Evx1 and Evx2 specify excitatory neurotransmitter fates and suppress inhibitory fates through a Pax2-independent mechanism
© Juárez-Morales et al. 2016
Received: 14 December 2015
Accepted: 4 February 2016
Published: 19 February 2016
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 which the transcription factors specifying this 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.
In this paper we use single and double mutant zebrafish embryos to identify the spinal cord functions of Evx1 and Evx2.
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.
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.
KeywordsSpinal cord Interneuron Zebrafish Evx Pax2 Glutamatergic Neurotransmitter CNS Transcription factor V0
Hundreds of millions of people across the world are affected by neurological diseases and injuries. Understanding how functional neuronal circuitry is established in the vertebrate central nervous system (CNS) is essential for developing better treatments for these conditions. How neuronal circuitry develops is also a fundamental question in developmental neuroscience. To answer this question, we need to identify how the functional properties of distinct neurons are specified; since these properties determine which circuits the neurons participate in, the functional roles that the neurons have within those circuits and the resulting outputs of the circuitry. The spinal cord is a powerful system for establishing fundamental principles of neuronal fate specification, function and circuit assembly, as it is relatively simple and experimentally tractable compared to the brain. This has enabled considerable progress in establishing the functions of different ventral spinal cord interneurons in locomotor circuitry (e.g. [1–9]). However, we still know relatively little about how the functional properties of these cells are determined.
For neurons to function correctly they must synthesize and utilize correct neurotransmitters. Within neuronal circuitry, if they inhibit rather than excite their synaptic partners, or vice versa, then the behaviors and functional outputs of those circuits will be dramatically disturbed, and may give rise to pathological conditions. For example, disruptions in the balance of excitatory and inhibitory neurons in the CNS have been implicated in epilepsy, autism, Alzheimer’s and many other neurological disorders (e.g. [10–13]). 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.
Many neuronal properties are determined by the transcription factors that cells express as they start to differentiate. We already know a few transcription factors (e.g. Ptf1a, Lhx1, Lhx5, Lbx1, Pax2) that specify the inhibitory (GABAergic and/or glycinergic) fates of several subsets of spinal interneurons [14–18]. Strikingly, most of these transcription factors function in dorsal spinal neurons and all of them act through Pax2 [14–21]. In contrast, we only know two transcription factors, Tlx1 and Tlx3, that are required for the specification of excitatory (glutamatergic) fates and these are only expressed in dorsal dI3, dI5 and DILB cells [15, 16, 22]. Interestingly, Tlx1 and Tlx3 determine the glutamatergic fates of dI3 and dI5 cells at least in part by down-regulating Pax2 . These results suggest that Pax2 is a crucial player in neurotransmitter fate specification with its presence being required for inhibitory fates and its absence required for excitatory fates. However, we still do not know which transcription factors regulate the neurotransmitter fates of many excitatory spinal neurons, including those in the ventral spinal cord, whose correct functional specification is essential for locomotion.
In this paper we identify two transcription factors, Evx1 and Evx2, which are required for a subset of excitatory fates in the ventral spinal cord. In mammals, the spinal cord expression of Evx1 and Evx2 is restricted to a population of cells located in an intermediate dorso-ventral position corresponding to V0 cells (e.g. [23–28]). V0 cells are post-mitotic cells that form from the p0 (Dbx1-positive, Nkx6.2-negative) progenitor domain [23, 27–29]. These cells develop into interneurons that are important components of locomotor circuitry and they can be subdivided into an Evx1-positive sub-population called V0v cells and an Evx1-negative sub-population called V0D cells. These names reflect the fact that V0v cells form more ventrally than V0D cells (e.g. [23–28, 30–34]). Evx2 is expressed in a similar pattern to Evx1 in the mouse CNS, suggesting that it may also be expressed by V0v cells. This is consistent with the observation that Evx2 spinal cord expression is lost in mouse Evx1 mutants . However, co-expression of Evx1 and Evx2 in the mouse spinal cord has not yet been demonstrated .
In mammals, both V0v and V0D interneurons are crucial for correct left-right alternation during locomotion, with V0v cells in particular being required for hindlimb left-right alternation during fast locomotion [9, 34]. While the functions of V0 cells in specific behaviors have so far only been assayed in mouse, these cells have highly conserved commissural axon trajectories in all animals examined so far ([23–28, 32, 33, 35, 36]; this paper), suggesting that their functional properties are likely to be highly conserved across the vertebrate lineage. However, when we started this work the neurotransmitter phenotype of V0v cells had not been identified.
In zebrafish, evx1 and evx2 are expressed in a similar intermediate dorsal-ventral spinal cord position to that observed in other vertebrates [26, 32, 33], although again, co-expression of these two genes has not previously been demonstrated. In this paper, we confirm that evx1 and evx2 are co-expressed by V0v cells and we show that V0v cells are glutamatergic and have a Commissural Ascending (Comissural Secondary Ascending or CoSA) morphology. We also provide the first analysis of evx1;evx2 double mutants in any vertebrate and the first analysis of the spinal cord phenotype of evx2 mutants. Significantly, we demonstrate that Evx1 and Evx2 are required, partially redundantly, to specify the glutamatergic fates of V0v cells. Given that we know so little about how excitatory fates are specified in the spinal cord and particularly the ventral spinal cord, these findings add considerably to our understanding of CNS circuit development.
In the absence of both Evx1 and Evx2, V0v cells lose their glutamatergic fates but other functional characteristics like soma/cell body morphology and axon trajectory are unchanged. In addition, and in contrast to a previously described mouse Evx1 mutant , these cells do not express markers of neighboring cell types. This suggests that V0v cells are not transfating into a different class of neuron; they have just changed some of their functional properties. Strikingly, in evx1;evx2 double mutants V0v cells become inhibitory, but they do not express Pax2, suggesting that they are acquiring their inhibitory fates through a novel Pax2-independent mechanism.
All zebrafish experiments in this research were approved either by the UK Home Office or by the Syracuse University IACUC committee.
Zebrafish husbandry and fish lines
Zebrafish (Danio rerio) were maintained on a 14 h light/10 h dark cycle at 28.5 °C and embryos were obtained from natural, paired and/or grouped spawnings of wild-type (WT) adults (AB, TL or AB/TL hybrids), identified heterozygous or homozygous Tg(slc17a6:EGFP) (used to be called Tg(vGlut2a:EGFP); [36, 37]), Tg(evx1:EGFP) SU1 or Tg(evx1:EGFP) SU2 adults, double heterozygous evx1 i232 ;evx2 sa140 mutants or double heterozygous evx1 i232 ;evx2 sa140 mutants that also carried one of the Tg(evx1:EGFP) lines (see below). Embryos were reared at 28.5 °C and staged by hours post fertilization (h), days post or prim staging/or prim staging .
Genotyping of mutant alleles was performed on both live adults and fixed embryos using DNA extracted from fin clips and dissected heads respectively. Fin clipping and evx1 genotyping were performed as in . To extract DNA from embryos, heads were removed in 80 % glycerol / 20 % PBS with insect pins. Embryonic trunks were stored in 70 % glycerol / 30 % PBS at 4 °C for later analysis. Heads were incubated in 50 μL of Proteinase K buffer solution (1 M Tris-HCl, pH 8.2; 0.5 M EDTA; 1 M NaCl; 20 % SDS; 10 mg/ml Proteinase K) for 2 h at 55 °C. Proteinase K was heat inactivated at 100 °C for 10 min and tubes were centrifuged for 20 min at 13,000 rpm. DNA was precipitated with 100 % ethanol at -20 °C overnight, centrifuged to pellet the DNA and re-suspended in 20 μL of water. 2 μL of DNA was used for each PCR.
The evx2 sa140 mutation creates a BfaI recognition site. A genomic region flanking the mutation site was PCR amplified using the following conditions: 94 °C for 60 s, followed by 5 cycles of 92 °C for 30 s, 54 °C for 30 s, 72 °C for 60 s; followed by 40 cycles of 92 °C for 20 s, 52 °C for 30 s, and 72 °C for 60 s, followed by a final extension at 72 °C for 5 min. Forward primer: GTAATGCGATCCCAAAACG. Reverse primer: TTATTTTAGATTTGGCAATGG.
PCR products were digested with BfaI and analysed on a 1 % agarose gel. The WT product is 454 bp, whereas the mutant product is cut into 218 bp and 236 bp fragments. These fragments are close enough in size that they are usually detected as one band on an agarose electrophoresis gel (Fig. 1b).
Creation of Tg(evx1:EGFP) lines
Potential evx1 enhancer regions were identified by multispecies sequence comparisons using the global alignment program Shuffle-LAGAN  and visualized using VISTA . Zebrafish (Danio rerio) evx1 genomic sequence (ENSDARG00000099365) and orthologous sequences from human (ENSG00000106038) and mouse (ENSMUSG00000005503) were obtained from Ensembl (http://www.ensembl.org). Zebrafish sequence was used as the baseline and annotated using exon/intron information from Ensembl. The alignment was performed using a 100 bp window and a cutoff score of 70 % identity. A multi-species comparison of approximately 23Kb of Danio rerio genomic DNA sequence containing evx1 and extending 5 Kb into flanking regions revealed high conservation in both coding and non-coding sequences among compared species. We identified three Conserved Non-coding Elements (CNEs) located 5’ and 3’ to evx1. The first is located 79 bp upstream of zebrafish evx1 coding sequence and extends over 100 bp. The other two are located 3' to evx1. One is 2354 bp downstream of the stop codon and is 184 bp long whereas the other is 2979 bp downstream of the stop codon and extends over 140 bp (Fig. 1d). One amplicon encompassing these two 3’ CNEs and the intervening sequence (1.34 Kb) was PCR-amplified from genomic DNA. Forward primer: AAGATTGGAATGGAATGTCT. Reverse primer: GCATTTTCGCCTTTGCATCA.
The Tg(evx1:EGFP) SU1 line was generated by cloning this 3’ CNE amplicon into the pDONRTMP4-P1R vector from Invitrogen using Gateway technology [42, 43]. The final construct was assembled using the pENTRbasegfp plasmid and the pCSDest2 vector . This resulted in a vector containing Tol2:1.3Kb 3’ zfish evx1:ßcarp minimal promoter:EGFP:Tol2.
The same 3’ CNE amplicon was used to generate the Tg(evx1:EGFP) SU2 line. In this case, the GAL4VP16;UAS-EGFP cassette was taken from the pBGAL4VP16;UAS-EGFP plasmid  and cloned into a middle entry vector from Invitrogen [42, 43]. An oligonucleotide containing the cfos minimal promoter sequence  plus 17 bp of the 5’ arm of GAL4 was synthesized and used with a RVeGFPAttb2 primer to PCR amplify the GAL4VP16;UAS-EGFP cassette.
FWcfosGAL4VP16 primer: CACTCATTCATAAAACGCTTGTTATAAAAGCAGTGGCTGCGGCGCCTCGTACTCCAACCGCATCTGCAGCGAGCAACTGAGAAGCCAAGACTGAGCCGGCGGCCTTTGTACAAAAAAGCAG
RVeGFPAttb2 primer: GGGGACCACTTTGTACAAGAAAGCTGGGTTTACTTGTACAGCTCGTCCA.
This PCR product was used to generate a second PCR product using the primer FWattB1cfos: GGGGACAAGTTTGTACAAAAAAGCAGGCTCACTCATTCATAAAATCGCTT and the RVeGFPAttb2 primer.
The final amplicon was cloned into pDONR™221 using gateway technology. This middle entry vector was used to generate a final vector containing Tol2:1.3Kb 3’ zfish evx1:cfos minimal promoter:GAL4VP16;UAS-EGFP:Tol2.
Each of these plasmids was separately co-injected with transposase mRNA into 1-2 cell embryos as described by . Embryos were raised to adulthood and out-crossed to identify founders. In each case one stable transgenic line was generated. The Tg(evx1:EGFP) SU2 or Tg(1.3 kb evx1:cfos:GAL4-UAS:EGFP) line has the advantage that it contains a GAL4-UAS cassette to amplify EGFP expression. This facilitates visualization of axons. However, this line has a slightly more variegated expression than the Tg(evx1:EGFP) SU1 or Tg(1.3Kb evx1:ßcarp:EGFP) line, presumably because of stochastic silencing of the construct due to the GAL4-UAS sequences . In contrast the Tg(evx1:EGFP) SU1 line labels all V0v cells more consistently, but the EGFP expression is slightly weaker and we were never able to obtain evx1 -/- ;evx2 -/- double mutant embryos that contained this transgene, even though we could obtain evx1 -/- ;evx2 +/+ and evx1 -/- ;evx2 +/- embryos. This suggests that the Tg(1.3Kb evx1:ßcarp:EGFP) construct integrated close to the WT evx2 allele.
Approximately 5 nl of a 1:1 combination of two Evx2 ATG Morpholino antisense oligonucleotides (MOs) at 1.25 mg/ml each were injected into 1-2 cell embryos (evx2-1 MO: TTCTTTTCTTATCCTCTCCATCATG; evx2-2 MO: AATCCAAAGTCCCAGGGCTGGTGCT). In all cases, we confirmed that the MOs had completely knocked down Evx2 using immunohistochemistry for zebrafish Evx2.
Expression profiling V0v cells
To determine which neurotransmitters V0v cells express and to identify additional transcription factors expressed by these cells, different combinations of spinal cord and trunk cells were extracted from live transgenic zebrafish embryos at 27 h using fluorescence activated cell-sorting (FACS). Prior to FACS, embryos were prim-staged, deyolked, dissected and dissociated as in [49, 50]. In all cases, the heads were removed to ensure that only trunk or spinal cord cells were collected. Pure populations of cells were obtained using combinations of the following transgenic lines: Tg(elav13:EGFP), Tg(evx1:EGFP) SU1 , Tg(pax2a:GFP), Tg(Xla.Tubb:DsRed (formerly Tg(NBT:DsRed)), Tg(vsx2:DsRed) and Tg(gata1:GFP) [8, 51–54]. Trunk samples correspond to FAC-sorted trunk cells (spinal cord and other tissues). All neuron samples are EGFP-positive cells from Tg(elav13:EGFP) trunks. V0v neurons are EGFP-positive cells from Tg(evx1:EGFP) SU1 trunks. V1 neurons are double-positive EGFP-positive, DsRed-positive cells from Tg(pax2a:GFP);Tg(Xla.Tubb:DsRed) trunks. V2a neurons are double-positive DsRed-positive, EGFP-positive cells from Tg(vsx2:DsRed);Tg(elavl3:EGFP) trunks. V2b + KA neurons are double-positive EGFP-positive, DsRed-positive cells from Tg(gata1:GFP);Tg(Xla.Tubb:DsRed) trunks. Total RNA was extracted using an RNeasy Micro Kit (Qiagen, 74004). RNA quality and quantity was assayed on an Agilent 2100 Bioanalyser (RNA 6000 Pico Kit, Agilent, 5067-1513), before converting to fluorescently-labelled cDNA (Ovation Pico WTA System V2, Pico, 3302) and hybridizing to a custom-designed Agilent microarray (EMBL Genomics Core, Heidelberg). Details of this microarray will be described elsewhere, along with the characterization of additional genes identified from these analyses. Data pre-processing and normalization was performed using Bioconductor software (https://www.bioconductor.org/). Two-class eBayes and three-class ANOVA analyses were performed using GEPAS software (Tárraga, (2008)). All reported statistics were corrected for multiple testing (Benjamini and Hochberg (1995)).
in situ hybridization
Embryos were fixed in 4 % paraformaldehyde and single and double in situ hybridizations were performed as previously described [55, 56]. RNA probes were prepared using the following templates, dbx1a and dbx1b , evx1 , evx2 , eve1 , pax2a, pax2b, pax8  and eng1b . To determine neurotransmitter phenotypes we used probes for genes that encode proteins that transport or synthesize specific neurotransmitters. A mixture of two probes (glyt2a and glyt2b) for slc6a5 (previously called glyt2) was used to label glycinergic cells [61, 62]. slc6a5 encodes for a glycine transporter necessary for glycine reuptake and transport across the plasma membrane. A mixture of two probes to gad1b (previously called gad67, probes used to be called gad67a and gad67b) and one probe to gad2 (previously called gad65) was used to label GABAergic cells [61, 62]. gad1b and gad2 encode for glutamic acid decarboxylases, necessary for the synthesis of GABA from glutamate. A mixture of slc17a6b (formerly called vglut2.1) and slc17a6a (formerly called vglut 2.2) probes was used to label glutamatergic cells [61, 62]. These genes encode proteins responsible for transporting glutamate to the synapse. In all of these cases, a mix of equal concentrations of the relevant probes was used [61, 62]. We also used slc32a1 (formerly called viaat), which encodes for a vesicular inhibitory amino acid transporter, to label all inhibitory cells .
The DNA template for the skor2 (ZDB-GENE-060825-57) probe was generated by PCR-amplifying the 3’ region of skor2 from cDNA using a reverse primer containing a T3 promoter sequence at the 5’ end (indicated in italics below). Total RNA was extracted by homogenizing 50-100 mg of 27hpf wild-type zebrafish embryos in 1 mL of TRIzol reagent (Ambion, 15596-026). cDNA was synthesized using Bio-Rad iScript Reverse Transcription Supermix kit (Bio-Rad, 170-8891). A 50 μL PCR was assembled containing 5 μL cDNA and 1 unit of Phusion High-Fidelity DNA Polymerase (NEB, M0530L). PCR conditions were: 94 °C for 3 min followed by 35 cycles of 94 °C for 30 s, 56.5 °C for 30 s, 72 °C for 1.5 min and then a final extension step of 72 °C for 10 min. PCR product was purified by phenol:chloroform extraction. Forward primer: CGCAAGACGCTTTTTATCC
Reverse primer: AATTAACCCTCACTAAAGGGAAAATGGAGAGCTGCCTTTCAG.
ZFIN Identification numbers are provided for all genes in Additional file 1: Table S2.
Primary antibodies used were rabbit anti-Evx2 (a kind gift from Dr Higashijima, described in Satou et al., 2012, raised against the first 168 amino acids of zebrafish Evx2, a region with no significant homology to zebrafish Evx1, 1:300), mouse anti-GFP (Roche Applied Science, 11814460001, 1:500), rabbit anti-GFP (Molecular Probes A6465, 1:500) and mouse anti-Pax2 (Covance PRB-276P 1:300). The Pax2 antibody recognizes both Pax2a and Pax2b in zebrafish . Antibodies used for fluorescent in situ hybridization were mouse anti-Dig (Jackson ImmunoResearch 200-002-156, 1:5000) and rabbit anti-Flu (Invitrogen A889, 1:2500). These were detected with Invitrogen Tyramide kits #12 and #5. Secondary antibodies used were Alexa Fluor 568 goat anti-rabbit (Molecular Probes A11036, 1:500), Alexa Fluor 488 goat anti-rabbit (Molecular Probes A11034, 1:500) and Alexa Fluor 488 goat anti-mouse (Molecular Probes A11029, 1:500).
Embryos for immunohistochemistry were treated with acetone for 15 min (24 h embryos) or 20 min (30 h embryos) to permeabilize them, washed for 5 min in distilled water, then washed 2 x 10 min in PBS. Embryos were treated with Image-iT Signal Enhancer (Invitrogen, I36933) for 30 min, then incubated in block solution (2 % goat serum, 1 % BSA, 10 % DMSO and 0.5 % Triton) for 1 h at room temperature followed by incubation in primary antibody in fresh block solution at 4 °C overnight. Embryos were washed with PBT (PBS + 0.1 % Triton) for 2 h at room temperature and incubated with secondary antibody in block solution at 4 °C overnight. Embryos were then washed with PBT for at 2 h at room temperature and stored in 2 % DABCO (Acros Organics, AC11247-1000).
For 3,3’- diaminobenzidine (DAB) staining, after incubation with primary antibody, samples were incubated in fresh blocking solution with goat anti-rabbit IgG (Covance SMI-5030C, 1:200) at 4 °C overnight. Embryos were then washed with PBT for 2 h and incubated with rabbit PAP (Covance SMI-4010 L, 1:200) in block solution at 4 °C overnight. Embryos were then washed in PBT for 2 h. Staining was performed using Sigma Fast 3,3’- diaminobenzidine tablets (Sigma, D4293).
Embryos were mounted in 70 % glycerol, 30 % PBS and DIC pictures were taken using an AxioCam MRc5 camera mounted on a Zeiss Axio Imager M1 compound microscope. Fluorescent images were taken on a Zeiss LSM 710 confocal microscope. Images were processed using Adobe Photoshop software (Adobe, Inc) and Image J software (Abramoff et al., 2004). In some cases different focal planes were merged to show labeled cells at different medial lateral positions in the spinal cord.
Cell counts and statistics
In all cases, cells counts are for both sides of a five-somite length of the spinal cord adjacent to somites 6-10. Most values are an average of at least 5 embryos. Exceptions are the skor2 + Tg(evx1:EGFP) double-labeling experiments, the skor2 + Tg(slc17a6:EGFP) double-labeling experiments and the pax2a and eng1b in situ hybridization results. In all of these cases 4 embryos were counted. Results were analyzed using the student’s t-test; Error bars indicate standard deviation.
Zebrafish V0v cells express evx1 and evx2
In mouse, Evx1 is expressed in V0v cells and while double-labeling experiments have not yet been performed, the data suggest that Evx2 is probably co-expressed by these same cells [23, 24, 27, 28]. Previous reports described evx1 and evx2 expression in a similar region of zebrafish spinal cord [32, 33] but didn’t determine whether these genes are co-expressed or the specific cell types that express them.
V0 cells develop from the p0 progenitor domain, which expresses dbx1 [28, 63]. Therefore, to confirm that evx1/2-expressing cells are V0v cells we performed EGFP immunohistochemistry and in situ hybridization for dbx1a and dbx1b in Tg(evx1:EGFP) SU1 embryos. We found that zebrafish evx genes are expressed lateral to cells expressing both of these dbx1 genes, as would be predicted for cells developing from the p0 domain. In addition, dbx1a and dbx1b expression persists in some EGFP-positive cells (Fig. 1h), suggesting that these genes continue to be expressed by V0v cells for a short while after they become post-mitotic.
Zebrafish V0v cells develop into commissural ascending interneurons
V0v cells are glutamatergic
Consistent with these analyses, when we FAC-sorted and expression profiled EGFP-labeled V0v cells using the Tg(evx1:EGFP) SU1 line we found that these cells express markers of glutamatergic fates (slc17a6a (formerly called vglut2.2) and slc17a6b (formerly called vglut2.1)) and do not express either glycinergic markers (slc6a9 (formerly called glyt1) or slc6a5 (formerly called glyt2)) or GABAergic markers (gad1b or gad2) (Fig. 5d & e).
Zebrafish V0v cells do not express Pax2
evx2 sa140 is a null allele
To identify the functions of evx1 and evx2 in zebrafish V0v cells we used evx1 and evx2 mutants (see Methods). Our previous analyses suggest that evx1 i232 is a null allele . The evx2 sa140 mutation introduces a premature stop codon just before the homeodomain, suggesting that if a truncated protein is synthesized it will have no DNA binding activity. However, it is possible that a truncated protein might retain some function in the embryo. To determine if any Evx2 protein is made in mutant embryos, we used an Evx2 antibody that was made against the first 168 amino acids of zebrafish Evx2 (which corresponds exactly to the region upstream of the premature stop codon in the evx2 sa140 mutant allele). We found that all of the WT (21/51) and heterozygous embryos (20/51) had Evx2 antibody staining but all of the homozygous mutants (10/51) did not (Fig. 1c). This strongly suggests that the evx2 mutant allele does not produce any protein and is a null allele.
evx2 sa140 homozygous mutants are not viable
Unlike the zebrafish evx1 mutant, which is homozygous viable , we never identified an adult evx2 homozygous mutant (n = 262 fish from incrosses of heterozygous evx2 fish, P < 0.0001 using chi-squared test). However, we did obtain fish homozygous for evx1 and heterozygous for evx2 (17 fish identified from a total of 191 fish from incrosses of heterozygous double mutants; P = 0.13 using chi-squared test). Our analyses of incrosses from identified evx2 heterozygous fish suggest that evx2 mutant embryos have no obvious morphological defects for the first few days of development but that most of them die by larval stages (see Additional file 1: Results).
V0v cells still form in evx1;evx2 double mutants
In mouse Evx1 mutants, expression of Evx2 is lost and there is an increase in the number of cells expressing the V1 marker En1. In addition, many of the cells that would normally have expressed Evx1 develop axon trajectories similar to V1 cells. Most strikingly their axons change from being commissural to ipsilateral . This suggests that in the absence of Evx1, most mouse V0v cells transfate to V1 cells.
Number of cells expressing particular genes and proteins in WT and mutant embryos
33.0 + /-2.0
31.0 + /-3.3
31.3 + /-2.5
17.4 + /-2.6
28.6 + /-1.6
22.4 + /-50
13.5 + /-3.5
33.6 + /-5.2
23.0 + /-4.6
43.3 + /-2.5
43.6 + /-2.0
43.5 + /-4.0
37.2 + /-6.0
63.0 + /-3.0
62.0 + /-2.5
63.0 + /-2.5
65.2 + /-2.0
89.0 + /-10.0
71.3 + /-4.3
74.6 + /-8.0
60.8 + /-10
157.4 + /-8.0
157.3 + /-10.0
158.2 + /-4.0
178.6 + /-7.0
50.0 + /-3.0
49.0 + /-4.0
51.6 + /-2.6
50.6 + /-2.2
81.0 + /-3.3
93.0 + /-9.0
87.6 + /-4.5
109 + /-10.0
50.6 + /-2.6
50.4 + /-2.0
49.5 + /-30
50.8 + /-2.5
40.3 + /-3.8
39.8 + /-5.7
36.9 + /-6.9
38.8 + /-5.3
24.3 + /-3.0
15.0 + /-5.0
19.8 + /-4.0
skor2 Total cell counts
46.7 + /-3.7
38.7 + /-3.3
42.4 + /-1.7
22.6 + /-1.7
Consistent with this persistence of V0v cells, we also found no change in the number of eng1b-expressing spinal cord cells in evx1 and evx2 single or double mutants compared to WT embryos at 24 h. If anything, we observed a slight reduction in the number of eng1b cells in the double mutants, although this was not statistically significant (Fig. 2f & o, Table 1). To further confirm that V0v cells were not adopting a V1 fate and turning on eng1b expression, we repeated this experiment at 30 h. We still found no change in eng1b expression in either single or double mutants when compared to WT embryos (Fig. 2h & p, Table 1).
Taken together, these results suggest that V0v cells are not transfating into V1 cells in zebrafish, even in the absence of both Evx1 and Evx2. Instead at least most of these cells are maintaining their V0v identities. In addition, these data suggest that Evx1 and Evx2 act partially redundantly to maintain each other’s expression, although only evx2 expression requires Evx1/Evx2 activity as more than half of V0v cells still express evx1 in evx1;evx2 double mutants.
Evx1 and Evx2 are required for skor2 expression in V0v cells
Evx1 and Evx2 are required to specify the glutamatergic fates of V0v cells
V0v cells become inhibitory in evx1;evx double mutant embryos
Given that V0v cells lose their excitatory phenotype in evx1;evx2 double mutants, we asked whether they are acquiring an inhibitory neurotransmitter fate instead. When we examined expression of slc32a1, which is expressed by all inhibitory neurons [8, 68, 69], there was no significant difference in the number of cells expressing this gene between either of the single mutants and WT embryos (Fig. 8e, g, h & p, Table 1). However, interestingly, there was a significant increase (approximately 21 cells) in the number of slc32a1-expressing cells in evx1;evx2 double mutants (Fig. 8f & p, Table 1). This suggests that Evx1 and Evx2 act redundantly to repress the inhibitory fate in V0v cells.
To further confirm that V0v cells are switching to an inhibitory fate, we performed in situ hybridization for slc32a1 plus immunohistochemistry for EGFP in embryos from a cross of double heterozygous parents that carry the Tg(evx1:EGFP) SU2 transgene. In WT embryos we see no co-expression of slc32a1 and EGFP (Fig. 5c). However, in double mutant embryos most V0v cells express slc32a1 (77 % of EGFP-positive V0v cells (30/39 cells counted in 2 embryos); Fig. 5f).
To determine whether V0v cells are becoming GABAergic and/or glycinergic we examined expression of markers of these two fates. We see no significant difference in the number of cells expressing GABAergic markers in single or double mutant embryos (Fig. 8m, n & r). In contrast, there is an increase in the number of cells expressing glycinergic markers. Interestingly, and in contrast to the slc32a1 (viaat) result, we see a slight increase in both single mutants as well as a more pronounced increase in double mutants (Fig. 8i-l & q, Table 1). The increase in double mutants (approximately 28 cells) suggests that all V0v cells are becoming glycinergic.
V0v cells become glycinergic through a novel Pax2-independent mechanism
All of the transcription factors that have been identified so far as specifying inhibitory spinal fates act through Pax2 [14–21]. In addition, Tlx1 and Tlx3, the only other transcription factors that have been identified as specifying excitatory spinal cord fates [4, 5, 8], work at least in part by down-regulating Pax2 . Therefore, we decided to test if V0v cells turn on Pax2 expression in evx1;evx2 double mutants. However, when we analyzed pax2a expression there was no significant difference in the number of cells expressing this gene in either the single or double mutants compared to WT embryos (Fig. 6b & e; Table 1). To further confirm this result, we performed immunohistochemistry using a Pax2 antibody that recognizes Pax2a and Pax2b . Again, we saw no significant change in the number of cells expressing Pax2 protein in single or double mutants (Fig. 6d & f; Table 1). Finally, we also performed double-labeling experiments for Pax2 and EGFP in WT and mutant embryos that carried the Tg(evx1:EGFP) SU2 transgene, using either the Pax2 antibody or in situ hybridization with a mix of pax2a, pax2b and pax8 probes. In each case we examined at least two WT embryos and two double homozygous mutants and we did not observe any double-labeled cells (Fig. 6h and data not shown).
Taken together these results show that V0v cells are becoming glycinergic through a Pax2-independent mechanism. This is the first time that a Pax2-independent mechanism of glycinergic specification has been identified in spinal cord neurons.
Evx genes are found in a wide range of animals ranging from corals to humans . They encode transcription factors that contain both DNA-binding homeobox and C-terminal repressor domains [73–77]. Amniotes have two Evx genes (Evx1 and Evx2) and teleosts, including zebrafish, have three (evx1, evx2 and eve1), although only evx1 and evx2 are expressed in the spinal cord ([26, 32, 33, 59, 64]; this paper Figs. 2, 3, Additional file 1: Results and Figure S1). Interestingly, the genomic positions of these evx genes (adjacent to specific Hox clusters) along with phylogenetic analyses strongly suggest that the third evx gene in teleosts (eve1) is not the result of the extra genome duplication in the teleost lineage ([26, 78, 79], Additional file 1). Instead, it is likely that all three of these genes originated from the two rounds of whole genome duplication that occurred early in the vertebrate lineage  and eve1 was later lost in the tetrapod lineage ([26, 78], Additional file 1).
Here we provide the first comprehensive analysis of the functions of Evx1 and Evx2 in spinal cord interneuron development in any vertebrate. We demonstrate that, within the spinal cord, both of these transcription factors are expressed exclusively by V0v cells. We also show that V0v cells are glutamatergic. These findings complement and extend those of Satou and colleagues , who reported that Evx2-expressing cells that develop from the dbx1b progenitor domain in zebrafish express the glutamateric maker slc17a6b and Talpalar and colleagues , who showed that mouse V0v cells express the glutamatergic marker slc17a6 (vglut2). In addition, we confirm and extend previous reports that suggested that V0v neurons extend commissural axons [23, 27, 35, 36] and we identify these neurons as CoSA interneurons. Interestingly, while Satou and colleagues  observed similar V0v cell morphologies at the stages that we have examined, at later stages of development they also saw descending and bifurcating commissural excitatory V0 cells, suggesting that V0v cells may diversify morphologically at later developmental stages .
In mouse Evx1 mutants, most V0v cells completely change their fate and acquire characteristics of V1 cells, the cell-type that normally forms ventral to V0v cells. Cells that would have formed V0v interneurons lose expression of Evx1 and Evx2 and instead express the V1 marker Engrailed1 (En1) and develop axon trajectories and migration patterns characteristic of V1 interneurons . Most notably, their axon trajectories change from being contralateral to ipsilateral . In addition, experiments in chick embryos revealed that ectopic Evx is sufficient to suppress Engrailed expression and therefore presumably V1 cell fate . The role of Evx2 in mouse V0v cells is less well understood. Evx2 expression is dependent on Evx1, suggesting that it may be involved in the specification events described above , but spinal cord phenotypes of Evx2 mutants have not been described in mouse and before this study, Evx1;Evx2 double mutants had not been described in any vertebrate.
These amniote data suggest that Evx1 is required to inhibit the V1 fate in post-mitotic V0v cells. This global cell fate change is unusual for a transcription factor expressed in post-mitotic cells: it is more commonly seen with transcription factors expressed in spinal progenitor domains (e.g. [27–29, 81, 82]). For example, Nkx2.2 is a transcription factor expressed in the p3 progenitor domain and in Nkx2.2 mutant mice, cells that would have formed V3 interneurons change their fates (transfate) and become motoneurons instead . Similarly, Dbx1 is expressed in the progenitor domain (p0) from which V0 cells develop and in Dbx1 mutant mice, cells that would have become V0v cells assume the characteristics of V1 cells [27, 28].
Our results show that in zebrafish, Evx1 and Evx2 act partially redundantly to specify the glutamatergic fate of V0v cells and inhibit an alternative glycinergic fate in these cells. Given that the only spinal cord cells that express evx1 and evx2 are V0v cells and that the only other trunk tissue that expresses either of these genes is the posterior gut, which expresses evx1, we consider that this requirement for Evx1 and Evx2 function is likely to be cell-autonomous. Interestingly, while there is a reduction of glutamatergic cells in both single and double mutants, expression of the inhibitory marker slc32a1 is only increased in double mutants, suggesting that the specification of glutamatergic fates and the inhibition of glycinergic fates may be independent processes which require different levels of Evx activity. However, in contrast to slc32a1, the number of cells expressing the glycinergic marker slc6a5 was slightly increased in single mutants, which suggests that the expression of different neurotransmitter transporter proteins is regulated independently and by distinct levels of Evx activity. These results are intriguing as they suggest that the regulation of neurotransmitter transporters and enzymes might be complex, with different components being regulated by distinct mechanisms.
V0v interneurons are a crucial part of locomotor circuitry as they are required for hindlimb left-right alternation during fast locomotion [9, 27, 34]. Therefore, changing the neurotransmitter fate of these cells might be expected to impair fast movements. Unfortunately, as evx2 mutants die by larval stages, we were not able to assess whether evx2 single mutants or evx1;evx2 double mutants have locomotion defects. In addition, evx1 single mutants lack joints in their fins , making it impossible to evaluate if any difference in evx1 single mutant behavior is due to this fin phenotype or a locomotive defect. Interestingly, we did not observe any obvious changes in V0v cell morphology or axon trajectory in evx1;evx2 double mutants. Given the changed neurotransmitter phenotype of V0v cells in these animals this might be considered surprising, although it is consistent with our previous analysis of V1 cells, that maintain their ipsilateral axon trajectories even when they lose their inhibitory fates in the absence of Pax2 and Pax8 . It is still possible though that there are subtle changes in V0v cell wiring and/or changes in V0v cell connectivity in evx1;evx2 double mutants as a result of their change in neurotransmitter fate. As there are fewer GFP-labelled V0v cells in evx1;evx2 double mutants it is also possible that V0v cells with inappropriate neurotransmitter fates eventually die, although alternatively this reduction in the number of GFP-positive cells may just reflect autoregulation of Evx expression.
In this paper, we also describe the expression of a different transcription factor gene expressed by V0v cells, skor2. Skor2 expression has also been reported in the mouse spinal cord but the cells that express it were not identified . Our results show that skor2 is expressed by a subset of V0v cells as well as at least one population of more dorsal excitatory spinal cord cells. We also demonstrate that expression of skor2 in V0v cells requires Evx1 and Evx2 activity. Given that skor2 is predominantly expressed by excitatory cells, it is possible that it acts downstream of Evx1 and Evx2 in V0v cells in either the specification of glutamatergic fates and/or the inhibition of glycinergic fates and that it might also have this function in other cells. However it is also possible that Skor2 acts downstream of Evx1 and Evx2 in some other as-yet-unidentified aspect of V0v cell specification. These alternatives can be tested by future loss-of-function analyses of Skor2.
Excitingly, in addition to demonstrating the roles of Evx1 and Evx2 in neurotransmitter specification, our data also show that these transcription factors function independently of Pax2 in specifying glutamatergic fates and inhibiting glycinergic fates. This is the first time that a Pax2-independent mechanism of inhibitory fate specification has been identified in the spinal cord. While several transcription factors have been identified that specify the inhibitory fates of particular spinal cord neurons, so far all of these act upstream of Pax2 [14–18]. In addition, as mentioned before, the only other transcription factors that have been identified as specifying excitatory spinal cord fates, Tlx1 and Tlx3 [4, 5, 8], work at least in part by down-regulating Pax2 . Therefore, our finding that V0v cells become inhibitory in evx1;evx2 double mutants but do not express Pax2 is a significant one as it demonstrates that there must be an additional Pax2-independent mechanism for specifying inhibitory neurons in the spinal cord.
In conclusion, in this paper we demonstrate that zebrafish V0v cells express evx1 and evx2 and develop into excitatory (glutamatergic) CoSA interneurons. We also show that Evx1 and Evx2 are required, partially redundantly for expression of skor2 and glutamatergic markers and inhibition of glycinergic markers in V0v cells and that in the absence of Evx1 and Evx2 function V0v cells become glycinergic through a novel Pax2-independent mechanism. Taken together, our data significantly increase our understanding of how neurotransmitter fates are specified and the genetic networks involved in these processes.
conserved non-coding element
central nervous system
commisural secondary ascending
days post fertilization
fluorescent activated cell sorting
glutamic acid decarboxylases
hours post fertilization
left hand side
right hand side
We thank Dr Shinichi Higashijima for kindly providing us with Evx2 antibody, Dr Derek Stemple and everyone working on the zebrafish mutation project at the Wellcome Trust Sanger Centre for the evx2 mutant allele, ZFIN for providing information on nomenclature and other essential zebrafish resources, Nigel Miller at the Flow Cytometry Facility, Department of Pathology, University of Cambridge, Cambridge, for his expert FAC-sorting, Tomi Ivacevic and Vladimir Benes at the Genomics Core Facility at EMBL, Heidelberg, for RNA amplification, labelling, and hybridization, Uwe Strähle, Olivier Armant and Jasmin Lampert for help with designing microarrays, Nicole Santos for help with genotyping, Henry Putz, Jessica Bouchard, Annika Swanson and several SU undergraduate fish husbandry workers for help with maintaining zebrafish lines and Dr Santanu Banerjee for helpful comments on a previous version of this manuscript.
This work was supported by NSF IOS-1257583, NIH NINDS R21NS073979, the Spinal Cord Injury Trust Fund through New York State Department of Health Contract #C030177, a Royal Society University Research Fellowship and Syracuse University start-up funds awarded to K.E.L; BBSRC, Cambridge Trust, DAAD and Daimler-Benz-Foundation funding awarded to C.J.S; NSF HRD-0703452 LSAMP and Syracuse University Ruth Meyer funding awarded to S.A.P and NSF HRD-1202480 LSAMP and Syracuse University Ruth Meyer funding awarded to G.K.V.
Open AccessThis 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.
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