Edri T et al. (2023), Alcohol induces neural tube defects by reducing...

XB-ART-60471

Front Cell Dev Biol 2023 Jan 01;11:1282273. doi: 10.3389/fcell.2023.1282273.

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Alcohol induces neural tube defects by reducing retinoic acid signaling and promoting neural plate expansion.

Edri T , Cohen D , Shabtai Y , Fainsod A .

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Introduction: Neural tube defects (NTDs) are among the most debilitating and common developmental defects in humans. The induction of NTDs has been attributed to abnormal folic acid (vitamin B9) metabolism, Wnt and BMP signaling, excess retinoic acid (RA), dietary components, environmental factors, and many others. In the present study we show that reduced RA signaling, including alcohol exposure, induces NTDs. Methods: Xenopus embryos were exposed to pharmacological RA biosynthesis inhibitors to study the induction of NTDs. Embryos were treated with DEAB, citral, or ethanol, all of which inhibit the biosynthesis of RA, or injected to overexpress Cyp26a1 to reduce RA. NTD induction was studied using neural plate and notochord markers together with morphological analysis. Expression of the neuroectodermal regulatory network and cell proliferation were analyzed to understand the morphological malformations of the neural plate. Results: Reducing RA signaling levels using retinaldehyde dehydrogenase inhibitors (ethanol, DEAB, and citral) or Cyp26a1-driven degradation efficiently induce NTDs. These NTDs can be rescued by providing precursors of RA. We mapped this RA requirement to early gastrula stages during the induction of neural plate precursors. This reduced RA signaling results in abnormal expression of neural network genes, including the neural plate stem cell maintenance genes, geminin, and foxd4l1.1. This abnormal expression of neural network genes results in increased proliferation of neural precursors giving rise to an expanded neural plate. Conclusion: We show that RA signaling is required for neural tube closure during embryogenesis. RA signaling plays a very early role in the regulation of proliferation and differentiation of the neural plate soon after the induction of neural progenitors during gastrulation. RA signaling disruption leads to the induction of NTDs through the mis regulation of the early neuroectodermal network, leading to increased proliferation resulting in the expansion of the neural plate. Ethanol exposure induces NTDs through this mechanism involving reduced RA levels.

???displayArticle.pubmedLink??? 38116205
???displayArticle.pmcLink??? PMC10728305
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Species referenced: Xenopus laevis
Genes referenced: aqp3 bmp4 chrd cyp26a1 foxd4l1 gmnn hoxa1 krt12.4 msx1 pax3 pax8 slc35b1 sox2 sox3 zic1
GO keywords: cell proliferation [+]
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???displayArticle.disOnts??? myelomeningocele [+]

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	FIGURE 1. Reduced RA signaling induces neural tube closure defects. Embryos were treated with DEAB (60 µM), citral (50 µM), or EtOH (0.5% or 1.5% vol/vol) from mid-blastula stages (NF8), and were allowed to develop to neural tube closure stages (NF 19-20). Experimental and control embryos were processed for whole-mount in situ hybridization with markers of the neural plate and notochord, (A–E’) sox3 and chrd.1 and (F–J’) aqp3 and pax8. (A–E), (F–J) Dorsal view of embryos, anterior to the top. (A’-E’), (F’J’) Cross section of the same embryo shown in the panel above, dorsal to the top. Brackets mark NTC defects, black arrowheads mark open neural tubes, blue dotted outline marks the closed neural tube, red dotted outline around the notochord, and green arrowheads mark the pax8 expression.
	FIGURE 2. The incidence of NTC defects at different axial levels by reduced RA signaling is concentration-dependent. (A) Analysis of the rostral-caudal distribution of NTDs with the different pharmacological inhibitors of RA biosynthesis. (B) EtOH concentration dependence in the induction of NTC defects. (C) Additive effect of combinations of RA biosynthesis inhibitors. Embryos were treated with DEAB (60 µM), citral (50 µM), or EtOH (0.5%) alone or in combination and subsequently analyzed for NTC defect induction. The percent of embryos with a NTC defect is shown. Sample sizes are shown as well as the distribution of the biological replicates analyzed. , p < 0.05; , p < 0.01; , p < 0.001 **, p < 0.0001; ns, not significant. Lines denote samples compared; no line, compared to the control sample.
	FIGURE 3. The EtOH-induced NTC defects can be rescued by the addition of RA or its precursors. Embryos were treated with 0.5% (A,C) or 1.5% (B,D) EtOH to induce NTC defects. Together with the EtOH treatment, the embryos were supplemented with retinol (ROL, 10 µM), retinaldehyde (RAL, 1 µM) (A, B), or RA (50 nM or 100 nM) (C, D). The frequency of NTC defects was calculated relative to control samples. Sample sizes are shown inside each bar. ****, p < 0.0001; ns, not significant. Brackets denote samples compared, no bracket, compared to the control sample.
	FIGURE 4. RA signaling is required in the prospective ectoderm at the onset of gastrulation to prevent NTC defects. (A–C) Embryos were exposed to DEAB (A), 0.5% EtOH (B), or 1.5% EtOH (C), starting at different developmental stages (NF8, 10.25, 11, 14). The frequency of NTC defects was calculated relative to control samples. (D,E) Embryos were treated with 0.5% (D) and 1.5% EtOH (E) to induce NTC defects. RAL (1 µM) was then added at different stages to rescue the reduction in RA levels. (F) Embryos were injected with mRNA encoding the Cyp26a1 enzyme. Injections targeted unilaterally either the prospective neuroectoderm (B-tier) or dorsal mesoderm (C-tier). The injection site was corroborated by the co-injection of FITC-dextran. (G–I) Embryos were injected with cyp26a1 mRNA targeting the prospective anterior neural plate. FITC-dextran was co-injected to label the injection site. At NTC stages (NF19) the embryos were monitored for NTC defect induction, the localization of the NTC defects, and the overlap with the lineage tracer. The orientation of the embryos, the injected side, and the NTD are shown. Sample sizes are shown within each bar. , p < 0.05; , p < 0.01; , p < 0.001; **, p < 0.0001; ns, not significant. Brackets denote samples compared, no bracket, sample compared to the control sample.
	FIGURE 5. Regulation of the neuroectodermal regulatory network by RA. Embryos were treated with EtOH (0.5% and 1.5%), DEAB (60 µM), or citral (50 µM) from the midblastula stage (NF8) and incubated until early/mid gastrula (NF10.5) for qPCR analysis. (A–F) Relative expression for hoxa1.L, foxd4l1.1.L, gmnn, sox3, pax3.L, and zic1, respectively. Expression levels were normalized to the control expression level at NF 10.5. , p < 0.05; , p < 0.01; , p < 0.001; **, p < 0.0001; ns, not significant. Samples compared to the control sample.
	FIGURE 6. Reduced RA signaling results in the expansion of the neural plate. Embryos were treated with EtOH (0.5% and 1.5%), DEAB (60 µM), and citral (50 µM) and collected for analysis during early neurula stages (NF 14). (A–E) The neural plate was visualized by in situ hybridization with the early marker, sox3. (F) The width of the neural plate was measured in all experimental samples and normalized to the overall width of the embryo to account for individual size changes. (G) The changes in sox3 expression levels were determined by qPCR with gene-specific primers. , p < 0.05; , p < 0.001; **, p < 0.0001; ns, not significant. Samples compared to the control sample.
	FIGURE 7. Reduced RA signaling promotes increased proliferation of neural precursors. Embryos were injected in one blastomere at the 2-cell stage to affect only half of the embryo. Injections included cyp26a1 mRNA with FITC-dextran or FITC-dextran alone. At the onset of gastrulation, the embryos were sorted based on the injected side and further incubated until late gastrula. Embryos were stained by immunohistochemistry with antibodies against pHH3. The positive cells were counted on both sides of the midline, averaging 24 pHH3 positive cells on the control side in each embryo. (A) Control embryo injected with FITC-dextran on the right side. (B) Embryo injected with cyp26a1 mRNA + FITC-dextran on the left side. (C) Relative proliferation analysis based on the number of pHH3-positive cells on both sides. The distribution of all embryos analyzed is shown. The number of embryos analyzed is shown. ****, p < 0.0001 compared to the control (FITC-dextran) sample ratio.
	Supplemental Figure S1. Inhibition of RA biosynthesis induces neural tube closure defects. Inhibition of RA biosynthesis was induced by treatment with DEAB (60 µM), citral (50 µM), or EtOH (0.5% or 1.5% vol/vol). Treatments were initiated during midblastula stages (NF8), and embryos were allowed to develop to neural tube closure stages (NF19-20). Experimental and control embryos were processed for whole-mount in situ hybridization with the neural plate marker pax3. (A-E) Dorsal view of embryos, anterior to the top. (A’-E’) Anterior view of the same embryo shown in the panel above, dorsal to the top. Brackets mark NTC defects and arrowheads open neural tubes.
	Supplemental Figure 2. Developmental progression as a result of EtOH exposure. (A, B) Embryos were treated during midblastula (NF8), early gastrula (NF10.25), mid gastrula (NF11), or early neurula (NF14) with low (0.5%) or high (1.5%) EtOH. The stage reached by the treated embryos was determined at the onset of neurula stages (NF13)(A) and neural tube closure stages (NF19-20)(B). The sample size and the percent embryos at each stage are shown. (C) Previously staged EtOH-treated or control embryos were measured to determine their length and width. The length-towidth ratio was calculated and plotted for EtOH-treated (0.5% and 1.5%) and control embryos for either NF19 and NF20. The ratio distributions are shown. , p<0.05; ***, p<0.0001; ns, not significant.
	Supplemental Figure S3. RA is required for the regulated activation of the neuroectodermal transcriptional network. Embryos were treated to reduce the level of RA signaling by exposing them to EtOH, DEAB, or citral from blastula stages (NF8). RNA was extracted during early/mid gastrula (NF10.5), and the relative expression level of multiple components of the early neural differentiation network was determined by qPCR with gene-specific primers. , p<0.05; , p<0.01; , p<0.001; **, p<0.0001; ns, not significant. Samples compared to the control sample.

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	FIGURE 1. Reduced RA signaling induces neural tube closure defects. Embryos were treated with DEAB (60 µM), citral (50 µM), or EtOH (0.5% or 1.5% vol/vol) from mid-blastula stages (NF8), and were allowed to develop to neural tube closure stages (NF 19-20). Experimental and control embryos were processed for whole-mount in situ hybridization with markers of the neural plate and notochord, (A–E’) sox3 and chrd.1 and (F–J’) aqp3 and pax8. (A–E), (F–J) Dorsal view of embryos, anterior to the top. (A’-E’), (F’J’) Cross section of the same embryo shown in the panel above, dorsal to the top. Brackets mark NTC defects, black arrowheads mark open neural tubes, blue dotted outline marks the closed neural tube, red dotted outline around the notochord, and green arrowheads mark the pax8 expression.
	FIGURE 2. The incidence of NTC defects at different axial levels by reduced RA signaling is concentration-dependent. (A) Analysis of the rostral-caudal distribution of NTDs with the different pharmacological inhibitors of RA biosynthesis. (B) EtOH concentration dependence in the induction of NTC defects. (C) Additive effect of combinations of RA biosynthesis inhibitors. Embryos were treated with DEAB (60 µM), citral (50 µM), or EtOH (0.5%) alone or in combination and subsequently analyzed for NTC defect induction. The percent of embryos with a NTC defect is shown. Sample sizes are shown as well as the distribution of the biological replicates analyzed. , p < 0.05; , p < 0.01; , p < 0.001 **, p < 0.0001; ns, not significant. Lines denote samples compared; no line, compared to the control sample.
	FIGURE 3. The EtOH-induced NTC defects can be rescued by the addition of RA or its precursors. Embryos were treated with 0.5% (A,C) or 1.5% (B,D) EtOH to induce NTC defects. Together with the EtOH treatment, the embryos were supplemented with retinol (ROL, 10 µM), retinaldehyde (RAL, 1 µM) (A, B), or RA (50 nM or 100 nM) (C, D). The frequency of NTC defects was calculated relative to control samples. Sample sizes are shown inside each bar. ****, p < 0.0001; ns, not significant. Brackets denote samples compared, no bracket, compared to the control sample.
	FIGURE 4. RA signaling is required in the prospective ectoderm at the onset of gastrulation to prevent NTC defects. (A–C) Embryos were exposed to DEAB (A), 0.5% EtOH (B), or 1.5% EtOH (C), starting at different developmental stages (NF8, 10.25, 11, 14). The frequency of NTC defects was calculated relative to control samples. (D,E) Embryos were treated with 0.5% (D) and 1.5% EtOH (E) to induce NTC defects. RAL (1 µM) was then added at different stages to rescue the reduction in RA levels. (F) Embryos were injected with mRNA encoding the Cyp26a1 enzyme. Injections targeted unilaterally either the prospective neuroectoderm (B-tier) or dorsal mesoderm (C-tier). The injection site was corroborated by the co-injection of FITC-dextran. (G–I) Embryos were injected with cyp26a1 mRNA targeting the prospective anterior neural plate. FITC-dextran was co-injected to label the injection site. At NTC stages (NF19) the embryos were monitored for NTC defect induction, the localization of the NTC defects, and the overlap with the lineage tracer. The orientation of the embryos, the injected side, and the NTD are shown. Sample sizes are shown within each bar. , p < 0.05; , p < 0.01; , p < 0.001; **, p < 0.0001; ns, not significant. Brackets denote samples compared, no bracket, sample compared to the control sample.
	FIGURE 5. Regulation of the neuroectodermal regulatory network by RA. Embryos were treated with EtOH (0.5% and 1.5%), DEAB (60 µM), or citral (50 µM) from the midblastula stage (NF8) and incubated until early/mid gastrula (NF10.5) for qPCR analysis. (A–F) Relative expression for hoxa1.L, foxd4l1.1.L, gmnn, sox3, pax3.L, and zic1, respectively. Expression levels were normalized to the control expression level at NF 10.5. , p < 0.05; , p < 0.01; , p < 0.001; **, p < 0.0001; ns, not significant. Samples compared to the control sample.
	FIGURE 6. Reduced RA signaling results in the expansion of the neural plate. Embryos were treated with EtOH (0.5% and 1.5%), DEAB (60 µM), and citral (50 µM) and collected for analysis during early neurula stages (NF 14). (A–E) The neural plate was visualized by in situ hybridization with the early marker, sox3. (F) The width of the neural plate was measured in all experimental samples and normalized to the overall width of the embryo to account for individual size changes. (G) The changes in sox3 expression levels were determined by qPCR with gene-specific primers. , p < 0.05; , p < 0.001; **, p < 0.0001; ns, not significant. Samples compared to the control sample.
	FIGURE 7. Reduced RA signaling promotes increased proliferation of neural precursors. Embryos were injected in one blastomere at the 2-cell stage to affect only half of the embryo. Injections included cyp26a1 mRNA with FITC-dextran or FITC-dextran alone. At the onset of gastrulation, the embryos were sorted based on the injected side and further incubated until late gastrula. Embryos were stained by immunohistochemistry with antibodies against pHH3. The positive cells were counted on both sides of the midline, averaging 24 pHH3 positive cells on the control side in each embryo. (A) Control embryo injected with FITC-dextran on the right side. (B) Embryo injected with cyp26a1 mRNA + FITC-dextran on the left side. (C) Relative proliferation analysis based on the number of pHH3-positive cells on both sides. The distribution of all embryos analyzed is shown. The number of embryos analyzed is shown. ****, p < 0.0001 compared to the control (FITC-dextran) sample ratio.
	Supplemental Figure S1. Inhibition of RA biosynthesis induces neural tube closure defects. Inhibition of RA biosynthesis was induced by treatment with DEAB (60 µM), citral (50 µM), or EtOH (0.5% or 1.5% vol/vol). Treatments were initiated during midblastula stages (NF8), and embryos were allowed to develop to neural tube closure stages (NF19-20). Experimental and control embryos were processed for whole-mount in situ hybridization with the neural plate marker pax3. (A-E) Dorsal view of embryos, anterior to the top. (A’-E’) Anterior view of the same embryo shown in the panel above, dorsal to the top. Brackets mark NTC defects and arrowheads open neural tubes.
	Supplemental Figure 2. Developmental progression as a result of EtOH exposure. (A, B) Embryos were treated during midblastula (NF8), early gastrula (NF10.25), mid gastrula (NF11), or early neurula (NF14) with low (0.5%) or high (1.5%) EtOH. The stage reached by the treated embryos was determined at the onset of neurula stages (NF13)(A) and neural tube closure stages (NF19-20)(B). The sample size and the percent embryos at each stage are shown. (C) Previously staged EtOH-treated or control embryos were measured to determine their length and width. The length-towidth ratio was calculated and plotted for EtOH-treated (0.5% and 1.5%) and control embryos for either NF19 and NF20. The ratio distributions are shown. , p<0.05; ***, p<0.0001; ns, not significant.
	Supplemental Figure S3. RA is required for the regulated activation of the neuroectodermal transcriptional network. Embryos were treated to reduce the level of RA signaling by exposing them to EtOH, DEAB, or citral from blastula stages (NF8). RNA was extracted during early/mid gastrula (NF10.5), and the relative expression level of multiple components of the early neural differentiation network was determined by qPCR with gene-specific primers. , p<0.05; , p<0.01; , p<0.001; **, p<0.0001; ns, not significant. Samples compared to the control sample.