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Fig. 3.
Folate receptor 1 knockdown in medial neural plate induces neural tube defects. (A,B) Two-cell-stage embryos were bilaterally microinjected with standard control morpholino (CMO) or morpholino against Folr1 (Folr1-MO). Rescue experiments were performed by either microinjecting along with Folr1-MO, 250 pg folr1 mRNA resistant to Folr1-MO (resistant-folr1 mRNA) or by incubating Folr1-MO-injected embryos with 150 μM folinic acid (FA). (A) Representative examples of control and experimental sibling embryos during neural tube closure (20 hpf) from four out of six experiments in which the severe NTD phenotype prevails in Folr1-MO-injected embryos. Arrowheads indicate open neural tube. Graph shows incidence of severe and nonsevere defective neural tube. Mean±s.e.m.; (number of embryos): 20 pmol CMO (89), 20 pmol Folr1-MO (68), 20 pmol Folr1-MO+150 μM FA (54), Folr1-MO+folr1 mRNA (46); ****P<0.0001; ns, not significant; two-tailed Mann–Whitney U-test (Wilcoxon rank-sum test). (B) Folr1-MO-induced moderate NTD phenotype results in open neural tube. Shown are representative examples of whole embryos at stage 21 and transverse sections of the neural tissue from Folr1-MO-injected embryos exhibiting a moderate NTD phenotype (arrowheads; two out of six experiments) and their siblings injected with CMO or Folr1-MO and FA. Embryos were then processed for β-tubulin (green) and E-cadherin (cadherin 1; red) immunostaining and nuclear labeling (blue, DAPI). Graph shows percentage of embryos with open neural tube; number of embryos: CMO (21), Folr1-MO (21), Folr1-MO+FA (20); ****P<0.0001, ***P<0.0005, *P<0.01; Mann–Whitney two-tailed U-test (Wilcoxon rank-sum test). (C) Dorsal medial and lateral (medial+lateral neural plate, NP) or only dorsal medial (medial neural plate, Med NP) animal blastomeres from 16-cell-stage embryos were microinjected with 3 pmol/blastomere CMO or Folr1-MO along with Alexa 594-dextran conjugate (in red). Embryos were fixed and photomicrographed under a macroscope. Red indicates CMO- or Folr1-MO-containing tissue. Graph shows incidence of severe NTD phenotype (%). Number of embryos with severe neural tube defects out of total in each group was: 0/33 CMO-NP, 42/42 Folr1-MO-NP and 44/44 Folr1-MO-Med NP; ****P<0.0001; Mann–Whitney two-tailed U-test (Wilcoxon rank-sum test). |
