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. 2020 Oct 13;21(20):7534.
doi: 10.3390/ijms21207534.

MiR-302 Regulates Glycolysis to Control Cell-Cycle during Neural Tube Closure

Rachel A Keuls  1   2 , Karin Kojima  2 , Brittney Lozzi  3 , John W Steele  4   5 , Qiuying Chen  6 , Steven S Gross  6 , Richard H Finnell  4   5 , Ronald J Parchem  1   2   4
Affiliations

MiR-302 Regulates Glycolysis to Control Cell-Cycle during Neural Tube Closure

Rachel A Keuls et al. Int J Mol Sci. .

Abstract

Neural tube closure is a critical early step in central nervous system development that requires precise control of metabolism to ensure proper cellular proliferation and differentiation. Dysregulation of glucose metabolism during pregnancy has been associated with neural tube closure defects (NTDs) in humans suggesting that the developing neuroepithelium is particularly sensitive to metabolic changes. However, it remains unclear how metabolic pathways are regulated during neurulation. Here, we used single-cell mRNA-sequencing to analyze expression of genes involved in metabolism of carbon, fats, vitamins, and antioxidants during neurulation in mice and identify a coupling of glycolysis and cellular proliferation to ensure proper neural tube closure. Using loss of miR-302 as a genetic model of cranial NTD, we identify misregulated metabolic pathways and find a significant upregulation of glycolysis genes in embryos with NTD. These findings were validated using mass spectrometry-based metabolite profiling, which identified increased glycolytic and decreased lipid metabolites, consistent with a rewiring of central carbon traffic following loss of miR-302. Predicted miR-302 targets Pfkp, Pfkfb3, and Hk1 are significantly upregulated upon NTD resulting in increased glycolytic flux, a shortened cell cycle, and increased proliferation. Our findings establish a critical role for miR-302 in coordinating the metabolic landscape of neural tube closure.

Keywords: cell cycle; glycolysis; hyperglycemia; maternal diabetes; miR-302; microRNA; neural tube closure; neural tube defects; oxidative stress; proliferation.

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Conflict of interest statement

R.H.F. formerly held a leadership position in the now dissolved consulting company TeratOmic Consulting LLC.

Figures

Figure 1
Figure 1
Metabolism and differentiation are coupled during neurulation. (A) Differential expression of ectoderm derived cell populations grouped together and compared to the non-ectoderm-derived cells at E8.25. (B) Bar plot showing the top up- and downregulated genes at E8.25. Arrows point out metabolic genes. (C) Dot plot showing molecular gene ontology of differentially expressed genes at E8.25. (D) Differential expression of ectoderm-derived cell populations grouped together and compared to the non-ectoderm-derived cells at E9.5. (E) Bar plot showing top up- and downregulated genes at E9.5. Arrows point out metabolic genes. (F) Dot plot showing molecular gene ontology analysis of differentially expressed genes at E9.5.
Figure 2
Figure 2
Single-cell sequencing reveals metabolic contributions to neural tube closure. (A) Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) plot showing the ectoderm-derived cell populations that contribute to neural tube closure obtained from single-cell sequencing of the cranial region at E8.25 and E9.5. (B) Bar plot showing gene expression of metabolic pathways in ectoderm-derived cell at E8.25 and (C) E9.5. (D) Circle plot showing the change in expression of metabolic pathways from E8.25 to E9.5 for each ectoderm-derived cell population. The white exterior region of the plot represents upregulation while the grey interior represents downregulation. (E) Line graphs showing change in expression for each metabolic pathway during neural tube closure in the neural tube, neural crest, and non-neural ectoderm.
Figure 3
Figure 3
Upper glycolysis is regulated by miR-302. (A) DAPI staining of wildtype and miR-302 knockout embryos in both wholemount and transverse cross section to show the resulting cranial neural tube closure defect. (B) UMAP plot comparing ectoderm-derived cell of wildtype and miR-302 knockout embryos at E9.5. (C) Bar plot and (D) circle plot showing the misregulation of metabolic pathways in ectoderm-derived populations upon miR-302 deletion. (E) Bar plot summarizing the misregulation of genes in each metabolic process upon miR-302 deletion. (F) Schematic showing the process of glycolysis including glucose import, the pentose phosphate pathway, and upper and lower glycolysis. (G) Boxplot of the misregulation of the glycolysis pathway of the miR-302 knockout. (H) Violin plots to show population specific misregulation of the glycolysis pathway in the miR-302 knockout.
Figure 4
Figure 4
Loss of miR-302 leads to accumulation of glycolytic intermediates. (A) Schematic showing sample collection for mass spectrometry. (B) Heatmap of global misregulation of metabolites upon miR-302 deletion. (C) Dot plot showing how metabolic pathways were changed in the miR-302 knockout. (D) Bar plot of the top significantly up- and downregulated metabolites. (E) Heatmap showing the upregulation of the glycolysis pathway. (F) Boxplot showing upregulation of pyruvate and (G) ATP in the miR-302 knockout.
Figure 5
Figure 5
miR-302 targets Pfkp, Pfkfb3, and Hk1 to regulate upper glycolysis. (A) Schematic showing miR-302 targeting of the upper glycolysis steps that control the rate of glycolytic flux. (B) Box plot of misregulated miR-302 targets within the glycolysis pathway. Each miR-302 target is represented as a data point. (C) Volcano plot showing the only significantly upregulated glycolysis metabolite with a fold change >1.5 is that produced by upregulated miR-302 target Pfkfb3. (D) Schematic showing protein extraction and PFK assay to measure PFK activity differences between wildtype and miR-302 knockout embryos. (E) Line graph of representative replicate from PFK assay showing change in absorbance over time. (F) Linear region of the curve plotted for representative replicate to show slope calculation to represent PFK activity. (G) Fold change of PFK activity between wildtype and miR-302 knockout embryos.
Figure 6
Figure 6
miR-302 targets Pfkp, Pfkfb3, and Hk1 to regulate cell proliferation. (A) Representative differentially expressed genes and (B) biological gene ontology analysis on the differentially expressed genes obtained from comparing the cells within ectoderm-derived populations expressing Pfkp, Pfkfb3, or Hk1 to ectoderm-derived and non-ectoderm-derived cells at E9.5. (C) Bar plot of misregulation of genes upon miR-302 deletion that promote the G2/M and G1/S phase transitions. (D) Violin plot of misregulation of Cyclin and Cdk genes of the miR-302 knockout. (E) Bar plots showing number of cells and cyclin expression in cells that co-express G1/S and G2/M genes. (F) Blended UMAP plot and bar plot to show number of cells co-expressing Pfkpfb3 with both Cdk1 and Cdk4. (G) Schematic of co-expression of cell cycle transition genes and increased proliferation of the miR-302 knockout.
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