Int J Stem Cells.  2023 Nov;16(4):385-393. 10.15283/ijsc23012.

Transcriptional Signature of Valproic Acid-Induced Neural Tube Defects in Human Spinal Cord Organoids

• Lee JH
• Shaker MR
• Park SH
• Sun W

• KMID: 2548002
• DOI: http://doi.org/10.15283/ijsc23012

Abstract

In vertebrates, the entire central nervous system is derived from the neural tube, which is formed through a conserved early developmental morphogenetic process called neurulation. Although the perturbations in neurulation caused by genetic or environmental factors lead to neural tube defects (NTDs), the most common congenital malformation and the precise molecular pathological cascades mediating NTDs are not well understood. Recently, we have developed human spinal cord organoids (hSCOs) that recapitulate some aspects of human neurulation and observed that valproic acid (VPA) could cause neurulation defects in an organoid model. In this study, we identified and verified the significant changes in cell–cell junctional genes/proteins in VPA-treated organoids using transcriptomic and immunostaining analysis. Furthermore, VPA-treated mouse embryos exhibited impaired gene expression and NTD phenotypes, similar to those observed in the hSCO model. Collectively, our data demonstrate that hSCOs provide a valuable biological resource for dissecting the molecular pathways underlying the currently unknown human neurulation process using destructive biological analysis tools.

Keyword

Organoid; Neurulation; Neurodevelopmental disorders; Neural tube defects; Valproic acid

Figure

• 

  Fig. 1 Modeling of valproic acid (VPA)–induced neural tube defects with human spinal cord organoids (hSCOs). (A) Experimental model to study the effect of duration of VPA treatment on morphogenesis. (B) Re-presentative time-lapse images of neural tube morphogenesis in VPA-treated groups compared with the control. Each image was acquired using an automated imaging system. Scale bar= 200 μm. (C) Quantification of morphogenesis with different durations of VPA treatment. The color of each box indicates the cumulative proportion of neural folding (upper) or neural tube (bottom). Stage of hSCOs at the indicated culture time. (D) Three-dimensional neural tube morphology with different VPA treatment durations on day 15. The structure was visualized using an apical marker and ZO1 staining. Scale bar=100 μm.

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  Fig. 2 Transcriptome analysis in human spinal cord organoids (hSCOs) exposed to valproic acid (VPA). (A) Scatter plots of transcriptome levels in hSCOs treated with VPA compared with the controls. Red circles denote the genes whose expression levels were significantly altered (≥2-fold) in VPA-treated vs control groups. (B) Biological processes associated with significantly upregulated genes. (C) Network plot of the upregulated genes enriched in VPA-treated hSCOs. Interactive network is generated using GeneMANIA plugin in Cytoscape. (D) Immunohistochemical evaluation of the upregulation of tight junction proteins, Occludin (OCLN), and ZO2. Average intensity of tight junction proteins was measured along the apico-basal axis. Scale bar=20 μm. (E) Perturbation of polarized localization of ZO1 and pMLC proteins in the neuroepithelial cells of VPA-treated hSCOs. White arrows indicate the ectopic expression of proteins in the VPA-treated group. Bar graphs show the number of ectopic ZO1 and pMLC punctas in neural-plate layers. Unpaired Student’s t-test was used for comparing two groups (**p<0.01 and ***p<0.01). Scale bar=20 μm. FC: fold change, Ctrl: control.

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  Fig. 3 Effect of tight junction proteins expression/distribution in valproic acid (VPA)-treated human spinal cord or-ganoid. (A) Immunohistochemistry of upregulation of tight junction proteins, ZO2 (red) and ZO1 (green) caused by VPA treatments for different durations. Scale bar=10 μm. (B) Line plots denoting the intensity of ZO1 and ZO2 staining along the closed/opened neural tube. (C) Pertur-bation of polarized localization of ZO1 proteins in neuroepithelium. The neuroepithelial cells were identified as SOX2 (magenta) staining and cellular arrangement surrounding apical marker, ZO1 (green). Scale bar=10 μm. (D) Quantification of the number of ectopic ZO1 punctas within neuroepithelial cell layers. Bar graphs represent mean±SEM, with all the points plotted. Unpaired Student’s t-test was used for comparing two groups (*p<0.05).

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  Fig. 4 Effect of valproic acid (VPA) on neural tube in the mouse em-bryo. Immunohistochemistry of VPA-induced neural tube defect mouse models. Various markers were used to observe changes in the expre-ssion/distribution of actin filaments (A, F-actin and pMLC) and tight junction proteins (B, Zo1, Zo2, and Ocln). Dissolved VPA was administrated three times to pregnant female mice at the time of primary neuru-lation. Scale bars=20 μm. Min: minimum, Max: maximum.

Reference

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