Two novel sites of expression of NADPH cytochrome P450 reductase during murine embryogenesis: Limb mesenchyme and developing olfactory neuroepithelia
1999; Wiley; Volume: 216; Issue: 4/5 Linguagem: Inglês
10.1002/(sici)1097-0177(199912)216
ISSN1097-0177
AutoresDiane S. Keeney, Michael R. Waterman,
Tópico(s)Neurogenesis and neuroplasticity mechanisms
ResumoDevelopmental DynamicsVolume 216, Issue 4-5 p. 511-517 Brief CommunicationFree Access Two novel sites of expression of NADPH cytochrome P450 reductase during murine embryogenesis: Limb mesenchyme and developing olfactory neuroepithelia Diane S. Keeney, Corresponding Author Diane S. Keeney [email protected] Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee Department of Medicine/Dermatology, Vanderbilt University School of Medicine, Nashville, TennesseeDepartment of Biochemistry, Vanderbilt University, 607 Light Hall, Nashville, TN 37232-0146Search for more papers by this authorMichael R. Waterman, Michael R. Waterman Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TennesseeSearch for more papers by this author Diane S. Keeney, Corresponding Author Diane S. Keeney [email protected] Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee Department of Medicine/Dermatology, Vanderbilt University School of Medicine, Nashville, TennesseeDepartment of Biochemistry, Vanderbilt University, 607 Light Hall, Nashville, TN 37232-0146Search for more papers by this authorMichael R. Waterman, Michael R. Waterman Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TennesseeSearch for more papers by this author First published: 11 June 2007 https://doi.org/10.1002/(SICI)1097-0177(199912)216:4/5 3.0.CO;2-HCitations: 12AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract While all cells in eukaryotic organisms probably express the gene encoding NADPH cytochrome P450 reductase, we identified two novel sites which have the highest local concentrations of P450 reductase transcripts during murine embryogenesis. One site is in developing limbs, including lateral limb bud mesenchyme and condensing mesenchyme in the footplate which will form precartilage. A second site is in primitive neuroepithelia, including future olfactory epithelia and olfactory lobes of the brain. These high, local concentrations of P450 reductase transcripts revealed by in situ hybridization were transient and most prominent between embryonic (E) days 12.5–15.5. They cannot be explained by the known functions for P450 reductase. The precursor nature of the highest reductase-expressing cells suggests that differentiation-specific mechanisms regulate P450 reductase gene transcription during organogenesis. The data suggest this multifunctional protein might serve an important role in the formation of precartilage models from condensing limb mesenchyme and in the early development of joints that will form at apposed surfaces of these models. Dev Dyn 1999;216:511–517. ©1999 Wiley-Liss, Inc. INTRODUCTION NADPH cytochrome P450 reductase is a biomarker of endoplasmic reticulum. This flavoprotein contains one mole of FAD and one mole of FMN and serves to transfer electrons, one at a time, from NADPH through FAD to FMN and on to an electron acceptor (Iyanagi and Mason, 1979; Masters and Okita, 1980; Shen and Kasper, 1993). P450 reductase is probably present in the endoplasmic reticulum of all nucleated, eukaryotic cells, including animals, plants, fungi, insects, fishes, and protozoa (Shen and Kasper, 1993). Its best known electron acceptors are the microsomal forms of the cytochrome P450 superfamily (Fig. 1). Beyond its essential role in the activities of P450s, P450 reductase supports the activities of heme oxygenases (Noguchi, Yoshida, and Kikuchi, 1979; Schacter et al., 1972) and squalene epoxidase (Ono et al., 1977). It is also reported to participate in fatty acid elongation (Ilan, Ilan, and Cinti, 1981), fatty acid CoA desaturation (Dailey and Strittmatter, 1980; Enoch and Strittmatter, 1979), thyroid hormone biosynthesis (Yamamoto and DeGroot, 1975), and the reduction of cytochrome b5 (Enoch and Strittmatter, 1979; Ilan et al., 1981). Figure 1Open in figure viewerPowerPoint The catalytic activities of microsomal P450s, heme oxygenases, and squalene epoxidase require reducing equivalents from NADPH, which are transferred by NADPH cytochrome P450 reductase to the active sites of these enzymes. Even though P450 reductase is a remarkably multifunctional flavoprotein, its obligatory role in supporting microsomal P450 activities is best understood. Most microsomal P450s in humans and mice are represented in the EST databases. The recent discovery of P450s expressed solely in differentiated, skin keratinocytes (Keeney et al., 1998a,b), which are not represented in expressed sequence tag (EST) databases, suggests that other novel microsomal P450s remain to be discovered. We asked whether in situ hybridization might be used to map specific sites of expression of P450 reductase during mouse embryogenesis, which in turn might suggest locations of expressed, novel forms of P450. P450 reductase transcripts were most concentrated in limb mesenchyme and developing olfactory neuroepithelia between E12.5 and E15.5. While microsomal P450s might be associated with P450 reductase at these sites, the data suggest that another function of P450 reductase is important. RESULTS AND DISCUSSION Overview of P450 Reductase Expression Patterns in Mouse Embryos At E7.5, the earliest developmental stage examined, antisense P450 reductase [35S]-cRNA hybridized strongly and specifically to steroidogenic, trophoblastic giant cells. Only background levels of hybridization were observed over embryos at E7.5, and at E9.5 and E10.5 (data not shown). P450 reductase transcripts were not absent in these embryonic tissues because they could be amplified by PCR (data not shown). This corroborates previous data demonstrating expression of P450 reductase and CYP51, the P450 required for cholesterol biosynthesis, in preimplantation mouse blastocysts (E4.0) (Strömstedt et al., 1996). In embryos aged E12.5–E15.5, remarkably high concentrations of P450 reductase transcripts were detected in mesenchymal cells in developing limbs and certain epithelial and neural cells. This was transient because after E16 all embryonic tissues appeared to have the same, relatively low expression levels. Elevated P450 Reductase Expression During Limb Development In the developing limbs of E13.5–E15.5 embryos, P450 reductase transcripts were highly concentrated in mesenchymal cells located between closely apposed surfaces of precartilage (Fig. 2). The reductase-expressing cells marked sites where joints will form between the developing cartilaginous elements of the future bones. In the forelimbs, for example, P450 reductase mRNAs localized to mesenchymal cells between the future humerus, ulna and radius and between the future phalanges and metacarpal bones (Fig. 2A–G). After E15.5, the intensity of the hybridization signal decreased, proximally to distally, as chondrification and subsequently ossification ensued. The precartilage models in Fig. 2G give the impression of a relief map, in which differentiating chondrocytes of future bone appear raised and relatively devoid of P450 reductase mRNA. The P450 reductase-expressing cells appear localized in the "clefts" between the models, but only in those mesenchymal cells at apposed surfaces of two or more precartilages where a joint will form. The lowest grain densities were observed in mesenchymal cells bordering the long aspects of the precartilage models and in the chondrifying models themselves (Fig. 2H). Mesenchymal cells delineating these different precartilage surfaces could not be distinguished morphologically, but they were clearly distinguished by their P450 reductase gene expression patterns. Because P450 reductase-expressing cells were never found to circumscribe the precartilage models, they are unlikely of osteoblastic origin. Figure 2Open in figure viewerPowerPoint Localization of NADPH cytochrome P450 reductase mRNA, and comparison with collagen types I and II mRNAs, in developing limbs of fetal mice. Embryonic tissues were hybridized with radiolabeled antisense and sense cRNAs encoding P450 reductase (A-H), collagen type I (I-J) and collagen type II (K-L); results for sense cRNA are not shown. Cellular morphology is shown in brightfield (A,C, E, G, H, I, K). White silver grains in darkfield illumination (B, D, F, J, L) represent specific hybridization. A-B: E13.5 mouse forelimb. Reductase mRNA localized in situ to mesenchymal cells between apposed surfaces of precartilage models (asterisks). C-D: E14.5 forelimb. Same results as A-B. E-F: E15.5 forelimb. Same results as A-B. G: Higher magnification of E15.5 forelimb in E. The dashed lines indicate positions of reductase-expressing mesenchymal cells. H: Higher magnification of precartilage in E. Arrows indicate mesenchymal cells bordering the lateral aspect (long axis) of a chondrifying precartilage model (ch) which had background grain densities. I-J: E13.5 forelimb. Very high levels of collagen type I mRNA localized to most tissues, except chondrocytes of future bones. Silver grains (section is overexposed) appear black in brightfield illumination and white in darkfield. K-L: E13.5 forelimb. Very high levels of collagen type II mRNA localized to the chondrocytes of future bones (this section is also overexposed). Magnification: 12.5×, except G and H are 100×. No similarities were observed between expression patterns for P450 reductase and collagens type I or type II. Collagen type I mRNA localized ubiquitously to mesenchyme surrounding (but excluded from) the precartilage models (Fig. 2I–J). Collagen type II mRNAs exhibited the inverse pattern. They were restricted to differentiating chondrocytes and excluded from the surrounding mesenchyme (Fig. 2K–L). While the identity of the P450 reductase-expressing cells is not known, these data suggest they are not chondrocytes. These in situ hybridization data prompted closer examination of earlier stages of limb development. The distal forelimb or handplate is pentagonal at E12.5 and exhibits five radiations of condensed mesenchyme representing the metacarpal precartilages (Fig. 3A). These are relatively devoid of P450 reductase transcripts, like the chondrifying precartilages in older embryos. P450 reductase transcripts were highly concentrated at the distal tips of each of these radiations and marked the sites of condensing mesenchyme that will form the next precartilage—the future phalanges precartilages (Fig. 3B,C). P450 reductase transcripts also localized to, but were less abundant in, the digital interzones where mesenchymal cells are programmed for cell death. A retinoid metabolizing cytochrome P450 (CYP26A) was previously shown to be expressed in the digital interzones (Fujii et al., 1997). Hence, one function for P450 reductase in these cells is to support CYP26A catalysis. While many microsomal CYP proteins must participate in cellular differentiation during organogenesis, few have been localized to specific, embryonic cell types like CYP26A, thought to catabolize retinoic acid (Fujii et al., 1997), and CYP2B19 in stratifying fetal epidermis thought to bioactivate arachidonic acid (Keeney et al., 1998a). Even CYP51 and CYP4A P450s, presumably required for cholesterol and fatty acid biosynthesis and metabolism, have not been mapped to specific cell types during embryogenesis. The high expression levels of P450 reductase might support catalysis of yet unidentified P450 enzymes. A new P450 subfamily (CYP26B) was recently discovered in the EST databases (Nelson, 1999). Close homology between CYP26A and CYP26B suggests that the uncharacterized CYP26B proteins might have roles in metabolism of retinoids or other signaling molecules during fetal development. Figure 3Open in figure viewerPowerPoint Localization of NADPH cytochrome P450 reductase mRNA in fetal mouse limb mesenchyme, future respiratory, and olfactory epithelia and future olfactory lobes of the brain. Serial sections of embryonic tissues were hybridized with radiolabeled antisense and sense cRNA encoding P450 reductase, except D–F where nonradioactive reductase cRNAs were used for whole-mount in situ hybridization. Results for sense cRNA are shown only for the whole-mount method (E-F). Cellular morphology is shown in paraffin sections illuminated in brightfield (A,G,H,I). Specific hybridization is represented by white silver grains in darkfield illumination (radioactive cRNA: B,C,J,K,L) or by purple reaction product (nonradioactive cRNA: D,E,F). A–C: E12.5 footplate, adjacent sections. The dashed lines indicate five radiations of condensed mesenchyme, the metacarpal precartilage models. These are relatively devoid of reductase mRNAs. Asterisks mark condensing mesenchyme at the distal tips of each metacarpal precartilage. The reductase-expressing mesenchymal condensations mark the sites of formation of phalanges precartilages. Lower levels of reductase mRNAs also localized to the digital interzones. D: E10.5 whole-mount embryos. Reductase mRNAs localized specifically to the lateral surfaces of forelimb and hindlimb buds (arrows). Head and forelimb bud are missing from the lower embryo. E, E10.5 embryos. Lower left embryo (head missing) was hybridized with sense (s) reductase cRNA and lacks reaction product in the forelimb bud. Upper right embryo was hybridized with antisense (as) cRNA and shows intense reaction product on lateral aspects of the forelimb bud, but not at the apical ectodermal ridge (AER). F: E10.5 embryo hybridized with sense reductase cRNA only. Forelimb and hindlimb buds (arrows) lack reaction product. G and J: E13.5 embryo. Reductase mRNA localized to future respiratory epithelium in the external nares (arrows) and nasal passages. H and K: E13.5 embryo. Reductase mRNA localized to epithelium of developing nasal cavity (n.e.) including the future olfactory epithelia and future olfactory lobes (f.o.l.) of the brain. I and L: E14.5 embryo. Same results as H and K. Magnifications: A–C, 12.5×; G and J, 25×; H, I, K, and L, 6.25×. Whole-mount in situ hybridization revealed P450 reductase transcripts in E10.5 limb buds that were not otherwise detected in paraffin sections in situ. The lateral limb bud mesenchyme stained intensely and specifically, while the apical ectodermal ridge appeared relatively devoid of P450 reductase transcripts (Fig. 3D–F). The picture emerging from our time course studies of limb development suggests an inverse relationship between P450 reductase gene expression and mesenchymal cell differentiation. Reductase mRNAs were highly concentrated in condensing limb mesenchyme, precursors of precartilage. Differentiation into precartilage results in loss of P450 reductase expression in the models themselves, concomitant with progressive and preferential expression in mesenchymal cells between apposed precartilages (future joint surfaces). This pattern is repeated as the next (distal) mesenchymal condensation forms, following limb outgrowth from the apical ectodermal ridge, until the full complement of precartilages is formed. These results do not reveal why P450 reductase localizes to specific mesenchymal cells during limb development. They do suggest an unidentified function for NADPH cytochrome P450 reductase in the formation of precartilage models from condensing limb mesenchyme and in the early development of joints that will form at apposed surfaces of these models. Elevated P450 Reductase Expression in Developing Olfactory Neuroepithelia Developing respiratory and olfactory neuroepithelia also exhibited high levels of P450 reductase transcripts between E13.5–E16.5. This includes epithelia lining the external nares, developing nasal airways, primitive nasal cavities (future olfactory epithelium), and future olfactory lobes of the brain (Fig. 3G–L). Except for the olfactory lobes of the brain, these sites in adult animals are noted for unusually high levels of P450 reductase activities and P450-dependent drug metabolism activities (Ding and Coon, 1992; Voigt, Guengerich, and Baron, 1985). P450 reductase transcripts were less concentrated but greater than background levels in neural tissues surrounding the lateral ventricles and the third ventricle at the level of the hypothalamus (data not shown). The spinal cord consistently showed specific hybridization that was slightly above background but less than that detected in the central nervous system. Relatively high levels of P450 reductase message were also observed (E13.5–E16.5) in the epithelium covering the stomach wall (future mucosal surface) and the lobular bronchi of the developing lungs (not shown). This was not surprising because immunoreactive P450 reductase has been described at these sites in adults of several species (Chichester et al., 1991; Hall et al., 1989; Lee and Dinsdale, 1995; Strum et al., 1990). P450 reductase expression in the liver was consistently at background levels, even though CYP3A11 was highly expressed and detected in situ by E13.5 (data not shown). This raises the question whether low expression levels of P450 reductase might be sufficient to support P450 catalysis in all tissues. We suspect that P450 reductase is present in all cells and tissues, and that in situ hybridization has revealed sites having the highest local concentrations in developing embryos. CONCLUSIONS The flavoprotein NADPH cytochrome P450 reductase is probably expressed ubiquitously in fetal and adult tissues to serve as electron donor for cytochromes P450 and possibly other enzymes (Fig. 1). This is evidenced in mouse embryos by unusually high background levels of silver grain development generated by P450 reductase antisense riboprobes. P450 reductase transcripts were detectable by PCR in every embryonic tissue examined (e.g. liver, bone, skin, heart, and muscle in Fig. 4), at every age examined. This was corroborated by Western immunobloting (Fig. 4). The relative abundance of immunoreactive P450 reductase in different embryonic tissues paralleled that observed for its mRNAs. Both are greater in extrahepatic than in hepatic tissues. Figure 4Open in figure viewerPowerPoint P450 reductase mRNA and immunoreactivity in fetal mouse tissues. Top, total RNA was isolated from microdissected tissues (E16.5–E17.5) and amplified by reverse transcriptase PCR. Oligonucleotides specific for NADPH cytochrome P450 reductase yielded a product of predicted size (567 bp) only in the presence of reverse transcriptase (RT). Bottom, E14.5 fetal tissues (50 μg protein/lane) were analyzed by SDS-PAGE and immunoblotting using antiserum raised against rat NADPH cytochrome P450 reductase. Immunoreactive protein migrated slightly slower than a 79.5 molecular size marker (prestained, Bio-Rad broad range standards). Contrary to our objectives, the novel sites of transient, high level expression of P450 reductase in mouse embryos did not lead us to identify novel P450s at these sites or novel functions for P450 reductase activities. The two most puzzling questions are: why are reductase transcripts so highly concentrated in the few, specific cell types; and, why do these patterns appear transiently between E12.5 and E15.5? None of the known functions for P450 reductase seem to explain its expression patterns in limb mesenchyme. For example, CYP51 and P450 reductase are required for cholesterol biosynthesis, and cholesterol is linked covalently to hedgehog proteins post-translationally (Porter, Young, and Beachy, 1996). Yet, P450 reductase expression patterns are quite different from those of hedgehog proteins in limb buds (Echelard et al., 1993). Likewise, none of the proposed roles for P450 reductase in olfactory neuroepithelia seem relevant in midgestation embryos (e.g., odorant bioactivation or clearance and protection from inhaled xenobiotics) (Ding and Coon, 1992; Voigt et al., 1985). Functions served by P450 reductase in olfactory epithelia might be conserved during evolution because in Drosophila melanogaster highest P450 reductase levels were also observed in embryos and in adult antennae (Hovemann, Sehlmeyer, and Malz, 1997). The transient nature of these novel sites of high reductase expression suggests differentiation-specific, transcriptional regulatory mechanisms. Whatever its function, its expression seems related to the precursor nature of cells at these novel sites. Condensing limb mesenchyme is precursor to precartilage (future bones). Mesenchymal cells at apposed precartilage surfaces might be precursors to synovial cells, which will line future joint surfaces. Primitive olfactory neuroepithelia are precursors to future olfactory epithelia and olfactory lobes of the brain. Commitment of reductase-expressing precursor cells to specific cell lineages might obviate the function(s) served by high levels of P450 reductase gene expression at these sites. These data suggest that P450 reductase has an as yet unidentified role in the differentiation of precursor cells during murine organogenesis. EXPERIMENTAL PROCEDURES The in situ hybridization studies utilized radioactive cRNA probes and paraffin-embedded embryos (E7.5–E18.5; gestation is 19 days) collected from timed-pregnant mice (CD-1 outbred strain, Charles Rivers). Embryos were immersion-fixed overnight in 4% buffered paraformaldehyde, and in situ hybridization was done as described (Keeney, 1999). E7.5 embryos remained inside the uterus; all others were removed and dissected free of placental tissues. [35S-UTP]-cRNAs were produced by in vitro transcription from a 1 kb fragment of murine NADPH cytochrome P450 reductase cDNA (573–1620 bp, GenBank D17571). Specific hybridization was distinguished by comparing silver grain development produced by sense and antisense cRNAs applied to consecutive sections on the same slide. Entire embryos were sectioned serially, in sagittal and transverse orientations, to compare results for all tissue types at each developmental stage. Whole-mount in situ hybridization was done as described (Wilkinson, 1992), using a Genius 4 nonradioactive RNA labeling kit (Boehringer Mannheim) and RNA color kit (Amersham). Total RNA was isolated as described (Keeney et al., 1995). 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