Juárez-Morales et al. Neural Development (2016)11:5 DOI 10.1186/s13064-016-0059-9 RESEARCH ARTICLE Open Access Evx1 and Evx2 specify excitatory neurotransmitter fates and suppress inhibitory fates through a Pax2- independent mechanism José L. Juárez-Morales1, Claus J. Schulte2, Sofia A. Pezoa1, Grace K. Vallejo1, William C. Hilinski1,3, Samantha J. England1, Sarah de Jager2 and Katharine E. Lewis1* Abstract Background: For neurons to function correctly in neuronal circuitry they must utilize appropriate neurotransmitters. However, even though neurotransmitter specificity is one of the most important and defining properties of a neuron we still do not fully understand how neurotransmitter fates are specified during development. Most neuronal properties are determined by the transcription factors that neurons express as they start to differentiate. While we know a few transcription factors that specify the neurotransmitter fates of particular neurons, there are still many spinal neurons for whichthetranscriptionfactorsspecifyingthis critical phenotype are unknown. Strikingly, all of the transcription factors that have been identified so far as specifying inhibitory fates in the spinal cord act through Pax2. Even Tlx1 and Tlx3, which specify the excitatory fates of dI3 and dI5 spinal neurons work at least in part by down-regulating Pax2. Methods: In this paper we use single and double mutant zebrafish embryos to identify the spinal cord functions of Evx1 and Evx2. Results: We demonstrate that Evx1 and Evx2 are expressed by spinal cord V0v cells and we show that these cells develop into excitatory (glutamatergic) Commissural Ascending (CoSA) interneurons. In the absence of both Evx1 and Evx2, V0v cells still form and develop a CoSA morphology. However, they lose their excitatory fate and instead express markers of a glycinergic fate. Interestingly, they do not express Pax2, suggesting that they are acquiring their inhibitory fate through a novel Pax2-independent mechanism. Conclusions: Evx1 and Evx2 are required, partially redundantly, for spinal cord V0v cells to become excitatory (glutamatergic) interneurons. These results significantly increase our understanding of the mechanisms of neuronal specification and the genetic networks involved in these processes. Keywords: Spinal cord, Interneuron, Zebrafish, Evx, Pax2, Glutamatergic, Neurotransmitter, CNS, Transcription factor, V0 Background question in developmental neuroscience. To answer this Hundreds of millions of people across the world are af- question, we need to identify how the functional proper- fected by neurological diseases and injuries. Understand- ties of distinct neurons are specified; since these proper- ing how functional neuronal circuitry is established in ties determine which circuits the neurons participate in, the vertebrate central nervous system (CNS) is essential the functional roles that the neurons have within those for developing better treatments for these conditions. circuits and the resulting outputs of the circuitry. The How neuronal circuitry develops is also a fundamental spinal cord is a powerful system for establishing funda- mental principles of neuronal fate specification, function * Correspondence: [email protected] and circuit assembly, as it is relatively simple and experi- 1Department of Biology, Syracuse University, 107 College Place, Syracuse, NY mentally tractable compared to the brain. This has en- 13244, USA abled considerable progress in establishing the functions Full list of author information is available at the end of the article © 2016 Juárez-Morales et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Juárez-Morales et al. Neural Development (2016)11:5 Page 2 of 20 of different ventral spinal cord interneurons in loco- an Evx1-negative sub-population called V0D cells. motor circuitry (e.g. [1–9]). However, we still know rela- These names reflect the fact that V0v cells form more tively little about how the functional properties of these ventrally than V0D cells (e.g. [23–28, 30–34]). Evx2 is cells are determined. expressed in a similar pattern to Evx1 in the mouse For neurons to function correctly they must CNS, suggesting that it may also be expressed by V0v synthesize and utilize correct neurotransmitters. cells. This is consistent with the observation that Within neuronal circuitry, if they inhibit rather than Evx2 spinal cord expression is lost in mouse Evx1 excite their synaptic partners, or vice versa, then the mutants [23]. However, co-expression of Evx1 and behaviors and functional outputs of those circuits will Evx2 in the mouse spinal cord has not yet been dem- be dramatically disturbed, and may give rise to patho- onstrated [24]. logical conditions. For example, disruptions in the In mammals, both V0v and V0D interneurons are cru- balance of excitatory and inhibitory neurons in the cial for correct left-right alternation during locomotion, CNS have been implicated in epilepsy, autism, Alzhei- with V0v cells in particular being required for hindlimb mer’s and many other neurological disorders (e.g. left-right alternation during fast locomotion [9, 34]. [10–13]). However, even though neurotransmitter spe- While the functions of V0 cells in specific behaviors cificity is one of the most important and defining have so far only been assayed in mouse, these cells have properties of a neuron we still do not fully under- highly conserved commissural axon trajectories in all stand how neurotransmitter fates are specified during animals examined so far ([23–28, 32, 33, 35, 36]; this development. paper), suggesting that their functional properties are Many neuronal properties are determined by the likely to be highly conserved across the vertebrate transcription factors that cells express as they start to lineage. However, when we started this work the differentiate. We already know a few transcription neurotransmitter phenotype of V0v cells had not been factors (e.g. Ptf1a, Lhx1, Lhx5, Lbx1, Pax2) that spe- identified. cify the inhibitory (GABAergic and/or glycinergic) In zebrafish, evx1 and evx2 are expressed in a similar fates of several subsets of spinal interneurons [14–18]. intermediate dorsal-ventral spinal cord position to that Strikingly, most of these transcription factors function in observed in other vertebrates [26, 32, 33], although dorsal spinal neurons and all of them act through Pax2 again, co-expression of these two genes has not previ- [14–21]. In contrast, we only know two transcription fac- ously been demonstrated. In this paper, we confirm that tors, Tlx1 and Tlx3, that are required for the specification evx1 and evx2 are co-expressed by V0v cells and we of excitatory (glutamatergic) fates and these are only show that V0v cells are glutamatergic and have a Com- expressed in dorsal dI3, dI5 and DILB cells [15, 16, 22]. missural Ascending (Comissural Secondary Ascending Interestingly, Tlx1 and Tlx3 determine the glutamatergic or CoSA) morphology. We also provide the first analysis fates of dI3 and dI5 cells at least in part by down- of evx1;evx2 double mutants in any vertebrate and the regulating Pax2 [15]. These results suggest that Pax2 is a first analysis of the spinal cord phenotype of evx2 mu- crucial player in neurotransmitter fate specification with tants. Significantly, we demonstrate that Evx1 and Evx2 its presence being required for inhibitory fates and its are required, partially redundantly, to specify the gluta- absence required for excitatory fates. However, we still matergic fates of V0v cells. Given that we know so little do not know which transcription factors regulate the about how excitatory fates are specified in the spinal neurotransmitter fates of many excitatory spinal cord and particularly the ventral spinal cord, these neurons, including those in the ventral spinal cord, findings add considerably to our understanding of CNS whose correct functional specification is essential for circuit development. locomotion. In the absence of both Evx1 and Evx2, V0v cells In this paper we identify two transcription factors, lose their glutamatergic fates but other functional Evx1 and Evx2, which are required for a subset of ex- characteristics like soma/cell body morphology and citatory fates in the ventral spinal cord. In mammals, axon trajectory are unchanged. In addition, and in the spinal cord expression of Evx1 and Evx2 is re- contrast to a previously described mouse Evx1 mutant stricted to a population of cells located in an inter- [23], these cells do not express markers of neighbor- mediate dorso-ventral position corresponding to V0 ing cell types. This suggests that V0v cells are not cells (e.g. [23–28]). V0 cells are post-mitotic cells that transfating into a different class of neuron; they have form from the p0 (Dbx1-positive, Nkx6.2-negative) just changed some of their functional properties. progenitor domain [23, 27–29]. These cells develop Strikingly, in evx1;evx2 double mutants V0v cells into interneurons that are important components of become inhibitory, but they do not express Pax2, locomotor circuitry and they can be subdivided into suggesting that they are acquiring their inhibitory an Evx1-positive sub-population called V0v cells and fates through a novel Pax2-independent mechanism. Juárez-Morales et al. Neural Development (2016)11:5 Page 3 of 20 Methods The evx2sa140 mutation creates a BfaI recognition site. A Ethics approval genomic region flanking the mutation site was PCR ampli- All zebrafish experiments in this research were approved fied using the following conditions: 94 °C for 60 s, followed either by the UK Home Office or by the Syracuse Uni- by 5 cycles of 92 °C for 30 s, 54 °C for 30 s, 72 °C for 60 s; versity IACUC committee.