Evolution of lbx spinal cord expression and function

Abstract Ladybird homeobox (Lbx) transcription factors have crucial functions in muscle and nervous system development in many different animals. Amniotes have two Lbx genes, Lbx1 and Lbx2, but only Lbx1 is expressed in the spinal cord. In contrast, teleosts have three lbx genes, lbx1a, lbx1b and lbx2. In this study, we characterize the spinal cord expression of zebrafish lbx1a, lbx1b and lbx2 and show that each of these genes is expressed by distinct cell types. Our data suggest that lbx1a is expressed by dI4, dI5 and dI6 spinal interneurons, whereas lbx1b and lbx2 are primarily expressed in different spinal cord progenitor domains. We investigated the evolution of Lbx spinal cord expression patterns by examining Lbx1 and Lbx2 expression in the lesser spotted dogfish, Scyliorhinus canicula and Lbx1 expression in the tetrapod, Xenopus tropicalis. Our results suggest that zebrafish lbx1a spinal cord expression is conserved with that of Lbx1 in other vertebrates, whereas lbx1b spinal cord expression probably evolved in teleosts after the duplication of lbx1 into lbx1a and lbx1b. lbx2 spinal expression was probably acquired somewhere in the ray-finned lineage, as this gene is not expressed in the spinal cords of either amniotes or S. canicula. Consistent with its conserved spinal cord expression pattern, we also show that the spinal cord function of zebrafish lbx1a is conserved with mouse Lbx1. In zebrafish lbx1a mutants, there is a reduction of inhibitory spinal neurons and an increase in excitatory neurons, similar to the phenotype of mouse Lbx1 mutants. Interestingly, we also see a reduction of inhibitory spinal neurons in lbx1b mutants, although in this case there is not a corresponding increase in the number of excitatory neurons and lbx1a;lbx1b double mutants do not have a more severe spinal cord phenotype than lbx1a single mutants, suggesting that lbx1a and lbx1b do not act redundantly in spinal neuron development. This suggests that lbx1b and lbx1a may be required in succession for correct specification of inhibitory dI4 and dI6 interneurons, although only lbx1a is required for suppression of excitatory fates in these cells.