Fig. 7.

GlyR α puncta are reduced in glra1 morphants and absent in glrbb morphants. Transverse sections through fast-frozen 24 hpf (left; A–D) and 48 hpf (right; A′–D′) larvae were stained for GlyR α subunits (red), gephyrin (green), and nuclei (blue). Diagrams above the micrographs show the tight packing of spinal cord cells. In these diagrams, cells with purple cytoplasm represent the differentiated neurons: primary motor neurons (PMNs) that exit the spinal cord to innervate muscle, early differentiating interneurons, and dorsal sensory Rohon Beard neurons. These differentiated neurons form in a rind that surrounds the medial stem cells, outlined in gray. By 48 hpf more neurons have differentiated and become incorporated into the circuit as reflected by an increased synaptic staining on lateral neurons. Representative images for each stage and morpholino treatment are displayed as groupings of four images with the leftmost image giving a low magnification view of the entire spinal cord with the motor/inter-neuron region boxed in white magnified in the subsequent three images. A. In 24 hpf control morphants, GlyR α and gephyrin staining are non-overlapping. GlyR puncta decorate precursor cells near the spinal cord midline (red arrowheads) as well as lateral neurons (red arrows). Gephyrin puncta (green arrows) are solely associated with lateral neurons, but do not co-localize with GlyRs at this developmental time point. B, C. In both glra1 morphants, GlyR α subunit staining is absent in precursors near the midline. Curiously, in glra1 morphants, residual GlyR α subunits did co-localize with gephyrin (yellow arrows) in lateral neurons. D. In 24 hpf glrbbMOex5 morphants, gephyrin puncta (green arrows) occur in the absence of GlyR α subunit puncta. A′. In 48 hpf control morphants, GlyR α subunit and gephyrin puncta commonly co-localized. B′, C′. In 48 hpf glra1 morphants, a subset of GlyR α subunit and gephyrin puncta commonly co-localized but these co-localized puncta are positioned more laterally than in control morphants. D′. 48 hpf glrbbMOex5 morphants lacked clear GlyR α subunit puncta, but exhibited smaller gephyrin puncta that tended towards the lateral neuropil rather than circum-nuclear as in control morphants. Scale bars: 10 μm.