Generation of BAC Transgenic Tadpoles Enabling Live Imaging of Motoneurons by Using the Urotensin II-Related Peptide (<i>ust2b</i>) Gene as a Driver

<div><p><i>Xenopus</i> is an excellent tetrapod model for studying normal and pathological motoneuron ontogeny due to its developmental morpho-physiological advantages. In mammals, the urotensin II-related peptide (<i>UTS2B</i>) gene is primarily expressed in motoneurons of the brainstem and the spinal cord. Here, we show that this expression pattern was conserved in <i>Xenopus</i> and established during the early embryonic development, starting at the early tailbud stage. In late tadpole stage, <i>uts2b</i> mRNA was detected both in the hindbrain and in the spinal cord. Spinal <i>uts2b⁺</i> cells were identified as axial motoneurons. In adult, however, the <i>uts2b</i> expression was only detected in the hindbrain. We assessed the ability of the <i>uts2b</i> promoter to drive the expression of a fluorescent reporter in motoneurons by recombineering a green fluorescent protein (GFP) into a bacterial artificial chromosome (BAC) clone containing the entire <i>X. tropicalis uts2b</i> locus. After injection of this construction in one-cell stage embryos, a transient GFP expression was observed in the spinal cord of about a quarter of the resulting animals from the early tailbud stage and up to juveniles. The GFP expression pattern was globally consistent with that of the endogenous <i>uts2b</i> in the spinal cord but no fluorescence was observed in the brainstem. A combination of histological and electrophysiological approaches was employed to further characterize the GFP⁺ cells in the larvae. More than 98% of the GFP⁺ cells expressed choline acetyltransferase, while their projections were co-localized with α-bungarotoxin labeling. When tail myotomes were injected with rhodamine dextran amine crystals, numerous double-stained GFP⁺ cells were observed. In addition, intracellular electrophysiological recordings of GFP⁺ neurons revealed locomotion-related rhythmic discharge patterns during fictive swimming. Taken together our results provide evidence that <i>uts2b</i> is an appropriate driver to express reporter genes in larval motoneurons of the <i>Xenopus</i> spinal cord.</p>