/ AChR subunit KO zebrafish lines

We generated a subunit gene KO zebrafish (KO) using CRISPR-Cas9 (Fig. 1A) and an subunit gene KO zebrafish (KO) using transcription activatorlike effector nucleases (TALEN) (Fig. 1A). The KO zebrafish did not show obvious phenotypes during development and matured in a fashion indistinguishable from wild-type (WT) siblings (fig. S1). In contrast, KO fish generally failed to form swim bladders, and most of them died prematurely within 2 weeks after fertilization. However, a fraction of KO fish (approximately 25%) survived to adulthood. A double KO (DKO) line was generated by crossing KO and K lines. DKO larvae also failed to form swim bladders (Fig. 1B) and died within 2 weeks after fertilization.

(A) Schematic diagram of targeted genes. Arrowheads indicate targeted regions of genome editing. Each box and line indicates an exon and an intron, respectively. Alignment of genomic DNA sequences of WT and KO lines showed a 7base pair (bp) insertion in the AChR subunit gene chrng and a 1-bp insertion in the AChR subunit gene chrne. (B) Photograph showing WT and / DKO larva at 6 dpf. Notice the lack of swim bladder (arrowheads) in DKO. Scale bar, 1 mm. (C) Trunk regions of a WT larva (6 dpf) and a DKO larva (6 dpf) were stained with -BTX conjugated with Alexa Fluor 488 (green). In WT, AChRs were distributed in myoseptal regions (arrows) and in punctae in middle regions (arrowhead). DKO had -BTX signals only in myoseptal regions. Scale bars, 100 m.

We histologically analyzed the expression of AChRs in the trunk region of 6 days post-fertilization (dpf) larvae by using -bungarotoxin (-BTX) conjugated with Alexa Fluor 488, a toxin that specifically binds to the assembled AChR (Fig. 1C). AChR clusters in DKOs were observed only in boundary regions between body segments (Fig. 1C), where slow muscles form NMJs (16). We initially expected that AChRs in fast muscles of DKO larvae would convert to the slow muscletype AChRs, comprising only , , and subunits. This conversion of subunit composition would not cause a change in AChR distribution visualized by -BTX, because both types of AChRs bind to -BTX. However, -BTX signals were absent in fast muscles, which suggested that fast muscles could not express AChRs composed of , , and subunits.

To correlate the AChR expression pattern observed by the -BTX staining with the synaptic function, we analyzed synaptic activities of fast and slow muscles in the DKO line at 6 dpf. We recorded spontaneous synaptic currents from muscle cells using the whole-cell patch clamp technique (Fig. 2, A to C). Traces show miniature endplate currents (mEPCs) from muscles of WT or DKO larvae (Fig. 2A). Slow muscles in the DKO line exhibited mEPCs. The frequency (14.5 3.1 Hz in WT, 15.5 3.2 Hz in DKO) and the amplitude of slow muscle mEPCs (260.0 74.1 pA in WT, 491.7 105.2 pA in DKO) showed no differences between WT and DKO lines (Fig. 2, B and C). However, fast muscles in DKO failed to produce mEPCs. To confirm that the lack of mEPCs is caused by the absence of functional receptors, we recorded currents in muscles generated by puff application of ACh (Fig. 2D). While fast muscles in WT larvae showed ACh-induced currents (756.4 138.6 pA), those in DKO larvae failed to show any response (0 0 pA). These results, in conjunction with the -BTX staining (Fig. 1C), showed that fast muscles of DKO larvae do not express any AChRs and receive no synaptic input.

(A) mEPC traces from fast or slow muscles of WT and DKO larvae (6 dpf) by whole-cell patch-clamp recordings. Fast muscle cells in DKO failed to exhibit mEPCs. (B and C) Frequencies (B) and amplitudes (C) of mEPCs were plotted for each muscle (n = 8 cells). (D) Representative traces of voltage-clamped slow and fast muscles in DKO larvae in response to the application of 30 M ACh. Calibration: 1 s, 500 pA. Amplitudes of ACh-induced currents in slow (n = 7 cells) and fast muscles (n = 7 cells) are shown. Each dot represents a muscle cell. (E) Construct used for Ca2+ imaging. Top: The GCaMP7a coding sequence was fused to the promoter region of the -actin promoter pact. Bottom: Schematic illustration showing the experimental procedure. The gene construct was injected into eggs of DKO at the one cell stage. Ca2+ response was analyzed at 6 dpf. Representative traces showing the increase of F/F in a fast muscle (black line) and a slow muscle (red line) during spontaneous contractions. (F) Overexpression of the subunit fused with an EGFP (-EGFP) in WT (3 dpf). Top panels: -EGFPs were expressed under the control of a slow musclespecific promoter, psmyhc. EGFP signals (green), expressed in the superficial slow muscles, filled the cytoplasm and did not colocalize with -BTX (magenta) signals. Bottom panels: -EGFPs were expressed under the regulation of pact. In deeper layer fast muscles, the clusters of EGFP and -BTX colocalized (arrowheads). Scale bars, 50 m.

We performed in vivo Ca2+ imaging in the DKO larvae at 6 dpf to further support the result of synaptic current recordings. We designed a gene construct in which a pan-muscle promoter, -actin promoter, drives the expression of a Ca2+ indicator, GCaMP7a (17), and injected the construct into fertilized eggs (Fig. 2E). In DKOs, we recorded Ca2+ response associated with spontaneous locomotion activities, induced by the application of N-methyl-d-aspartate (50 M) (18). The results showed that slow muscle cells exhibited Ca2+ transients, while fast muscle cells did not generate any Ca2+ response.

Considering that fast muscles do not allow composition of , , and subunits, we next examined whether slow muscles conversely allow incorporation of subunits in the AChR pentamer, by overexpressing the subunit in slow muscles. We designed a gene construct that expressed an subunit fused with enhanced green fluorescent protein (-EGFP) under the regulation of a slow musclespecific promoter, psmyhc (19). We injected the construct into fertilized WT eggs and observed the expression of EGFP at 3 to 4 dpf. EGFP signals typically filled the cytoplasm of the slow muscle cells and never colocalized with -BTX signals (Fig. 2F). In a control experiment, in which -EGFP was driven by the pan-muscle promoter (-actin promoter), the -EGFP signals made clusters in fast muscles, colocalizing with -BTX signals in deeper layers of the trunk region where fast muscles form NMJs. Together, fast muscles and slow muscles express specific types of AChR, and the alternate composition of subunits is prohibited.

To examine how silencing of synapses in fast muscles affect locomotion, we next analyzed swimming of WT and DKO larvae at 6 dpf. We induced escape responses by gentle tactile stimuli. Locomotion was recorded with a high-speed camera, and we measured angles between head and tail trajectories throughout each escape response (Fig. 3A and movie S1). WT fish turned their heads 120 to 140 in the initial stage of escape. The typical startle response of teleosts generally begins with a large turn of the head (termed C-bend), followed by a robust forward propulsion as described in previous studies (20).

(A) Escape behaviors in WT and DKO lines at 6 dpf in response to tactile stimuli. Images of representative larva on the left show superimposed frames of the complete escape response (the duration of movement is indicated in the top right corner). Scale bars, 2 mm. Kinematics for representative traces of 10 larvae are shown for the initial 50 ms of the response. Middle panels represent averaged traces. In the right panels, each trace represents a different larva. Body angles are shown in degrees, with 0 indicating a straight body, and positive and negative values indicating body bends in opposite directions. Scale bars, 10 ms. (B to D) Maximum turn angles, time to reach the maximum angle, and post-startle swimming speed were calculated for each group of fish (6 dpf). In DKO, the turn angle and the swimming speed were notably reduced, and it took longer to reach maximum angles (n = 10 fish). (E and F) Analyses of spontaneous locomotion. Images of representative larva (left) for WT or DKO showed superimposed frames of spontaneous swim bouts (the duration of movement indicated in the bottom right corner). Swimming speed was calculated for WT (n = 5 fish) and DKO (n = 5 fish), which showed no significant difference. Scale bars, 2 mm.

The initial turns of the DKO larvae were in sharp contrast to WT. Averaged maximum head turn angles in DKOs were markedly smaller compared to WT larvae (116.0 5.8 in WT, 20.2 4.0 in DKO; P < 0.001) (Fig. 3B), and time to reach the maximum angle was increased (8.7 0.2 ms in WT, 15.8 0.8 ms in DKO; P < 0.001) (Fig. 3C). In addition to the absence of C-bends, the post-startle swimming speed of the DKO line was also notably slower (84.9 8.1 mm/s in WT, 12.8 1.3 mm/s in DKO; P < 0.001) (Fig. 3D).

In addition to the escape response, we also analyzed spontaneous locomotion, which corresponds to the slow swim described by Budick and OMalley (21) or scoot reported by Burgess and Granato (22) (Fig. 3, E and F). Significant difference in swimming speed was not observed between WT and DKO (16.1 1.60 mm/s in WT, 13.2 0.9 mm/s in DKO; P = 0.20) (Fig. 3F). Thus, the contribution of fast muscles in spontaneous swimming is relatively small. These results strongly suggest that fast muscles in larval zebrafish play a key role in executing quick escape responses including the C-bend and fast forward propulsion behaviors, which corroborate earlier studies (23).

DKO fish die prematurely and do not develop into adults. However, KOs that reached the adult stage are expected to lack both and subunits, because subunit expression terminates early in development.

To dismiss the possibility of compensatory up-regulation of the subunit in adult KOs, we analyzed the expression of subunit mRNA with digital droplet polymerase chain reaction (ddPCR). Subunit mRNA was not detected in adult KOs, which were 3 to 5 months old (Fig. 4A). Interestingly, subunit mRNA was strongly up-regulated in larval KOs (Fig. 4B), which may account for functional escape response behavior at 6 dpf (fig. S1). Thus, our findings suggest that compensation by the subunit expression occurs only in larval KOs and not in adults.

(A) Quantification of or subunit mRNA in adult muscles. Subunit was not detected in WT. or subunit mRNA was not detected in KO (n = 6 fish in WT, n = 5 fish in KO). Sample numbers are shown in parentheses. (B) mRNA expression of subunit in 1-dpf larvae. Subunit was highly up-regulated in the KO (n = 5 fish) compared to WT (n = 5 fish). Sample numbers are shown in parentheses. (C) Schematic illustration of a transverse section of the trunk region. The area shown in micropictograms is indicated with a box. The distribution of AChRs in adults, WT or KO, was visualized by -BTX conjugated with Alexa Fluor 488 (green). Broken lines indicate the boundary of fast muscle area (arrowheads). Fast muscles in the KO fish lack -BTX signals. (D) Sections of adult fast muscles of WT and KO, stained with the fast musclespecific F310 antibody. Fast muscles in KO fish did not display atrophy. In the right panel, diameters of fast muscles in WT and KO were calculated (87 fibers, n = 3 fish). There was no significant difference. Scale bars, 100 m.

The expression of AChR in adult KO fish, visualized by -BTX, was consistent with the lack of compensation (Fig. 4C). Transverse sections of the trunk region were labeled with -BTX. Slow, intermediate, and fast muscles are spatially segregated (11). Slow muscles are located closest to the surface. WT fish displayed universally distributed, positive -BTX signals. In sharp contrast, -BTX signals in the KO fish were detected only in shallow, lateral regions, and fast muscles of the adult KO lacked AChR expression.

In spite of the absence of -BTXpositive signals, fast muscle fibers in KO fish unexpectedly lacked signs of prominent atrophy (24). A fast musclespecific F310 antibody used via immunohistochemistry allowed the visualization and diameter measurements of fast muscle fibers. Statistical analysis revealed no difference between KO and WT fiber size (58.7 0.5 m in WT, 58.3 0.7 m in KO; P = 0.945) (Fig. 4D).

We observed escape responses induced by objects dropping on water and subsequently analyzed C-bend angles and the swimming speed during escape (Fig. 5A) (25). We compared the maximum C-bend angles between the focal genetic lines. Similar to WT larvae (Fig. 3), WT adults start the escape response with the initial extreme head turn. Unexpectedly, we found that KO adult fish also display robust C-bends (Fig. 5, A and B). Although smaller in amplitude (103.0 7.5 in WT, 53.4 2.5 in KO), their time course did not exhibit any delay compared to WT. This is in sharp contrast to the complete loss of C-bend behavior observed in larval DKOs (Fig. 3). The duration of first turn also showed no significant difference between WTs and KOs (38.9 3.8 ms in WT, 46.6 4.9 ms in KO).

(A) Escape behaviors in WT and KO adults (3 to 4 months old). The startle response was induced by dropping objects on water. Images of representative fish to the left show superimposed frames of the complete escape response (the duration of movement is indicated in the bottom right corner). Kinematics for representative traces from 10 or 9 fish are shown for the initial 50 ms of response. Middle panels represent averaged traces. In right panels, each trace represents a different fish. Body angles are shown in degrees, with 0 indicating a straight body. Positive and negative values indicate body bends in opposite directions. (B) First turn angles were calculated for each group of fish (n = 10 fish in WT, n = 9 fish in KO). Turn angles were reduced in the KO fish. Sample numbers are shown in parentheses. (C) Post-startle swimming speed and total distance traveled were calculated for the first 120 ms. There was no significant difference between WT (n = 10 fish) and KO (n = 9 fish) adults.

Furthermore, the forward propulsion during escape of the KO adult zebrafish was almost intact. When the distance traveled was plotted against the time after stimulation, the curves for WT and KO nearly overlapped (Fig. 5C). The swimming speed (31.7 1.3 cm/s in WT, 25.5 3.0 cm/s in KO; P = 0.08) and total distance traveled (4.0 0.2 cm in WT, 3.2 0.4 cm in KO; P = 0.08) were similar between WT and KO adults.

Suspecting that compensation of locomotion occurred at the level of neural projection, we examined the projections of motor neurons by retrograde labeling using a fluorescent tracer, dextran conjugated with Alexa Fluor 488 (Fig. 6, A to C). We injected the tracer into muscles of WT and K fish following a method described in a previous report (26). Spinal motor neurons in adult zebrafish are classified on the basis of morphological features. Dorsomedial motor neurons with larger cell somas, which are called primary motor neurons (pMNs), specifically innervate fast muscles. Ventrolateral motor neurons with smaller somas, called secondary motor neurons (sMNs), are grouped in distinct populations depending on the innervation target: fast, intermediate, and slow muscles (2729). We analyzed the location of motor neuron somas in the spinal cord (Fig. 6B) by measuring the distance from the center of spinal cord to cell somas. In WT adults, fast muscles were innervated mainly by dorsomedial motor neurons (located close to the center), and slow muscles were innervated by ventrolateral motor neurons (Fig. 6, A and B).

(A) Schematic illustration of a transverse section of the trunk region showing the sites of dye injections. Right panels showing cell bodies of labeled motor neurons (arrowheads) in spinal cords. Broken lines indicating outlines of spinal cords. Scale bars, 50 m. (B) A graph showing the distance from the center of the spinal cord to cell bodies of motor neurons. In WT, motor neurons located close to the center innervate fast muscles, and ventrolateral motor neurons innervate slow muscles. In KO, slow muscles were innervated by motor neurons located close to the center. Numbers of labeled cells are shown in parentheses. (C) Graph showing the size of cell somas of motor neurons. In WT, large motor neurons innervate fast muscles, and smaller neurons innervate slow muscles. In KO, slow muscles were innervated by large motor neurons. (D) Schematic illustration of a transverse section of the trunk region showing the locations of the DiI crystal insertion. The right panel displays cell body of labeled pMN (arrowhead) in the spinal cord. The broken line indicates the outline of the spinal cord. Scale bar, 50 m. (E) Presynaptic structures were visualized by SV2A antibody. Broken lines indicate the boundary of slow muscle area (left side). Note the reduced signal in the fast muscles of the KO fish. Scale bars, 100 m. (F and G) Fast musclespecific myosins labeled by F310 antibody in WT (F) and KO (G). In (G), the boxed area is enlarged in the right panel. Broken lines indicate the boundary of slow muscle area (left side). Arrowheads indicate muscle cells with F310 signals in the slow muscle region. While a small number of slow muscle cells in WT sometimes showed immunoreactivity, the cell number was markedly increased in KO. Scale bars, 100 m. (H and I) Glycolytic muscle fibers were visualized by GPD staining in WT (H) and KO (I). Black broken lines indicate the boundary between slow and intermediate muscles, and the red broken line indicates the boundary between intermediate and fast muscles. Fast, intermediate, and slow muscle areas are labeled with F, I, and S, respectively. Note that the intermediate muscle region in KO is hard to distinguish from the fast muscle region, blurring the boundary (I). Arrowheads in the right panel indicate muscle cells with GPD signals in the slow muscle region. Scale bars, 100 m. (J) Schematic illustration showing the rerouted innervation of pMNs. In KO adults, synaptic silencing of fast muscles led to the innervation of fast musclespecific pMNs on slow muscle. This reinnervation caused conversion of slow to fast muscles. The projections of sMNs that innervate fast muscles may not change.

Both the location and the size of motor neuron somas suggested that slow muscles in KO adults were innervated by large motor neurons, which innervate only fast muscles in WT adults (Fig. 6C). Ventrolateral neurons did not seem to innervate slow muscles in KOs, as they were absent in retrograde labeling (Fig. 6, B and C). When we injected the tracer into fast muscles of KO adults, pMNs were not labeled (fig. S2). Motor neurons labeled in these preparations were presumably fast sMNs (26).

To rule out the possibility that pMN axons are inadvertently damaged by dye injections into slow muscles of KO adults, we used another method of retrograde labeling using a lipophilic tracer DiI (or DiIC18), which has a minimal possibility of causing pressure injection damage (30). After gently placing crystals of DiI onto slow muscles of KO adults, we found that pMNs were labeled in spinal cords of KO adults (Fig. 6D). We also analyzed the presynaptic input in muscles of WT and KO adults using SV2A antibody to visualize presynaptic proteins (Fig. 6E). The results showed that positive signals within fast muscles were reduced in KO compared to WT adults. Thus, fewer motor neurons innervated fast muscles in KO fish.

The muscle cell type is determined by the motor neuron input (31). Suspecting the signals from pMNs may convert the properties of slow muscles into those of fast muscles in adult KO fish, we examined the characteristics of slow muscle fibers. To do so, we analyzed the F310 antibody immunohistochemistry in adult KO fish, which labels fast musclespecific myosin (Fig. 6, F and G) (19). We also examined the -glycerophosphate dehydrogenase (-GPD) activity, which is a well-established method to visualize glycolytic muscles, i.e., fast muscles (Fig. 6, H and I) (32). Some tissue located in slow muscle regions stained positive for F310 (n = 3 fish; Fig. 6G) and -GPD signals (n = 3 fish; Fig. 6I), suggesting that some slow muscles expressed the fast muscletype isoform of myosin light chain and obtained glycolytic ability. Intermediate muscle fibers in KO also showed higher glycolytic ability compared to WT (Fig. 6, H and I). Thus, a subpopulation of slow and intermediate muscles was converted to fast muscles, presumably due to the innervation of fast muscle motor neurons (31).

In summary, the absence of AChRs in developing KOs is presumed to drive motor neuron axon innervation of fast muscles to instead reroute to slow muscles. These rewired pMNs presumably predominate over original axons in slow muscles, as a result of synaptic competition, and convert some slow and intermediate muscles to fast muscles (Fig. 6J).

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Synaptic silencing of fast muscle is compensated by rewired innervation of slow muscle - Science Advances