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Evx1 and Evx2 specify excitatory neurotransmitter fates and suppress inhibitory fates through a Pax2-independent mechanism.

Juárez-Morales JL, Schulte CJ, Pezoa SA, Vallejo GK, Hilinski WC, England SJ, de Jager S, Lewis KE - Neural Dev (2016)

Bottom Line: 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.Interestingly, they do not express Pax2, suggesting that they are acquiring their inhibitory fate through a novel Pax2-independent mechanism.These results significantly increase our understanding of the mechanisms of neuronal specification and the genetic networks involved in these processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Syracuse University, 107 College Place, Syracuse, NY, 13244, USA.

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 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.

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.

No MeSH data available.


Related in: MedlinePlus

skor2 is expressed by V0v cells and this expression is lost in evx1;evx2 double mutants. Lateral views of zebrafish spinal cord at 27 h (a) and 30 h (b-d). Anterior left, dorsal top. a & b Merged images on top followed by single-channel views. Panels on RHS are single confocal planes of white dashed-box regions. Stars indicate double-positive cells. a Expression of skor2 (red) and EGFP (green) in Tg(evx1:EGFP)SU1 embryo. In this example, all ventral skor2-expressing cells co-express EGFP. On average, 97 % of ventral skor2-expressing cells co-express EGFP (73/75 cells counted in 4 embryos). In contrast, about 57.5 % of V0v cells co-express skor2 (73/127 cells counted in 4 embryos). b Expression of skor2 (red) and EGFP (green) in Tg(slc17a6:EGFP) embryo that labels glutamatergic cells. Crosses indicate cells that are only clearly positive for skor2. On average 93.5 % of skor2-expressing cells co-express EGFP (201/215 cells counted in 4 embryos). As there is usually a delay in EGFP expression it is possible that all ventral skor2-expressing cells are excitatory V0v cells, as the small number of ventral skor2-positive EGFP-negative cells may be just starting to express EGFP. c & d Expression of skor2 (blue) in both WT (c) and evx1;evx2 double mutant (d) embryos. The ventral row of skor2 expression is lost in double mutants. e Average number of cells (y-axis) expressing skor2 in spinal cord region adjacent to somites 6-10 in WT embryos and evx1 and evx2 single and double mutants (x-axis) at 30 h. Results are shown for the ventral (V0v) domain of skor2 expression and for the whole skor2 expression domain (total cell counts). Values are mean +/- standard deviation (also see Table 1). In each case at least 5 embryos were counted. Statistically significant differences (P < 0.05) from WT values are indicated with stars. P values for all comparisons are provided in Table 1. Scale bar: 50 μm (a & b); 40 μm (c & d)
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Fig7: skor2 is expressed by V0v cells and this expression is lost in evx1;evx2 double mutants. Lateral views of zebrafish spinal cord at 27 h (a) and 30 h (b-d). Anterior left, dorsal top. a & b Merged images on top followed by single-channel views. Panels on RHS are single confocal planes of white dashed-box regions. Stars indicate double-positive cells. a Expression of skor2 (red) and EGFP (green) in Tg(evx1:EGFP)SU1 embryo. In this example, all ventral skor2-expressing cells co-express EGFP. On average, 97 % of ventral skor2-expressing cells co-express EGFP (73/75 cells counted in 4 embryos). In contrast, about 57.5 % of V0v cells co-express skor2 (73/127 cells counted in 4 embryos). b Expression of skor2 (red) and EGFP (green) in Tg(slc17a6:EGFP) embryo that labels glutamatergic cells. Crosses indicate cells that are only clearly positive for skor2. On average 93.5 % of skor2-expressing cells co-express EGFP (201/215 cells counted in 4 embryos). As there is usually a delay in EGFP expression it is possible that all ventral skor2-expressing cells are excitatory V0v cells, as the small number of ventral skor2-positive EGFP-negative cells may be just starting to express EGFP. c & d Expression of skor2 (blue) in both WT (c) and evx1;evx2 double mutant (d) embryos. The ventral row of skor2 expression is lost in double mutants. e Average number of cells (y-axis) expressing skor2 in spinal cord region adjacent to somites 6-10 in WT embryos and evx1 and evx2 single and double mutants (x-axis) at 30 h. Results are shown for the ventral (V0v) domain of skor2 expression and for the whole skor2 expression domain (total cell counts). Values are mean +/- standard deviation (also see Table 1). In each case at least 5 embryos were counted. Statistically significant differences (P < 0.05) from WT values are indicated with stars. P values for all comparisons are provided in Table 1. Scale bar: 50 μm (a & b); 40 μm (c & d)

Mentions: To identify additional transcription factors that might be required for specification of V0v functional characteristics, we expression-profiled FAC-sorted V0v cells (see Methods; [50]). From these analyses, we identified skor2 as a transcription factor gene potentially expressed by V0v neurons. Our subsequent in situ hybridization experiments demonstrated that skor2 has two clear domains of spinal cord expression, a ventral domain and a more dorsal domain (Fig. 7c). Double labeling experiments show that in the ventral domain, at least most of the skor2-expressing cells are V0v cells (Fig. 7a). On average, 97 % of ventral skor2-expressing cells co-express EGFP in Tg(evx1:EGFP)SU1 embryos (73/75 cells counted in 4 embryos). Given this high number of double positive cells and the fact that there is usually a delay in EGFP expression it is possible that all of the ventral skor2-expressing cells are V0v cells. Interestingly, double labeling experiments with skor2 and Tg(slc17a6:EGFP) demonstrated that both the ventral and dorsal skor2-expressing cells are excitatory cells (Fig. 7b), suggesting that Skor2 may play in role in specifying excitatory fates. As skor2 is expressed by V0v cells, we tested whether it is regulated by Evx1 and Evx2. We found that the number of cells expressing skor2 in the ventral spinal cord is reduced in evx1 and evx2 single mutants compared to WT embryos. More strikingly, ventral skor2 expression is completely abolished in double evx1;evx2 mutants (Figs 7d & e; Table 1), demonstrating that Evx1 and Evx2 are required, partially redundantly for skor2 expression in V0v cells. In contrast, there was no change in the dorsal expression of skor2 (Fig. 7d & e; Table 1).Fig. 7


Evx1 and Evx2 specify excitatory neurotransmitter fates and suppress inhibitory fates through a Pax2-independent mechanism.

Juárez-Morales JL, Schulte CJ, Pezoa SA, Vallejo GK, Hilinski WC, England SJ, de Jager S, Lewis KE - Neural Dev (2016)

skor2 is expressed by V0v cells and this expression is lost in evx1;evx2 double mutants. Lateral views of zebrafish spinal cord at 27 h (a) and 30 h (b-d). Anterior left, dorsal top. a & b Merged images on top followed by single-channel views. Panels on RHS are single confocal planes of white dashed-box regions. Stars indicate double-positive cells. a Expression of skor2 (red) and EGFP (green) in Tg(evx1:EGFP)SU1 embryo. In this example, all ventral skor2-expressing cells co-express EGFP. On average, 97 % of ventral skor2-expressing cells co-express EGFP (73/75 cells counted in 4 embryos). In contrast, about 57.5 % of V0v cells co-express skor2 (73/127 cells counted in 4 embryos). b Expression of skor2 (red) and EGFP (green) in Tg(slc17a6:EGFP) embryo that labels glutamatergic cells. Crosses indicate cells that are only clearly positive for skor2. On average 93.5 % of skor2-expressing cells co-express EGFP (201/215 cells counted in 4 embryos). As there is usually a delay in EGFP expression it is possible that all ventral skor2-expressing cells are excitatory V0v cells, as the small number of ventral skor2-positive EGFP-negative cells may be just starting to express EGFP. c & d Expression of skor2 (blue) in both WT (c) and evx1;evx2 double mutant (d) embryos. The ventral row of skor2 expression is lost in double mutants. e Average number of cells (y-axis) expressing skor2 in spinal cord region adjacent to somites 6-10 in WT embryos and evx1 and evx2 single and double mutants (x-axis) at 30 h. Results are shown for the ventral (V0v) domain of skor2 expression and for the whole skor2 expression domain (total cell counts). Values are mean +/- standard deviation (also see Table 1). In each case at least 5 embryos were counted. Statistically significant differences (P < 0.05) from WT values are indicated with stars. P values for all comparisons are provided in Table 1. Scale bar: 50 μm (a & b); 40 μm (c & d)
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Fig7: skor2 is expressed by V0v cells and this expression is lost in evx1;evx2 double mutants. Lateral views of zebrafish spinal cord at 27 h (a) and 30 h (b-d). Anterior left, dorsal top. a & b Merged images on top followed by single-channel views. Panels on RHS are single confocal planes of white dashed-box regions. Stars indicate double-positive cells. a Expression of skor2 (red) and EGFP (green) in Tg(evx1:EGFP)SU1 embryo. In this example, all ventral skor2-expressing cells co-express EGFP. On average, 97 % of ventral skor2-expressing cells co-express EGFP (73/75 cells counted in 4 embryos). In contrast, about 57.5 % of V0v cells co-express skor2 (73/127 cells counted in 4 embryos). b Expression of skor2 (red) and EGFP (green) in Tg(slc17a6:EGFP) embryo that labels glutamatergic cells. Crosses indicate cells that are only clearly positive for skor2. On average 93.5 % of skor2-expressing cells co-express EGFP (201/215 cells counted in 4 embryos). As there is usually a delay in EGFP expression it is possible that all ventral skor2-expressing cells are excitatory V0v cells, as the small number of ventral skor2-positive EGFP-negative cells may be just starting to express EGFP. c & d Expression of skor2 (blue) in both WT (c) and evx1;evx2 double mutant (d) embryos. The ventral row of skor2 expression is lost in double mutants. e Average number of cells (y-axis) expressing skor2 in spinal cord region adjacent to somites 6-10 in WT embryos and evx1 and evx2 single and double mutants (x-axis) at 30 h. Results are shown for the ventral (V0v) domain of skor2 expression and for the whole skor2 expression domain (total cell counts). Values are mean +/- standard deviation (also see Table 1). In each case at least 5 embryos were counted. Statistically significant differences (P < 0.05) from WT values are indicated with stars. P values for all comparisons are provided in Table 1. Scale bar: 50 μm (a & b); 40 μm (c & d)
Mentions: To identify additional transcription factors that might be required for specification of V0v functional characteristics, we expression-profiled FAC-sorted V0v cells (see Methods; [50]). From these analyses, we identified skor2 as a transcription factor gene potentially expressed by V0v neurons. Our subsequent in situ hybridization experiments demonstrated that skor2 has two clear domains of spinal cord expression, a ventral domain and a more dorsal domain (Fig. 7c). Double labeling experiments show that in the ventral domain, at least most of the skor2-expressing cells are V0v cells (Fig. 7a). On average, 97 % of ventral skor2-expressing cells co-express EGFP in Tg(evx1:EGFP)SU1 embryos (73/75 cells counted in 4 embryos). Given this high number of double positive cells and the fact that there is usually a delay in EGFP expression it is possible that all of the ventral skor2-expressing cells are V0v cells. Interestingly, double labeling experiments with skor2 and Tg(slc17a6:EGFP) demonstrated that both the ventral and dorsal skor2-expressing cells are excitatory cells (Fig. 7b), suggesting that Skor2 may play in role in specifying excitatory fates. As skor2 is expressed by V0v cells, we tested whether it is regulated by Evx1 and Evx2. We found that the number of cells expressing skor2 in the ventral spinal cord is reduced in evx1 and evx2 single mutants compared to WT embryos. More strikingly, ventral skor2 expression is completely abolished in double evx1;evx2 mutants (Figs 7d & e; Table 1), demonstrating that Evx1 and Evx2 are required, partially redundantly for skor2 expression in V0v cells. In contrast, there was no change in the dorsal expression of skor2 (Fig. 7d & e; Table 1).Fig. 7

Bottom Line: 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.Interestingly, they do not express Pax2, suggesting that they are acquiring their inhibitory fate through a novel Pax2-independent mechanism.These results significantly increase our understanding of the mechanisms of neuronal specification and the genetic networks involved in these processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Syracuse University, 107 College Place, Syracuse, NY, 13244, USA.

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 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.

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.

No MeSH data available.


Related in: MedlinePlus