The identification of a 9-cis retinol dehydrogenase in the mouse embryo reveals a pathway for synthesis of 9-cis retinoic acid

Abstract

The ligand-controlled retinoic acid (RA) receptors and retinoid X receptors are important for several physiological processes, including normal embryonic development, but little is known about how their ligands, all-trans and 9-cis RA, are generated. Here we report the identification of a stereo-specific 9-cis retinol dehydrogenase, which is abundantly expressed in embryonic tissues known to be targets in the retinoid signaling pathway. The membrane-bound enzyme is a member of the short-chain alcohol dehydrogenase/reductase superfamily, able to oxidize 9-cis retinol into 9-cis retinaldehyde, an intermediate in 9-cis RA biosynthesis. Analysis by nonradioactive in situ hybridization in mouse embryos shows that expression of the enzyme is temporally and spatially well controlled during embryogenesis with prominent expression in parts of the developing central nervous system, sensory organs, somites and myotomes, and several tissues of endodermal origin. The identification of this enzyme reveals a pathway in RA biosynthesis, where 9-cis retinol is generated for subsequent oxidation to 9-cis RA.

Figure 1

(A) Alignment of the amino acid sequence of RDH4 with bovine 11-cis RDH (b11-cis RDH) and three all-trans RDHs expressed in rat liver (ra-t RDHs). Residues identical to RDH4 are boxed and gaps have been introduced to optimize the alignment. The numbered bars indicate the positions of the oligonucleotide mixtures used in the first (#1), and in the second (#2) round of PCR amplifications of mRNA from mouse gd 10 embryos. (B) Phylogenetic analysis of the amino acid sequences of several RDHs. RDH4 is closely related to bovine and human 11-cis RDHs (t11-cis RDH and h11-cis RDH, respectively) suggesting that the cis-specific RDHs and the all-trans-specific RDHs form distinct subgroups within the RDHs of the SDR family. The analysis was done by using the PAM 250 distance table.

Figure 2

(A) Immunoblotting analysis of total membrane fractions from baculovirus-infected Sf9 insect cells expressing RDH4 or from mock-infected Sf9 insect cells. The results show that RDH4 is expressed as a 32-kDa membrane-associated polypeptide. (B-F) Reversed-phase HPLC profiles on the formation of 9-cis retinaldehyde (9-cis RAl) from 9-cis retinol (9-cis ROH) in membrane fractions from Sf9 insect cells overexpressing RDH4 in the presence of NAD⁺ (B), NADP (C), or from mock-infected cells in the presence of NAD⁺ (D). Identical analyses using all-trans retinol as the substrate when using membrane fractions from cells overexpressing RDH4 (E) or from mock-infected cells (F) show that all-trans retinol is not a preferred substrate for RDH4. The results demonstrate that RDH4 is a NAD⁺-dependent 9-cis RDH.

Figure 3

(A) Northern blotting analysis of transcripts for RDH4 in several adult mouse tissues. Expression of a single 1.4-kb transcript is seen in liver, kidney, and brain whereas expression levels in other tissues are much lower. (B) RT-PCR amplifications of a 420-bp fragment derived from RDH4 transcripts in mouse gd 9, 11, 13, and 15 embryos show that the enzyme is expressed during a large part of mouse embryonic development.

Figure 4

Nonradioactive in situ hybridization analysis of RDH4 expression in mouse embryos during gd 10 and 11. (A) Sagittal section of a gd 10 embryo, at the level of the heart, showing expression of RDH4 in the alar plate of the spinal cord. (B) Section, parallel to that of A, hybridized with a sense probe showing absence of staining, thus verifying the specificity of the in situ hybridization technique. (C) Transverse section of a gd 11 embryo, at the level of the genital ridge, showing the expression of RDH4 in myotome and migrating muscle progenitor cells in the body wall. Expression also was seen in cells of the genital ridge, spinal ganglion, and the liver. (D) Sagittal section of a gd 11 embryo showing expression of RDH4 in the basal cells of the neuroepithelium of the mesencephalic flexure, and in collections of cells in the pons. (E) Frontal section of a gd 11 embryo, showing expression of RDH4 in the retina and lens, and also in the neural crest-derived cells of the anterior eye segment. (F) Frontal section of a gd 11 embryo, showing a dorso-ventral gradient of expression of RDH4 in the mesencephalon and a distinct expression in the floor plate. (G) Section through the hind part (just behind the hind limb) of a gd 11 embryo showing strongly stained cells in the spinal cord and notochord, and in the cells of the dermomyotome in a pattern consistent with an expression in a myogenetic cell lineage. Note the lack of expression in the sclerotome. ap, Alar plate; bp, basal plate; cp, neural crest-derived corneal precursor mesenchyme; dm, dermomyotome; fp, floor plate; l, liver; m, myocytes; mf, mesencephalic flexure; my, myotome; pe, pigment epithelium; p, pons; n, notochord; r, retina; s, sclerotome; sc, spinal cord; sg, spinal ganglia; gr, genital ridge. Original magnifications were ×80 (A–E and G) and ×40 (F).

Figure 5

Proposed metabolic pathways for synthesis of all-trans, 9-cis, and 11-cis retinoids. The existence of a 9-cis RDH suggests that 9-cis retinol is generated from all-trans retinol by using an evolutionary conserved mechanism similar to that involved in the generation of 11-cis retinol in the visual system. The proposed metabolic route generating 9-cis RA from 9-cis retinol is similar to that generating all-trans RA from all-trans retinol.