Zebrafish transgenic constructs label specific neurons in Xenopus laevis spinal cord and identify frog V0v spinal neurons

Abstract

A correctly functioning spinal cord is crucial for locomotion and communication between body and brain but there are fundamental gaps in our knowledge of how spinal neuronal circuitry is established and functions. To understand the genetic program that regulates specification and functions of this circuitry, we need to connect neuronal molecular phenotypes with physiological analyses. Studies using Xenopus laevis tadpoles have increased our understanding of spinal cord neuronal physiology and function, particularly in locomotor circuitry. However, the X. laevis tetraploid genome and long generation time make it difficult to investigate how neurons are specified. The opacity of X. laevis embryos also makes it hard to connect functional classes of neurons and the genes that they express. We demonstrate here that Tol2 transgenic constructs using zebrafish enhancers that drive expression in specific zebrafish spinal neurons label equivalent neurons in X. laevis and that the incorporation of a Gal4:UAS amplification cassette enables cells to be observed in live X. laevis tadpoles. This technique should enable the molecular phenotypes, morphologies and physiologies of distinct X. laevis spinal neurons to be examined together in vivo. We have used an islet1 enhancer to label Rohon-Beard sensory neurons and evx enhancers to identify V0v neurons, for the first time, in X. laevis spinal cord. Our work demonstrates the homology of spinal cord circuitry in zebrafish and X. laevis, suggesting that future work could combine their relative strengths to elucidate a more complete picture of how vertebrate spinal cord neurons are specified, and function to generate behavior.

Reconciling the functions of even-skipped interneurons during crawling, swimming, and walking

“…These neurons are integrated into zebrafish motor networks early during development and are recruited during spontaneous, whole-body bends [16], which resemble escape bends used in response to threats in older fish. Recent work from the Lewis lab has revealed that V0v neurons in frog embryos are also primarily ascending [17,18], which is a time of life when frogs engage axially-based escape movements [19,20]. These results are consistent with the idea of a conserved function for early-born Eve/Evx interneurons during powerful escape behaviors.…”

Evolution of lbx spinal cord expression and function

“…Plasmid DNA and transposase mRNA for microinjection was prepared as in (Juárez‐Morales et al, 2017; Kwan et al, 2007). Approximately 10 nl of a combination of plasmid DNA (60–80 ng/μl) and transposase mRNA (30 ng/μl) was injected into both blastomeres of 1–2‐cell stage zebrafish embryos.…”

Evolution oflbxspinal cord expression and function

“…Consistent with these conserved expression patterns, the types of neurons found in the spinal cord, and the functions of particular transcription factors in specifying these neuronal subtypes has also been highly conserved throughout vertebrate evolution (e.g. England et al, 2014;Gosgnach et al, 2006;Goulding and Pfaff, 2005;Juarez-Morales et al, 2017;Lewis, 2006;Li et al, 2004)}. However, most of these comparative analyses have so far been limited to ventral spinal cord and it is still unclear whether dorsal spinal cord neurons are as highly conserved.…”

“…Plasmid DNA and transposase mRNA for microinjection was prepared as in (Juarez-Morales et al, 2017;Kwan et al, 2007…”

“…Plasmid DNA and transposase mRNA for microinjection was prepared as in (Juarez-Morales et al, 2017;Kwan et al, 2007). Approximately 10 nl of a combination of plasmid DNA [60-80 ng/μL] and transposase mRNA [30 ng/μL] was injected into both blastomeres of 1-2-cell stage zebrafish embryos.…”