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Glycine, a major inhibitory neurotransmitter in the vertebrate nervous system, not only functions in synaptic signaling, but has also been implicated in regulating neuronal differentiation, neuronal proliferation, synaptic modeling, and neural network stability. Elements of the glycinergic phenotype include the membrane-bound glycine transporters (GLYT1 and GLYT2), which remove glycine from the synaptic cleft, and the vesicular inhibitory amino acid transporter (VIAAT or VGAT), which sequesters both glycine and GABA into synaptic vesicles. Here, we describe the spatial and temporal expression patterns of xGlyT1, xGlyT2, and xVIAAT during early developmental stages of Xenopus laevis. In situ hybridization reveals that xGlyT1 is first expressed in early tailbud stages in the midbrain, hindbrain, and anterior spinal cord; it extends posteriorly through the spinal cord and appears in the forebrain, retina, between the somites, and in the blood islands by swimming tadpole stages. xGlyT2 and xVIAAT initially appear in late neurula stages in the anterior spinal cord. By swimming tadpole stages, the expression of these genes appears in the forebrain, midbrain, and hindbrain and extends posteriorly through the spinal cord; xVIAAT is also expressed in the retina. Confocal analysis of multiplex fluorescent in situ hybridization signal in the spinal cord reveals that xGlyT1 and xGlyT2 share little cellular colocalization. While there is significant coexpression between xVIAAT and xGlyT2, xVIAAT and the GABAergic marker glutamic acid decarboxylase (xGAD67), and xGlyT2 and xGAD67, each gene also appears to have discrete, non-colocalized areas of expression.
Fig. 1. Spatial and temporal expression patterns of xGlyT1, xGlyT2, and xVIAAT in developing embryos using whole mount in situ hybridization. Embryos are viewed laterally, with anterior to the left and dorsal to the top. (A–C) Stages 21–22 (late neurula). (D–F) Stages 24–25 (early tailbud). (G–I) Stages 27–29 (late tailbud). (J–L) Stages 31–32 (early hatching). (M–O) Stages 33–34 (late hatching). (P–R) Stages 35–37 (swimming tadpole). (A, D, G, J, M, P) xGlyT1 expression; (B, E, H, K, N, Q) xGlyT2 expression; (C, F, I, L, O, R) xVIAAT expression. Hollow arrowheads denote telencephalic xVIAAT expression; solid arrowheads denote diencephalic xVIAAT expression. Abbreviations: fb, forebrain; hb, hindbrain; mb, midbrain; sc, spinal cord; so, somite. Scale bars represent 1.0 mm.
Fig. 2. Histological analysis of xGlyT1 mRNA expression in stages 33–34 (late hatching) and stages 35–37 (swimming tadpole) X. laevis using whole mount in situ hybridization. (A–G) Transverse 10-μm sections along the anterior–posterior axis, with dorsal to the top. (A–B) Stages 33–34 (late hatching). (C–G) Stages 35–37 (swimming tadpole). (B′, D′, E′) Magnified images of the neural tube from the above sections. (H) Transverse 10-μm section of the eye of a stage 35–37 (swimming tadpole) embryo. (I) Transverse 10-μm section of the somites of a stage 33–34 (late hatching) embryo. Abbreviations: cg, cement gland; e, eye; fb, forebrain; fg, foregut; hb, hindbrain; mb, midbrain; no, notochord; ot, otic vesicle; sc, spinal cord; so, somite. Scale bars represent 1.0 mm for A–G, 0.4 mm for B′, D′, E′, and H, and 0.2 mm for I.
Fig. 3. Histological analysis of xGlyT2 mRNA expression in stages 35–37 (swimming tadpole) X. laevis using whole mount in situ hybridization. (A–H) Transverse 10-μm sections along the anterior–posterior axis, with dorsal to the top. (E′–H′) Magnified images of the neural tube from the above mentioned sections. Abbreviations: cg, cement gland; e, eye; fb, forebrain; fg, foregut; hb, hindbrain; mb, midbrain; no, notochord; ot, otic vesicle; sc, spinal cord; so, somite. Scale bars represent 1.0 mm for A–H and 0.25 mm for E′–H′.
Fig. 4. Histological analysis of xVIAAT mRNA expression in stages 35–37 (swimming tadpole) X. laevis using whole mount in situ hybridization. (A–H) Transverse 10-μm sections along the anterior–posterior axis, with dorsal to the top. (E′–H′) Magnified images of the neural tube from the above mentioned sections. Abbreviations: cg, cement gland; e, eye; fb, forebrain; fg, foregut; hb, hindbrain; mb, midbrain; no, notochord; ot, otic vesicle; sc, spinal cord; so, somite. Scale bars represent 1.0 mm for A–H and 0.25 mm for E′–H′.
Xenopus solute carrier family 6 (neurotransmitter transporter, glycine), member 5 / slc6a5 expression in stage 33 embryo
