Sequerra EB et al. (2018), NMDA Receptor Signaling Is Important for Neural...

XB-ART-54853

J Neurosci 2018 May 16;3820:4762-4773. doi: 10.1523/JNEUROSCI.2634-17.2018.

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NMDA Receptor Signaling Is Important for Neural Tube Formation and for Preventing Antiepileptic Drug-Induced Neural Tube Defects.

Sequerra EB , Goyal R , Castro PA , Levin JB , Borodinsky LN .

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Failure of neural tube closure leads to neural tube defects (NTDs), which can have serious neurological consequences or be lethal. Use of antiepileptic drugs (AEDs) during pregnancy increases the incidence of NTDs in offspring by unknown mechanisms. Here we show that during Xenopus laevis neural tube formation, neural plate cells exhibit spontaneous calcium dynamics that are partially mediated by glutamate signaling. We demonstrate that NMDA receptors are important for the formation of the neural tube and that the loss of their function induces an increase in neural plate cell proliferation and impairs neural cell migration, which result in NTDs. We present evidence that the AED valproic acid perturbs glutamate signaling, leading to NTDs that are rescued with varied efficacy by preventing DNA synthesis, activating NMDA receptors, or recruiting the NMDA receptor target ERK1/2. These findings may prompt mechanistic identification of AEDs that do not interfere with neural tube formation.SIGNIFICANCE STATEMENT Neural tube defects are one of the most common birth defects. Clinical investigations have determined that the use of antiepileptic drugs during pregnancy increases the incidence of these defects in the offspring by unknown mechanisms. This study discovers that glutamate signaling regulates neural plate cell proliferation and oriented migration and is necessary for neural tube formation. We demonstrate that the widely used antiepileptic drug valproic acid interferes with glutamate signaling and consequently induces neural tube defects, challenging the current hypotheses arguing that they are side effects of this antiepileptic drug that cause the increased incidence of these defects. Understanding the mechanisms of neurotransmitter signaling during neural tube formation may contribute to the identification and development of antiepileptic drugs that are safer during pregnancy.

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Species referenced: Xenopus laevis
Genes referenced: adm elavl1 h2bc21 map2k1 mapk1 pcna slc5a5 sox2
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	Fig 1 Ca2+ dynamics and glutamate signaling are present during neural tube formation. A, Developmental stages of X. laevis neural tube formation. hpf: hours post fertilization, hpf at 23°C. Adapted from the study by Nieuwkoop and Faber (1994). B, Representative trace of in vivo spontaneous changes in the Ca2+ sensor GCaMP6s fluorescence in a neural plate cell from a neurulating stage 14 embryo. C, Frequency of Ca2+ transients (per 40 min period, percentage of total number of transients in 2 h period, stages 12.5–14); values are the mean ± SEM, n = 12 embryos, p < 0.05, ANOVA followed by Dunnett's test, compared with first 40 min period. St., Stage. D, Distribution of Ca2+ transients (per 2 h period) in the dorsal ectoderm (percentage of the total number of transients per 2 h period, stages 12.5–14); values are the mean ± SEM, n = 15 embryos. **p < 0.0001, ANOVA followed by Dunnett's test compared with the most lateral region. E, RT-PCR from the dorsal half of stage 13 embryos. F, Western blot assay from whole-embryo lysates at neural plate (stage 18) and tailbud (stage 28) stages (top), and from neural plate stage (stage 18) embryos injected at the two-cell stage with C-Mo or GluN1-Mo2 (bottom). Similar results were obtained in three independent experiments. G, Traces from stage 15-dissociated neural plate cells loaded with the Ca2+-sensitive dye Fluo4-AM and imaged during the addition of 10 μm NMDA or 5 mm glutamate. Lines under the traces indicate the time of incubation with NMDA or glutamate. Samples were time-lapse imaged at a rate of 0.2 Hz for 5 min. Regions of interest were selected to measure the fluorescence intensity over time for individual neural plate cells with NIS Elements software (Nikon). Fluorescence (F) is normalized to the baseline for each cell (F0) as a percentage. H, Examples of GCaMP6s-expressing embryos imaged in the absence (vehicle) or presence of the indicated drugs for 2 h starting at stage 12.5. Circles indicate the occurrence of Ca2+ transients. Ten micromolar NMDA was preincubated for 30 min before the addition of 1 mm VPA (NMDA+VPA). I, Frequency of Ca2+ transients (per 2 h period, stages 12.5–14); values are the mean ± SEM, numbers between parentheses are the number of embryos per treatment. p < 0.05, one-way ANOVA, followed by Dunnett's test compared with vehicle-treated samples.
	Fig 2 NMDAR-mediated signaling is necessary for neural tube formation. A, B, Embryos were incubated with vehicle (0 μm d-AP5) or 5–500 μm d-AP5 starting at stage 13 until stage 24 (∼11 h incubation). A, Immunostained transverse sections from stage 24 embryos incubated with vehicle or d-AP5. Scale bar, 20 μm. B, Incidence of NTDs is the percentage of embryos with open neural tube (NT), n ≥ 15 embryos per group. p < 0.05, p < 0.001, χ2 test compared with vehicle-incubated embryos. C, Western blot assays from whole, stage 18 embryos previously injected at the two-cell stage with GluN1-Mo1 or Control-morpholino (C-Mo). Similar results were obtained in three independent experiments. D, Examples of stage 21 embryos previously injected at the two-cell stage with control-Mo and GluN1-Mo1. Arrowheads indicate the failure of neural fold convergence in the midline. A comparable phenotype was obtained with GluN1-Mo2; hence, GluN1-Mo1 was used for the remainder of this study. E, Incidence of NTDs is the percentage of embryos with open neural tubes; values are the mean ± SEM for n > 10 embryos per group, N ≥ 5 experiments. p < 0.01, *p < 0.001, *p < 0.0001, χ2 test. F, Images illustrate embryos bilaterally expressing GCaMP6s and unilaterally containing GluN1-Mo1 or control-Mo and a fluorescent tracer. Circles indicate Ca2+ transients during a 2 h period (stages 12.5–14). Graphs show Ca2+ transient frequency (per 2 h period) in GluN1-Mo- or control-Mo-containing and contralateral (WT) neural plate in individual stage 12.5–14 embryos; n = 7 GluN1-Mo and control-Mo embryos. p < 0.05, ns, not significant, paired t test. Lines connect paired data from the same embryo.
	Fig 3 NMDA receptor signaling regulates neural plate cell proliferation and migration. A, Whole-mount, immunostained stage 13 through stage 18 embryos were imaged coronally and analyzed using Imaris 3D software. Top image shows a single plane of a representative z-stack image of the dorsal half of a stage 17 embryo. Bottom images are volume renderings of virtual transverse sections of the neural plate in which Sox2+ cells were identified and analyzed for PCNA expression. ne, Neural ectoderm; non-ne, non-neural ectoderm. The graph shows the percentage of neural plate cells (identified by Sox2 expression) immunopositive for PCNA; values are the mean ± SEM; n = 4–7 embryos per stage. The line connects data points at sequential developmental stages (bottom, x-axis) corresponding to different times postfertilization (top, x-axis). B, C, Images illustrate immunostained transverse sections from vehicle and d-AP5-treated embryos (5 h incubation starting at stage 13; B) or from unilaterally GluN1-Mo-injected embryos during neural plate folding (stage 18; C). Numbers on the top of the images in B are the number of PH3+ cells in 10 30-μm-thick sections of neural plate per embryo; values are the mean ± SEM, n = 9 embryos per group. p < 0.05, t test. C, Graph shows the numbers of PH3+ cells in 10 30-μm-thick sections of WT and GluN1-Mo-containing halves of neural plate in individual embryos (stages 16–18). p < 0.05, paired t test. D, E, Embryos were unilaterally injected with GluN1-Mo or control-Mo, and bilaterally with H2B-GFP mRNA at the two-cell stage and time-lapse imaged starting at stage 13 for 1.5 (D) and 2 (E) h. D, Image on left shows a single frame from a time-lapse series (Movie 2) of an embryo bilaterally expressing H2B-GFP (green) and unilaterally containing GluN1-Mo + tracer (red). Magnified images to the right depict a cell division (arrowheads). Graphs show the number of cell divisions during 1.5 h starting at stage 13 in contralateral and ipsilateral halves of neural plate as the percentage of total H2B-GFP cells in each side; n = 6 embryos per group. p < 0.05, ns, not significant, paired t test. Scale bars: A–D, 20 μm. Lines in C and D connect paired data from the same embryo. E, The trajectories of individual H2B-GFP-labeled cells were measured in both halves of the neural plate of embryos unilaterally containing GluN1-Mo or control-Mo during a 2 h period starting at stage 13 (Movie 2). Drawing on the left depicts the trajectories of individual cells (circles with straight arrows), and the lateromedial movement of these same cells (segments) that is reported in the graph. Schematic in the middle shows an example of the trajectories of pairs of cells initially equidistant from the midline (dashed line) in WT and GluN1-Mo-containing halves of neural plate over 2 h with numbers positioned at time 0 of the trajectory. Graphs show lateromedial displacement for individual WT and GluN1-Mo- or Control-Mo-containing cells and the mean ± SEM; n ≥ 50 cells, N = 3 embryos per group. **p < 0.001, t test. P, Posterior; A, anterior.
	Fig 4 The antiepileptic drug VPA induces neural tube defects by interfering with glutamate signaling and by increasing neural plate cell proliferation. A, B, Embryos were incubated with vehicle (control) or 0.1–1 mm VPA starting at stage 13 or were preincubated with 10 μm NMDA starting at the four-cell stage until stages 22–24. A, Immunostained transverse sections from stage 22 vehicle-, VPA-, and VPA + NMDA-treated embryos. Sox2 and E-cadherin immunostaining label neural and non-neural ectoderm, respectively. B, Severity of NTDs is the percentage of transverse sections of the entire anteroposterior axis with open neural tube per embryo; values are the mean ± SEM; between parentheses are the number of embryos per group, from at least three independent experiments. p < 0.005, *p < 0.0001, ANOVA followed by Tukey's test. C, D, Dissociated neural plate cells from stage 15 embryos were loaded with the Ca2+-sensitive dye Fluo4-AM and imaged during the addition of 5 mm glutamate or 10 μm NMDA in the absence and presence (preincubation with VPA for 30 min) of 100 μm VPA. Cells were washed with saline for 10 min between recordings. Samples were time-lapse imaged at 0.2 Hz. Regions of interest were selected to measure the fluorescence intensity over time for individual neural plate cells with NIS Elements software (Nikon). C, Shown is an example of a neural plate cell responsive to glutamate only in the absence of VPA. Lines under the traces indicate the time of incubation of the culture with glutamate or VPA. Arrows indicate the time when washes with saline were performed for 10 min. Arrowhead represents a 30 min preincubation with VPA. Fluorescence (F) is normalized to the baseline for each cell (F0) in percentage. D, Graphs show the numbers of cells responsive to 5 mm glutamate and 10 μm NMDA as a percentage of the total number of cells in the field of view in the absence or presence of VPA for the same field of view; n = 6 (glutamate, 70 cells recorded) and n = 8 (NMDA, 217 cells recorded) independent experiments, p < 0.05, paired t test. Lines connect paired data from the same field of view. E–H, Embryos were incubated starting at stage 13 with vehicle (control) or 0.1 mm VPA for 5 h. In E and F, BrdU was added to control and experimental groups during the 5 h of treatment. E, Transverse sections immunolabeled for BrdU. Scale bars: A, E, 20 μm. F–H, Graphs show the number of BrdU+ (F), PCNA+ (G), and DAPI+ (H) cells per 10 30-μm-thick sections of neural plate per embryo; values are the mean ± SEM; n = 9 embryos per group. p < 0.05, t test. I, Embryos were incubated with vehicle or 0.1 mm VPA starting at stage 13 and/or preincubated with HUA: 20 mm hydroxyurea + 150 μm aphidicolin from stage 12.5 (30 min before VPA treatment) until stage 24. The severity of NTDs is the percentage of transverse sections of the entire anteroposterior axis with open neural tube per embryo, mean ± SEM; n ≥ 10 embryos per group. p < 0.05, ANOVA followed by Tukey's test.
	Fig 5 ERK1/2 activation is downstream of glutamate-NMDAR signaling in the promotion of neural tube closure. A, Whole-mount stage 13 through stage 18 embryos immunostained for pERK1/2 and Sox2 were imaged coronally and analyzed using Imaris 3D software. Images show a single plane of a representative z-stack of the dorsal half of a stage 17 embryo. ne, Neural ectoderm. The graph shows the percentage of pERK1/2-immunopositive neural plate cells identified by Sox2 expression; values are the mean ± SEM, n = 4–7 embryos per stage. The line connects data points at sequential developmental stages (bottom x-axis) corresponding to different times postfertilization (top x-axis). A′, To confirm specificity of the pERK1/2 antibody, embryos were incubated with either vehicle (control) or 10 μm PD0325901 (MEK1/2 inhibitor) from stage 11.5 to stage 15, then were processed for whole-mount immmunostaining. B, Stage 13 embryos were incubated with 10 μm NMDA or vehicle (control) for 5 min followed by processing for pERK1/2 immunostaining. Shown are immunostained transverse sections of neural plate and number of pERK1/2-immunopositive cells per 10 30 μm sections of neural plate per embryo; values are the mean ± SEM; n = 14 embryos per treatment. p < 0.005, t test. C, Western blot assay from whole-embryo lysates of WT and CA-MEK1-ERα expressing (CA-MEK) stage 15 embryos treated with 500 nm 4-OHT from stage 11. Similar results were obtained in three independent experiments. D, Activation of ERK1/2 rescues d-AP5 or VPA-induced NTD. WT or CA-MEK-injected embryos were incubated with 4-OHT from stage 11 followed by incubation with vehicle or with 500 μm d-AP5 or 1 mm VPA from stage 13, then fixed and processed for transverse sectioning and immunostaining at stage 22, as shown in images. Graph shows the severity of NTDs, which is the percentage of transverse sections of the entire anteroposterior axis with open neural tube per embryo; values are the mean ± SEM; between parentheses are the number of embryos per group, from at least three independent experiments. **p < 0.0001, one-way ANOVA followed by Tukey's test. Scale bars, 20 μm.

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	Fig 1 Ca2+ dynamics and glutamate signaling are present during neural tube formation. A, Developmental stages of X. laevis neural tube formation. hpf: hours post fertilization, hpf at 23°C. Adapted from the study by Nieuwkoop and Faber (1994). B, Representative trace of in vivo spontaneous changes in the Ca2+ sensor GCaMP6s fluorescence in a neural plate cell from a neurulating stage 14 embryo. C, Frequency of Ca2+ transients (per 40 min period, percentage of total number of transients in 2 h period, stages 12.5–14); values are the mean ± SEM, n = 12 embryos, p < 0.05, ANOVA followed by Dunnett's test, compared with first 40 min period. St., Stage. D, Distribution of Ca2+ transients (per 2 h period) in the dorsal ectoderm (percentage of the total number of transients per 2 h period, stages 12.5–14); values are the mean ± SEM, n = 15 embryos. **p < 0.0001, ANOVA followed by Dunnett's test compared with the most lateral region. E, RT-PCR from the dorsal half of stage 13 embryos. F, Western blot assay from whole-embryo lysates at neural plate (stage 18) and tailbud (stage 28) stages (top), and from neural plate stage (stage 18) embryos injected at the two-cell stage with C-Mo or GluN1-Mo2 (bottom). Similar results were obtained in three independent experiments. G, Traces from stage 15-dissociated neural plate cells loaded with the Ca2+-sensitive dye Fluo4-AM and imaged during the addition of 10 μm NMDA or 5 mm glutamate. Lines under the traces indicate the time of incubation with NMDA or glutamate. Samples were time-lapse imaged at a rate of 0.2 Hz for 5 min. Regions of interest were selected to measure the fluorescence intensity over time for individual neural plate cells with NIS Elements software (Nikon). Fluorescence (F) is normalized to the baseline for each cell (F0) as a percentage. H, Examples of GCaMP6s-expressing embryos imaged in the absence (vehicle) or presence of the indicated drugs for 2 h starting at stage 12.5. Circles indicate the occurrence of Ca2+ transients. Ten micromolar NMDA was preincubated for 30 min before the addition of 1 mm VPA (NMDA+VPA). I, Frequency of Ca2+ transients (per 2 h period, stages 12.5–14); values are the mean ± SEM, numbers between parentheses are the number of embryos per treatment. p < 0.05, one-way ANOVA, followed by Dunnett's test compared with vehicle-treated samples.
	Fig 2 NMDAR-mediated signaling is necessary for neural tube formation. A, B, Embryos were incubated with vehicle (0 μm d-AP5) or 5–500 μm d-AP5 starting at stage 13 until stage 24 (∼11 h incubation). A, Immunostained transverse sections from stage 24 embryos incubated with vehicle or d-AP5. Scale bar, 20 μm. B, Incidence of NTDs is the percentage of embryos with open neural tube (NT), n ≥ 15 embryos per group. p < 0.05, p < 0.001, χ2 test compared with vehicle-incubated embryos. C, Western blot assays from whole, stage 18 embryos previously injected at the two-cell stage with GluN1-Mo1 or Control-morpholino (C-Mo). Similar results were obtained in three independent experiments. D, Examples of stage 21 embryos previously injected at the two-cell stage with control-Mo and GluN1-Mo1. Arrowheads indicate the failure of neural fold convergence in the midline. A comparable phenotype was obtained with GluN1-Mo2; hence, GluN1-Mo1 was used for the remainder of this study. E, Incidence of NTDs is the percentage of embryos with open neural tubes; values are the mean ± SEM for n > 10 embryos per group, N ≥ 5 experiments. p < 0.01, *p < 0.001, *p < 0.0001, χ2 test. F, Images illustrate embryos bilaterally expressing GCaMP6s and unilaterally containing GluN1-Mo1 or control-Mo and a fluorescent tracer. Circles indicate Ca2+ transients during a 2 h period (stages 12.5–14). Graphs show Ca2+ transient frequency (per 2 h period) in GluN1-Mo- or control-Mo-containing and contralateral (WT) neural plate in individual stage 12.5–14 embryos; n = 7 GluN1-Mo and control-Mo embryos. p < 0.05, ns, not significant, paired t test. Lines connect paired data from the same embryo.
	Fig 3 NMDA receptor signaling regulates neural plate cell proliferation and migration. A, Whole-mount, immunostained stage 13 through stage 18 embryos were imaged coronally and analyzed using Imaris 3D software. Top image shows a single plane of a representative z-stack image of the dorsal half of a stage 17 embryo. Bottom images are volume renderings of virtual transverse sections of the neural plate in which Sox2+ cells were identified and analyzed for PCNA expression. ne, Neural ectoderm; non-ne, non-neural ectoderm. The graph shows the percentage of neural plate cells (identified by Sox2 expression) immunopositive for PCNA; values are the mean ± SEM; n = 4–7 embryos per stage. The line connects data points at sequential developmental stages (bottom, x-axis) corresponding to different times postfertilization (top, x-axis). B, C, Images illustrate immunostained transverse sections from vehicle and d-AP5-treated embryos (5 h incubation starting at stage 13; B) or from unilaterally GluN1-Mo-injected embryos during neural plate folding (stage 18; C). Numbers on the top of the images in B are the number of PH3+ cells in 10 30-μm-thick sections of neural plate per embryo; values are the mean ± SEM, n = 9 embryos per group. p < 0.05, t test. C, Graph shows the numbers of PH3+ cells in 10 30-μm-thick sections of WT and GluN1-Mo-containing halves of neural plate in individual embryos (stages 16–18). p < 0.05, paired t test. D, E, Embryos were unilaterally injected with GluN1-Mo or control-Mo, and bilaterally with H2B-GFP mRNA at the two-cell stage and time-lapse imaged starting at stage 13 for 1.5 (D) and 2 (E) h. D, Image on left shows a single frame from a time-lapse series (Movie 2) of an embryo bilaterally expressing H2B-GFP (green) and unilaterally containing GluN1-Mo + tracer (red). Magnified images to the right depict a cell division (arrowheads). Graphs show the number of cell divisions during 1.5 h starting at stage 13 in contralateral and ipsilateral halves of neural plate as the percentage of total H2B-GFP cells in each side; n = 6 embryos per group. p < 0.05, ns, not significant, paired t test. Scale bars: A–D, 20 μm. Lines in C and D connect paired data from the same embryo. E, The trajectories of individual H2B-GFP-labeled cells were measured in both halves of the neural plate of embryos unilaterally containing GluN1-Mo or control-Mo during a 2 h period starting at stage 13 (Movie 2). Drawing on the left depicts the trajectories of individual cells (circles with straight arrows), and the lateromedial movement of these same cells (segments) that is reported in the graph. Schematic in the middle shows an example of the trajectories of pairs of cells initially equidistant from the midline (dashed line) in WT and GluN1-Mo-containing halves of neural plate over 2 h with numbers positioned at time 0 of the trajectory. Graphs show lateromedial displacement for individual WT and GluN1-Mo- or Control-Mo-containing cells and the mean ± SEM; n ≥ 50 cells, N = 3 embryos per group. **p < 0.001, t test. P, Posterior; A, anterior.
	Fig 4 The antiepileptic drug VPA induces neural tube defects by interfering with glutamate signaling and by increasing neural plate cell proliferation. A, B, Embryos were incubated with vehicle (control) or 0.1–1 mm VPA starting at stage 13 or were preincubated with 10 μm NMDA starting at the four-cell stage until stages 22–24. A, Immunostained transverse sections from stage 22 vehicle-, VPA-, and VPA + NMDA-treated embryos. Sox2 and E-cadherin immunostaining label neural and non-neural ectoderm, respectively. B, Severity of NTDs is the percentage of transverse sections of the entire anteroposterior axis with open neural tube per embryo; values are the mean ± SEM; between parentheses are the number of embryos per group, from at least three independent experiments. p < 0.005, *p < 0.0001, ANOVA followed by Tukey's test. C, D, Dissociated neural plate cells from stage 15 embryos were loaded with the Ca2+-sensitive dye Fluo4-AM and imaged during the addition of 5 mm glutamate or 10 μm NMDA in the absence and presence (preincubation with VPA for 30 min) of 100 μm VPA. Cells were washed with saline for 10 min between recordings. Samples were time-lapse imaged at 0.2 Hz. Regions of interest were selected to measure the fluorescence intensity over time for individual neural plate cells with NIS Elements software (Nikon). C, Shown is an example of a neural plate cell responsive to glutamate only in the absence of VPA. Lines under the traces indicate the time of incubation of the culture with glutamate or VPA. Arrows indicate the time when washes with saline were performed for 10 min. Arrowhead represents a 30 min preincubation with VPA. Fluorescence (F) is normalized to the baseline for each cell (F0) in percentage. D, Graphs show the numbers of cells responsive to 5 mm glutamate and 10 μm NMDA as a percentage of the total number of cells in the field of view in the absence or presence of VPA for the same field of view; n = 6 (glutamate, 70 cells recorded) and n = 8 (NMDA, 217 cells recorded) independent experiments, p < 0.05, paired t test. Lines connect paired data from the same field of view. E–H, Embryos were incubated starting at stage 13 with vehicle (control) or 0.1 mm VPA for 5 h. In E and F, BrdU was added to control and experimental groups during the 5 h of treatment. E, Transverse sections immunolabeled for BrdU. Scale bars: A, E, 20 μm. F–H, Graphs show the number of BrdU+ (F), PCNA+ (G), and DAPI+ (H) cells per 10 30-μm-thick sections of neural plate per embryo; values are the mean ± SEM; n = 9 embryos per group. p < 0.05, t test. I, Embryos were incubated with vehicle or 0.1 mm VPA starting at stage 13 and/or preincubated with HUA: 20 mm hydroxyurea + 150 μm aphidicolin from stage 12.5 (30 min before VPA treatment) until stage 24. The severity of NTDs is the percentage of transverse sections of the entire anteroposterior axis with open neural tube per embryo, mean ± SEM; n ≥ 10 embryos per group. p < 0.05, ANOVA followed by Tukey's test.
	Fig 5 ERK1/2 activation is downstream of glutamate-NMDAR signaling in the promotion of neural tube closure. A, Whole-mount stage 13 through stage 18 embryos immunostained for pERK1/2 and Sox2 were imaged coronally and analyzed using Imaris 3D software. Images show a single plane of a representative z-stack of the dorsal half of a stage 17 embryo. ne, Neural ectoderm. The graph shows the percentage of pERK1/2-immunopositive neural plate cells identified by Sox2 expression; values are the mean ± SEM, n = 4–7 embryos per stage. The line connects data points at sequential developmental stages (bottom x-axis) corresponding to different times postfertilization (top x-axis). A′, To confirm specificity of the pERK1/2 antibody, embryos were incubated with either vehicle (control) or 10 μm PD0325901 (MEK1/2 inhibitor) from stage 11.5 to stage 15, then were processed for whole-mount immmunostaining. B, Stage 13 embryos were incubated with 10 μm NMDA or vehicle (control) for 5 min followed by processing for pERK1/2 immunostaining. Shown are immunostained transverse sections of neural plate and number of pERK1/2-immunopositive cells per 10 30 μm sections of neural plate per embryo; values are the mean ± SEM; n = 14 embryos per treatment. p < 0.005, t test. C, Western blot assay from whole-embryo lysates of WT and CA-MEK1-ERα expressing (CA-MEK) stage 15 embryos treated with 500 nm 4-OHT from stage 11. Similar results were obtained in three independent experiments. D, Activation of ERK1/2 rescues d-AP5 or VPA-induced NTD. WT or CA-MEK-injected embryos were incubated with 4-OHT from stage 11 followed by incubation with vehicle or with 500 μm d-AP5 or 1 mm VPA from stage 13, then fixed and processed for transverse sectioning and immunostaining at stage 22, as shown in images. Graph shows the severity of NTDs, which is the percentage of transverse sections of the entire anteroposterior axis with open neural tube per embryo; values are the mean ± SEM; between parentheses are the number of embryos per group, from at least three independent experiments. **p < 0.0001, one-way ANOVA followed by Tukey's test. Scale bars, 20 μm.