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. 2021 Oct 20;9:747969. doi: 10.3389/fcell.2021.747969

FIGURE 3.

FIGURE 3

Transient physiological manipulation of RA levels for robustness analysis. (A) Embryos were treated with increasing concentrations of all-trans RA from 1 nM to 1 μM. Treatments were initiated at st. 8.5 and RNA samples were collected at tailbud stages (st. 32–33) for phenotypic analysis. To exemplify the induced developmental malformations, lines depicting the size of the head domain (blue), the trunk (red), and the tail (purple), were drawn on the control embryo (-RA) and then copied unto the treated embryos (10–1,000 nM). (B) Distribution of the severity of the developmental defects induced by the retinoic acid manipulations. Treated embryos were scored for the induction of moderate (as in 25 or 50 nM) or severe phenotypes (as in 100 or 1,000 nM), or normal looking (as in -RA). (C–H) RA treated embryos were collected at early (st. 10.25) and late (st. 12) gastrula for analysis of the changes in expression level. The response of RA metabolic and target genes was studied by qPCR. (C) hoxb1 (D) cyp26a1 (E) dhrs3 (F) aldh1a2 (G) aldh1a3 (H) rdh10. Statistical significance (Student’s t-test) was calculated compared to the control group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.