DEV004713 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Thapsigargin treatment impacts asymmetric gene expression. Arrowheads denote bilateral domain of spaw expression in the tailbud of (A) wt and (B) treated embryos. Arrows denote left-sided lefty2 expression in (C) wt and (D) bilateral in treated embryos. Arrows denote left-sided pitx2c expression in (E) wt and (F) bilateral in treated. Dotted arrows denote left-sided pitx2c expression in the brain of (G) wt and (H) bilateral in treated.

• Supplemental Figure S2 -

  Fig. S2. Thapsigargin treatment impacts organ laterality without disrupting midline integrity. ntl RNA expression in (A) wt and (B) treated embryos. Ntl protein expression in (C) wt and (D) treated. goosecoid (gsc)-GFP in (E) wt and (F) treated embryos. (G) Summary of midline expression of lefty1, shh, ntl, Ntl, and gsc in wt, control and thapsigargin-treated embryos. Thap, thapsigargin.

• Supplemental Figure S3 -

  Fig. S3. Valproate treatment results in LR patterning defects. Left-sided lefty1 expression in (A) wt and (B) bilateral in valproate-treated embryos. (C) Summary of lefty1 LPM expression after exposure to valproate.

• Supplemental Figure S4 -

  Fig. S4. Molecular marker evaluation of DFC migration/cohesion and Syto-11 uptake after thapsigargin treatment. Arrowheads denote lrdr1 expression 80% epiboly in the DFC region in (A) wt and (B) thapsigargin-treated embryos. lrdr1 expression at 4-somite stage in the KV region in (C) wt and in (D) thapsigargin-treated embryos. sox17 expression at 4-somite stage in the KV region in (E) wt and in (F) thapsigargin-treated embryos. Syto-11 fluorescence at 17-somite stage in the tail bud region in (G) wt and in (H) thapsigargin-treated embryos. Acetylated tubulin (acTub) immunostaining denotes cilia at 10-somite stage in the KV region in (I) wt and in (J) thapsigargin-treated embryos. (K) Summary of DFC distribution. Thap, thapsigargin.

• Supplemental Figure S5 -

  Fig. S5. Increased β-catenin reporter activity after thapsigargin treatment. (A) TopFlash (top) vs FopFlash (fop) luciferase reporter constructs analyzed for relative luminescence in wt or thap-treated embryos 20 minutes after treatment, normalized to renilla luciferase. TOPdGFP reporter transgenic line, WMISH with GFP probe, dorsal side shown, GFP expression is excluded from DFC region in (B) wt, but detected in (C) thapsigargin-treated embryos. Typical lateral domains of TOPdGFP reporter expression are present in both untreated and treated embryos. Arrows note DFC region. (D) Summary of lefty1/2 expression in DFC-targeted embryos. Axin1-MO injected had a P value <0.01 by Chi-square analysis. (E) Summary table of KV alterations of DFC-targeted MOs and Ca²⁺/ PI cycle inhibitors. Thap, thapsigargin; WT, wild type; Ctrl, control DMSO; Thap low, low concentration; Thap high, high concentration; VPA, valproate.

• Supplemental Figure S6 -

  Fig. S6. Thapsigargin treatment in Xenopus results in increased nuclear β-catenin. Confocal images of both sides of wild-type and thapsigargin-treated embryos at stage 17, showing β-catenin immunostaining. Black boxes on top panels are enlarged in bottom panels to show nuclear β-catenin in wild-type and treated embryos. Thap, thapsigargin.

• Movie 1 -

  Movie 1. Ratio images from a lateral view spanning 65-80% epiboly. The dorsal side is shown, with Shield/DFC region located to the left. Images were collected at 15-second intervals playing at 5 frames per second in a Quicktime movie.

• Movie 2 -

  Movie 2. Images from Dusp6:d2EGFP transgenic embryo spanning 80% epiboly-5 somites. DFC/KV region is shown. Images were collected at 5-minute intervals playing at 6 frames per second in a Quicktime movie.

• Movie 3 -

  Movie 3. Images from Dusp6:d2EGFP transgenic embryo spanning 80% epiboly-5 somites after thapsigargin treatment. DFC/KV region is shown. Images were collected at 5-minute intervals playing at 6 frames per second in a Quicktime movie.