Abstract
Xenopus 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 (UTS2B) gene is primarily expressed in motoneurons of the brainstem and the spinal cord. Here, we show that this expression pattern was conserved in Xenopus and established during the early embryonic development, starting at the early tailbud stage. In late tadpole stage, uts2b mRNA was detected both in the hindbrain and in the spinal cord. Spinal uts2b+ cells were identified as axial motoneurons. In adult, however, the uts2b expression was only detected in the hindbrain. We assessed the ability of the uts2b 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 X. tropicalis uts2b 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 uts2b 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 uts2b is an appropriate driver to express reporter genes in larval motoneurons of the Xenopus spinal cord.
Highlights
Generation of transgenic lines with promoter-specific fluorescent reporter proteins has significantly advanced neurobiological research by enabling the visualization of neuronal subsets in vivo
The first uts2b signal could be detected in the rostral spinal cord from stage 24, pre-hatching early tailbud embryos (Fig. 2A)
A few uts2b+ cells were still observable in the spinal cord of a small proportion of stage 66 juveniles, but no longer in adults, whereas the uts2b gene was still strongly expressed in the hindbrain during
Summary
Generation of transgenic lines with promoter-specific fluorescent reporter proteins has significantly advanced neurobiological research by enabling the visualization of neuronal subsets in vivo. For genetic engineering of such animals, mice have long been the most frequently used experimental vertebrate model [1] Organisms such as zebrafish and Xenopus have recently emerged as alternative systems and are being widely exploited to study human neurological diseases as well as to screen for potential therapeutics [2,3,4,5]. Several features of these species make them amenable for neurodevelopmental and neurophysiological investigations including their rapid development rate as free-living larvae, their transparency allowing easy visualization of internal structures and cells in live animals, and the general organization of their central nervous system (CNS) which is quite similar to other vertebrate species, including human. This proximity is especially important when studying the neural control of processes that have no genuine equivalent in fish, such as legged terrestrial locomotion [12]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
