Saili KS, Zurlinden TH, Antonijevic T, Shah I, Deisenroth C, Knudsen TB. Endodermal differentiation trajectories diverge with increasing all-trans retinoic acid (ATRA) exposure. Poster at Society of Toxicology Annual Meeting, Baltimore, MD, March 2019.
A narrow range of endogenous retinoic acid (RA) levels is required for proper anteroposterior patterning of the early embryo. Insufficient or overabundant RA levels disrupt the expression of HOX and other genes important for axis patterning and can lead to birth defects such as cleft palate. We hypothesized that the expression of development marker genes is measurably altered by exogenous concentrations of all-trans retinoic acid (ATRA) that affect endogenous RA signaling. To confirm molecular markers of altered embryogenesis, a pilot study was conducted in differentiating endoderm exposed to ATRA. Induced pluripotent stem cells derived from adult female fibroblasts were directed to a differentiating endodermal trajectory using low serum and activin. Differentiating cells were then exposed daily to five concentrations of ATRA (0.001 to 10 µM) or 0.1% DMSO control and collected at three timepoints (6h, 96h, and 192h) for high content imaging (HCI) and RNA-sequencing analysis. HCI analysis quantitated the cell counts and percentage of cells that were dead, metabolically active, or expressing FOXA2 (a definitive endoderm marker). These results were compared to the differentially expressed genes (DEGs; padj < 0.05 and abs[log2 fold change] >1) at each time and concentration. Using system trajectory and dynamics analysis of the DEGs, a tipping point defined by the persistence of effects at both 96h and 192h timepoints was identified at 0.01 µM. Global gene expression at concentrations above and below the tipping point followed two diverging trajectories reflected in a principal components analysis, with concentrations above 0.01 µM resulting in an increase in cell counts and reduced FOXA2 expression compared to time-matched DMSO controls. Approximately 100 genes were selected as developmental markers to include the primitive streak, definitive endoderm, yolk sac endoderm, foregut, midgut, and hindgut. The expression profile of these marker genes suggested that cells exposed to concentrations above 0.01 µM shifted from an anterior to posterior molecular phenotype compared to cells exposed to concentrations below the tipping point. The molecular markers of this phenotypic shift will be used to establish a testing platform to screen chemicals for potential disruption to early embryonic development through altered RA signaling. This work does not represent US EPA policy.