Calcium Influx and Release Cooperatively Regulate AChR Patterning and Motor Axon Outgrowth during Neuromuscular Junction Formation

Article

Calcium Influx and Release Cooperatively Regulate AChR Patterning and Motor Axon Outgrowth during Neuromuscular Junction Formation Graphical Abstract

Authors Mehmet Mahsum Kaplan, Nasreen Sultana, Ariane Benedetti, ..., Anamika Dayal, Manfred Grabner, Bernhard E. Flucher

Correspondence [email protected]

In Brief Motor neurons innervate skeletal muscles at a single synapse in the center of each fiber. Kaplan et al. use combinations of mouse models in which influx and release of calcium in developing skeletal muscle are abolished or enhanced to demonstrate that these calcium signals control where neuromuscular junctions are formed.

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CaV1.1-mediated Ca2+ signals are critical for central patterning of NMJ Either L-type Ca2+ currents or SR Ca2+ release is sufficient for NMJ patterning Ca2+ signal loss causes aberrant MuSK expression, AChR clustering, and nerve branching Without EC coupling, accuracy of NMJ patterning correlates with size of LTCCs

Kaplan et al., 2018, Cell Reports 23, 3891–3904 June 26, 2018 ª 2018 The Authors. https://doi.org/10.1016/j.celrep.2018.05.085

Cell Reports

Article Calcium Influx and Release Cooperatively Regulate AChR Patterning and Motor Axon Outgrowth during Neuromuscular Junction Formation Mehmet Mahsum Kaplan,1 Nasreen Sultana,1 Ariane Benedetti,1 Gerald J. Obermair,1 Nina F. Linde,2 Symeon Papadopoulos,2 Anamika Dayal,3 Manfred Grabner,3 and Bernhard E. Flucher1,4,* 1Department

of Physiology and Medical Physics, Medical University Innsbruck, 6020 Innsbruck, Austria of Physiology and Pathophysiology, Institute of Vegetative Physiology, University of Cologne, 50931 Cologne, Germany 3Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University Innsbruck, 6020 Innsbruck, Austria 4Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2018.05.085 2Center

SUMMARY

Formation of synapses between motor neurons and muscles is initiated by clustering of acetylcholine receptors (AChRs) in the center of muscle fibers prior to nerve arrival. This AChR patterning is considered to be critically dependent on calcium influx through L-type channels (CaV1.1). Using a genetic approach in mice, we demonstrate here that either the L-type calcium currents (LTCCs) or sarcoplasmic reticulum (SR) calcium release is necessary and sufficient to regulate AChR clustering at the onset of neuromuscular junction (NMJ) development. The combined lack of both calcium signals results in loss of AChR patterning and excessive nerve branching. In the absence of SR calcium release, the severity of synapse formation defects inversely correlates with the magnitude of LTCCs. These findings highlight the importance of activity-dependent calcium signaling in early neuromuscular junction formation and indicate that both LTCC and SR calcium release individually support proper innervation of muscle by regulating AChR patterning and motor axon outgrowth. INTRODUCTION To establish functional synaptic connections, outgrowing neuronal processes are guided by environmental cues to their specific targets. During the formation of neuromuscular junctions (NMJs) in zebrafish, the incoming motor nerve is directed to the center of the postsynaptic muscle fibers containing preformed acetylcholine receptor (AChR) clusters (Flanagan-Steet et al., 2005; Panzer et al., 2006). Expression and clustering of AChRs in a central endplate band are regulated by muscleintrinsic processes prior to the arrival of the nerve as was first shown in mouse models lacking motor neurons or the neuronderived synaptogenic factor agrin (Yang et al., 2000, 2001; Lin et al., 2001, 2008). Although the innervating nerves are capable of inducing and stabilizing postsynaptic AChR clusters apart

from pre-formed aneural AChR clusters (Lin et al., 2001, 2008; Flanagan-Steet et al., 2005), failure of AChR pre-patterning results in abnormal growth and branching of the motor nerves and in multiple innervation of the muscle fibers (Chen et al., 2011; Pacifici et al., 2011; Vock et al., 2008). Notwithstanding their crucial role in NMJ formation, the cellular mechanisms controlling AChR patterning are still incompletely understood (Burden et al., 2018). Current models assume an interplay of AChR-cluster-promoting activity in the center and of AChR-cluster-dispersing activity in the periphery of the muscle fiber (Kummer et al., 2006). The muscle-intrinsic mechanisms promoting AChR patterning involve muscle-specific kinase (MuSK) signaling and the local transcription of AChR and MuSK in the central band of the muscle fibers (Kim and Burden, 2008). ACh and muscle activity have AChR-cluster-dispersing activity and suppress AChR and MuSK expression outside the endplate band (Misgeld et al., 2005; Lin et al., 2005). In this mechanism, a central role of calcium signaling—and more specifically of calcium influx through L-type calcium channels— has been proposed (Chen et al., 2011; Li et al., 2018). However, the nature and exact function of the calcium signal in the initial steps of NMJ formation are still elusive. In skeletal muscle, activity-dependent calcium signaling is highly specialized to accomplish the efficient control of contraction. The excitation-contraction (EC) coupling mechanism involves the tight interaction of two calcium channels. The voltage-gated calcium channel CaV1.1 (also called dihydropyridine receptor [DHPR]) functions as voltage sensor for EC coupling (Rı´os and Pizarro, 1991). It is located in the triads— junctions between the sarcoplasmic reticulum (SR) and t-tubules—where CaV1.1 interacts with the SR calcium release channel (type 1 ryanodine receptor [RyR1]; Block et al., 1988; Franzini-Armstrong et al., 1999; Flucher et al., 1990). Upon membrane depolarization, conformational changes of CaV1.1 directly translate into the activation of the RyR1, thus triggering depolarization-induced calcium release from the SR (Melzer et al., 1995). Remarkably, in mature skeletal muscle, L-type calcium currents (LTCCs) through the CaV1.1a channel variant are small and not essential for EC coupling. However, in developing muscles, exclusion of an alternatively spliced exon gives rise to a channel splice variant (CaV1.1e) that supports sizeable LTCCs as well as depolarization-induced SR calcium release (Tuluc et al., 2009;

Cell Reports 23, 3891–3904, June 26, 2018 ª 2018 The Authors. 3891 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Benedetti et al., 2015). These currents are still not essential for EC coupling but may play important roles in the regulation of developmental and adaptive processes in skeletal muscles (Flucher and Tuluc, 2011; Sultana et al., 2016). Consistent with this notion, Chen et al. (2011) proposed that calcium influx through CaV1.1 is required for the regulation of AChR patterning independently of CaV1.1’s function in EC coupling. Here, we tested this hypothesis directly in a recently published knockin mouse model expressing a non-conducting CaV1.1 (DHPRnc/nc; Dayal et al., 2017) and further examined the importance of calcium influx and release for AChR patterning and motor axon outgrowth using a range of genetic mouse models lacking specific components of the skeletal muscle calcium signaling machinery. Whereas mice lacking either LTCCs or SR calcium release showed normal early NMJ development, AChR patterning failed and motor neuron branching was excessive in double mutants lacking both calcium sources. Moreover, in the absence of RyR1, the severity of the AChR patterning defects inversely correlated with the magnitude of LTCCs in double mutants with different compositions of CaV1.1 variants. Together, these results demonstrate that (1) activity-dependent calcium signaling in developing skeletal muscle is critical for regulation of AChR patterning and normal innervation, (2) either one of the calcium sources—influx through CaV1.1 or release through RyR1—is sufficient to accomplish this initial step in NMJ development, and (3) that this regulatory mechanism can function independently of skeletal muscle EC coupling. Our results suggest that activity-dependent calcium signals act in repressing expression and clustering of MuSK and AChR in the periphery of developing muscle fibers and thus induce correct AChR patterning and innervation in the muscle center. RESULTS The Lack of CaV1.1, but Not of RyR1, Results in Failed AChR Patterning and Excessive Motor Nerve Branching in E14.5 Mouse Diaphragm At the onset of NMJ formation in mouse diaphragm muscle, AChRs are expressed and cluster exclusively in a central band perpendicular to the orientation of the muscle fibers, and the branches of the ingrowing phrenic nerve are restricted to this region (Figures S1A–S1C). At embryonic day 14½ (E14.5) about half of the AChR clusters have been contacted by the nerve (neural AChR clusters), whereas the other half is not yet colocalized with nerve terminals (aneural AChR clusters; Figures S1D and

S1E). We used Alexa-488-conjugated bungarotoxin (BTX) and anti-synapsin to label AChRs and the phrenic nerve branches, respectively, in whole-mount preparations of E14.5 mouse diaphragm. Images and analysis of AChR patterns are from the dorsal quadrant of the left hemi-diaphragm (Figure S1A), whereas the entire corresponding right hemi-diaphragms are presented to display the overall nerve-branching patterns (Figures 1, 2, and 3). In embryonic skeletal muscle, the CaV1.1 a1 subunit functions as voltage sensor for EC coupling and as L-type calcium channel (Melzer et al., 1995; Tuluc et al., 2009). Knockout of the auxiliary b1a channel subunit results in perturbed CaV1.1 expression and loss of EC coupling and recently has been demonstrated to cause failure of AChR pre-patterning. This NMJ defect occurred also in aneural diaphragm of Cacnb1 / /Hb9 / double mutants and in Cacnb1 / mice could be rescued by a muscle-specific Cacnb1 transgene, indicative of a muscle-intrinsic mechanism that is independent of the motor neuron (Chen et al., 2011). Consistent with this NMJ phenotype, early descriptions of the phenotype of dysgenic (CaV1.1-null) mice reported extensive nerve outgrowth and abnormal distribution of AChRs (Rieger et al., 1984; Powell et al., 1984). In order to explore the specific role of the pore-forming CaV1.1 a1 subunit in the early NMJ development, we therefore analyzed AChR patterning and motor neuron growth pattern using double-fluorescence staining of diaphragms from E14.5 dysgenic mice. Compared to wildtype control littermates, the width of the endplate band was significantly widened in dysgenic diaphragm (Figures 1A–1D). This was accompanied by excessive branching and a wider distribution of the phrenic nerve. Not only were the AChR clusters distributed wider along myofibers, but also the number of AChR clusters per fiber was increased in CaV1.1 / mice (Figures 1E and 1F). Together, these data corroborate previous findings in different DHPR mutant mice and demonstrate that CaV1.1 is essential for normal patterning of nascent AChR clusters as well as for the subsequent innervation of diaphragm muscle. Although both CaV1.1 and RyR1 are essential components of the skeletal muscle EC coupling apparatus, the NMJ phenotype of RyR-deficient mice differs from that of CaV1.1 / mice. Actually, published results are contradictory: in a RyR1/RyR3 doubleknockout mouse (Barone et al., 1998), the AChR band was shown to be widened, although not to the same extent as in CaV1.1 b1a-null mice (Chen et al., 2011). In contrast, in a single RyR1 knockout mouse (Buck et al., 1997) and in a non-functional

Figure 1. Dysgenic CaV1.1-Null Mice, but Not Dyspedic RyR1 Knockout Mice, Show Defects in AChR Patterning and Motor Nerve Branching (A and G) Representative segments from dorsal left quadrant of diaphragms from wild-type and CaV1.1 / (A) or RyR1 / (G) mice at E14.5 stained with Alexa488-a-BTX (green) and anti-synapsin (red). (B and H) Motor nerve branches in right half-diaphragms labeled with anti-synapsin. (A, G, B, and H) In wild-type AChRs, clusters form a narrow endplate band and motor nerve branches are restricted to the center of the muscle fibers. In CaV1.1 / diaphragms, AChR clusters are distributed in a wider region and the phrenic nerve is excessively branched over the entire width of the diaphragm, whereas in RyR1 / diaphragms, AChR patterning and nerve branching are not different from wild-type controls. The scale bars represent 200 mm (A and G) and 400 mm (B and H). (C, D, I, and J) Quantification of AChR cluster distribution (C and I) and motor axon extension (D and J) along the length of the muscle fibers with representative images aligned below the graphs. (E, F, K, and L) Mean endplate band width (E and K) and mean number of AChR clusters per myofiber (F and L) of wild-type (black) and CaV1.1 / or RyR1 / mice (red) at E14.5. N R 6 diaphragms from at least 4 litters for CaV1.1 / , N = 3 from 3 litters for RyR1 / ; mean ± SEM; t test, ***p < 0.001, p = 0.962 for (K), and p = 0.909 for (L).

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Figure 2. L-type Calcium Currents through DHPRs Are Not Required for Proper AChR Patterning and Motor Nerve Branching (A) Representative fluorescence micrographs of E14.5 diaphragm segments from non-conducting DHPRnc/nc mice and their wild-type littermates double-labeled with a-BTX (green) and anti-synapsin (red). (B) Motor nerve branches in right half-diaphragms labeled with anti-synapsin. (A and B) Patterning of AChR clusters and branching of the motor nerve in DHPRnc/nc mice lacking L-type calcium currents are indistinguishable from wild-type controls. The scale bars represent 200 mm (A) and 400 mm (B). (C and D) Quantification of (C) AChR cluster distribution and (D) motor axon extension along the length of the muscle fibers with representative images aligned below the graphs. (E and F) Mean endplate band width (E) and mean number of AChR clusters per myofiber (F) of wild-type (black) and DHPRnc/nc mice (red) at E14.5. N = 4 diaphragms from 4 litters for each genotype; mean ± SEM; t test, difference not significant, p = 0.884 for (E) and p = 0.302 for (F).

RyR1 mutant, AChR clustering was reported to be reduced and the AChR band was narrower than in normal controls (Hanson and Niswander, 2014; Gartz Hanson and Niswander, 2015). To resolve this issue and to further investigate whether RyR1 is involved in the regulation of AChR patterning and innervation, we next analyzed AChR patterning and nerve branching in diaphragm of dyspedic RyR1 (Buck et al., 1997) knockout mice. Contrary to CaV1.1 / mice, knockout of RyR1 did not cause failure of AChR patterning or nerve branching in E14.5 diaphragm (Figures 1G and 1H). The band width of the centrally localized AChR clusters was not significantly different from that of wildtype controls (Figures 1I and 1J), and the number of AChR clusters per myofibers was identical in RyR1 / and wild-type littermates (Figures 1K and 1L). Accordingly, at E14.5, the ingrowing nerve branches were restricted to the central endplate band as in wild-type controls. Thus, our analysis supports the conclusion that the RyR1 is not essential for the early patterning of AChRs in the center of muscle fibers.

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The Selective Loss of LTCC Does Not Disrupt AChR Patterning and Muscle Innervation Similar findings in CaV1.1 b1a knockout and RyR1/RyR3 doubleknockout mice led to the conclusion that the channel function of CaV1.1, but not EC coupling, is necessary for the regulation of AChR patterning (Chen et al., 2011). So far, our results obtained in CaV1.1- and RyR1-null mutants are fully consistent with this interpretation. To test this hypothesis directly, we next analyzed NMJ formation in a knockin mouse model expressing a non-conducting CaV1.1. DHPRnc/nc mice carry a mutation in the Cacna1s gene that totally abolishes ion conductivity of CaV1.1 without affecting its function in EC coupling (Dayal et al., 2017). If currents through CaV1.1, but not RyR1 calcium release, are necessary for AChR patterning, it should be disrupted in DHPRnc/nc mice. However, our analysis of E14.5 diaphragm preparations of DHPRnc/nc mice revealed that NMJ formation is indistinguishable from that in their wild-type littermates. Neither the width of the central band of AChR clusters, nor branching of the phrenic

Figure 3. Combined Loss of SR Calcium Release through RyR1 and L-type Calcium Currents through DHPRs Disrupts AChR Patterning and Results in Excessive Motor Nerve Branching (A) AChR clusters and motor axons double labeled with a-BTX (green) and anti-synapsin (red), respectively, in representative diaphragm segments from E14.5 littermates with the following genotypes: RyR1+/ ; DHPR+/nc (control); RyR1 / ; DHPR+/nc; and RyR1 / ; DHPRnc/nc. (B) Motor nerve branches labeled with anti-synapsin in right half-diaphragms of the same genotypes. (A and B) In RyR1+/ ; DHPR+/nc control mice, AChR clusters and motor axons are restricted to the central region of the muscle as in wild-type mice (cf. Figures 1A and 1B). In contrast, RyR1 / ; DHPRnc/nc mice show complete disruption of AChR patterning and hyper-branching of the motor nerve. Notably, RyR1 / ; DHPR+/nc mice display an intermediate phenotype. The scale bars represent 200 mm (A) and 400 mm (B). (C and D) Quantification of (C) AChR cluster distribution and (D) motor axon extension along the length of the muscle fibers with representative images aligned below the graphs. Black, RyR1+/ ; DHPR+/nc (control); blue, RyR1 / ; DHPR+/nc; red, RyR1 / ; DHPRnc/nc. (E and F) Mean endplate band width (E) and mean number of cluster per myofiber at E14.5 (F). N R 3 diaphragms from at least 3 litters for each genotype; mean ± SEM; one-way ANOVA: F(2,8) = 196.1; p < 0.0001 for (E) ANOVA: F(2,6) = 17.72; p < 0.01 for (F); Newman-Keuls post hoc test: *p < 0.05; **p < 0.01; and ***p < 0.001.

nerve (Figures 2A–2D), nor the number of AChR clusters per myofibers (Figures 2E and 2F) were different in DHPRnc/nc mice compared to normal controls. This finding conclusively demon-

strates that LTCCs through CaV1.1 are not essential for normal AChR patterning and innervation. It does, however, not exclude the possibility that LTCCs may contribute to early NMJ formation

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and even may be sufficient for its regulation in the absence of RyR1 calcium release. Alternatively, the participation of CaV1.1 in protein-protein interactions with downstream signaling proteins rather than its channel function might explain its importance in regulating AChR patterning and innervation. Combined Loss of LTCC and RyR1 Disrupts AChR Patterning and Causes Excessive Branching of Motor Neurons To distinguish between these two possibilities and to examine whether either one of the two calcium signals—LTCC or SR calcium release—by itself is sufficient for regulating AChR patterning, we cross-bred DHPRnc/nc and RyR1+/ mice to generate double mutants lacking LTCC and RyR1. If an activity-dependent calcium signaling is required and if, in the individual DHPRnc/nc and RyR1 / mice, the remaining other calcium channel supported AChR patterning, AChR patterning is expected to fail in double-homozygous offspring lacking both calcium sources. Indeed, although in double-heterozygous RyR1+/ ; DHPR+/nc controls AChR patterning and nerve branching were normal, in semi-homozygous RyR1 / ; DHPR+/nc and in double-homozygous RyR1 / ; DHPRnc/nc mice, NMJ development was differentially strongly affected. In double-homozygous RyR1 / ; DHPRnc/nc mice AChR patterning was totally disrupted and branching of motor neurons was excessive (Figures 3A–3E). Also, the number of AChR clusters per muscle fiber was increased (Figure 3F). Because RyR1 / ; DHPRnc/nc mice still express the non-conducting CaV1.1 in the triads (Figure S2), it excludes the possibility that non-channel functions of CaV1.1 regulate the AChR patterning. Instead, together with the normal AChR patterning observed in the DHPRnc/nc and RyR1 / mice, the observed disruption of AChR patterning in the double mutant RyR1 / ; DHPRnc/nc mice indicates that a calcium signal is critical for regulation of this initial step of NMJ formation and that sufficient calcium to support normal AChR patterning and to control synaptic targeting of the motor axons can be supplied either by LTCCs through CaV1.1 or by SR calcium release through RyR1. Notably, the severity of the NMJ phenotype in RyR1 / ; DHPRnc/nc mice was greater than that in dysgenic (CaV1.1 / ) mice (cf. Figure 1), in that AChR clusters in RyR1 / ; DHPRnc/nc mice were scattered along the entire length of the muscle fibers and the nerve branches grew across the entire width of the diaphragm. This indicates a difference between the situation in CaV1.1 / , where RyR1 is expressed but no longer activated in response to depolarization, and the situation in RyR1 / ; DHPRnc/nc mice, where RyR1 is lacking. Possibly, increased leak of calcium through RyR1 in dysgenic myotubes (Eltit et al., 2011) partially offsets the defects caused by the loss of activity-dependent calcium release. Importantly, semi-homozygous E14.5 RyR1 / ; DHPR+/nc mice also displayed an intermediate NMJ phenotype. The central band containing AChR clusters and nerve branches was wider than in unaffected controls but narrower than in CaV1.1 / or RyR1 / ; DHPRnc/nc mice (Figures 3A–3D; cf. Figure 1). Muscles of heterozygous DHPR+/nc mice express LTCCs with half the current density compared to wild-type controls (Dayal et al., 2017). Therefore, in the absence of EC coupling, this reduction

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of LTCCs is sufficient to affect AChR patterning and nerve branching in E14.5 diaphragm of RyR1 / ; DHPR+/nc mice. Thus, regulation of AChR patterning and innervation appears to be highly sensitive to the magnitude of the calcium influx, and in the absence of RyR1, the size of the LTCCs through CaV1.1 is limiting for this function. The Combined Lack of LTCC and SR Calcium Release Causes Peripheral Expression of AChR and MuSK in E14.5 Diaphragm Muscle The crucial role of calcium signals in AChR patterning and innervation can either be mediated by repression of AChR transcription in peripheral myonuclei or by restraining AChR clustering in the peripheral membrane domain. To reveal possible effects on AChR expression, we performed in situ hybridization of AChR a subunit mRNA in the various channel mutants. Whereas at E14.5, expression of aAChR transcripts in wild-type and mutant mice with normal AChR patterning (RyR1 / , DHPRnc/nc, and RyR1+/ ; DHPR+/nc) was restricted to the center of the muscle fibers, in CaV1.1 / and in double mutant RyR1 / ; DHPRnc/nc mice, aAChR transcripts were broadly expressed in the muscle fibers (Figure 4A). This indicates that combined, but not individual, lack of LTCC and SR calcium release disrupts the confined expression of AChR in the center of the muscle fibers. MuSK is the master regulator of AChR expression and clustering in the endplate band. Its expression in the center of muscle fibers is necessary for AChR patterning, and conversely, ectopic expression of MuSK induces clustering of AChRs at these sites (Kim and Burden, 2008). Therefore, we analyzed whether our calcium channel mutants affect the expression and distribution of MuSK in developing diaphragm muscle. qRT-PCR analysis demonstrated that expression of MuSK mRNA is significantly increased in E14.5 diaphragm of CaV1.1 / and RyR1 / ; DHPRnc/nc mice, consistent with an inhibiting role of activitydependent calcium signals on MuSK expression (Figure 4B). Immunofluorescence analysis in E14.5 diaphragm demonstrated that MuSK is colocalized with AChR clusters inside and in the genotypes with disrupted AChR patterning (CaV1.1 / and RyR1 / ; DHPRnc/nc) also outside the central domain. In addition, a continuous MuSK staining was observed in the plasma membrane. In wild-type and non-affected genotypes, this continuous MuSK staining was restricted to the center of the fibers. On the contrary, in diaphragm muscles of CaV1.1 / and RyR1 / ; DHPRnc/nc mice, the continuous MuSK labeling intensity was substantially increased and uniformly present in the center and in the periphery of the fibers (Figures 4C–4F). Thus, MuSK membrane expression in the fiber periphery is negatively regulated by LTCC and SR calcium release independently of its aggregation in nascent AChR clusters. The upregulation of MuSK in peripheral regions of muscles lacking both LTCC and SR calcium release could explain the elevated expression and ectopic clustering of AChR outside the central endplate band. Disrupted Early AChR Patterning in Calcium Channel Mutant Mice Leads to Aberrant Innervation at Late Fetal Stages As innervation proceeds, nascent AChR clustering mechanisms are gradually replaced by agrin-mediated mechanisms for AChR

Figure 4. Lack of Both L-type Calcium Currents and RyR1 Calcium Release Leads to Ectopic Expression of AChR mRNA and Increased Expression and Peripheral Clustering of MuSK Protein (A) In situ hybridization of aAChR mRNA in diaphragms from calcium channels mutant mice at E14.5. In wild-type, RyR1 / , DHPRnc/nc, and RyR1+/ ; DHPR+/nc mice aAChR mRNA expression is restricted to the center of the muscle fibers, whereas in Cav1.1 / and RyR1 / ; DHPRnc/nc mice, aAChR mRNA is detected throughout the whole width of the fibers. Notably, in RyR1 / ; DHPR+/nc mice, expression of aAChR mRNA is wider than RyR1+/ ; DHPR+/nc controls but narrower than CaV1.1 / and RyR1 / ; DHPRnc/nc mice. (B) Expression of MuSK mRNA (relative to the GapDH) is elevated in E14.5 diaphragms of the affected genotypes (CaV1.1 / and RyR1 / ; DHPRnc/nc). N R 4 diaphragms from 4 litters for CaV1.1 / and 2 litters for litters RyR1 / ; DHPRnc/nc; mean ± SEM; t test; **p < 0.01. (C–F) Representative fluorescence micrographs of E14.5 diaphragm segments of wild-type and indicated calcium channel mutants double labeled with Alexa488-aBTX (green) and anti-MuSK (red). The line scans show the fluorescence intensity along the lines (24 mm) drawn perpendicular to the fiber orientation as indicated in the images above. In all genotypes, MuSK is co-aggregated with AChR both in the central endplate band (wild-type and RyR1+/ ; DHPR+/nc) and in the periphery (CaV1.1 / and RyR1 / ; DHPRnc/nc) of the muscle fibers. In addition, continuous sarcolemmal MuSK staining is restricted to the central endplate band in wild-type and RyR1+/ ; DHPR+/nc mice but observed throughout the fibers in CaV1.1 / and RyR1 / ; DHPRnc/nc muscles. The scale bars represent 100 mm (A) and 10 mm (C–F).

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stabilization (Ruegg and Bixby, 1998). To examine whether disrupted AChR patterning and innervation in E14.5 diaphragm of CaV1.1 / and in RyR1 / ; DHPRnc/nc mice are maintained throughout fetal development or become rectified following innervation, we examined AChR distribution, nerve branching, and innervation in mouse diaphragm at E18.5. At this developmental stage, wild-type diaphragm muscles typically are innervated at a single NMJ in the center of each muscle fiber. Aneural AChR clusters are absent (cf. Figure S1E) and nerve branches are restricted to a narrow central endplate band (Figure 5A). This normal phenotype was also observed in double-heterozygous RyR1+/ ; DHPR+/nc controls (Figure 5B). In contrast, E18.5 diaphragm of CaV1.1 / and RyR1 / ; DHPRnc/nc mice showed severely disrupted AChR patterning and innervation (Figures 5A and 5B). Furthermore, in both genotypes, the muscle fibers were multiply innervated with up to eight NMJs (Figures 5C–5F). Thus, calcium-channel-mediated defects of AChR patterning first observed at the onset of NMJ development progress into severely distorted innervation patterns late in fetal development. Because the affected mouse genotypes die of respiratory failure at birth, the postnatal pathology of these innervation defects could not be assessed in these mouse models. Increased Expression of the Highly Calcium-Conducting CaV1.1e Isoform Rescues Aberrant AChR Patterning and Nerve Outgrowth in E18.5 RyR1 / Diaphragm Consistent with previous results (Chen et al., 2011), RyR1 / mice, which showed no AChR patterning defects at E14.5, developed innervation defects late in fetal development. At E18.5, RyR1 / mice expressed ectopic AChR clusters in the periphery of the muscle fibers in addition to the central band of AChR clusters (Figure 6A). These ectopic AChR clusters were innervated by excessively growing motor neurons, but their BTX-labeling intensity was markedly weaker than that of the centrally localized AChR clusters. This developmentally delayed NMJ defect in RyR1 / mice indicates that, although LTCCs are sufficient for the normal regulation of AChR patterning at E14.5, this is no longer the case at E18.5. Consistent with this notion, qRT-PCR analysis showed that, during fetal development, expression of the highly conducting embryonic CaV1.1e splice variant declined relative to that of the weakly conducting adult CaV1.1a splice variant from 69.5% ± 1.6% at E14.5 to 20.3% ± 2.6% at E18.5 in wildtype and from 68.8% ± 5.7% at E14.5 to 33.8% ± 1.4% at E18.5 in RyR1 / diaphragm muscle (N numbers: E14.5, wildtype [WT] = 8, RyR1 / = 3; E18.5: WT = 4, RyR1 / = 4; Chi2: p < 0.001 between E14.5 and E18.5 for wild-type and for RyR1 / mice). If the resulting developmental decline of LTCCs is responsible for the late-onset AChR patterning and innervation defects, this phenotype should be rescued by interfering with the developmentally regulated alternative splicing of CaV1.1 exon 29. To test this, we crossed RyR1 / mice with a knockin mouse model in which Cacna1s exon 29 had been deleted (CaV1.1DE29/DE29), and thus, only the highly conducting embryonic CaV1.1e variant is expressed (Sultana et al., 2016). As hypothesized, in homozygous RyR1 / ; CaV1.1DE29/DE29 mice, the RyR1 / phenotype was largely rescued in that AChR patterning in the center of the fibers was essentially

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restored and excessive nerve growth was significantly reduced (Figures 6A–6C). Interestingly, semi-homozygous RyR1 / ; CaV1.1+/DE29 mice revealed an intermediate phenotype, indicating that the extent of rescue corresponds to the magnitude of LTCCs expected in the different genotypes (Sultana et al., 2016). This strongly supports the strict dependence of AChR patterning and innervation on muscle calcium signaling and implicates the highly conducting embryonic CaV1.1e splice variant in this process. DISCUSSION The central finding of this study is that the proper patterning of nascent AChR clusters in the center of developing muscle fibers and the subsequent innervation by the motor nerve critically depend on muscle calcium signals and that either calcium influx through CaV1.1e or SR calcium release through the RyR1 is sufficient for the proper organization of NMJs. Both the CaV1.1 and RyR1 isoforms are specifically expressed in skeletal muscle, excluding the possibility that one or the other gene acted presynaptically in the motor neuron. The essential role of the muscle calcium signal in the regulation of neuromuscular synapse formation is established by aberrant AChR patterning and excessive nerve branching in CaV1.1 / and in RyR1 / ; DHPRnc/nc double mutant mice, both of which lack activity-dependent LTCC and EC coupling. On the other hand, normal AChR patterning in RyR1 / as well as in the non-conducting DHPRnc/nc mice demonstrates that the activity of either one of the two calcium channels is sufficient to regulate this initial step in NMJ formation (Figure 7A). A requirement of functional DHPRs in neuromuscular synaptic patterning has previously been suggested by Chen et al. (2011). These authors concluded that the calcium channel function, but not EC coupling, is essential for regulating AChR patterning. Using a knockin mouse model with a non-conducting CaV1.1 (Dayal et al., 2017), we unambiguously demonstrate here that AChR patterning and proper innervation can readily be accomplished in the absence of LTCCs when the calcium signal exclusively comes from the RyR1. This finding is consistent with an isolated observation in a similar mouse model (Lee et al., 2015). However, whereas these authors suggested that the findings by Chen et al. (2011) may be caused by the non-conventional role of the calcium channel b1a subunit in transcriptional regulation, our results obtained in RyR1 / ; DHPRnc/nc double-mutant mice exclude the possibility that regulation of AChR patterning is mediated by a non-channel function of CaV1.1 (i.e., signaling via proteinprotein interaction) and substantiate the central role of muscle calcium signaling in AChR patterning. Based on the combined evidence from the three tested calcium channel mutants and double knockouts thereof, we further conclude that, in DHPRnc/nc mice, activity-dependent calcium release from the SR (i.e., EC coupling) regulates AChR patterning and innervation. Thus, the signaling pathway regulating muscle contraction in differentiated skeletal muscle cells serves an additional important function in NMJ formation during fetal development. Conversely, in RyR1 / mice, which lack EC coupling, activity-dependent calcium influx (i.e., LTCCs) is sufficient for establishing normal AChR patterning and innervation.

Figure 5. In Late Fetal Development (E18.5), Lack of DHPR-Dependent Postsynaptic Calcium Signals Results in Distorted Localization of Neuromuscular Synapses and in Aberrant Innervation (A and B) AChR clusters and motor axons double labeled with a-BTX (green) and anti-synapsin (red), respectively, in diaphragm of wild-type and CaV1.1 / , RyR1+/ ; DHPR+/nc, and RyR1 / ; DHPRnc/nc mice at E18.5. In both genotypes lacking activity-dependent calcium signals, the territory of motor nerve innervation is massively expanded and NMJs are formed throughout the entire width of the muscle fibers. The scale bar represents 400 mm. (C and E) Double fluorescence labeling synapses with Alexa-488-BTX together with either anti-synapsin or anti-MuSK reveals mostly single innervation in wildtype and RyR1+/ ; DHPR+/nc controls but poly-innervation in diaphragms of CaV1.1 / and RyR1 / ; DHPRnc/nc mice. The scale bars represent 10 mm for BTX/Syn and 50 mm for BTX/MuSK images. (D and F) Quantification of synapses per muscle fiber in CaV1.1 / and RyR1 / ; DHPRnc/nc diaphragm and littermate controls. AChR clusters were counted in laminin-labeled muscle fibers, which could be traced from end to end. N = 55–60 myofibers from R3 diaphragms from 2 litters for each genotype.

At the onset of NMJ formation, this calcium influx most likely originates from the embryonic CaV1.1e splice variant, which— at variance with the adult CaV1.1a variant—activates at lower

depolarizing membrane potentials and conducts sizable LTCCs (Tuluc et al., 2009; Flucher and Tuluc, 2011). Our data indicate that, at E14.5, LTCCs through CaV1.1e are sufficiently large to

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Figure 6. Rescue of NMJ Patterning and Nerve Branching Defects in RyR1 ducting Embryonic CaV1.1e Splice Variant

/

Diaphragms by Maintaining Expression of the Highly Con-

(A) Double labeling of NMJs with Alexa-488-BTX (green) and anti-synapsin (red) in E18.5 diaphragms shows that, in RyR1 / mice, ectopic AChR clusters emerge outside the clearly visible central endplate band and motor axons collaterally grow well beyond its perimeter. These AChR clusters are fully innervated. In RyR1 / ; CaV1.1DE29/DE29 mice, comparatively rare ectopic AChR clusters are detected and motor axons grow over a shorter distance (ends indicated by arrows) than that in RyR1 / mice. Semi-homozygous RyR1 / ; CaV1.1+/DE29 mice show an intermediate phenotype. Scale bar, 400 mm. (B and C) Quantification of the number of ectopic synapses per image frame (1.55 3 1.55 mm) and of the width of innervation territory shows a significant reduction of both parameters with the number of DE29 alleles. N R 4 diaphragms from 4 litters for RyR1 / , wild-type and RyR1 / ; CaV1.1+/DE29 and 2 litters for RyR1 / ; CaV1.1DE29/DE29; mean ± SEM; one-way ANOVA: F(3,15) = 41.04; p < 0.0001 for (B) ANOVA: F(3,16) = 57.85; p < 0.0001 for (C); Newman-Keuls post hoc test: **p < 0.01; ***p < 0.001; n.s., p > 0.5.

fully compensate for the loss of calcium release via the EC coupling mechanism. However, when the size of LTCCs is gradually reduced by the developmental shift from the embryonic to the adult CaV1.1 splice variant or experimentally halved in semihomozygous RyR1 / ; DHPRnc/+ mice, AChR patterning and nerve branching become impaired. Thus, under these conditions, the size of LTCCs is relevant and limiting. During fetal and early postnatal development, the highly conducting CaV1.1e splice variant is gradually replaced by the poorly conducting CaV1.1a splice variant (Tuluc et al., 2009; Flucher and Tuluc, 2011). Whereas, in normal muscle, the emerging SR calcium release dominates and masks the developmental decline of LTCCs, in RyR1 / mice, this declining calcium influx leads to the appearance of ectopic AChR clusters and a second wave of motor axon outgrowth, as observed by us and others at late fetal stages (Chen et al., 2011). The substantial rescue of this phenotype observed in RyR1 / ; DHPRDE29/DE29 mice, which do not undergo the developmental splicing of CaV1.1 exon 29, highlights the exquisite calcium dependence of the mechanism regulating AChR patterning and nerve branching and suggests a privileged position of the embryonic CaV1.1e isoform in activating this signaling pathway (Figure 7B).

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Thus, normal AChR patterning and innervation are regulated by two distinct calcium signals that are both initiated by the voltage-dependent calcium channel CaV1.1 in response to electrical activity of the muscle fibers. This conclusion is consistent with the similarity of the CaV1.1 / phenotype with that of mouse models deficient in neuromuscular synaptic transmission. Like CaV1.1 / mice, mouse models lacking acetylcholine (ACh) release (Misgeld et al., 2002; Lin et al., 2005) or expressing inactive AChRs (Pacifici et al., 2011) show AChR clusters outside the central band, excessive nerve branching, and ultimately innervation of muscle fibers with multiple NMJs. ACh is regarded as the chief nerve-derived signal responsible for elimination of extrasynaptic AChR clusters (Kummer et al., 2006). Our present results identify activity-dependent calcium influx and release as the downstream signals mediating this process from the earliest steps of NMJ formation on. Prior to innervation, at the stage when the AChR pre-patterning takes place, spontaneous electrical activity and subsequent calcium influx might be supported by the interplay of two embryonic channel variants with increased open probability: AChRs containing the g-subunit and CaV1.1e (Koenen et al., 2005; Sultana et al., 2016; Tuluc et al., 2009). Because all of our experiments were performed in the presence

Figure 7. Importance of Activity-Induced Calcium Signals for Early NMJ Development (A) Calcium influx or release is necessary and sufficient for regulation of AChR patterning and targeting of motor axons to the central endplate band. In wild-type muscle, spontaneous electrical activity induces calcium influx through the DHPR as well as SR calcium release through RyR1. These calcium signals confine nascent AChR clusters to the center of the myofibers. In CaV1.1 / mice, lack of DHPR causes the lack of both calcium influx and release, which in turn results in a failure of AChR patterning and excessive nerve outgrowth. In RyR1 / and in DHPRnc/nc mice, either influx or release of calcium is sufficient for regulating AChR patterning. Abolishing both calcium signals simultaneously in RyR1 / ; DHPRnc/nc causes severe defects in AChR patterning and motor nerve branching. (B) Stepwise increase of LTCCs in genetic mouse models inhibits motor axon outgrowth and peripheral synapse formation in RyR1 / background at E18.5. With two non-conducting CaV1.1 alleles (RyR1 / ; DHPRnc/nc), neuromuscular synapses are uniformly distributed across the muscle and motor nerve branching pattern is disrupted. With one non-conducting allele (RyR1 / ; DHPR+/nc), a central accumulation of endplates is formed next to frequent peripheral synapses. In RyR1 / muscles containing two wild-type CaV1.1 alleles, a discrete central endplate band is formed and ectopic AChR clusters appear in the periphery; although innervated, ectopic AChR clusters are smaller than those in the endplate band. Expression of a single allele of the embryonic high-calcium-conducting CaV1.1e (RyR1 / ; CaV1.1+/DE29) reduces the innervation territory of the motor axons and significantly inhibits ectopic synapse formation. Expression of two CaV1.1e alleles further inhibits motor axon growth and fully rescues AChR patterning in the center of the muscle fibers. Right column: representative images of E18.5 diaphragm double labeled with Alexa-488-BTX (green) to show AChR clusters and anti-synapsin (red) to label motor axon branches are shown. The scale bar represents 400 mm.

of motor neurons, we cannot rule out that the function of the calcium signal in restricting AChR clustering to the muscle center is dependent on nerve-induced muscle activity. However, the previous report of similar AChR patterning defects observed in Cacnb1 / /Hb9 / double mutants, which lack motor neurons (Chen et al., 2011), supports the notion that also pre-patterning of nascent AChR is regulated by these calcium signals in spontaneously active muscle fibers. Downstream, the activity-dependent calcium signals negatively regulate expression of MuSK and AChR in extrasynaptic regions via the CaMKII/myogenin pathway and cause the calpain/CDK5-dependent dispersal of aneural AChR clusters (Wu

et al., 2010; Tang et al., 2006; Macpherson et al., 2002; Lin et al., 2005). Consistent with a central role of calcium signaling in suppressing AChR and MuSK expression (Lomo and Rosenthal, 1972; Cohen and Fischbach, 1973; Birnbaum et al., 1980; Pezzementi and Schmidt, 1981; Klarsfeld et al., 1989; Walke et al., 1994; Adams and Goldman, 1998), CaV1.1 / and RyR1 / ; DHPRnc/nc mice displayed upregulation of aAChR mRNA and MuSK protein in the peripheral domains of the muscle fibers. Importantly, in wild-type and unaffected calcium channel mutants, we observed diffuse membrane expression of MuSK near the center, but not in the periphery of the muscle fiber. Because the local concentration of MuSK is important for

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AChR clustering (Kim and Burden, 2008), this MuSK gradient may be instrumental in the patterning of nascent AChR clusters in the center of the muscle fibers prior to innervation. As expression of MuSK is inhibited by calcium (Valenzuela et al., 1995; Tang et al., 2006; Chen et al., 2011), the observed loss of calcium-dependent inhibition of MuSK expression in the periphery of the muscle fibers may cause the disruption of AChR patterning and of the signals directing the nerve to the prospective endplate band. After innervation, activation of MuSK by nerve-derived agrin stabilizes neural AChR clusters, whereas the regular calcium signals during EC coupling keep extrasynaptic expression of MuSK and AChR in check (Lin et al., 2001; Yang et al., 2001). Thus, in the absence of LTCC and SR calcium release, the NMJ phenotype becomes progressively worse, resulting in the total breakdown of AChR patterning, excessive nerve branching, and hyperinnervation of muscle fibers at late fetal stages. Neuronally evoked or spontaneous electrical activity of primary myotubes regulates the formation of secondary myotubes (Ashby et al., 1993), and central NMJ patterning has been suggested to originate from myoblast fusion at the growing ends of myotubes (Kim and Burden, 2008). Therefore, CaV1.1-dependent calcium signals might also be involved in the regulation of primary and secondary myotube formation, and alterations of calcium-dependent muscle differentiation may contribute to the observed loss of NMJ patterning. In conclusion, the combined evidence from the individual and combined calcium channel mutant mouse models demonstrates the central role of CaV1.1-mediated calcium signals for proper NMJ formation. Calcium influx through CaV1.1e and SR calcium release mediate spontaneous and nerve-induced electrical activity, respectively, to suppress MuSK expression and AChR clustering in the periphery of muscle fibers and thus confine the patterning of AChR and the growth of motor axons to the center of muscle fibers.

by Dr. S. Burden (New York University). For further details, see Supplemental Experimental Procedures. Statistical Analysis Student’s t test was applied to assess statistical differences between two genotypes. One-way ANOVA with Newman-Keuls post hoc test was applied to assess statistical significance for the differences between more than two genotypes. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and two figures and can be found with this article online at https://doi.org/ 10.1016/j.celrep.2018.05.085. ACKNOWLEDGMENTS We thank S. Burden for providing the aAChR in situ hybridization plasmid and €egg for providing the rbMuSK antibody. We thank M. Offterdinger for M. Ru support in the Biooptic Facility of the Medical University Innsbruck and K. Heinz and M. Heitz for competent technical assistance. This work was supported by grants from the Austrian Science Fund (FWF) P27031 and W1101 to B.E.F., P27392 to M.G., and a grant from the German Research Fund (DFG) PA801-6 to S.P. AUTHOR CONTRIBUTIONS B.E.F. supervised all experiments. B.E.F. and M.M.K. designed the experiments and wrote the manuscript. M.M.K., N.S., A.B., and G.J.O. collected and analyzed data. A.D., M.G., N.F.L., and S.P. generated and provided mutant mouse models and tissue samples. DECLARATION OF INTERESTS The authors declare no competing interests. Received: April 9, 2018 Revised: May 7, 2018 Accepted: May 25, 2018 Published: June 26, 2018

EXPERIMENTAL PROCEDURES REFERENCES Mice All animal protocols conformed to the guidelines of the European Community (86/609/EEC) and were approved by the Austrian Ministry of Science (BMWFW-66.011/0002-WF/V/3b/2015). Cav1.1 / (Tanabe et al., 1988), RyR1 / (Buck et al., 1997), DHPRnc/nc (Dayal et al., 2017), and Cav1.1DE29/DE29 (Sultana et al., 2016) mice have been described previously. Homozygous mutant mice for Cav1.1 / , RyR1 / , DHPRnc/nc, and their wild-type littermates were obtained from heterozygous matings. RyR1+/ ; DHPR+/nc, RyR1 / ; DHPR+/nc, and RyR1 / ; DHPRnc/nc embryos were obtained by crossing RyR1+/ ; DHPR+/nc with RyR1+/ ; DHPRnc/nc mice. RyR1 / ; Cav1.1+/DE29 and RyR1 / ; Cav1.1DE29/DE29 mice were obtained by crossing RyR1+/ ; Cav1.1+/DE29 with RyR1+/ ; Cav1.1DE29/DE29 mice. Sperm plugs were checked daily during mating periods at 8:00 a.m. and 5:30 p.m. The day on which a sperm plug was detected was counted as embryonic day E0.5. Embryos were collected at the indicated days of pregnancy by cesarean section of sacrificed pregnant mice. Labeling Procedures Fluorescence labeling of dissected diaphragms was essentially performed as described in Yang et al. (2001). Primary antibodies are as follows: rbSynapsin (1/10,000; Synaptic System); rbMuSK (194 T [Nsk-2]; 1/2,000); mDHPRa1 (1/1,000; Thermo Scientific); mDHPRb1 (1/1,000 cl. N7/18; NeuroMab); or rbRyR1 (Flucher et al., 1999). In situ hybridization was performed as described in DeChiara et al. (1996) and Herbst et al. (2002). aAChR plasmid was provided

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