Retinoid activation of retinoic acid receptor but not retinoid X receptor is sufficient to rescue lethal defect in retinoic acid synthesis

Abstract

Two isomers of retinoic acid (RA) may be necessary as ligands for retinoid signaling: all-trans-RA for RA receptors (RARs) and 9-cis-RA for retinoid X receptors (RXRs). This was explored by using retinaldehyde dehydrogenase (Raldh)2-/- mouse embryos lacking mesodermal RA synthesis that display early growth arrest unless rescued by all-trans-RA administration. Because isomerization of all-trans-RA to 9-cis-RA can occur, it is unclear whether both ligands are needed for rescue. We show here that an RAR-specific ligand can rescue Raldh2-/- embryos as efficiently as all-trans-RA, whereas an RXR-specific ligand has no effect. Further, whereas all-trans-RA was detected in embryos, 9-cis-RA was undetectable unless a supraphysiological dose of all-trans-RA was administered, revealing that 9-cis-RA is of pharmacological but not physiological significance. Because 9-cis-RA is undetectable and unnecessary for Raldh2-/- rescue, and others have shown that 4-oxo-RA is unnecessary for mouse development, all-trans-RA emerges as the only ligand clearly necessary for retinoid receptor signaling.

Fig. 1.

Structures and receptor specificities of heteroarotinoids. Heteroarotinoids are a class of retinoids that exhibit decreased toxicity in animal models due to the insertion of a heteroatom in the cyclic ring of the arotinoid structure. Specific structural alterations of the heteroarotinoid structure have been shown to alter the receptor specificities of the compounds. The synthesis and receptor specificity of each heteroarotinoid indicated here was described previously as follows. SHet100 (previously compound 18; ref. 31) activates all RARs in the nanomolar range but does not activate RXRs up to 10 μM. OHet72 (previously compound 6; ref. 32) activates RXRs in the nanomolar range but is inactive with RARs up to 10 μM. SHetA2 does not activate either RARs or RXRs (33).

Fig. 2.

Efficiency of Raldh2^-/- embryonic rescue with natural and synthetic retinoids. E10.5 wild-type (WT) and Raldh2^-/- embryos are shown from pregnant mice that were untreated (A and B) or treated on E7.25–8.25 with 2.5 mg/kg all-trans-RA (atRA, C and D), 2.5 mg/kg 9-cis-RA (9cRA, E and F), 10 mg/kg 9-cis-RA (G and H), 0.5 mg/kg RAR-specific SHet100 (I and J), and 2.5 mg/kg RXR-specific OHet72 (K and L). Also, the extent of RARE-lacZ transgene expression is compared for each embryo as an indicator of where retinoid signaling is occurring. It can be seen that treatment with 2.5 mg/kg all-trans-RA, 10 mg/kg 9-cis-RA, and 0.5 mg/kg SHet100 results in a very similar rescued phenotype. However, it should be stressed that, whereas rescued Raldh2^-/- embryos appear grossly normal, forelimb outgrowth is severely retarded (see D, H, and J), and other developmental abnormalities such as those reported for rhombomere segmentation (43) and cardiac development (44) may still exist.

Fig. 3.

Detection of all-trans-RA and 9-cis-RA in E10.5 wild-type and Raldh2^-/- embryos. HPLC chromatograms at 355 nm show the characteristic retention times for 1 ng each of all-trans-retinol (peak 1), 9-cis-RA (peak 2), and all-trans-RA (peak 3) (A) as well as detection of these retinoids in 10 pooled wild-type embryos untreated (B); 9 pooled wild-type embryos (C) and 9 pooled Raldh2^-/- embryos (D) collected on E10.5 from four litters of mice subjected only to the RA-rescue protocol (three doses of all-trans-RA from E7.25–8.25); and 10 pooled wild-type embryos (E) and 10 pooled Raldh2^-/- embryos (F) collected on E10.5 from four litters of mice subjected to the RA-rescue protocol, then administered 10 mg/kg 9-cis-RA on E10.5 2 h before collection.