Identification of Endogenous Retinoids, Enzymes, Binding Proteins, and Receptors during Early Postimplantation Development in Mouse: Important Role of Retinal Dehydrogenase Type 2 in Synthesis of All-trans-Retinoic Acid

Abstract

Specific combinations of nuclear retinoid receptors acting as ligand-inducible transcription factors mediate the essential role of retinoids in embryonic development. Whereas some data exist on the expression of these receptors during early postimplantation development in mouse, little is known about the enzymes controlling the production of active ligands for the retinoid receptors. Furthermore, at early stages of mouse development virtually no data are available on the presence of endogenous retinoids. In the present study we have used a recently developed high-performance liquid chromatographic (HPLC) technique to identify endogenous retinoids in mouse embryos down to the egg cylinder stage. All-trans-retinoic acid, a ligand for the retinoic acid receptors, was detected in embryos dissected as early as 7.5 dpc (i.e., a combination of midstreak until late allantoic bud stage embryos). At these stages, we detected mRNA coding for all the retinoid receptors, retinoid binding proteins, and two enzymes able to convert retinol to retinal (retinol dehydrogenase 5 (RDH5) and alcohol dehydrogenase 4 (ADH4)). We also detected retinal dehydrogenase type 2 (RALDH2), an enzyme capable of oxidising the final step in the all-trans-retinoic acid synthesis. In egg cylinder stage mouse embryos no all-trans-retinoic acid was detected. However, at this stage its precursor all-trans-retinal was present. In accordance with these HPLC observations, RDH5 and ADH4 were expressed, but no transcripts coding for enzymes that oxidise retinal to retinoic acid. Therefore, our results suggest that RALDH2 is a key regulator in initiating retinoic acid synthesis sometime between the mid-primitive streak stage and the late allantoic bud stage in mouse embryos.

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

“…Even though a targeted mutation of the RXR␣ AF-2 ligand-binding domain shows that this protein domain is important for mouse development (14), there is no evidence that AF-2 needs to bind 9-cis-RA to perform its function. Also, although all-trans-RA is easily detectable in many mammalian tissues (15)(16)(17)(18), detection of 9-cis-RA as originally reported (6) has not been confirmed. Nevertheless, in vitro studies pursuing the mechanism of retinoid signaling suggest that transcriptional activation by RXR homodimers depends on 9-cis-RA (13), and that RAR/RXR heterodimers may in some contexts depend solely on binding of all-trans-RA to the RAR partner, so-called RXR subordination (19); but in other contexts RXR subordination is overcome, and binding of 9-cis-RA to RXR is involved in the transcriptional mechanism (20).…”

“…Because RXRs exhibit binding constants for 9-cis-RA ranging from 14 to 18 nM plus EC 50 values for 9-cis-RA transactivation ranging from 7 to 20 nM (7), our finding of a value Ͻ0.7 nM for 9-cis-RA indicates that there is no evidence that 9-cis-RA is present in embryos at a concentration high enough to regulate RXR. Other studies that have detected all-trans-RA in mouse embryos have also mentioned that 9-cis-RA is undetectable (15,17). Whereas it remains possible that a small population of cells in the embryo may contain sufficient 9-cis-RA to activate RXR, no evidence for this has been reported.…”

Retinoids Regulate the Repairing Process of the Podocytes in Puromycin Aminonucleoside-induced Nephrotic Rats

“…Because it is difficult to measure the intracellular concentration of ATRA and/or other retinoids in the podocytes, different strategies will be required to address this question. It is reported that the distribution of RALDH2 provides the most accurate guide to the localization of ATRA (41)(42)(43)(44). Therefore, studying the transcriptional regulation of RALDH2 gene might be informative to further understand the temporal change of intracellular concentration of ATRA.…”

Quantitative axial profiles of retinoic acid in the embryonic mouse spinal cord: 9‐Cis retinoic acid only detected after all‐trans‐retinoic acid levels are super‐elevated experimentally

“…cDNA synthesis and PCR (40 cycles) was performed as described by Ulven et al (1998). The specific primer pairs are listed in a previous report (Ulven et al, 2000).…”