Embryonic exposure to propylthiouracil disrupts left-right patterning in Xenopus embryos

Abstract

Antithyroid medications are the preferred therapy for the treatment of Graves' disease during pregnancy. Propylthiouracil (PTU) is favored over methimazole (MMI) due to potential teratogenic concerns with MMI. This study was to determine the teratogenic potential of MMI and PTU using a validated Xenopus tropicalis embryo model. Embryos were exposed to 1 mM PTU (EC(50)=0.88 mM), 1 mM MMI, or vehicle control (water) from stages 2 to 45. Treated embryos were examined for gross morphological defects, ciliary function, and gene expression by in situ hybridization. Exposure to PTU, but not MMI, led to cardiac and gut looping defects and shortening along the anterior-posterior axis. PTU exposure during gastrulation (stage 8-12.5) was identified as the critical period of exposure leading to left-right (LR) patterning defects. Abnormal cilia polarization, abnormal cilia-driven leftward flow at the gastrocoel roof plate (GRP), and aberrant expression of both Coco and Pitx2c were associated with abnormal LR symmetry observed following PTU exposure. PTU is teratogenic during late blastula, gastrulation, and neurulation; whereas MMI is not. PTU alters ciliary-driven flow and disrupts the normal genetic program involved in LR axis determination. These studies have important implications for women taking PTU during early pregnancy.

Figure 1.

PTU induces embryonic malformations. Embryos were treated from st.8 to 45 with vehicle (n=212), 1 mM PTU (n=122), or 1 mM MMI (n=196). Each experiment was carried out in triplicate, and embryos were examined at st.45. A) PTU treatment led to several developmental defects in the embryo, including heart defects, L-looped hearts, and gut defects, whereas vehicle and MMI treatment had no adverse effects. B) PTU also inhibited tail elongation. C, E, G) Vehicle-treated embryos displayed normal heart looping to the right, counterclockwise gut coiling, and proper tail elongation. D, F, H) Treatment with PTU caused specific malformations, including L-looping of the heart, gut defect (miscoiling), and inhibition of tail elongation. E, F) PTU-treated embryos were immunostained with antibodies to troponin-I, a heart muscle marker, to visualize heart looping. G, gut; n, number of embryos scored; OFT, outflow tract; V, ventricle. *P < 0.001.

Figure 2.

The critical window of vulnerability to PTU is from st. 8 to 12.5. Embryos were exposed to PTU (1 mM) or vehicle over different stages of development and scored at st. 45 for gut defects (A), heart defects (B), and L-looping (C); see Table 3. Tadpoles (st. 45) treated with PTU (1 mM) during st. 8–12.5 showed 3 types of abnormal phenotypes: gut miscoiling (E), L-looped heart and gut miscoiling (F), and complete situs inversus (G). Note that the vehicle picture was taken from a different angle to visualize the OFT and note that one embryo can have different malformations. All malformations were scored and counted individually. G, gut; OFT, outflow tract; V, ventricle. *P < 0.0001; 2-way ANOVA with Bonferroni posttest.

Figure 3.

PTU impairs normal Pitx2c and Coco expression. A) PTU resulted in predominately absent Pitx2c expression in the left LPM. Note that parallel left-sided knockdown of Coco rescued left LPM expression of Pitx2c. PTU prevented left-sided down-regulation of Coco in postflow neurula stage embryos. Numbers in parentheses represent number of analyzed specimens, derived in each case from ≥3 independent experiments. B) ISH (st. 19) of dorsal explants for the early LR marker Coco. Embryos were treated with vehicle or PTU from st.8 to 19. Vehicle treatment resulted in normal expression of Coco (R>L). PTU treatment caused different expression of Coco. R > L, normal Coco expression; R = L, equal Coco expression; R < L, reversed Coco expression. ***P < 0.001; 1-way ANOVA.

Figure 4.

Aberrant flow in PTU-treated GRP explants. A, B) Flow as displayed by GTTs of 25 s length (cf. color bar; A) from representative control (A) and PTU-incubated (B) specimens. C) Bead velocity. Note that beads in PTU samples moved only at 60% of the velocity of control specimens, which were set to 1. D, E) Frequency distribution of trajectory angles (in 8 segments of 45° each), calculated from 9 control and 14 PTU-incubated time-lapse movies. F) Box plot of directionality, as indicated by the dimensionless number ρ. A value of 1 represents a case in which all beads move within the same 45° sector, while ρ = 0 indicates net random displacements of beads. a, anterior; co, control; l, left; p, posterior; r, right. Numbers in parentheses represent number of analyzed movies. *P < 0.05; ***P < 0.001.

Figure 5.

Ciliation defects in the GRP of PTU-treated embryos. A–B″) Cilia and cell boundaries were visualized in dorsal explants from control (A–A″) and PTU-incubated specimens (B–B″) by immunohistochemistry (acetylated α-tubulin, red) and phalloidin staining (actin, green), respectively. C–E) Note that posterior polarization of cilia was significantly reduced (cf. quantification in C), while cilia length (D) and cell surface area (E) were unaltered. F–G′) GRP marker gene Tekt2 was markedly reduced in PTU-treated (G, G′) vs. control (F, F′) explants.