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Acetylcholinesterase is required for neuronal and muscular development in the zebrafish embryo

Nature Neuroscience volume 5, pages 111–118 (2002)Cite this article

Abstract

The neurotransmitter acetylcholine (ACh) has a crucial role in central and neuromuscular synapses of the cholinergic system. After release into the synaptic cleft, ACh is rapidly degraded by acetylcholinesterase (AChE). We have identified a mutation in the ache gene of the zebrafish, which abolishes ACh hydrolysis in homozygous animals completely. Embryos are initially motile but subsequently develop paralysis. Mutant embryos show defects in muscle fiber formation and innervation, and primary sensory neurons die prematurely. The neuromuscular phenotype in ache mutants is suppressed by a homozygous loss-of-function allele of the α-subunit of the nicotinic acetylcholine receptor (nAChR), indicating that the impairment of neuromuscular development is mediated by activation of nAChR in the mutant. Here we provide genetic evidence for non-classical functions of AChE in vertebrate development.

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Figure 1: A recessive mutation abolishes AChE enzymatic activity in the zebrafish.
Figure 2: Embryos deficient in AChE activity carry a point mutation in the ache gene changing Ser226 to an Asn.
Figure 3: ache mutants have severely impaired motility at 48 hpf.
Figure 4: Lack of AChE activity disrupts organization of myofibers.
Figure 5: Development of neuromuscular junctions is impaired in achesb55 mutant embryos.
Figure 6: RB sensory neurons are affected by the achesb55 mutation.
Figure 7: The muscle defects of ache mutants are suppressed by mutations in the α-subunit of nAChR (nic1).
Figure 8: The phenotype of morpholino knock-down embryos is the same as that of achesb55 mutants.

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Acknowledgements

We thank V. Korzh, H.G. Frohnhöfer, B. Ticho, J. Grassi, E. Krejci, H. Blau, B. Trevarrow, M. Westerfield and the Developmental Studies Hybridoma Bank (University of Iowa) for mutants and materials. We thank A. Gansmüller and M. Digelmann for help with electron microscopy, and P. Blader, F. Müller and M. König for critically reading the manuscript. We also thank A. Karmin and O. Nkundwa, for care of the fish and to D. Hentsch, M. Bogelin for help with the confocal microscope. We thank N. Fischer for technical assistance and INRA, INSERM, CNRS, HUS, AFM, ARC and ACI for support.

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  1. Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France

    Martine Behra, Jean-Luc Vonesch, Dominique Biellmann & Uwe Strähle

  2. Unite Différenciation Cellulaire et Croissance—INRA, 2 place Viala, Montpellier, 34060, France

    Xavier Cousin, Christelle Bertrand & Arnaud Chatonnet

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Supplementary information

Animations.

3D reconstructions were calculated from confocal optical sections of a wild-type (wt), mutant ache -/- (ache) and ache morpholino injected (Mo-ache) embryo. The sections cover half of the width of the embryos, at the level of the hindgut extension. Bound znp1 antibody was revealed with a secondary coupled to cy3, staining the motor neurons and the neural tube (red). The a-bungarotoxin coupled to alexa green, visualizes the AChRs in somites (green). The overlay of the two shows the distribution of the neuromuscular junctions. Animation 4 is a explanatory scheme, showing the projections of the three primary motor neurons (RoP, MiP and CaP) in wild-type (red) compared to ache-/- and MO-ache injected embryos (blue). The projections of RoP projecting along the horizontal myoseptum are not affected by the mutation at 27hpf. The MiP does not project as far caudally in the mutant as in the wild-type somite. The CaP projecting ventrally, runs more rostrally in the mutant when compared to the wild type. The arc of the motor innervation by MiP, and CaP covering the entire somite in the wild type is significantly shallower in the mutant and in the Mo-ache injected embryos. (MPG 331 kb)

Animation 2 (MPG 373 kb)

Animation 3 (MPG 233 kb)

Animation 4 (MPG 166 kb)

Supplementary Figure 1.

Computer model of the active site in the wild-type (a) and mutant AChE (b). A potential hydrogen bond between Ser226 and Glu327, which is one of the three amino acids directly involved in hydrolysis of ACh, may be disrupted by the Asn in the mutant. (JPG 133 kb)

Supplementary Figure 2.

Electron micrographs of wild-type and ache mutant muscle fibers at 27 hpf. The ultrastructure of differentiating muscle fibers is the same in mutant and wild-type embryos. In contrast to older stages, necrosis cannot be detected in the mutant muscle at this stage. Sections are close to the medial midline and thus represent fast muscle fibers. (JPG 110 kb)

Supplementary Figure 3.

Slow muscle cells express less slow muscle myosin, form striated muscle fibrils and are positioned correctly at the surface of the somite in ache mutants. Wild-type (a, c, e) and ache mutant somites (b, d, f) stained with F59 antibody. F59 staining of slow muscle cells is reduced but not completely abolished in the mutant. Furthermore, as seen in cross sections (right panels in a, c), the slow muscle cells reside at the periphery of the somite (arrowhead). (d, e) DAPI staining of sections to outline the somite and the position of the F59 staining in (c) and (d). Thus, migration of slow muscle cells does not appear to be affected by the mutation. Furthermore, high-power magnification reveals striated muscle fibrils in this superficial muscle layer (e, f). The fibrils in the mutant are as abundant as in wild type. Note the detection system of the confocal microscope was twice and 4 times more sensitive in (b) and (d), respectively. Lateral views and cross section in (a) and (b) are projections of stacks of confocal sections collected through half the embryo. Scale bars, 30 mm (a-d), 2 mm (e, f). (JPG 82 kb)

Supplementary Figure 4.

Ubiquitous alexa-bungarotoxin staining in ache mutant somites is specific. Wild-type (wt) and ache mutant embryos were stained with alexa conjugated bungarotoxin alone (1/0) or with increasing concentrations of unlabeled bungarotoxin (1/0.2), (1/0.4), (1/0.6). Equal molar ratios (1/1) abolished both staining of synapses and aberrant staining throughout the mutant somite. (JPG 74 kb)

Supplementary Figure 5.

Cap and MiP motor axons of achesb55 mutants and MO-ache injected embryos cover a smaller area of the somite. The width of area covered by the CaP and MiP axons on the somite (hindgut area of 27 h old embryos) was measured in wild-type (wt, 30 somites, 10 embryos), achesb55 mutants (ache, 30 somites, 10 embryos), control morpholino-injected embryos (co, 24 somites, 8 embryos) and embryos injected with MO-ache (27 somites, 9 embryos). Bars, averages in mm; standard deviations are indicated. (JPG 40 kb)

Supplementary Figure 6.

Quantification of length of RB dendrites in wild-type (wt) and ache mutant embryos and embryos that were exposed to 10 mM tetrodotoxin (TTX). Dendritic extensions were grouped into three categories longer than 120 mm, between 120 and 60 mm and below 60 mm. Length of dendrites was measured in the posterior trunk over the hindgut extension and in the tail of 27 hpf embryos. Embryos were bathed in 10 mM TTX from the 16 somite stage onwards. This treatment abolished motility completely but was not toxic, as wild-type embryos recovered motility after removal of TTX even if they had been treated until 48 hpf. Number of long dendrites (longer than 120 mm) is reduced by approximately twofold in the ache mutant. The frequency of intermediate length dendrites (between 120 and 60 mm) were reduced only slightly. Short dendrites (below 60 mm) are not present in 27 hpf wild-type embryos at all but are very abundant in the ache mutant. TTX did not suppress the ache phenotype, and dendrite extensions in wild-type embryos were not affected by the mutation. Bars, average from 4 wild-type, 4 ache mutant 15 TTX-treated wild-type and 5 TTX-treated ache mutants; standard deviations of measurements are indicated. (JPG 54 kb)

Supplementary Figure 7.

Quantification of apoptosis in Rohon Beard (RB) cells in wild-type, ache mutant and MO-ache injected embryos. Each bar represents the average number of TUNEL-positive RB cells per somite. The average is calculated from at least 19 somites per embryo and n = 10 for wild-type and ache mutant, respectively, and n = 4 for MO-ache injected and control, respectively. The standard deviation is indicated. (JPG 44 kb)

Supplementary Figure 8.

Double staining to confirm identity of TUNEL-positive cells at the dorsal neural tube. Dorsal view of ache mutant embryo stained with the TUNEL procedure (blue black nuclei) to reveal dying RB cells. Embryos were immunohistochemically (brown) re-stained with the antibody Zn12, which recognizes the HNK1/L2 epitope on RB cells. Arrowheads indicate TUNEL-positive/Zn12-positive nuclei, arrows point at TUNEL-negative, Zn12-positive RB cells and asterisks indicate TUNEL-positive and Zn12-negative cells. RB cells are recognized by their large cell bodies (light brown staining). (JPG 120 kb)

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Behra, M., Cousin, X., Bertrand, C. et al. Acetylcholinesterase is required for neuronal and muscular development in the zebrafish embryo. Nat Neurosci 5, 111–118 (2002). https://doi.org/10.1038/nn788

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