Glycolytic activity is required for the onset of neural plate folding during neural tube closure in mouse embryos

Abstract

Physiological hypoxia is critical for placental mammalian development. However, the underlying mechanisms by which hypoxia regulates embryonic development remain unclear. We discovered that the expression of glycolytic genes partially depends on hypoxia in neuroepithelial cells of E8.25 mouse embryos. Consistent with this finding, inhibiting glycolysis during the early phase of neural tube closure (E8.0-8.5) resulted in a neural tube closure defect. In contrast, inhibiting the electron transport chain did not affect neural tube formation. Furthermore, inhibiting glycolysis affected cell proliferation, but not differentiation and survival. Inhibiting glycolysis repressed the phosphorylation of myosin light chain 2, and consequent neural plate folding. Our findings revealed that anaerobic glycolysis regulates neuroepithelial cell proliferation and apical constriction during the early phase of neural tube closure.

Keywords: glycolysis; hypoxia; metabolism; mouse; neural tube closure.

FIGURE 1

Hypoxia-dependent expression of glycolytic genes in the mouse embryo. (A) Expression of Aldoa, Ldha, and Pfkfb3 mRNA was detected in whole-mount (Dorsal view) and cryosection (Section) in situ hybridization of E8.25 (six to eight somite stage) embryos. Scale bars, 500 μm (Dorsal view) and 100 μm (Section). Data are representative of six independent experiments. NP, neural plate; NNE, non-neural ectoderm; M, cranial mesenchyme. (B) Expression of Aldoa, Ldha, Pfkfb3, and Nqo1 after the whole-embryo culture with 5% or 20% O₂ determined by RT-qPCR. Messenger RNA levels of genes were normalized to that of Gapdh, and the relative values are presented as white (5% O₂) and gray (20% O₂) bars. Data are shown as means ± S.E.M of five embryos. Statistical differences were assessed using Student’s t-tests, **p < 0.001. (C) Embryos were cultured under hypoxia (5% O₂) or normoxia (20% O₂) from E8.0 (three to five somite stage) to E8.25 (six to eight somite stage). Expression of Aldoa, Ldha, and Pfkfb3 mRNA was detected by in situ hybridization. Neural plates are indicated by gray dotted lines. Scale bars, 100 μm. Data are representative of four independent experiments. NP, neural plate; M, cranial mesenchyme.

FIGURE 2

Inhibition of glycolytic activity results in neural tube closure defects. (A) Schema of exo utero whole-embryo culture with glycolysis inhibitors. Cellular events occurring during the neural tube closure process are shown above. NP, neural plate; NTC, neural tube closure. (B) Morphology of head and whole body of embryos incubated with PBS (control), 0.1 mM 2-DG (2-DG), 28 mM oxamate (oxamate), and normoxia (20% O₂). Yellow arrowheads, defective neural tube closure. Scale bars, 100 μm (head), 500 μm (whole). Images are representative of 33 (control), 21 (2-DG), 15 (oxamate), and 8 (20% O₂) embryos after exo utero whole-embryo cultur.

FIGURE 3

Inhibition of ETC, results in severe growth retardation, but not neural tube closure defect. Head and whole body morphology of embryos incubated with oligomycin and 3-NP. Scale bars, 100 μm (head), 500 μm (whole). Images are representative of 23 (oligomycin) and 20 (3-NP) embryos after exo utero whole-embryo culture.

FIGURE 4

Inhibition of glycolytic activity prevents neural plate folding. (A) Schematic transverse sections of four stages of neural tube closure. (B) Neuroepithelial cells were detected by immunofluorescence staining Sox2 (green) at indicated times after incubation without (control) or with 2-DG. Scale bars, 100 μm. Images are representative of 8 (6 h in control), 9 (12 h in control), 8 (24 h in control), and 10 (6, 12, and 24 h in 2-DG) independent experiments. (C) Ratios (%) of embryos with normal morphology (white bar) and open neural tube (ONT, Gy bar) after incubation without (−) or with 2-DG (+) at indicated somite stage (ss). Data are shown of 14 (control, 4 somite stage), 16 (2-DG, 4 somite stage), 22 (control, 5 somite stage), 26 (2-DG, 5 somite stage), 10 (control, 6 somite stage), 14 (2-DG, 6 somite stage), 13 (control. Seven somite stage) and, 13 (2-DG 7 somite stage) embryos after incubation. Statistical differences were assessed using Chi-Square tests, **p < 0.001.

FIGURE 5

Effect of glycolytic inhibition on mitosis. (A) Mitotic cells detected by immunofluorescence staining phospho-histone H3 (Red) in transverse sections of the neural plate (unilateral) at E8.25 (six to eight somite stage). Directional planes are shown (Ap; apical, Ba; basal). (B) Percentage of phospho-histone H3⁺ mitotic cells among all neuroepithelial cells after incubation without (−) or with 2-DG (+). Data are shown as means ± S.E.M of six histological sections from three embryos. Statistical differences were assessed using Student’s t-tests, *p < 0.05.

FIGURE 6

Substantially reduced pMLC2, and consequent failure of apical constriction of neuroepithelial cells caused by 2-DG. (A) Localization of tubulin, F-actin, and pMLC2 in transverse sections of the neural plate at E8.25 (six to eight somite stage). White arrowheads indicate reduced pMLC2 in embryos incubated with 2-DG. Scale bars, 50 μm. Images are representative of three independent experiments. White arrowheads reduced pMLC2. NP, neural plate. (B) Fluorescence intensity of pMLC2 in neural plate after incubation without (−) or with 2-DG (+). Data are shown as means ± S.E.M of eight histological sections from four embryos. Statistical differences were assessed using Student’s t-tests, **p < 0.001. (C) Localization of pMLC2 in transverse sections of the neural plate at E8.25 (6-8 somite stage) cultured under normoxia and hypoxia. Scale bars, 50 μm. Images are representative of eight independent experiments from four embryos. White arrowheads indicate reduced pMLC2. NP, neural plate. (D) F-actin rings at the apical side of NP visualized by staining with Phalloidin-488 at E8.5 (9–11 somite stage). (E) Graph shows numbers of neuroepithelial cells with different apical areas. Cells were counted in five histological sections from five embryos. The total cell number was 197 in control, and 195 in 2-DG, respectively. Blue, control; Red, 2-DG.

FIGURE 7

Apical localization of glycolytic enzymes in neuroepithelial cells.Localization of Aldoa, Ldha, and Pfkfb3 in transverse sections of the neural plate at E8.25 (six to eight somite stage). Apical surface visualized by pMLC2 staining. Scale bars, 20 μm. Images are representative of five independent experiments. Directional planes were shown (Ap; apical, Ba; basal). White arrowheads, punctate structures. NP, neural plate.