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Figure 5. Switch from glutamate to acetylcholine receptors in hindlimb muscles. A.
Examples of hindlimb innervation patterns and distribution of ACh nicotinic receptors in a
whole-mount hindlimb bud, revealed by fluorescence immunolabeling of neurofilament
associated protein (Neurofil., red), glutamate receptor (NR2b, blue) and α-bungarotoxin
labeling (α-bungaroTx, yellow) respectively, at stages 52 (upper panels) and 57 (lower
panels). Inset drawings at bottom left of each panel show bud morphology at the two
representative larval stages. Scale bars: 200μm for stage 52, 100μm for stage 57. B. Examples
of fluorescence immunolabeling against neurofilament associated protein (red), synaptophysin
(Synaptoph., green) and NMDA glutamate receptor subunit 1 (NR1, blue) in whole-mount
limb bud at stage 52. White arrowheads indicate sites of apposition of all 3 markers. Scale
bar: 20μm. C. Examples of fluorescence immunolabeling against neurofilament associated
transporter 1 (VGluT1, yellow) and the glutamate
receptor subunit NR2b (blue) in whole-mount limb at stage 52. Scale bar: 50μm. |
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Figure 5 supplement 1. VGluT1 expression appendicular MNs. A. Fluorescence
immunolabeling against ChAT (red) and VGluT1 (yellow) in limb MNs retrogradely labeled
with Alexa Fluor dextran 647 (AD 647, cyan) at stage 52. Whereas ChAT is absent from
retrogradely labeled MNs, VGluT1 is expressed throughout almost the entire ventral horn,
including in limb MN somata. Bottom images are magnifications of the area (a) delineated by
the dotted line square in the merged image at top right. Punctiform VGluT1 expression
occurred around MN somata (indicating labeling of glutamatergic presynaptic terminals) and
a more diffuse VGluT1 signal also appeared in MN cytoplasm surrounding cell nuclei. B.
Fluorescence immunolabeling against synaptophysin (Synaptoph., green), NMDA glutamate
receptor subunit 1 (NR1, blue), VGluT1 (red) and neurofilament (Neurofil., grey) in hindlimb
muscle tissue at stage 52 (B1) and stage 55 (B2). VGluT1 expression colocalized with
synaptophysin and postsynaptic glutamate receptors were detected at stage 52, but not at stage
54. The bottom images in B1 are magnifications of the area (a) indicated in the merged image
at top right. Scale bars = 20μm. |