical Institute, Boston, MA 02115; and bDivision of Matrix Biology, Beth Israel Deaconess Hospital and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Harvard-MIT Division of Health Sciences and Technology, Boston, MA 02115

Contributed by Louis M. Kunkel, April 22, 2009 (sent for review February 10, 2009) Skeletal muscle is formed via fusion of myoblasts, a well-studied nephrin expression in developing zebrafish results in fish unable process in Drosophila. In vertebrates however, this process is less to swim with abnormal myosepta length. Accordingly, muscle well understood, and whether there is evolutionary conservation cells isolated from nephrinKO mice exhibit a secondary fusion with the studied in flies is under investigation. Sticks and defect and are unable to form mature myotubes. NephrinKO stones (Sns), a cell surface protein found on Drosophila myoblasts, myoblast cultures maintain MAPK/ERK pathway activity during has structural homology to nephrin. Nephrin is a protein expressed differentiation, but this activation does not affect expression of in that is part of the filtration barrier formed by . myogenin, which appears normal. Pharmacological inhibition of No previous study has established any role for nephrin in skeletal the MAPK/ERK pathway does not restore normal myotube muscle. We show, using two models, zebrafish and mice, that the formation, suggesting that this pathway is likely not causative of absence of nephrin results in poorly developed muscles and in- the observed fusion deficiency. In cell mixing experiments, completely fused myotubes, respectively. Although nephrin- human myoblasts are able to fuse to nephrinKO nascent myotubes knockout (nephrinKO) myoblasts exhibit prolonged activation of to form hybrid mature myotubes. Conversely, nephrinKO myo- MAPK/ERK pathway during myogenic differentiation, expression blasts provide little or no contribution to developing human of myogenin does not seem to be altered. Nevertheless, MAPK myotubes. These studies suggest a new role for nephrin in pathway blockade does not rescue myoblast fusion. Co-cultures of skeletal muscle, where its expression appears to be necessary for unaffected human fetal myoblasts with nephrinKO myoblasts or mononucleated myoblasts to fuse into myotubes. These findings myotubes restore the formation of mature myotubes; however, also highlight the possible functional conservation between the contribution of nephrinKO myoblasts is minimal. These studies nephrin and Drosophila Sns. suggest that nephrin plays a role in secondary fusion of myoblasts into nascent myotubes, thus establishing a possible functional Results conservation with Drosophila Sns. Nephrin Is Expressed in Embryonic, Adult Diseased, or Adult Injured Muscle. If nephrin is important for fusion of myoblasts into myoblast fusion ͉ sticks and stones myotubes, its expression should be up-regulated when myoblast fusion is occurring. Indeed, nephrin message was present by ertebrate skeletal muscle is a syncytium formed via two nested reverse transcription–polymerase chain reaction (RT- Vphases of myoblast fusion. During the first phase, myoblasts PCR) in embryonic murine skeletal muscle (Fig. 1A, E15.5), in fuse to generate nascent myotubes that serve as scaffolds for the skeletal muscle of young mice with muscular dystrophy, further growth; in the second phase, more myocytes fuse into which exhibit spontaneous muscle degeneration/regeneration ␦ these nascent myotubes to form mature myotubes (1, 2). Myo- (14) (Fig. 1A, mdx Y (dystrophin null); Scg Y ( -sarcoglycan- blast fusion is a highly regulated process in Drosophila, where null), both 3–4 weeks old) and in wild-type muscle after acute founder cells serve as the ‘‘seeds’’ in muscle formation and injury with cardiotoxin (Fig. 1A, Ctx). No message was detected express the membrane protein Kirre, also known as Dumb- in uninjured adult wild-type mouse skeletal muscle (Fig. 1A, ␦ founded (Duf) (3, 4). Fusion-competent myoblasts (FCM) ex- Wt), in 4-month old mdx (Fig. 1A, mdx O) or -sarcoglycan (Fig. press the transmembrane protein Sticks and stones (Sns) (5) 1A, Scg O) muscles. In multiple independent PCR reactions Kirre Duf followed by sequence analysis, the full nephrin message, includ- which interacts with / (6), bringing the two muscle cell Ј types together, to initiate a complex intra-cellular program that ing most of exon 1a through the 3 UTR was confirmed to be the leads to the fusion of the FCM into the myotubes. There has been same transcript as that found in kidney (data not shown). sparse evidence demonstrating whether the orthologues of these Nephrin was also transiently up-regulated in human fetal skel- proteins are conserved in vertebrate myoblasts (7), although etal myoblasts (isolated from 17-week-old fetus) undergoing recent studies reported a role for Kirrel (Kirre-like) in developing fusion in culture for 2 days (Fig. 1 C–E), after which nephrin zebrafish skeletal muscle (8). expression returned to the low baseline levels (Fig. 1D). Thus, Nephrin is a protein the function of which has been associated

with maintenance of the kidney filtration barrier (9). Mutations Author contributions: R.L.S., P.H., and E.G. designed research; R.L.S., P.H., G.K., M.M., and in nephrin cause the congenital nephrotic syndrome of the J.G. performed research; G.K., R.K., and L.M.K. contributed new reagents/analytic tools; Finnish type (10). Similarly, nephrin-knockout mice (nephrinKO) R.L.S., P.H., G.K., L.M.K., and E.G. analyzed data; and R.L.S., P.H., and E.G. wrote the paper. die at day 2 after birth of severe proteinuria (11–13). Despite the The authors declare no conflict of interest. structural similarities with Drosophila Sns, nephrin expression or 1R.L.S. and P.H. contributed equally to this work. function in vertebrate skeletal muscle has not yet been reported. 2Present address: PAREXEL, International, 200 West Street, Waltham, MA 02451. In the current study, nephrin was found expressed in developing 3Present address: National Marine Fisheries Service, Auke Bay Laboratories, Ted Stevens mouse skeletal muscle and in human fetal muscle cells under- Marine Research Institute, 17109 Pt Lena Loop Road, Juneau, AK 99801. going fusion. Nephrin expression is also up-regulated in the 4To whom correspondence should be addressed. E-mail: [email protected]. skeletal muscle of two murine models of human muscular This article contains supporting information online at www.pnas.org/cgi/content/full/ dystrophies, when myogenic repair is needed. Downregulation of 0904398106/DCSupplemental.

9274–9279 ͉ PNAS ͉ June 9, 2009 ͉ vol. 106 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904398106 Downloaded by guest on September 30, 2021 Y A O Y O A B C t M Mdx Ctx K E15.5 Mdx Scg Scg W Ctx Wt Wt Ctx -RT 398bp

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Fig. 2. Nephrin morpholino experiments in zebrafish reveal smaller muscles. (A) Light micrograph of variable phenotypes noted with nephrin morpholino in zebrafish embryos at 4 days post fertilization (dpf). Many of the embryos

Nephrin fold change appear curved and shorter (red arrows) compared with control (black arrow). nephrin GAPDH (B) Light micrograph of wild-type (top) mismatched (middle) and nephrin morpholino embryos (MO1) at 4 dpf. (C) RT-PCR analysis of mismatched and Fig. 1. Nephrin mRNA is present in low quantities in vertebrate skeletal nephrin morpholino-injected embryos confirms the expected splicing defect muscle. Nested RT-PCR was performed on RNA isolated from the skeletal leading to an in-frame deletion of the transmembrane domain, rendering a muscles of different mice. The expected PCR products (1.2 kb) were gel protein no longer able to anchor at the membrane; this is manifest by a purified, sequenced and confirmed to be nephrin. (A) RT-PCR product result- decrease in RT-PCR product size (from 398 bp to 272 bp). Lane 1, molecular ing from nested PCR, 1.2 kb (arrow). (B) Neph1, a protein associated with weight markers; lane 2, uninjected wild-type embryo; lane 3, 1.25 ng nephrin nephrin, is present throughout (expected product is 546 bp). Lanes: M, 1-kb morpholino MO1; lane 4, 1.25 ng mismatched morpholino; lane 5, 2.5 ng markers; 1, embryonic skeletal muscle, E15.5; 2, mdx skeletal muscle, 3 weeks nephrin morpholino MO1; lane 6, 2.5 ng mismatched morpholino. (D-I) Mor- (Mdx Y); 3, mdx skeletal muscle 4 months old (Mdx O); 4, ␦-sarcoglycan null 3.5 phometric analyses performed on whole-mount embryos stained with anti weeks (Scg Y); 5, ␦-sarcoglycan null 1 year (Scg O); 6, 8, and 10, WT gastroc- ␤-dystroglycan antibody confirm that the myosepta to myosepta distance of nemius, 3 months; 7, 9, and 11, contralateral WT gastrocnemius 5 days after embryos injected with nephrin morpholino MO1 (F, I) are smaller than those cardiotoxin injection; 12, no RT; 13, kidney. (C) Human fetal myoblasts (17 injected with mismatched morpholino (E, H) or wild-type uninjected embryo CELL BIOLOGY weeks) at the indicated stages of fusion. (D) Transient up-regulation of (D, G). (J) Bar graph of myosepta length measurements from wild-type, nephrin mRNA in nascent myotubes as determined by real-time quantitative mismatched MO1 and nephrin morpholino MO1 injected embryos. Asterisk PCR analysis. The fold change value in the myoblasts was set as 1, and the fold denotes a statistically significant P value by t test (P Յ 0.00000004). Individual change values in nascent and mature myotubes were plotted accordingly. measurements are listed in Table S1. Whole-mount control (K) and nephrin GAPDH was used as a reference in the amplification. Mb: myoblasts, Mt: morpholino (L) embryos stained with anti-laminin (red) and anti-myosin heavy myotubes. (E) Agarose gels of end products of qRT-PCR. Lanes: M, 1kb plus chain antibody (green); nuclei are stained in blue with DAPI. Nephrin mor- markers; 1, myoblasts; 2, nascent myotubes; 3, mature myotubes; 4, negative pholinos displayed shorter myosepta and presence of clustered nuclei (white control. arrows). (M-O) Whole-mount 1 dpf embryos are stained with anti-␤-catenin antibody (red), and nuclei are stained in blue with DAPI. (M) Wild-type control; (N) control mismatched morpholino (MISMO1); (O) nephrin morpholino nephrin expression is exquisitely controlled and is present when (MO1). Open arrows in (M) and (N) point to nuclei that appear regularly myoblast fusion is expected to occur. It was also confirmed that aligned, whereas arrows in (O) point to clustered nuclei. one nephrin-associated protein, Neph1 (), is ubiquitously present in all of the muscles tested, regardless of age, disease, or injury (Fig. 1B). and results in an in-frame deletion that prevents localization of nephrin to the membrane (15). The injected embryos were Downregulation of Nephrin Expression in Developing Zebrafish. Ze- examined at 1 and 4 dpf (days postfertilization). Morpholino brafish nephrin has been localized to the pronephros as in disruption of nephrin expression resulted in shorter, slightly mammalian kidneys (15). Loss of nephrin by morpholino knock- curved embryos that were unable to swim properly and that sank down results in altered morphology and nephrosis at to the bottom of the dish (Fig. 2 A and B). Analysis of 120 96 hpf. Whether the muscles were affected was not indicated. embryos at 1 dpf injected with the nephrin morpholino MO1 Nephrin morpholino experiments were therefore conducted. demonstrated that 55% of the embryos were curved, short, and Two morpholinos, one directed against nephrin (MO1) and a poor swimmers, whereas no embryos displayed this phenotype mismatched morpholino (MIS-MO1), were injected into fertil- when injected with the control MIS-MO1. At 4 dpf, 37% of the ized eggs. The morpholino MO1 (kindly provided by I. Drum- embryos injected with nephrin morpholino MO1 had died, and mond) is directed against the transmembrane domain of nephrin 10% were curved and short. The embryos injected with MIS-

Sohn et al. PNAS ͉ June 9, 2009 ͉ vol. 106 ͉ no. 23 ͉ 9275 Downloaded by guest on September 30, 2021 MO1 appeared to be normal (wild-type) (Fig. 2B). RT-PCR A B analysis confirmed the splicing defect in the embryos injected C with the nephrin morpholino MO1 (Fig. 2C). To study whether WT the abnormal length of MO1-injected embryos was caused by shorter individual myosepta, the distances between myosepta was determined by microscopic analysis on whole-mount em- bryos at 96 hpf stained for alpha-dystroglycan (Fig. 2 D–I). Ten embryos each were analyzed for myosepta length in non-injected D E F control, MIS-MO1, and nephrin MO1-injected embryos. Within KO each embryo, five myosepta were measured starting from the same anatomical position (Fig. 2 D–I,[supporting information (SI) Table S1]. Compared with MIS-MO1–injected embryos, the G HI 90 nephrin MO1-injected zebrafish displayed shorter myoseptum to 0.9 * ** 0.8 * * 0.8 * 80 * WT 0.7 * * 0.7 70 myoseptum distance, consistent with a muscle defect (Fig. 2 J; 0.6 KO

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0.6 + 0.5 Ͻ 0.5 50 P 4E-08 via t test). Comparison using a t test of the myoseptum 0.4 0.4 40 0.3 0.3 30 % Desmin to myoseptum distance in wild-type compared with MO1- Fusion Index>2 Fusion Index>5 0.2 0.2 20 0.1 0.1 10 Ͻ 0 injected embryos was also significant (P 2.08E-08), whereas no 0 0 significant difference was observed by comparing wild-type and Day1 Day2 Day3 Day4 Day1 Day2 Day3 Day4 Day1 Day2 Day3 Day4 MIS-MO1–injected embryos (P ϭ 0.75). Finally, whole-mount Fig. 3. Myoblasts isolated from nephrinKO neonatal mice are unable to form control, MIS-MO1–injected, or MO1-injected zebrafish em- mature myotubes. Light micrographs of wild-type (A, B) and knockout (D, E) bryos were stained at 1dpf using antibodies directed to laminin myocytes after 1 day (A, D) and 4 days (B, E) in differentiation medium. and myosin heavy chain (MHC, F59) (Fig. 2 K and L)orwith Immunofluorescence staining for desmin of wild-type (C) and knockout (F) KO anti-␤-catenin (Fig. 2 M–O). Wild-type (Fig. 2 K and M) and cultures demonstrates that the mononuclear cells in the nephrin cultures are myogenic (white arrows). (G, H) Quantification of the fusion indices for MIS-MO1-injected embryos (Fig. 2N) displayed regularly control and nephrinKO myoblasts. The fusion index (FI) is determined by aligned nuclei (Fig. 2 M and N open arrowheads) and compacted dividing the number of nuclei found within myotubes by the total number of myofibers (Fig. 2K). In contrast, zebrafish embryos injected with nuclei in a microscopic field (three to four microscopic fields per sample; three nephin morpholino MO1 exhibited a less organized structure in independent experiments). (G) Overall FI, counting nuclei in myotubes con- several (but not all) myosepta, with compacted clusters of nuclei taining two or more nuclei. (H) FI for myotubes containing five or more nuclei. (Fig. 2L and O, arrows) that were not observed in wild-type or (I) Percentage of desmin-positive mononuclear cells decreases in wild-type as MIS-MO1–injected embryos. Therefore, downregulation of the FI increases, whereas it remains the same in the nephrinKO cultures. nephrin expression in developing zebrafish results in a pheno- Asterisk indicates significant differences between wild-type (lilac) and knock- Ͻ type consistent with muscle defects. out (crimson), P 0.05.

Assessment of Myocyte Fusion in Nephrin-Knockout Myoblast Cul- cultured under proliferation conditions. Cell numbers were tures. To assess whether nephrin plays a role in myoblast fusion counted daily for 4 days and although there was no significant and determine when its expression is necessary, we turned to a difference during the first 2 days, nephrinKO cells subsequently murine model. Methods for propagation and differentiation of grew much faster than wild-type cells (Fig. S1A). Samples from muscle cells are well established for mouse compared with the cultures were immunostained at day 4 for desmin and the zebrafish tissue (16–19). Skeletal myoblasts were isolated from percentage of positive cells was similar in both cultures (Fig. neonate nephrinKO and wild-type mice, as nephrinKO mice die S1B). Cell cycle analyses were also performed using flow cy- within the first 2 days of life because of kidney failure (11). The tometry, and control cultures were found to contain half the fusion ability of nephrinKO and wild-type cultures were assessed number of cells in S phase compared with nephrinKO cultures in vitro. For each culture, equal numbers of mononuclear cells were plated, allowed to differentiate and the fusion indices (FI; (Fig. S1 C and D). number of nuclei in myotubes divided by total number of nuclei) Studies by others have suggested that the p42/44 (Erk1/Erk2) were calculated over 4 successive days. Wild-type and nephrinKO MAPK pathway is necessary for the activation and proliferation myoblasts began to fuse from day 1 (Fig. 3 A and D), but by day of satellite cells upon injury (20, 21). To address whether the decreased fusion ability of nephrinKO myoblasts could be caused 2 clear differences arose: more of the knockout myocytes KO remained mononuclear and the overall myotube size was small by a persistent activation of Erk1/Erk2, wild-type and nephrin (Fig. 3 E and F), whereas myotubes in the wild-type cultures were myoblasts differentiated cultures were analyzed by Western blot branching and twitching (Fig. 3 B and C, twitching not shown). using antibodies directed against total and phospho-p42/44 The nephrinKO myocytes had lower fusion indices (Fig. 3G) and MAPK (Fig. S1 E–G). Whereas the p42/44 MAPK pathway were also inefficient in forming large myotubes (containing five appeared nearly inactive during differentiation in control cul- KO or more nuclei) compared with wild-type myocytes (Fig. 3H). tures, it was persistently active in the nephrin samples Immunohistochemistry with an antibody against desmin, a myo- (Fig. S1E). genic specific marker, demonstrated that the mononuclear cells To address whether the constitutive activation of the MAPK in the knockout cultures were indeed myogenic, even if they were pathway could result in slowed myogenic differentiation and, not fusing (Fig. 3F, arrows). Moreover, the number of desmin- consequently, fusion of nephrinKO myocytes, wild-type and positive mononuclear cells was significantly higher in knockout nephrinKO myoblast cultures were differentiated over the course cultures compared with wild-type cultures at days 2, 3 and 4 in of 4 days, and myogenin expression was monitored (Fig. S2). It differentiation medium (Fig. 3I). These findings raised the was found that myogenin expression was similar in wild-type and question as to whether nephrinKO mononuclear cells had im- nephrinKO cultures, suggesting that early myogenic differentia- paired differentiation potential because of persistent prolifera- tion is not altered by the persistent activation of MAPK/ERK in tive activity. nephrinKO myoblasts. Next, the MAPK inhibitor PD98059 was applied during myoblast differentiation. Treatment of nephrinKO NephrinKO Myocytes Have Constitutively Activated MAPK/ERK Path- cells with 50 ␮M PD98059 markedly blocked the phosphoryla- way, but This Does Not Seem to Affect Myogenin Expression. Wild- tion of ERK1/2; however nephrinKO myotubes treated with the type and knockout myoblasts were plated at equal numbers and inhibitor remained small (Fig. S3), suggesting that the persistent

9276 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904398106 Sohn et al. Downloaded by guest on September 30, 2021 KO A Mouse WT or KO myoblasts Human myoblasts G, and J). In contrast, nephrin myoblasts initially formed hybrid myotubes with human cells 1 day after differentiation + (Fig. 4 D, E, and K), but by day 4 the contribution of human- derived cells was more predominant (Fig. 4 H, I, and K). Thus, Allow cells to fuse for 1 and 4 days, stain with anti-human nuclei Ab consistent with our previous findings, nascent myotube forma- tion is not affected by nephrin depletion, whereas lack of nephrin expression results in little additional contribution of murine WT KO WT knockout myoblasts to hybrid myotubes. 0.8 B D J KO To address whether nephrin expression is necessary in nascent 0.7 0.6 myotubes to allow fusion of myoblasts into them, wild-type or KO 0.5 nephrin nascent myotubes were co-cultured with human 0.4 A 0.3 mononuclear cells (Fig. 5 ). Both co-cultures gave rise to hybrid

Fusion index 0.2 myotubes (Fig. 5 B–E), demonstrating efficient cell fusion 0.1 between murine and human myocytes. The number of total C E 0 Day1 Day4 nuclei in the myotubes, regardless of their origin (murine or human), revealed no significant difference between wild-type and knockout co-cultures (Fig. 5F). However, there were more nuclei of human origin in knockout hybrid myotubes than in wild-type myotubes (37.4% vs. 17.5%, Fig. 5G). Thus, wild-type FHK myotubes recruit both murine and human myoblasts, while nephrinKO myotubes recruit primarily human myoblasts. The WT 0.9 * 0.8 KO converse experiment was also performed to confirm whether 0.7 nephrin must be present on myoblasts to complete fusion (Fig. 0.6 KO 0.5 5H). Here, wild-type (Fig. 5 I and J) or nephrin myoblasts (Fig. 0.4 5 K and L) were co-cultured with human nascent myotubes 0.3 hybrid myotubes

GI % Human nuclei in 0.2 resulting in chimeric myotubes of similar size in both co-cultures 0.1 0 (Fig. 5M). However, knockout myoblasts fused with human Day1 Day4 myotubes less efficiently. Only 39% of nuclei in knockout chimeric myotubes were from murine myotubes compared with 56% in wild-type chimeric myotubes (Fig. 5N), and these per- Ͻ Fig. 4. NephrinKO myoblasts fuse poorly to human myoblasts compared with centages were statistically significantly different by t test (P control mouse myoblasts. (A) Wild-type or nephrinKO myoblasts were labeled 0.01). These findings support the conclusion that absence of with a green fluorescent dye and mixed with human myoblasts for 1 and 4 days nephrin in myoblasts results in inefficient formation of second- in DM. Myotubes were then fixed and stained with anti-human nuclei anti- ary (mature) myotubes. body (red). Nuclei were stained with DAPI. The myotubes with dual labeling were analyzed for contribution of human and mouse nuclei. Representative Discussion KO images showing the fusion of wild-type (B, C, F, G) or nephrin (D, E, H, I) Nephrin is a cell surface protein expressed in the kidney myoblasts to human myoblasts after coculture for 1 day (B-E) and 4 days (F-I). glomerulus at the epithelial podocytes. Podocytes interdigitate The fusion index (number of fused nuclei, irrespective of their species/total number nuclei) (J), was similar at both days 1 and 4. The contribution of human with one another to form a filtration barrier that allows water versus mouse-derived nuclei to the myotubes was also assessed (K). At day 1, and ionic salts, but not proteins, to leave the bloodstream (9). there was no statistical difference between the number of wild-type or Although nephrin function in the kidney is still largely unknown, nephrinKO mouse myoblasts that had fused to human myoblasts; however, at it seems to be involved in the ‘‘outside-in’’ signaling that main- day 4, many more human-derived nuclei were detected within the myotubes tains the communication between podocyte foot processes and CELL BIOLOGY of the nephrinKO:human myoblast hybrid cultures compared with wild-type sustains the podocyte barrier function (24, 25). Despite struc- ϭ mouse myoblasts:human myoblasts (P 8.68204 E-11 by t test). White arrows tural similarities to Drosophila Sns, a cell-surface protein ex- in (B) through (I) point to nuclei of murine origin. pressed by fusion-competent myoblasts, nephrin has been re- ported as ‘‘absent’’ in skeletal muscle (26). The results reported activation of the MAPK pathway is likely not associated with the here indicate that process of myoblast fusion in Drosophila may fusion defect observed in nephrinKO myoblast cultures. be replicated by homologous players in mammalian cells. Nephrin is expressed in skeletal muscle when cell fusion is Nephrin Must Be Present on Myoblasts for Secondary Fusion to Occur. occurring, during development and during injury or disease that Studies in Drosophila suggest that Sns must be present in require myofiber regeneration. Overall, the level of nephrin fusion-competent myoblasts (FCM), but not in nascent myo- expression is low, and it is present in a narrow window of time. tubes, for myoblast fusion to occur. Our initial studies did not Nephrin is not detected in wild-type adult muscle, whereas it is clarify whether nephrin expression is required on mononuclear present in murine embryonic muscle and in the muscle of young, cells, nascent myotubes, or both, for myotubes to form. To but not old, mice with muscular dystrophy, which undergo a address this question, coculture experiments were performed by spontaneous phase of myofiber regeneration at Ϸ2–3 weeks of mixing murine myoblasts (wild-type or nephrinKO) with human age (14). Myofiber regeneration requires fusion of mononuclear myoblasts. First, murine wild-type and nephrinKO myoblasts cells into nascent myofibers, supporting the need for nephrin labeled with the green fluorescent dye CellTracker CMFDA (22, expression. Consistently, young patients (Ϸ1 year of age) af- 23) were co-cultured with human myoblasts. Fusion was allowed fected by Duchenne Muscular Dystrophy (DMD), express higher to occur for 1 and 4 days, after which the presence of mouse- and levels of nephrin mRNA in their muscle than unaffected indi- human-derived nuclei to the formed myotubes was quantified viduals or older DMD patients (27, 28). With age, both mice and (diagrammed in Fig. 4A and Fig. S4). Coculture of wild-type humans with muscular dystrophy exhibit impaired muscle re- mouse myoblasts with human myoblasts resulted in hybrid generation capacity accompanied by increased fibrosis, partly myotubes containing a similar proportion of mouse and human- because of exhaustion of resident satellite cells (29–31). There- derived nuclei after 1 day (Fig. 4 B and C) and day 4 (Fig. 4 F, fore, a decline of nephrin expression in aged mice is consistent

Sohn et al. PNAS ͉ June 9, 2009 ͉ vol. 106 ͉ no. 23 ͉ 9277 Downloaded by guest on September 30, 2021 A Mouse WT or KO myotubes Human myoblasts H Mouse WT or KO myoblasts Human myotubes

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FG22 MN 20 14 mouse 70 * human 18 90 mouse 12 60 16 80 human * 10 14 70 50 12 8 60 40 10 50 6 8 40 30 6 30 4 20 Nuclei / myotube Nuclei / myotube 4 20 nuclei in myotubes % Species-specific nuclei in myotubes % Species-specific 2 2 10 10 0 0 0 0 WT KO WT KO WT KO WT KO

Fig. 5. Human fetal myoblasts are able to fuse into nephrinKO nascent myotubes, whereas nephrinKO myoblasts fuse less efficiently into nascent human myotubes as compared with wild-type murine myoblasts. (A) Schematic: wild-type or nephrinKO nascent myotubes were labeled with a green fluorescent dye and mixed with human mononuclear myoblasts for 24 hours in DM. Myotubes were then fixed and stained with anti-human nuclei antibody. Myotubes with dual labeling were analyzed for contribution of human and mouse nuclei. (B-E) Representative confocal images showing fusion of human myoblasts (red nuclei, white arrows) with wild-type (B, C) or nephrinKO (D, E) mouse myotubes (green). Nuclei were stained with DAPI (blue). (F) The total number of nuclei in myotubes, regardless of their origin (mouse or human), does not differ in wild-type and knockout myotubes co-cultures. (G) The percentage of human nuclei in myotubes with dual labels was calculated and was found to be significantly increased when human myoblasts were mixed with knockout nascent myotubes compared with wild-type nascent myotubes. *P Ͻ 0.01. (H) Schematic representation of mixing of prelabeled mouse myoblasts (wild-type or knockout) with human nascent myotubes. (I-L) Representative confocal images showing the fusion of murine myoblasts (green, white arrowheads) with human myotubes (red nuclei). Nuclei were stained with DAPI (blue). Coculture of wild-type (I, J) or nephrinKO (K, L) murine myoblasts with human nascent myotubes. The total number of nuclei in myotubes, regardless of their origin, does not differ in the co-cultures (M); however, the percentage of mouse nuclei in myotubes was significantly decreased when knockout myoblasts were mixed with human myotubes compared with wild-type myoblasts (N). *P Ͻ 0.01 via t test.

with the hypothesis of a decline in the number of ‘fusion- mononucleated myoblasts and not in the nascent myotubes. competent‘‘ myoblasts in the chronic disease state. Conversely, human myoblasts fuse efficiently to nephrinKO nas- Lack of nephrin in myogenic cells unveils a defect in recruit- cent myotubes, suggesting that nephrin expression on nascent ment of fusion-competent myoblasts to nascent myotubes. Myo- myotubes is not necessary for wild-type myoblasts to fuse. blasts from nephrinKO mice are unable to form large myotubes Nephrin may act in concert with other transmembrane pro- and to maintain a constitutively active MAPK/ERK1/2 pathway, teins with similar motifs likely to be important for vertebrate even when in differentiation medium. However, the activated muscle fusion. For example, CDO and BOC are members of the MAPK/ERK1/2 pathway that has been associated with the Ig superfamily and have been found to regulate fusion (32–34). activation of satellite cells (20, 21) does not seem to interfere Mannose receptors have been shown to regulate myogenic with early differentiation of nephrinKO myoblasts or to be the motility preceding myoblast fusion (35). Whether these or other cause of inefficient myoblast fusion. In support of this, it was membrane-associated molecules interact with nephrin to result found that expression of myogenin did not differ in nephrinKO in myoblast fusion is currently unknown. cultures compared with control, and that pharmacological inhi- Nephrin in the vertebrate kidney is part of a complex network bition of MAPK/ERK pathway failed to rescue normal myotube of proteins that are necessary for the structural and signaling formation in nephrinKO cultures. Our results suggest that nephrin integrity of the podocyte slit diaphragm (9). Future work must may have a role in vertebrate myoblasts similar to Drosophila Sns. be done to determine other structural partners for nephrin in For secondary myotubes to form, nephrin must be present in the skeletal muscle and factors that modulate the expression of

9278 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904398106 Sohn et al. Downloaded by guest on September 30, 2021 nephrin. Studies suggest that nephrin expression can be in- Western blot analyses of ERK1/2 proteins can be found in SI Materials and creased in the kidney by PPAR-␥ agonists, such as pioglitazone, Methods. a drug for diabetes (36). Perhaps similar treatment with myo- blasts can also increase nephrin expression and thereby increase Co-Culturing Experiments of Murine and Human Myoblasts. Murine myoblasts were stained with 5 ␮M CellTracker green CMFDA (Molecular Probes). Labeled myoblast fusion. Thus, with more knowledge of the regulation of mouse and human myoblasts were plated together in equal numbers and nephrin in the skeletal muscle, it may become possible to allowed to differentiate for 1 and 4 days in differentiation medium. Cells were improve the efficiency of cell-based therapies for skeletal muscle then stained with anti-human nuclei antibody (Millipore, clone 235–1, MAB diseases such as Duchenne Muscular Dystrophy. 1281). Additional information can be found in SI Materials and Methods.

Materials and Methods Co-Cultures of Myoblasts with Nascent Myotubes. To form nascent myotubes, Ϸ RNA Isolation and RT-PCR Amplification of Nephrin mRNA. Total RNA was mouse or human myoblasts at 70% confluency were switched to differen- isolated from primary myocyte cultures using Qiagen RNeasy kit (Qiagen) and tiation medium (DM) for 24 hours. Meanwhile, 20–30% confluent myoblasts from gastrocnemius muscles of C57Bl6, mdx5cv, delta-sarcoglycan null mice as were cultured in DM for 24 hours to produce differentiated, mononucleated cells. After 24 hours, murine myoblasts or myotubes were labeled with 5 ␮M previously described (37). Detailed methods, including cardiotoxin injury, CellTracker green CMFDA. Murine and human cells were trypsinized, plated in primer sequences, and RT/PCR amplification conditions can be found in the SI 12-well plates at equal cell numbers, and co-cultured for another 24 hours. Materials and Methods. Cells were then fixed, permeabilized, and stained with anti-human nuclei antibody (Millipore) as detailed in SI Materials and Methods. Zebrafish Morpholino Experiments. Zebrafish experiments were performed using previously described procedures (38), with 1.25 and 2.5 ng of nephrin ACKNOWLEDGMENTS. The authors thank Ian Drummond for the nephrin morpholino or mismatched morpholino oligonucleotides. Morpholino se- morpholino (MO1). The authors also thank the MRDDRC flow cytometry quences, RT-PCR, and whole-mount immunostaining analyses are detailed in facility, the MRDDRC sequencing facility and MDDRC Imaging Core for con- SI Materials and Methods. focal microscopy, all supported by a grant from the National Institute of Health (5P30HD018655). The authors express their gratitude to the members of the Gussoni and Kunkel laboratories for helpful discussions and critical Primary Muscle Cell Culture and Fusion Experiments. Primary muscle cultures review of this manuscript. This work was supported by funding from the KO were derived from the limb muscles of 1–2-day-old wild-type and nephrin Muscular Dystrophy Association to E.G. (MDA 3589 and MDA 4146), the neonates as described (18). Differentiation experiments were performed by National Institutes of Health (RO1NS047727 to E.G. and 5P50NS040828 to plating the myocytes at a density of 8 ϫ 104 cells/well in 12-well plates. L.M.K.). L.M.K. is an Investigator supported by the Howard Hughes Medical Detailed information on culture conditions and determination of the fusion Institute (HHMI). R.L.S. was supported by a Mentored Clinical Scientist Devel- indices is described in SI Materials and Methods. opment Award from the National Heart, Lung, and Blood Institute (NIH- K08HLL04216). P.H. is supported by a Scientist Development Grant from American Heart Association (0730285N) and by the Joshua Frase Foundation. Cell Cycle Analysis. Flow-cytometry analyses of the cell cycle were performed R.K. is supported by the NIH grant DK 55001. The authors have no conflicts of according to a previously described protocol (39). Additional details including interest to declare.

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