Lmx1b is required for the glutamatergic fates of a subset of spinal cord neurons

Abstract

BackgroundAlterations in neurotransmitter phenotypes of specific neurons can cause imbalances in excitation and inhibition in the central nervous system (CNS), leading to diseases. Therefore, the correct specification and maintenance of neurotransmitter phenotypes is vital. As with other neuronal properties, neurotransmitter phenotypes are often specified and maintained by particular transcription factors. However, the specific molecular mechanisms and transcription factors that regulate neurotransmitter phenotypes remain largely unknown.MethodsIn this paper we use single mutant, double mutant and transgenic zebrafish embryos to elucidate the functions of Lmx1ba and Lmx1bb in the regulation of spinal cord interneuron neurotransmitter phenotypes.ResultsWe demonstrate that lmx1ba and lmx1bb are both expressed in zebrafish spinal cord and that lmx1bb is expressed by both V0v cells and dI5 cells. Our functional analyses demonstrate that these transcription factors are not required for neurotransmitter fate specification at early stages of development, but that in embryos with at least two lmx1ba and/or lmx1bb mutant alleles there is a reduced number of excitatory (glutamatergic) spinal interneurons at later stages of development. In contrast, there is no change in the numbers of V0v or dI5 cells. These data suggest that lmx1b-expressing spinal neurons still form normally, but at least a subset of them lose, or do not form, their normal excitatory fates. As the reduction in glutamatergic cells is only seen at later stages of development, Lmx1b is probably required either for the maintenance of glutamatergic fates or to specify glutamatergic phenotypes of a subset of later forming neurons. Using double labeling experiments, we also show that at least some of the cells that lose their normal glutamatergic phenotype are V0v cells. Finally, we also establish that Evx1 and Evx2, two transcription factors that are required for V0v cells to acquire their excitatory neurotransmitter phenotype, are also required for lmx1ba and lmx1bb expression in these cells, suggesting that Lmx1ba and Lmx1bb act downstream of Evx1 and Evx2 in V0v cells.ConclusionsLmx1ba and Lmx1bb function at least partially redundantly in the spinal cord and three functional lmx1b alleles are required in zebrafish for correct numbers of excitatory spinal interneurons at later developmental stages. Taken together, our data significantly enhance our understanding of how spinal cord neurotransmitter fates are regulated.

Tal1, Gata2a, and Gata3 Have Distinct Functions in the Development of V2b and Cerebrospinal Fluid-Contacting KA Spinal Neurons

“…To assess expression of Caspase3, rabbit Anti-Activated Caspase-3 (Fisher Scientific/BD, BDB559565, 1:500) was used as described previously (Hilinski et al, 2016 ). Twenty-four hours embryos were fixed in 4% PFA at 4°C overnight, washed 3 times in PBST, permeabilized with acetone for 20 min at −20°C and then washed 3 × 5 min with PBS.…”

“…We identified somites 6–10 in each embryo and counted the number of labeled cells in that stretch of the spinal cord. We adjusted the focal plane as we examined the embryo to count cells at all medial/lateral positions (both sides of the spinal cord; also see Batista and Lewis, 2008 ; Batista et al, 2008 ; England et al, 2011 ; Hilinski et al, 2016 ; Juárez-Morales et al, 2016 ). Cell counts for fluorescently-labeled cells were performed by analyzing all focal planes in a confocal stack of the appropriate region of the spinal cord.…”

“…Most studies so far, suggest that the development of distinct spinal neurons is regulated by the transcription factors that each cell type express. For example, some transcription factors control when cells differentiate, others determine the overall identity of the cell and some specify particular functional properties such as axon trajectory, neurotransmitter phenotype, and/or expression of particular neuropeptides (e.g., Moran-Rivard et al, 2001 ; Gross et al, 2002 ; Muller et al, 2002 ; Lanuza et al, 2004 ; Sapir et al, 2004 ; Cheng et al, 2005 ; Pillai et al, 2007 ; Batista and Lewis, 2008 ; Hilinski et al, 2016 ; Juárez-Morales et al, 2016 ). However, for many classes of spinal neurons, including several of those involved in locomotor circuitry, we still don't know which transcription factors regulate these different aspects of specification and differentiation.…”

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

“…In contrast, zebrafish are a powerful model system for investigating how spinal cord neurons are specified because loss‐of‐function and gain‐of‐function experiments are more easy to perform using mutant lines and antisense reagents (e.g., Lewis and Eisen, ; Varga et al, ; Lewis and Eisen, ; Lewis and Eisen, ; Lewis et al, ; Gribble et al, ; Batista and Lewis, ; Batista et al, ; Bonner et al, ; Gribble et al, ; Yang et al, ; England et al, ; Hilinski et al, ; Juárez‐Morales et al, ). Zebrafish embryos are also transparent making it relatively easy to identify genomic enhancers that drive expression in particular populations of zebrafish neurons (e.g., Higashijima et al, ; Bohm et al, ; Juárez‐Morales et al, ) and use these to correlate gene expression with neuronal morphology (e.g., Kimura et al, ; Batista et al, ; Satou et al, ; Satou et al, ; Juárez‐Morales et al, ).…”

Hmx3a has essential functions in zebrafish spinal cord, ear and lateral line development

“…We identified somites 6-10 in each embryo and counted the number of labeled cells in that stretch of the spinal cord. We adjusted the focal plane as we examined the embryo to count cells at all medial/lateral positions (both sides of the spinal cord; also see England et al, 2011;Hilinski et al, 2016;Juárez-Morales et al, 2016).…”