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Aldehyde Dehydrogenase 6, a Cytosolic Retinaldehyde Dehydrogenase Prominently Expressed in Sensory Neuroepithelia during Development

2000; Elsevier BV; Volume: 275; Issue: 52 Linguagem: Inglês

10.1074/jbc.m007376200

1083-351X

Felix Grün, Yukihiro Hirose, Shimako Kawauchi, Toshihiko Ogura, Kazuhiko Umesono,

Advanced Fluorescence Microscopy Techniques

We have isolated the chick and mouse homologs of human aldehyde dehydrogenase 6 (ALDH6) that encode a third cytosolic retinaldehyde-specific aldehyde dehydrogenase. In both chick and mouse embryos, strong expression is observed in the sensory neuroepithelia of the head. In situ hybridization analysis in chick shows compartmentalized expression primarily in the ventral retina, olfactory epithelium, and otic vesicle; additional sites of expression include the isthmus, Rathke's pouch, posterior spinal cord interneurons, and developing limbs. Recombinant chick ALDH6 has aK0.5 = 0.26 μm,Vmax = 48.4 nmol/min/mg and exhibits strong positive cooperativity (H = 1.9) toward all-trans-retinaldehyde; mouse ALDH6 has similar kinetic parameters. Expression constructs can confer 1000-fold increased sensitivity to retinoic acid receptor-dependent signaling from retinol in transient transfections experiments. The localization of ALDH6 to the developing sensory neuroepithelia of the eye, nose, and ear and discreet sites within the CNS suggests a role for RA signaling during primary neurogenesis at these sites. We have isolated the chick and mouse homologs of human aldehyde dehydrogenase 6 (ALDH6) that encode a third cytosolic retinaldehyde-specific aldehyde dehydrogenase. In both chick and mouse embryos, strong expression is observed in the sensory neuroepithelia of the head. In situ hybridization analysis in chick shows compartmentalized expression primarily in the ventral retina, olfactory epithelium, and otic vesicle; additional sites of expression include the isthmus, Rathke's pouch, posterior spinal cord interneurons, and developing limbs. Recombinant chick ALDH6 has aK0.5 = 0.26 μm,Vmax = 48.4 nmol/min/mg and exhibits strong positive cooperativity (H = 1.9) toward all-trans-retinaldehyde; mouse ALDH6 has similar kinetic parameters. Expression constructs can confer 1000-fold increased sensitivity to retinoic acid receptor-dependent signaling from retinol in transient transfections experiments. The localization of ALDH6 to the developing sensory neuroepithelia of the eye, nose, and ear and discreet sites within the CNS suggests a role for RA signaling during primary neurogenesis at these sites. retinoic acid response element retinoic acid aldehyde dehydrogenase mouse aldehyde dehydrogenase 6 chick aldehyde dehydrogenase 6 retinoic acid receptor retinoid X receptor polymerase chain reaction high pressure liquid chromatography retinaldehyde-specific aldehyde dehydrogenase Genes with promoters that contain retinoic acid response elements (RAREs)1 are subject to regulation by the ligand-dependent nuclear transcription factors, the retinoic acid receptors RARs and RXRs. Disturbances in vitamin A signaling, either by vitamin A deficiency, through teratogenic excess of the receptor ligand retinoic acid (RA), or by retinoic acid receptor knockout studies (1Kastner P. Grondona J.M. Mark M. Gansmuller A. LeMeur M. Decimo D. Vonesch J.L. Dolle P. Chambon P. Cell. 1994; 78: 987-1003Abstract Full Text PDF PubMed Scopus (610) Google Scholar, 2Lohnes D. Mark M. Mendelsohn C. Dolle P. Dierich A. Gorry P. Gansmuller A. Chambon P. Development. 1994; 120: 2723-2748Crossref PubMed Google Scholar, 3Mendelsohn C. Lohnes D. Decimo D. Lufkin T. LeMeur M. Chambon P. Mark M. Development. 1994; 120: 2749-2771Crossref PubMed Google Scholar), have shown that retinoid signals participate in vertebrate morphogenesis within specific temporal windows and target tissues. Affected tissues include the eye, craniofacial structures, heart, circulatory, urogenital, respiratory system, limbs, and the anterior-posterior axis of the central nervous system (4Wilson J.G. Roth C.B. Warkany J. Am. J. Anat. 1953; 92: 189-217Crossref PubMed Scopus (630) Google Scholar, 5Morriss-Kay G.M. Sokolova N. FASEB J. 1996; 10: 961-968Crossref PubMed Scopus (171) Google Scholar, 6Maden M. Gale E. Kostetskii I. Zile M. Curr. Biol. 1996; 6: 417-426Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 7Dickman E.D. Thaller C. Smith S.M. Development. 1997; 124: 3111-3121PubMed Google Scholar, 8Blumberg B. Bolado Jr., J. Moreno T.A. Kintner C. Evans R.M. Papalopulu N. Development. 1997; 124: 373-379PubMed Google Scholar). Precise control over the effective concentration of the receptor ligands, all-trans- and 9-cis-RA, is therefore a central requirement for proper receptor function and is maintained by the balance between synthesis and degradation. In retinoic acid-sensitive target tissues, ligands could be derived either by uptake from the low levels circulating in serum or through the in situ metabolism of the prohormone vitamin A (retinol) by alcohol dehydrogenases (including members of the short chain dehydrogenase/reductase and medium chain alcohol dehydrogenase families) and aldehyde dehydrogenases (reviewed in Refs. 9Napoli J.L. Prog. Nucleic Acids Res. Mol. Biol. 1999; 63: 139-188Crossref PubMed Google Scholar and 10Duester G. Adv. Exp. Med. Biol. 1999; 463: 311-319Crossref PubMed Scopus (15) Google Scholar). Such an intracrine mechanism has the advantage that ligand synthesis and signaling can be tightly coupled through the cell-specific regulation of the respective enzymes, a property that would be desirable in various developmental models where RA is thought to act locally as a morphogenic signal. Degradation of RA proceeds primarily through further oxidative metabolism by members of the cytochrome P450 family of enzymes such as CYP26 (11White J.A. Beckett-Jones B. Guo Y.D. Dilworth F.J. Bonasoro J. Jones G. Petkovich M. J. Biol. Chem. 1997; 272: 18538-18541Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). In vertebrates, phylogenetic analysis indicates 13 families of aldehyde dehydrogenases that fall into two main clades. The "class 3" group, which consists mostly of substrate-specific dehydrogenases, and the "class 1/2" dehydrogenases that have broader substrate specificity (12Perozich J. Nicholas H. Wang B.C. Lindahl R. Hempel J. Protein Sci. 1999; 8: 137-146Crossref PubMed Scopus (249) Google Scholar). The "class 1/2" group contains members that can utilize retinaldehyde at submicromolar concentrations (13Yoshida A. Hsu L.C. Dave V. Enzyme. 1992; 46: 239-244Crossref PubMed Scopus (136) Google Scholar, 14Penzes P. Wang X. Napoli J.L. Biochim. Biophys. Acta. 1997; 1342: 175-181Crossref PubMed Scopus (38) Google Scholar, 15Wang X. Penzes P. Napoli J.L. J. Biol. Chem. 1996; 271: 16288-16293Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 16Zhao D. McCaffery P. Ivins K.J. Neve R.L. Hogan P. Chin W.W. Drager U.C. Eur. J. Biochem. 1996; 240: 15-22Crossref PubMed Scopus (256) Google Scholar). Recent reports confirm the essential role of ALDH1 and RALDH2 in RA signaling in vivo. For instance, premature expression of ALDH1 or RALDH2 by mRNA injection into Xenopus embryos results in the induction of premature RA synthesis and teratogenic effects (17Haselbeck R.J. Hoffmann I. Duester G. Dev. Genet. 1999; 25: 353-364Crossref PubMed Google Scholar), while the targeted disruption of RALDH2 in mice has shown its essential and specific role in RA signaling during axial rotation (body turning) and heart and limb morphogenesis (18Niederreither K. Subbarayan V. Dolle P. Chambon P. Nat. Genet. 1999; 21: 444-448Crossref PubMed Scopus (886) Google Scholar). Studies using retinoic acid-sensitive β-galactosidase reporter transgenic mice or indicator cell lines (19Balkan W. Colbert M. Bock C. Linney E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3347-3351Crossref PubMed Scopus (149) Google Scholar, 20Colbert M.C. Linney E. LaMantia A.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6572-6576Crossref PubMed Scopus (93) Google Scholar, 21Moss J.B. Xavier-Neto J. Shapiro M.D. Nayeem S.M. McCaffery P. Drager U.C. Rosenthal N. Dev. Biol. 1998; 199: 55-71Crossref PubMed Scopus (186) Google Scholar) have demonstrated that RA signaling is restricted to specific tissues or regions during vertebrate embryogenesis and that many of these sites, but not all, co-localize to the expression pattern of ALDH1 and RALDH2. One notable discrepancy is the ventral neural retina, where evidence from in situ hybridization and zymographs of isoelectric focusing gels in both mouse and chick support the presence of an additional high activity retinaldehyde dehydrogenase that is distinct from ALDH1 or RALDH2 (22McCaffery P. Lee M.O. Wagner M.A. Sladek N.E. Drager U.C. Development. 1992; 115: 371-382Crossref PubMed Google Scholar, 23Mey J. McCaffery P. Drager U.C. J. Neurosci. 1997; 17: 7441-7449Crossref PubMed Google Scholar). Here we describe the cloning and characterization of a third cytosolic aldehyde dehydrogenase, ALDH6, that can synthesize RA and is expressed in the sensory neuroepithelia including the ventral retina. We also show that ALDH6 is able to specifically transactivate the RAR-dependent signaling pathway when transfected into cells and can sensitize cells to retinol by shifting the dose-response curve 1000-fold. Comparisons with other ALDHs suggest that ALDH6 is equivalent in efficacy to RALDH2 and 10-fold better than ALDH1 in supporting RA-dependent signaling. PCR amplicons of aldehyde dehydrogenases (1025 base pairs for mouse; 713 base pairs for chick) were amplified from 100 ng of oligo(dT)-primed mouse organ or chick embryonic day 4 (E4) ventral retina cDNA using 2.5 units of ExTaq polymerase (TaKaRa Shuzo, Kyoto, Japan) with 50 pmol each of forward and reverse degenerate primers: mouse ALDHdegF, 5′-GCW GGI TGG GCI GAY AAR ATY CAY GG-3′; mouse ALDHdegR, 5′-CCR TKI CCW GAC ATY TTR AAS CC-3′; chick ALDHdegF, 5′-CAR ATH ATH CCI TGG AAY TT-3′; chick ALDH6degR, 5′-AAI ATY TCY TCY TTI GCI AT-3′. Cycle conditions were 5 min at 95 °C and then 30 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 90 s. Amplicons were cloned into pCRII-TOPO vector (Invitrogen, Carlsbad, CA) and screened by restriction enzyme digestion pattern, and four clones were sequenced. The full-length open reading frame (1539 base pairs) of mouse ALDH6 (mALDH6) was amplified with 20 pmol of the gene-specific primers ALDH6ATG (5′-ATG GCT ACC ACC AAC GGG GCT-3′) and ALDH6STOP (5′-TCA GGG GTT CTT CTC CTC GAG-3′) deduced from mouse expressed sequence tag sequences AA790530 and AA499064; four independent clones were sequenced. In addition, three independent 3.26-kilobase pair ALDH6 cDNA clones that included 5′- and 3′-untranslated regions were amplified with 20 pmol each of ALDH6ESTA (5′-CCG GAG AGT GCG AAC CAG TTA-3′) and ALDH6ESTC (5′-CAC ACC ACA GGG GTA AAC CTT-3′) derived from expressed sequence tag sequences AA790530, AA560389, and AA848809. The sequence for mouse ALDH6 is available under GenBankTMaccession number AF152359. Chick ALDH6 5′ cDNA sequences were cloned with a 5′ rapid amplification of cDNA ends kit (Life Technologies, Inc). 600 ng of E4 ventral retina RNA was reverse transcribed with the gene-specific primer YHR3 (5′-TCA TCC ACA GGG ATA GTC CTG-3′), oligo(dC)-tailed, and then amplified by two rounds of PCR with 20 pmol of the kit oligo(dG) adapter primer and the nested gene-specific primers YHR2 (5′-CCT GGT GAA ACA GAC-3′) and YHR1 (5′-CAG AGA GCC AAT GTA CAG TGA TG-3′) (30 cycles each, annealing temperature was 55 °C for 30 s). Four independent 0.6-kilobase pair clones were sequenced. Chick ALDH6 3′ cDNA sequences (approximately 3 kilobase pairs) were cloned by sequential PCR with 20 pmol of oligo(dT)-M13-M4 primer and the nested gene-specific primers YHF4 (5′-GCC ATT GAA GAC AGA GGC CTG-3′), YHF6 (5′-TAC CGA GTA TGG ACT CAC-3′), and YHF7 (5′-AGC TTC TGC TTT GCA GTC G-3′). The sequence for chick ALDH6 (cALDH6) is available under GenBankTM accession number AF152358. The RARβ retinoic acid response element-thymidine kinase luciferase reporter construct (RARE×2-tk-Luc), thymidine kinase-luciferase (tk-Luc) control plasmid, MH-100×4-Luc reporter plasmid, GAL4-nuclear receptor effector constructs (GAL-RAR, GAL-RXR, GAL-VDR, and GAL-PPAR), and β-galactosidase (pCMX-βGal) plasmid have been previously described (24Umesono K. Murakami K.K. Thompson C.C. Evans R.M. Cell. 1991; 65: 1255-1266Abstract Full Text PDF PubMed Scopus (1497) Google Scholar, 25Forman B.M. Umesono K. Chen J. Evans R.M. Cell. 1995; 81: 541-550Abstract Full Text PDF PubMed Scopus (569) Google Scholar). The open reading frames of chick aldehyde dehydrogenases were subcloned into the mammalian expression vector pCAGGS (26Niwa H. Yamamura K. Miyazaki J. Gene ( Amst. ). 1991; 108: 193-199Crossref PubMed Scopus (4617) Google Scholar) to generate pCAGGS-cALDH1, -cRALDH2, and -cALDH6. JEG-3 cells were transfected by the calcium phosphate method in 24-well tissue culture plates for 8 h with ALDH constructs and reporter plasmids as indicated in the figure legends and then washed with phosphate-buffered saline and transferred to serum-free ITLB (5 μg/ml insulin, 5 μg/mlholo-transferrin, 1× Life Technologies, Inc. Defined Lipid Mix and 1.2 g/liter bovine serum albumin)-supplemented Dulbecco's modified Eagle's medium (Life Technologies, Inc.). Retinoids were added, and cells were incubated for 24–36 h before being assayed for luciferase and β-galactosidase activities. cDNA fragments encoding chick ALDH6 (nucleotides 433–1458 in sequenceAF152358), chick ALDH1 (nucleotides 597–1178 (27Godbout R. Exp. Eye Res. 1992; 54: 297-305Crossref PubMed Scopus (36) Google Scholar)), mouse ALDH1 (AHD-2), and ALDH6 (nucleotides 433–1458 in sequence AF152359) were used to prepare digoxygenin-labeled antisense RNA probes. Whole mountin situ hybridization was performed as described by Wilkinson (28Wilkinson D.G. Wilkinson D.G. In Situ Hybridization: A Practical Approach. IRL Press, Oxford1992: 75-83Google Scholar), and section in situ hybridization was performed as described by Ishii et al. (29Ishii Y. Fukuda K. Saiga H. Matsushita S. Yasugi S. Dev. Growth Differ. 1997; 39: 643-653Crossref PubMed Scopus (100) Google Scholar), except that both hybridization and posthybridization washes were carried out at 69 °C and RNase treatment was omitted. Chick embryos were embedded in O.C.T compound (Sakura Finetechnical Co., Ltd., Tokyo) for frozen sections. Mouse embryos and E4–E7 chick embryos were embedded in paraffin. The sections were cut at 6–12-μm thickness. Recombinant chick ALDH6 enzyme was prepared from the expression vector pBAD-cALDH6 in DH5α bacterial cultures induced with 0.2% arabinose. Purification from lysates was by sequential column chromatography with a 100 × 2.5-cm Sephacryl S-300 gel filtration column (Amersham Pharmacia Biotech) developed in phosphate-buffered saline. Active fractions were dialyzed against 20 mm HEPES, pH 7.5, applied to a preparative 10 × 2.5-cm Macroprep High Q column (Bio-Rad), and eluted with a gradient from 0 to 1 m NaCl in 60 min. Final purification was by a TSK-Gel DEAE-5PW (Tosoh, Tokyo, Japan) anion exchange column with a gradient of 0–0.3 m NaCl in 30 min. Aldehyde dehydrogenase activity was monitored by NADH-dependent formazan dye formation at 566 nm. Briefly, 1–10 μg of protein were assayed in 200 μl of enzyme buffer (50 mm Tris-Cl, pH 8.5, 200 mm KCl, 250 μm NAD, 250 μm nitro blue tetrazolium, 8 μm phenomethyl sulfonate, and 0.26% gelatin) with varying concentrations of substrate aldehydes. Retinaldehyde dehydrogenase activity was confirmed by HPLC assay as described below. Protein concentrations were assayed with the Bio-Rad Protein Kit, and purity was determined by SDS-polyacrylamide gel electrophoresis analysis. Kinetic parameters for aldehyde substrates and NAD were determined by HPLC quantitation of retinoic acid formation or NADH production at 340 nm. Retinaldehyde dehydrogenase activity was assayed with 0.1–0.4 μg of protein and 2 μm all-trans-retinaldehyde in 1 ml of enzyme buffer (50 mm HEPES, pH 8.0, 200 mm KCl, 2 mm NAD, 1 mmMgCl2, and 1 mm dithiothreitol) at 37 °C for 10 min. Retinoids were extracted by the addition of 250 μl of acetonitrile/butanol (1:1) and 200 μl of saturated potassium phosphate buffer according to the method of McLean (30McLean S.W. Rudel M.E. Gross E.G. DiGiovani J.J. Pede G.L. Clin. Chem. 1982; 28: 693-696Crossref PubMed Scopus (99) Google Scholar), separated using a 5 × 4.1-mm C18 reverse phase TSK-Gel SuperODS column (Tosoh, Tokyo, Japan), and quantitated by photodiode array detection. HPLC gradient conditions were as follows: flow rate, 1.5 ml/min; 100% 50 mm ammonium acetate, pH 6.9; linear change from 0–60% acetonitrile in 3.0 min; isocratic at 60% acetonitrile for 3.0 min; linear change from 60 to 100% acetonitrile in 1.5 min; isocratic at 100% acetonitrile for 2.5 min. Kinetic data were fitted using the nonlinear regression analysis program Prism (GraphPad Software, Inc., San Diego, CA). In the developing chick and mouse retina, ALDH1 is restricted to the dorsal neural retina, while RALDH2 is expressed in the pigmented retinal epithelium (31Berggren K. McCaffery P. Drager U. Forehand C.J. Dev. Biol. 1999; 210: 288-304Crossref PubMed Scopus (158) Google Scholar). However, an additional retinaldehyde dehydrogenase activity, named V1 in mouse and C-V in chick, that is biochemically distinct from ALDH1 and RALDH2 has been detected in the ventral neural retina (22McCaffery P. Lee M.O. Wagner M.A. Sladek N.E. Drager U.C. Development. 1992; 115: 371-382Crossref PubMed Google Scholar, 23Mey J. McCaffery P. Drager U.C. J. Neurosci. 1997; 17: 7441-7449Crossref PubMed Google Scholar). Since the small tissue sample size makes it impractical for classical protein purification, we isolated novel ALDH cDNA clones by designing degenerate oligonucleotide PCR primers to regions of high sequence conservation in an alignment of published human, mouse, and chick ALDH sequences for the nonallelic ALDH1/2/5/6 and RALDH2 genes. In addition to detecting the known aldehyde dehydrogenases, 4 of 26 clones from mouse and 5 of 120 clones from chick represented a new ALDH that by BLAST search of GenBankTM data base sequences shared closest homology with human ALDH6 (32Hsu L.C. Chang W.C. Hiraoka L. Hsieh C.L. Genomics. 1994; 24: 333-341Crossref PubMed Scopus (49) Google Scholar) and the recently described murine RALDH3 cloned from the lateral ganglionic eminence (33Li H. Wagner E. McCaffery P. Smith D. Andreadis A. Drager U.C. Mech. Dev. 2000; 95: 283-289Crossref PubMed Scopus (142) Google Scholar). On the basis of nucleotide and amino acid similarity, we assign this new ALDH as the mouse and chick homolog of human ALDH6. Fig. 1,A and B, shows the mouse and chick nucleotide sequences and deduced open reading frame of the longest cDNAs obtained by PCR as described under "Experimental Procedures." The phylogenetic relationship between various aldehyde dehydrogenases is shown in Fig. 2 and TableI. Human, mouse, and chick ALDH6 sequences share 85–94% amino acid conservation, equivalent to that seen between species for sequences of ALDH1 (83%) or RALDH2 (96%). ALDH6 is only 69% conserved between either ALDH1 or RALDH2. The murine ALDH6 is essentially identical (99%) to RALDH-3 (Ref. 33Li H. Wagner E. McCaffery P. Smith D. Andreadis A. Drager U.C. Mech. Dev. 2000; 95: 283-289Crossref PubMed Scopus (142) Google Scholar; GenBankTM accession number AF253409). The ALDH6 open reading frame contains the NAD binding motif (GXGXXXG) at residues 235–241 and the conserved cysteine (Cys314), which acts as the active site nucleophile (34Blatter E.E. Abriola D.P. Pietruszko R. Biochem. J. 1992; 282: 353-360Crossref PubMed Scopus (42) Google Scholar). In addition, residues that are found in all catalytically active ALDHs (12Perozich J. Nicholas H. Wang B.C. Lindahl R. Hempel J. Protein Sci. 1999; 8: 137-146Crossref PubMed Scopus (249) Google Scholar) are present in equivalent positions in an optimal alignment, suggesting that this enzyme is functional.Table ISequence pair distances of human, mouse, and chick ALDHs Open table in a new tab We performed in situ hybridization of chick and mouse embryos with antisense RNA probes of ALDH6. Figs. 3 and 4show the expression pattern of ALDH6 in whole mount and sections in chick from stage 10 to 18. No signal was detected with sense probes. ALDH6 is first detected at stage 10 in the surface epithelium anterior to the optic vesicle but is absent from ectoderm directly overlying the lens vesicle (Figs. 3 A and 4 F). By contrast, the expression of ALDH1 is first detectable as the optic vesicle makes contact with the overlying ectoderm and the lens placode begins to thicken at stage 12. ALDH1 expression is initiated in the proximal layer of the vesicle directly opposite the lens placode (Fig. 4 A). This region of strong ALDH1 expression involutes during optic cup formation and expands to form the dorsal neural retina (Fig. 4, A–E). In contrast, ALDH6 expression extends to both the proximal and distal ventral sides of the vesicle that are destined to become, respectively, the ventral halves of the neural and pigmented retina (Fig. 4, F–J). By embryonic day 4, ALDH1 and ALDH6 exhibit complementary nonoverlapping domains of expression in the dorsal and ventral neural retina (Fig. 4, E andJ). Separating these two expression domains is a region that transects the dorso-ventral axis of the retina where neither ALDH1 nor ALDH6 is expressed. Interestingly, this stripe represents the expression domain of CYP26, a cytochrome P450 enzyme that is involved in RA breakdown (11White J.A. Beckett-Jones B. Guo Y.D. Dilworth F.J. Bonasoro J. Jones G. Petkovich M. J. Biol. Chem. 1997; 272: 18538-18541Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 35Swindell E.C. Thaller C. Sockanathan S. Petkovich M. Jessell T.M. Eichele G. Dev. Biol. 1999; 216: 282-296Crossref PubMed Scopus (232) Google Scholar, 36McCaffery P. Wagner E. O'Neil J. Petkovich M. Drager U.C. Mech. Dev. 1999; 85: 203-214Crossref PubMed Google Scholar).Figure 4Chick ALDH6 and ALDH1 expression in the eye. The expression of cALDH1 (A–E) and cALDH6 (F–J) during early eye development in chick. At stage (st.) 12 (A and F), cALDH6 is expressed in the surface epithelium ventral to the optic vesicle. The dorsal limit corresponds roughly with the ventral limit of the lens placode (lp) (indicated by an arrow). This expression persists into the later stages (G–I). In the neural tissue at this stage, cALDH6 is expressed only at the junction of the eye vesicle and forebrain (arrowhead). cALDH1 is expressed in the lateral side of the eye vesicle underlying the lens placode. At stage 13 (B and G), cALDH6 is expressed in the prospective pigmented retina and ventral neural retina. cALDH1 is expressed broadly in the region of the eye cup beneath the lens placode. At stage 16 (C and H), cALDH6 expression remains basically the same as that at stage 13. cALDH1 expression becomes restricted to the dorsal portion of the neural retina. At stage 18 (D and I), cALDH6 and cALDH1 expression increase in intensity. cALDH6 expression can also be observed in the optic stalk. At E4 (E andJ), expression of both cALDH6 and cALDH1 remain basically unchanged, but the cALDH6 expression in the optic stalk is weaker than at stage 18 (indicated by an arrowhead). The central retina region where neither cALDH1 nor cALDH6 expression is detected represents the expression domain of CYP26, a P450 enzyme involved in RA breakdown.View Large Image Figure ViewerDownload (PPT) In later stages of eye development, neural retina differentiates into a well organized layered structure in which various types of cells reside at specific positions. At E10, both ALDH1 and ALDH6 were found to be expressed in the outer side of the inner nuclear layer, where amacrine cells are differentiating (Figs. 5,A, B, E, and F) (37Milam A.H. Possin D.E. Huang J. Fariss R.N. Flannery J.G. Saari J.C. Vis. Neurosci. 1997; 14: 601-608Crossref PubMed Scopus (14) Google Scholar). Both transcripts were also found in the ganglion cell layer, albeit faintly. By E16, expression of both ALDH1 and ALDH6 have become faint (Fig. 5,C, D, G, and H). Concurrent with its strong expression in retina, the posterior olfactory neuroepithelium and dorsal epithelium of the otic vesicle are early sites of expression (Figs. 3 C and 6, D andE). Additional sites in the developing head include a band of expression in the isthmus joining the midbrain and hind brain and Rathke's pouch (Fig. 6, C andD). Although we could not detect expression during early limb bud formation (stages 16–21), ALDH6 was observed later at E4 as diffuse staining in mesenchymal limb tissue and at E7 in the hind limb and fore limb interdigital zones and the perichondrial membranes of wing and leg buds (Fig. 6, A, B, andG). In the posterior trunk, ALDH6 staining was found in somites and a subset of spinal cord interneurons opposite the hind limb field (Fig. 6, G and H). This spinal cord expression co-localized with En-1, a specific marker for interneurons (38Burrill J.D. Moran L. Goulding M.D. Saueressig H. Development. 1997; 124: 4493-4503PubMed Google Scholar). The localization of ALDH6 expression to the sensory neuroepithelia is conserved between chick and mouse (Fig. 7 A), although some minor differences were observed. In mouse embryos, ALDH6 was not detected before E8.5 but became clearly visible by E9.5 in the dorsal and ventral margins of the optic vesicle and in a large area of overlying surface head ectoderm (Fig. 7 B). The dynamic nature of the expression pattern is identical to that reported for RALDH3 (33Li H. Wagner E. McCaffery P. Smith D. Andreadis A. Drager U.C. Mech. Dev. 2000; 95: 283-289Crossref PubMed Scopus (142) Google Scholar). By E10.5, ALDH6 expression in surface ectoderm is rapidly reduced (Fig. 7,A and C) but is reexpressed at E11.5, where the ectoderm invaginates at the dorsal and ventral margins of the optic cup (Fig. 7 D). During this period, expression in the ventral neural retina becomes more pronounced (Fig. 7, A,C, and D), and in contrast to the chick, some staining is also transiently seen in the dorsal neural retina through E11.5 (Fig. 7 D). However, by E12.5 ALDH1 and ALDH6 are separated into distinct dorsal and ventral domains in the neural retina (Fig. 7, E and F). Expression of ALDH6 in retina weakens slightly by E15.5 (Fig. 7 G). The conservation of expression between mouse and chick ALDH6 extended to the olfactory placode and otic vesicle, which give rise to the sensory neuroepithelia of the nose and ear. Whole mount and sectionin situ hybridization of mouse embryos showed initially strong uniform expression in the developing olfactory epithelium at E10.5 (Fig. 7, A and C). Starting at E11.5, ALDH6 expression became progressively more restricted to the dorso-lateral neuroepithelium (Fig. 7 D). By E15.5, transverse sections of olfactory structures exhibited a punctated cellular expression pattern in the sensory epithelium and the mesenchymal stroma directly underlying it that was restricted to discreet zones within the developing turbinates (Fig. 7 H). Expression in the otic vesicle was transient and not detected after E10.5. In coronal sections of brain at E15.5, we also confirmed expression in the lateral ganglionic eminence as described in detail by Li et al.(33Li H. Wagner E. McCaffery P. Smith D. Andreadis A. Drager U.C. Mech. Dev. 2000; 95: 283-289Crossref PubMed Scopus (142) Google Scholar). In order to confirm that ALDH6 is indeed the retinaldehyde-specific dehydrogenase of ventral retina, we constructed inducible bacterial expression vectors containing the open reading frame. Conversion of all-trans-retinal to retinoic acid was only detected in lysates from bacterial cultures containing pBAD-ALDH6 and was dependent on induction of the protein (Fig. 8). Purified chick and mouse ALDH6 enzymes were used for determination of kinetic parameters and substrate specificity (Table II). Activity was found to be strongly stimulated with increasing pH between 7 and 8 but was constant above pH 8.0. Enzyme assays were therefore carried out at pH 8.0. ALDH6, like other dehydrogenases in the "class 1/2" clade, can utilize a variety of aldehyde substrates preferring aliphatic longer chain aldehydes; Km values for acetaldehyde, benzaldehyde, and octanal were 3.2 mm, 4.3 μm, and 0.24 μm, respectively. By comparison, ALDH6 used all-trans-retinal as substrate with aVmax of 48.4 ± 0.4 nmol/mg/min andK0.5 of 0.26 μm ± 0.033 and displayed positive cooperativity (H = 1.9) (Fig. 9 A). Mouse ALDH6 had very similar kinetic parameters: Vmax = 58.1 ± 1.0 nmol/mg/min, K0.5 = 0.33 ± 0.040 μm, and H = 1.8 (n = 3). The submicromolar affinity for retinaldehyde and substantialVmax support the conclusion that ALDH6 can function as a retinaldehyde-specific dehydrogenase in vivo.Table IIKinetic parameters of chick and mouse ALDH6EnzymeSubstrateKm 2-aAssays were performed at pH 8.0 and 37 °C. Data represent means ± S.E.Vmax 2-bVmax values for aldehyde substrates (except retinaldehyde) were determined from rates of NADH formation at substrate concentrations 15 ×Km.HNμmnmol/mg/minChickRetinal0.26 ± 0.03348.4 ± 0.41.94MouseRetinal0.33 ± 0.04058.1 ± 1.01.83ChickOctanal 2-cFor alternate substrates,Ki equals Km. Values were determined from inhibition curves of RA synthesis at fixed retinaldehyde concentrations in the range of 2–5 μmaccording to the method in Ref. 58.0.24 ± 0.046163 ± 144ChickBenzaldehyde 2-cFor alternate substrates,Ki equals Km. Values were determined from inhibition curves of RA synthesis at fixed retinaldehyde concentrations in the range of 2–5 μmaccording to the method in Ref. 58.4.3 ± 0.5423 ± 1.33ChickAcetaldehyde 2-cFor alternate substrates,Ki equals Km. Values were determined from inhibition curves of RA synthesis at fixed retinaldehyde concentrations in the range of 2–5 μmaccording to the method in Ref. 58.3200 ± 300ND 2-dNot determined.3ChickCitral 2-cFor alternate substrates,