Fig. 8.
Pharmaceutical intervention of spinal NTDs in Pax3-Cre;Lrp6-cKOs by maternal supplementation of a Wnt/β-catenin signaling agonist. (A) The embryo numbers and Lrp6-cKO ratios are not significantly different between the control and lithium-treated groups at E18.5 as compared with the expected Mendelian ratio (25% cKOs) (P>0.05, chi-square test). (B) Dorsal–caudal body views of a double heterozygous (Het) Pax3Cre;Lrp6flox/+ embryo that shows no NTD and with normal tail (B1), an Lrp6-cKO embryo in the control group that shows the severest lumbosacral NTD (with ∼8 mm lesion length, bracket in B2), an Lrp6-cKO embryo treated with lithium that shows a fully closed or rescued spinal cord (0 mm lesion length in B3, asterisk shows partially rescued tail growth), and a mutant embryo treated with lithium that shows milder NTD (with 3 mm lesion length in B4) at E18.5. (C) Rescue effects of spinal NTDs in Lrp6-cKOs examined at E18.5 after maternal supplementation of lithium chloride (LiCl) from E7.5 to E9.5. The lesion lengths (mm) were measured under a microscope. *P=0.02 (Fisher exact test); after all samples combined and averaged in each group, P=0.01 (unpaired, two-tailed Student's t-test). (D) RT-qPCR results demonstrate significant restoration of Pax3 mRNA in the lithium-treated Lrp6-cKO PNPs at E9.5. n.s., no statistical significance (P>0.05); **P<0.01 (unpaired, two-tailed Student's t-test). (E) Illustrative summary of Lrp6-mediated β-catenin–Pax3/Cdx2/Cdx4 signaling underlying PNP closure/elongation and spinal NTD; the latter can be rescued by either genetic activation of β-catenin or maternal supplementation of lithium ion, which stabilizes intracellular β-catenin by inhibiting Gsk3 in the canonical Wnt signaling pathway, thus restoring a key downstream transcription factor Pax3 in Lrp6-deficient PNPs. Red font, mutants and phenotypes; green font and arrows, genetic or pharmacological rescues demonstrated in the current study.