An asymmetrically distributed protein in the embryonic mouse retina was identified as an aldehyde dehydrogenase through protein microsequencing. It was characterized as a cytosolic isoform with basic isoelectric point and preference for aliphatic substrates, features that resemble those of the isoform AHD-2 which is known to oxidize retinaldehyde to retinoic acid. Immunohistochemistry with aldehyde dehydrogenase antisera showed strong labeling of the dorsal retina from the early eye vesicle stage into adulthood. In addition, optic axons originating from the dorsal retina were transiently labeled during their outgrowth phase. Whereas in the embryo the enzyme was expressed in undifferentiated cells and in neurons, in the retina of the adult mouse the asymmetrically distributed isoform was mainly expressed in Müller glia, with the number of labeled glial cells varying with retinal position.

The neuronal connections from the vertebrate retina to central targets are laid out in the form of topographic maps. The mechanisms underlying the generation of this specificity are not understood, but it is likely that the initial steps of the process have a biochemical basis. It is believed that retinal positional determination takes place in a Cartesian coordinate system, eventually resulting in two sets of cell-surface properties asymmetrically distributed in the dorsoventral and anteroposterior dimensions of the retina, which enable retinal axons to search out their address by matching up with corresponding asymmetrically distributed properties in central targets (Bonhoeffer and Gierer, 1984; Sperry, 1963).

Although the early embryonic retina appears morphologically and biochemically homogeneous, several asymmetrically distributed properties have been found. For the anteroposterior dimension, differences in axon fasciculation and in response to a tectal repulsive factor have been detected in functional assays (Halfter et al. 1981; Stahl et al. 1990; Walter et al. 1987). For the dorsoventral axis, three factors have been localized to the dorsal retina: the as yet unidentified TOP antigen (Trisler et al. 1981); the JONES antigen, a modified GD3 ganglioside (Constantine-Paton et al. 1986); and an open conformation of the ribosome-associated protein p40, a protein previously called laminin receptor but now believed to be a factor involved in protein translation (Auth and Brawerman, personal communication; McCaffery et al. 1990; Rabacchi et al. 1990). Restricted to the ventral embryonic retina is the homeobox-containing transcript Pax-2, a murine homolog to transcripts from several pair-rule segmentation genes in Drosophila (Nornes et al. 1990). Here we show an asymmetry in an aldehyde dehydrogenase isoform whose characteristics resemble those of the isoforms ‘known to oxidize retinaldehyde to retinoic acid in the liver (Lee et al. 1990a,b, 1991). The spatiotemporal pattern in the expression of this enzyme is suggestive of a morphogenetic role in the positional determination of the retina.

Mouse embryos were staged according to Theiler (1972) and dissected in tissue culture medium. The tissue samples were washed in balanced salt solution and quickly frozen. Assessment of retinal coordinates was easy in young embryos, where the dorsal retina points dorsally in the head and where the optic fissure is an obvious indicator of the ventral retina; at this stage, the eye has a square shape with the fissure bisecting the lower side of the square. Later the eye undergoes an apparent rotation - clockwise left and counterclockwise right - and the fissure obliterates, which leaves only the attenuated square shape as criterion for the evaluation of retinal coordinates. In the adult eye, the dorsal coordinate deviates about 35° towards caudal from a vertical onto the line of the lid fissure; the dorsal coordinate corresponds optically to down in the visual field (Dräger and Olsen, 1980). The approximate border of the dorsal zone in the adult retina (Fig. 7A) corresponds optically to a horizontal line located about at the level of the horizon in a normal standing mouse.

Homogenizations and subcellular fractionations of the small tissue samples were done as described in McCaffery et al. (1990), and for gel electrophoresis we used standard protocols of Laemmli (1970). For silver staining of proteins in polyacrylamide gels (Fig. 1A), we applied the protocol of Morrissey (1981). Protein determinations were done with the Micro BCA kit (Pierce).

Fig. 1.

(A) Silver-stained gels of soluble fractions from dissected embryonic retinas. The left two lanes show a comparison of dorsal (D) and ventral (V) retinal retinal halves from embryonic day 13 (E13); for the central three lanes, E13.5 retinas were dissected into dorsal (D), middle (M) and ventral (V) thirds; and the right five lanes represent whole retinas from E12, E14.5, E16, E18 and newborn mouse (Pl). The dorsal protein at 53×103Mr is indicated by the thick arrows. Open arrows indicate relative molecular mass markers at 46×103Mr and 69x103Mr. (B) High-pressure liquid chromatography traces of the tryptic fragments of the 53×103Mr protein band from dorsal retina halves and the corresponding region from ventral halves. Filled in are the peptide peaks processed for microsequencing.

Fig. 1.

(A) Silver-stained gels of soluble fractions from dissected embryonic retinas. The left two lanes show a comparison of dorsal (D) and ventral (V) retinal retinal halves from embryonic day 13 (E13); for the central three lanes, E13.5 retinas were dissected into dorsal (D), middle (M) and ventral (V) thirds; and the right five lanes represent whole retinas from E12, E14.5, E16, E18 and newborn mouse (Pl). The dorsal protein at 53×103Mr is indicated by the thick arrows. Open arrows indicate relative molecular mass markers at 46×103Mr and 69x103Mr. (B) High-pressure liquid chromatography traces of the tryptic fragments of the 53×103Mr protein band from dorsal retina halves and the corresponding region from ventral halves. Filled in are the peptide peaks processed for microsequencing.

The protocol for the protein sequence analysis was as described in Tempst et al. (1990). Briefly, the gel-separated proteins were blotted onto nitrocellulose, stained and the bands of interest were excised with a razor blade. The proteins were digested with trypsin for 15 h at 37°C. The resulting peptides were reduced with β-mercapto ethanol, S-alkylated with 4-vinyl pyridine and immediately separated by narrow-bore reversed phase high-pressure liquid chromatography. Peptides were sequenced with the aid of an Applied Biosystems model 477A automated sequenator.

For the detection of aldehyde dehydrogenase, we tested five rabbit antisera. One was generated by Dr John Hilton against cytosolic aldehyde dehydrogenases from mouse (Russo and Hilton, 1988), and the other four were raised by Dr Ronald Lindahl against rat aldehyde dehydrogenases: against the phenobarbital-inducible (cytosolic) isoform, and against microsomal, mitochondrial and tumor-inducible isoforms, purified as described in Lindahl and Evces (1984a) and Lindahl and Evces (1984b). The different antisera were tested individually, which showed identical labeling patterns, and, in most experiments, two or three antisera were used in combination.

For native isoelectric focusing (Figs 3A and 8), the tissue samples were homogenized in phosphate homogenization buffer and run on IsoGel Agarose IEF plates pH 3–10 purchased from Hoefer Scientific Instruments. Proteins were press-blotted onto nitrocellulose following conditions recommended by Hoefer, and aldehyde dehydrogenase was visualized by immunoblotting using an alkaline-phosphatase-based detection system.

For the assay of enzyme activity, aldehyde dehydrogenase was immunopurified from cell lysates using the rabbit antialdehyde dehydrogenase antisera coupled to protein-A sepharose beads. An aliquot of beads was washed in homogenization buffer with 1 % Triton X-100. Incubations were done at room temperature, in the dark, and on a rotator in 1 ml of 60 mM phosphate buffer at pH 7.2. As substrate we used either 14 mM propionaldehyde or 20mM benzaldehyde, in combination with 1mg nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate (NAD or NADP), and 1 mg nitroblue tetrazolium and 0.1 mg phenazine methosulfate. After color development, which lasted up to 40 min, the reaction was stopped by washing the beads into phosphate buffer.

For immunohistochemistry mice from E7 to adulthood were fixed in a paraformaldehyde-picric acid mixture or in periodate-lysine-paraformaldehyde (McLean and Nakane, 1974), embryos by immersion and perinatal to adult ages by transcardiac perfusion. The tissue was cryo-protected with 25% sucrose, and serial sections, mostly in the coronal (=frontal) plane were cut on a cryostat. For the youngest ages, the entire embryo was examined, later only the head and in the adult only the retina. In addition to the sections, retinas from E16 to adult were dissected free and processed free-floating as whole mounts. Immunohistochemistry with aldehyde dehydrogenase antisera was done following standard procedures, using fluorescent detection systems. Of the tested five rabbit antisera (Lindahl and Evces, 1984a,b; Russo and Hilton, 1988) only the serum against the tumor-inducible form was negative; the others showed identical labeling patterns, indicating that in immunohistochemistry the antisera probably recognize all isoforms. In most experiments, a combination of the two strongest antisera was used: the sera against the cytosolic aldehyde dehydrogenases from mouse and against the phenobarbital-inducible isoform from rat. The aldehyde dehydrogenase pattern was compared to the labeling with the p40 monoclonal antibody Dolce, either by double-labeling with two different fluorochromes or by treating adjoining sections.

In subcellular fractionations of dissected portions from embryonic mouse retinas, we noticed a protein that was much more abundant in dorsal than ventral retina. The protein was present in the cytosolic fraction and it appeared as a single or in some cases double band around 53 x 103Mr, as seen in the silver-stained gel of retinal halves from embryonic day 13 (E13) in Fig. 1A, left. In retinas cut into three portions along the dorsoventral axis (Fig. 1A, middle), the 53×103Mr protein was highest in the dorsal, intermediate in the middle and lowest in the ventral samples. In an age comparison (Fig. 1A, right), the 53x103Mr protein, as a fraction of total soluble protein, was highest in the earliest sample from E12, and decreased to minor amounts at E18 and in the newborn (P1).

We separated the cytosolic fractions from 150 dorsal and ventral retina halves from E13 mice by denaturing gel electrophoresis, blotted the proteins onto nitrocellulose and stained with Ponceau S. The 53X103Mr band from the dorsal sample and the corresponding region from the ventral sample were cut out and digested with trypsin. The peptide fragments were separated by high-pressure liquid chromatography, and the peaks from the dorsal sample that are indicated in Fig. 1B were subjected to micro-sequencing by the Edman-degradation procedure (Tempst et al. 1990). A total of 131 amino acids were determined, which unambiguously identified the 53×103Mr protein as an aldehyde dehydrogenase. A comparison with three published aldehyde dehydrogenase sequences - a phenobarbital- inducible (cytosolic) form from rat, a human cytosolic and a human mitochondrial isoform - are shown in Fig. 2 (Dunn et al. 1989; Hempel et al. 1984, 1985; Lindahl and Hempel, 1990). Only four amino acids in the partial sequence of the retinal aldehyde dehydrogenase are not matched in any of the other three sequences, and three of these are conservative substitutions. The retinal aldehyde dehydrogenase is most similar to the cytosolic form from rat, and least similar to the human mitochondrial form.

Fig. 2.

Sequence for the aldehyde dehydrogenase from the early mouse retina (ret) and comparison to three known aldehyde dehydrogenase sequences: a rat phenobarbital-inducible (Pb), a human cytosolic (E1) and a human mitochondrial (E2) isoform (Dunn et al. 1989; Hempel et al. 1984, 1985; Lindahl and Hempel, 1990).

Fig. 2.

Sequence for the aldehyde dehydrogenase from the early mouse retina (ret) and comparison to three known aldehyde dehydrogenase sequences: a rat phenobarbital-inducible (Pb), a human cytosolic (E1) and a human mitochondrial (E2) isoform (Dunn et al. 1989; Hempel et al. 1984, 1985; Lindahl and Hempel, 1990).

Fig. 3.

Biochemical characterization of the aldehyde dehydrogenase from embryonic retina, compared to aldehyde dehydrogenase from liver. (A) Blots of isoelectric focusing gels of cytosolic fractions from dorsal (D) and ventral (V) E13.5 retina halves, and fetal (E15) and adult liver. (B) Activity tests of aldehyde dehydrogenase immunoprecipitated from dorsal (D) and ventral (V) E13 retina halves, adult liver and embryonic tongue, reacted with propionaldehyde (PA) or benzaldehyde (BA) as substrates. Propionaldehyde was tested with nicotinamide adenine dinucleotide (NAD) and benzaldehyde with nicotinamide adenine dinucleotide phosphate (NADP); the two right vertical lanes (labeled NAD and NADP) represent identical conditions of reaction except that the substrates were omitted. For the purpose of this figure, the beads were transferred at the end of the reactions from centrifuge tubes into a round-bottom well plate and photographed.

Fig. 3.

Biochemical characterization of the aldehyde dehydrogenase from embryonic retina, compared to aldehyde dehydrogenase from liver. (A) Blots of isoelectric focusing gels of cytosolic fractions from dorsal (D) and ventral (V) E13.5 retina halves, and fetal (E15) and adult liver. (B) Activity tests of aldehyde dehydrogenase immunoprecipitated from dorsal (D) and ventral (V) E13 retina halves, adult liver and embryonic tongue, reacted with propionaldehyde (PA) or benzaldehyde (BA) as substrates. Propionaldehyde was tested with nicotinamide adenine dinucleotide (NAD) and benzaldehyde with nicotinamide adenine dinucleotide phosphate (NADP); the two right vertical lanes (labeled NAD and NADP) represent identical conditions of reaction except that the substrates were omitted. For the purpose of this figure, the beads were transferred at the end of the reactions from centrifuge tubes into a round-bottom well plate and photographed.

As the different aldehyde dehydrogenase isoforms all have an apparent molecular mass around 53 x 103Mr but differ in charge, we used their isoelectric points for further characterization. Soluble fractions from dorsal and ventral E13.5 retina halves, and from adult and fetal livers, were separated by isoelectric focusing and the aldehyde dehydrogenases were detected by immunoblotting (Fig. 3A). While the dominant signals from the two liver samples focused as relatively sharp bands around neutral pH and in the acidic range, the aldehyde dehydrogenase from dorsal retina accumulated as a broad region around pH 8, within which three relative maxima at pH8.2, 8 and 7.6 could be discerned. Although the blur of the retinal signal might point to a structural rather than conformational heterogeneity, for brevity we will continue to refer to the embryonic retinal form as a single entity. An aldehyde dehydrogenase signal, identical to the retinal signal in broadness of focus and distribution of relative maxima, was also present in the adult liver, but here it was a minor component. No aldehyde dehydrogenase signal was detectable in isoelectric focusing blots from E14 cerebral hemispheres, while neutral and acidic aldehyde dehydrogenase isoforms were seen in preparations of adult intestine and kidney (not shown).

As the site taken to be essential for enzyme function (Lindahl and Hempel, 1990) was not contained in our determined sequence, we performed tests for enzymatic activity. Aldehyde dehydrogenase immunoprecipitated from dorsal halves of E13.5 retinas, as compared to precipitate from ventral halves, was functional, and it showed relative preference for aliphatic over cyclic substrates: NAD-dependent reduction of propionaldehyde as compared to NADP-dependent reduction of benzaldehyde (Fig. 3B). Aldehyde dehydrogenases immunoprecipitated from adult liver contained strong activities for both kinds of substrates, and immunoprecipitate from embryonic tongue, tissue that contained only trace amounts of aldehyde dehydrogenases by immunohistochemical criteria (see Fig. 5), was negative in these activity tests.

Fig. 4.

Series of coronal sections through eyes of mouse embryos at E9, E10, E12 and E16, double-labeled for aldehyde dehydrogenase (A-D) and with a monoclonal antibody to the ribosome-associated protein p40 (E-H). Scale: 200μm.

Fig. 4.

Series of coronal sections through eyes of mouse embryos at E9, E10, E12 and E16, double-labeled for aldehyde dehydrogenase (A-D) and with a monoclonal antibody to the ribosome-associated protein p40 (E-H). Scale: 200μm.

Fig. 5.

Coronal section through head of E15 mouse embryo labeled with aldehyde dehydrogenase antiserum; because the section is slightly oblique, only one eye is visible. Note the strong labeling of the optic axons which can be followed in other sections all the way to their targets. The brain at the level shown, which includes the diencephalon and the cerebral hemispheres, contains no distinct labeling. The only other tissues labeled more brightly here are the surface epithelium of the tongue and throat, and two small organs at the lateral base of the tongue, presumably glandular tissue. Further caudally and not visible here, the inner ear could be seen to be brightly labeled in a patchy way indicative of a topographical pattern. This pattern was in some places the same but differed in others from the labeling pattern of the p40 antibodies. Scale: 500 μm.

Fig. 5.

Coronal section through head of E15 mouse embryo labeled with aldehyde dehydrogenase antiserum; because the section is slightly oblique, only one eye is visible. Note the strong labeling of the optic axons which can be followed in other sections all the way to their targets. The brain at the level shown, which includes the diencephalon and the cerebral hemispheres, contains no distinct labeling. The only other tissues labeled more brightly here are the surface epithelium of the tongue and throat, and two small organs at the lateral base of the tongue, presumably glandular tissue. Further caudally and not visible here, the inner ear could be seen to be brightly labeled in a patchy way indicative of a topographical pattern. This pattern was in some places the same but differed in others from the labeling pattern of the p40 antibodies. Scale: 500 μm.

In order to resolve better the anatomical distribution of the enzyme, we applied aldehyde dehydrogenase antisera to sections of mice from E7 to adulthood. No distinct labeling was detectable at E7 and E8. Labeling started to appear early on E9 in the dorsal part of the eye vesicle and in the surface ectoderm overlying and extending slightly caudally to the eye region (Fig. 4A). The ocular labeling exceeded in brightness the labeling in two other neuronal sites - the ventral midline of the most rostral forebrain and the otic vesicle - and it was similar in intensity to the labeling of the fourth pharyngeal pouch, a region which gives rise to the parathyroid (not shown). The labeled region of the surface ectoderm develops into the lens, the cornea and the conjunctiva, which continued to be brightly labeled (Fig. 4A-D).

For Fig. 4 coronal sections through the eyes of mouse embryos at E9, E10, E12 and E16 were double-labeled with aldehyde dehydrogenase antisera (Fig. 4A-D) and with a monoclonal antibody to the exposed conformation of the ribosome-associated protein p40 (Fig. 4E-H). The aldehyde dehydrogenase appeared at E9 a few hours before the p40 labeling. Over the following three to four days the retinal patterns of the two reagents were very similar: both labeled every cell in the dorsal retina, and there was a relatively sharp boundary to the very weakly labeled ventral retina; the transition from dorsal to ventral occurred at exactly the same location, with almost perfect concordance for the two reagents up to the level of every cell. At the subcellular level, however, the two antigens differed, as the p40 antigen was granular and the aldehyde dehydrogenase filled the cytoplasm diffusely. Regional congruence between expression of aldehyde dehydrogenase and the p40 antigen was also seen in early embryos of rat and chick (not shown).

Like the p40 antigen (Rabacchi et al. 1990), the aldehyde dehydrogenase appeared in optic axons around E13. While all p40 antigen disappeared two to three days later, however, a few aldehyde dehydrogenase-positive axons were visible until several days after birth, that is, as long as axons from the last formed ganglion cells are growing out. An impression for the selectivity and intensity of the retinal and optic-nerve labeling by the enzyme antisera is given in the low-power view of a section through an E15 head (Fig. 5). In other sections, the labeled optic axons could be easily followed to the lateral geniculate nucleus and the optic tectum (not shown).

The retinal labeling patterns with the two reagents started to diverge around E14: whereas the p40 antigen in the older embryos slowly receded towards the dorsal retinal pole (Fig. 4H), the aldehyde dehydrogenase remained present in the entire dorsal retinal third (Fig. 4D), and within the weakly labeled remaining retina first an unlabeled region appeared just below the optic disk, and then a secondary minor focus developed at the ventral pole (Fig. 5). In sections through the optic disk and in whole mounts of E16 retinas labeled for aldehyde dehydrogenase (Fig. 6), brightly labeled optic axons could be seen emerging from the dorsal zone and a weakly labeled axon bundle from the ventral patch. In whole mounts from Pl retinas (not shown) only a handful of labeled axons were still visible, all originating from the dorsal retina and many ending in clubshaped formations on their way to the optic disk, presumably growth cones. The cell bodies of origin could not be distinguished. In sections through the early postnatal optic nerve, similar numbers of axons were labeled, indicating that the enzyme was present in distal portions of the outgrowing axons.

Fig. 6.

Aldehyde dehydrogenase labeling of E16 retina, shown as: (A) cross-section in the plane of the vertical meridian of the eye, (B) whole mount. Dorsal is up in the figure. Scales: (A) 200 μm; (B) 500 μm.

Fig. 6.

Aldehyde dehydrogenase labeling of E16 retina, shown as: (A) cross-section in the plane of the vertical meridian of the eye, (B) whole mount. Dorsal is up in the figure. Scales: (A) 200 μm; (B) 500 μm.

Fig. 7.

Aldehyde dehydrogenase labeling of the adult retina shown as whole mount (A), and in cross-section in the dorsoventral plane at low (B) and higher magnification (C). C is a view of the transition zone from dorsal to central retina. Scales: (A) 1mm; (B) 500μm: (C) 50μm.

Fig. 7.

Aldehyde dehydrogenase labeling of the adult retina shown as whole mount (A), and in cross-section in the dorsoventral plane at low (B) and higher magnification (C). C is a view of the transition zone from dorsal to central retina. Scales: (A) 1mm; (B) 500μm: (C) 50μm.

In the adult retina, the aldehyde dehydrogenase labeling remained highly asymmetrical with the strongest intensity in a dorsal zone, low levels in most of the central retina, and intermediate levels in the extreme retinal periphery (Fig. 7). While in the embryo the aldehyde dehydrogenase was contained in undifferentiated cells and in neurons, in the adult mouse retina the variation was mainly in the number of Miiller glial cells, each of which was labeled with similar intensity throughout all its processes (Fig. 7B,C). The pattern varied from apparently every glial cell labeled in dorsal retina, to a moderate number in the temporal periphery and an occasional one in central retina. In addition, some other retinal cell types such as ganglion cells, were weakly labeled, but in the mouse their intensity did not noticeably vary across the retina; however, because Millier cells extend processes throughout the retina, their very bright labeling in dorsal retina may obscure a weak asymmetry in other cells. In the adult rat retina, an asymmetry in aldehyde dehydrogenase labeling was evident both in neurons and glia (not shown). The presence of ganglion cells was not necessary for the asymmetry in aldehyde dehydrogenase labeling, as a similar pattern as in the normal was seen in mouse retinas lacking all ganglion cells following optic-nerve cut at postnatal day 1 (not shown).

We wondered whether the similarity in regional distributions of aldehyde dehydrogenase between embryo and adult extends to the isoform of the enzyme. In the early embryonic retina (∼E12,13), the only aldehyde dehydrogenase isoform detected by isoelectric focusing was the cytosolic basic form with pronounced dorsal preference, as shown in Fig. 3A. At later embryonic ages (∼E13.5 onward), a weak cytosolic band around pH5.6 started to appear, whose intensity was identical in dorsal and ventral retina halves. In the adult retina both cytosolic and membrane-bound aldehyde dehydrogenase signals were detectable in isoelectric focusing blots. The cytosolic fraction contained the same basic component as seen in the early embryonic retina and in addition bands at pH5.6 and pH4.7. In cytosolic fractions from adult retinas cut into dorsal and ventral halves only the basic isoform was found to be highly asymmetrically expressed (Fig. 8). Isoelectric focusing blots of cytosolic fractions from adult lens and cornea, tissues known for high aldehyde dehydrogenase activity (Holmes, 1988), are given for comparison (Fig. 8): the corneal aldehyde dehydrogenases were different, but the lens forms resembled those from retina. Not shown here is a weak aldehyde dehydrogenase signal from the membrane-bound fractions with isoelectric point around 6.5–7.5, which was only detectable in samples from dorsal retina halves; so far we have not been able to localize it more precisely. This form was of much lower abundance than the basic cytosolic form, which makes it unlikely to contribute significantly to the asymmetrical labeling in Fig. 7.

Fig. 8.

Isoelectric focusing blot of cytosolic aldehyde dehydrogenases from dorsal and ventral retina halves, and from cornea and lens of adult mouse. Note the much higher level of the basic isoform in the dorsal than ventral retina sample. A similar asymmetry in a basic cytosolic aldehyde dehydrogenase isoform was seen in the adult rat retina (not shown).

Fig. 8.

Isoelectric focusing blot of cytosolic aldehyde dehydrogenases from dorsal and ventral retina halves, and from cornea and lens of adult mouse. Note the much higher level of the basic isoform in the dorsal than ventral retina sample. A similar asymmetry in a basic cytosolic aldehyde dehydrogenase isoform was seen in the adult rat retina (not shown).

We have described here the identification of a soluble protein, present at high concentration in the dorsal part of the embryonic retina, as an aldehyde dehydrogenase. Aldehyde dehydrogenases are a structurally similar group of enzymes that catalyze the oxidation of a wide variety of aldehydes (Lindahl and Hempel, 1990; Manthey et al. 1990; Sladek et al. 1989). The isoforms differ with respect to subcellular localization - mitochondrial, microsomal and cytosolic -, and with respect to their physical and catalytic properties. Many forms oxidase a broad range of aldehydes, but others are relatively substrate-specific. From the liver, the organ with the highest levels of aldehyde dehydrogenases, twelve different isoforms have been isolated in the mouse (Manthey et al. 1990). Of these, seven are cytosolic forms, five with isoelectric points in the acidic to close-neutral pH range and two with basic pl, the forms AHD-2 and AHD-7. AHD-2 shows preference for aliphatic and AHD-7 for cyclic substrates, and both isoforms have recently been reported to catalyze the reaction of retinaldehyde to retinoic acid, with AHD-2 contributing the bulk of this activity in the liver (Lee et al. 1990a,b, 1991; Manthey et al. 1990). In isoelectric focusing blots, the aldehyde dehydrogenase from the dorsal retina lined up precisely with the basic component from adult liver, which is consistent with the assumption that it represents AHD-2, maybe with a minor contribution of AHD-7.

This raises the possibility that retinoic acid, which has been implicated in the morphogenesis of other systems (Blomhoff et al. 1990; Thaller and Eichele, 1987; Tickle et al. 1982), plays a role in the spatial determination of the retina. Several other observations suggest a function of the retinoids in retinal development. The cellular retinoic acid binding protein CRABP I is expressed in the central part of the embryonic retina (Dollé et al. 1990; Perez-Castro et al. 1989), at a location that appears to be just ventral to the aldehyde dehydrogenase boundary. By sequestering retinoic acid, CRABP is believed to act as a buffer, protecting cells from inappropriate actions of free retinoic acid (Hirschel-Scholz et al. 1989; Vaessen et al. 1990). The apparent localization of CRABP at the aldehyde dehydrogenase boundary might result in an even sharper step in free retinoic acid levels. Free retinoic acid exerts its effects by binding to nuclear receptors and turns these into specific transcription factors (Blomhoff et al. 1990; Petkovich et al. 1987). The early embryonic retina contains transcripts for the retinoic acid receptor-a and very high levels of the cellular retinol binding protein CRBP I, both homogeneously distributed (Dollé et al. 1990); the role of CRBP is believed to be the accumulation of retinol from the blood circulation. Both deprivation and excess of retinoids have pronounced teratogenic effects on the eye. The main defect in offspring of pregnant pigs and rats, kept on a vitamin-A deficient diet prior and during the early stages of pregnancy, consists of complete lack of eyes, microphthalmia or other gross ocular malformations (Hale, 1937; Warkany and Schraffenberger, 1946; Wilson et al. 1953). In experimental hypervitami-nosis-A-induced teratogenesis, as well as in accidental retinoic-acid embryopathy in humans, a wide range of ocular abnormalities were observed in addition to other malformations (Geelen, 1979; Lammer et al. 1985). Interestingly, these abnormalities include indications that the optic axons have lost their normal sense of directionality: in the eye, optic axons failed to grow towards the optic disk and, in the diencephalon, optic axons grew abnormally towards rostral rather than in the direction of their normal targets (Giroud et al. 1962). So far, there is no direct evidence for retinoic acid synthesis in the embryonic eye or for a higher level of retinoic acid in dorsal retina.

By immunohistochemistry, we found the highest levels of aldehyde dehydrogenase expression of the entire early embryo in the eye and in the fourth pharyngeal pouch. For the early embryonic eye, we estimate that the enzyme constitutes between one-half to one percent of soluble proteins in the dorsal retina, a level not much lower than in the adult liver. It is surprising that such an abundant protein has not been detected in the many attempts to find asymmetrically distributed factors through monoclonal antibodies, while antibodies against a much more elusive property - an open conformation of the ribosome-associated protein p40, which is considerably less abundant than the aldehyde dehydrogenase (unpublished observations) - have been generated repeatedly (McCaffery et al. 1990; Rabacchi et al. 1990). The very high level of the enzyme may point to a unique and critical dependence of the developing eye for its product(s). If these products do indeed include retinoic acid, the high enzyme levels are consistent with the observations that teratogenic effects of vitamin-A deprivation are most severe for the eye (Hale, 1937; Wilson et al. 1953).

A large range of experimental manipulations on the vertebrate visual system, such as eye rotations and displacement of retinal or target tissue fragments to inappropriate locations, have provided an operational demonstration for the existence of positional information: single retinal ganglion cells behave as if they remember from where in the retina they come from and what their proper target ought to be (O’Rourke and Fraser, 1988). This hypothetical positional information is established very early in eye development, when exactly is not known. As a biochemical basis for retinal specificity, Sperry postulated the existence of two sets of graded cell surface markers, orthogonally arranged in the anteroposterior and dorsoventral axes of the retina (Sperry, 1963). Aldehyde dehydrogenase, like the p40 antigen, is clearly not a cell surface marker of the kind postulated by Sperry, but the two factors might be involved in directing the expression of surface markers such as the TOP and the JONES antigens (Constantine-Paton et al. 1986; Trisler et al. 1981). The distribution of aldehyde dehydrogenase and p40 antigen is not in the form of a gradient, rather it resembles the delineation of a compartment, and it seems complementary to the Pax-2 transcript that appears to mark a ventral retinal compartment (Nornes et al. 1990). Of these three early retinal marker systems, the aldehyde dehydrogenase may be the earliest one; its product might suppress expression of Pax-2 in ventral retina, and positively influence the p40 antigen.

We have given here a brief description of the most prominent characteristics in the spatiotemporal distribution of the aldehyde dehydrogenase. Many of the details, in particular concerning the transition from the late embryonic to the adult pattern, remain to be worked out. In the early embryo, the enzyme demarcated the dorsal retina. Later and transiently a complex pattern developed which consisted of a dorsal compartment, optic axons emerging from it, and a very weakly labeled axon contribution from the ventral pole. The positional asymmetry in aldehyde dehydrogenase, which in the embryo was expressed in those cells that presumably need it - undifferentiated stem cells and retinal ganglion cells - persisted into adulthood, but in the mouse it was now mainly expressed in Müller glial cells. This observation confirms the impression that it is the spatial localization rather than the cell type that matters for the enzyme.

We are grateful to Dr John Hilton and Dr Ronald Lindahl for gifts of the aldehyde dehydrogenase antisera, and to Lise Riviere for help with the protein sequence analysis. This work was supported by grant EY 03819 from the National Eye Institute.

Blomhoff
,
R.
,
Green
,
M. H.
,
Berg
,
T.
and
Norum
,
K. R.
(
1990
).
Transport and storage of vitamin A
.
Science
250
,
399
404
.
Bonhoeffer
,
F.
and
Gierer
,
A.
(
1984
).
How do retinal axons find their targets on the tectum?
TINS
7
,
378
381
.
Constantine-Paton
,
M.
,
Blum
,
A. S.
,
Mendez-Otero
,
R.
and
Barnstable
,
C. J.
(
1986
).
A cell surface molecule distributed in a dorso-ventral gradient in the perinatal rat retina
.
Nature
324
,
459
462
.
Dollé
,
P.
,
Ruberte
,
E.
,
Leroy
,
P.
,
Morriss-Kay
,
G.
and
Chambón
,
P.
(
1990
).
Retinoic acid receptors and cellular retinoid binding proteins. I. A systematic study of their differential pattern of transcription during mouse organogenesis
.
Development
110
,
1133
1151
.
Dräger
,
U. C.
and
Olsen
,
J. F.
(
1980
).
Origins of crossed and uncrossed retinal projections in pigmented and albino mice
.
J. comp. Neurol
.
191
,
383
412
.
Dunn
,
T. J.
,
Koleske
,
A. J.
,
Lindahl
,
R.
and
Pitot
,
H. C.
(
1989
).
Phenobarbital-inducible aldehyde dehydrogenase in the rat. cDNA sequence and regulation of the mRNA by phenobarbital in responsive rats
.
J. biol. Chem
.
264
,
13057
13 065
.
Geelen
,
J. A. G.
(
1979
).
Hypervitaminosis A induced teratogenesis
.
CRC Crit. Rev. Toxicol
.
6
,
351
375
.
Giroud
,
A.
,
Martinet
,
M.
and
Roux
,
C.
(
1962
).
Migrations anormales des fibres optiques et considérations générales
.
Arch. Anat. Histol. Embryol
.
45
,
177
190
.
Hale
,
F.
(
1937
).
The relation of maternal vitamin A deficiency to microphthalmia in pigs
.
Texas State J. Med
.
33
,
228
232
.
Haleter
,
W.
,
Claviez
,
M.
and
Schwarz
,
U.
(
1981
).
Preferential adhesion of tectal membranes to anterior embryonic chick retina neurites
.
Nature
292
,
67
70
.
Hempel
,
J.
,
Kaiser
,
R.
and
Jornvall
,
H.
(
1985
).
Mitochondrial aldehyde dehydrogenase from human liver. Primary structure, differences in relation to the cytosolic enzyme and functional correlations
.
Eur. J. Biochem
.
153
,
13
28
.
Hempel
,
J.
,
Von Bahr-Lindstrom
,
H.
and
Jornvall
,
H.
(
1984
).
Aldehyde dehydrogenase from human liver. Primary structure of the cytosolic isoenzyme
.
Eur. J. Biochem
.
141
,
21
35
.
Hirschel-Scholz
,
S.
,
Siegenthaler
,
G.
and
Saurat
,
J.-H.
(
1989
).
Ligand-specific and non-specific in vivo modulation of human epidermal cellular retinoic acid binding protein (CRABP)
.
Eur. J. clin. Invest
.
19
,
220
227
.
Holmes
,
R. S.
(
1988
).
Alcohol dehydrogenases and aldehyde dehydrogenases of anterior eye tissues from humans and other mammals
.
In Biomedical and Social Aspects of Alcohol and Alcoholism
(Edited by
K.
Kuriyama
,
A.
Takada
and
H.
Ishii
)
51
57
.
Elsevier Science Publishers
.
Laemmli
,
U. K.
(
1970
).
Cleavage of structural proteins during the assembly of the head of bacteriophage T4
.
Nature
227
,
680
685
.
Lammer
,
E. J.
,
Chen
,
D. T.
,
Hoar
,
R. M.
,
Agnish
,
N. D.
,
Benke
,
P. J.
,
Braun
,
J. T.
,
Curry
,
C. J.
,
Fernhoff
,
P. M.
,
Grix
,
A. W.
,
Lott
,
I. T.
,
Richard
,
J. M.
and
Sun
,
S. C.
(
1985
).
Retinoic acid embryopathy
.
New England J. Med
.
313
,
837
841
.
Lee
,
M. O.
,
Dockham
,
P. A.
and
Sladek
,
N. E.
(
1990a
).
Identification of human liver aldehyde dehydrogenases that catalyze the oxidation of retinaldehyde to retinoic acid
.
The Pharmacologist
32
,
156
.
Lee
,
M. O.
,
Manthey
,
C. L.
and
Sladek
,
N. E.
(
1990b
).
Identification of mouse liver aldehyde dehydrogenases that catalyze the oxidation of retinaldehyde to retinoic acid
.
FASEB J
.
4
,
A659
.
Lee
,
M. O.
,
Manthey
,
C. L.
and
Sladek
,
N. E.
(
1991
).
Identification of mouse liver aldehyde dehydrogenases that catalyze the oxidation of retinaldehyde to retinoic acid
.
Biochem. Pharmacol
. In press.
Lindahl
,
R.
and
Evces
,
S.
(
1984a
).
Rat liver aldehyde dehydrogenase. I. Isolation and characterization of four high Km normal liver isozymes
.
J. biol. Chem
.
259
,
11986
11990
.
Lindahl
,
R.
and
Evces
,
S.
(
1984b
).
Rat liver aldehyde dehydrogenase. I. Isolation and characterization of four inducible isozymes
.
J. biol. Chem
.
259
,
11991
11996
.
Lindahl
,
R.
and
Hempel
,
J.
(
1990
).
Aldehyde dehydrogenases: what can be learned from a baker’s dozen sequences?
In Enzymology and Molecular Biology of Carbamyl Metabolism III
. (Edited by
H.
Weiner
,
B.
Wermuth
and
D. W.
Crabb
)
1
8
.
New York
:
Plenum Press
.
Manthey
,
C. L.
,
Landkamer
,
G. J.
and
Sladek
,
N. E.
(
1990
).
Identification of the mouse aldehyde dehydrogenases important in aldophosphamide detoxification
.
Cancer Res
.
50
,
4991
5002
.
McLean
,
I. W.
and
Nakane
,
P. K.
(
1974
).
Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy
.
J. Histochem. Cytochem
.
22
,
1077
1083
.
McCaffery
,
P.
,
Neve
,
R. L.
and
Drager
,
U. C.
(
1990
).
A dorsoventral asymmetry in the embryonic retina defined by protein conformation
.
Proc. natn. Acad. Sci. U.S.A
.
87
,
8570
8574
.
Morrissey
,
J. H.
(
1981
).
Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity
.
Analyt. Biochem
.
117
,
307
310
.
Nornes
,
H. O.
,
Dressler
,
G. R.
,
Knapik
,
E. W.
,
Deutsch
,
U.
and
Gruss
,
P.
(
1990
).
Spatially and temporally restricted expression of Pax2 during murine neurogenesis
.
Development
109
,
797
809
.
O’Rourke
,
N. A.
and
Fraser
,
S. E.
(
1988
).
Positional cues in the developing eyebud of Xenopus
.
In Cell Interactions in Visual Development
(Edited by
S. R
Hilfer
and
J. B.
Sheffield
)
47
68
.
New York
:
Springer-Verlag
.
Perez-Castro
,
A. V.
,
Toth-Rogler
,
L. E.
,
Wei
,
L.-N.
and
Nguyen-Huu
,
M. C.
(
1989
).
Spatial and temporal pattern of expression of the cellular retinoic acid-binding protein during mouse embryogenesis
.
Proc. natn. Acad. Sci. U.S.A
.
86
,
8813
8817
.
Petkovich
,
M.
,
Brand
,
N.
,
Krust
,
A.
and
Chambón
,
P.
(
1987
).
A human retinoic acid receptor which belongs to the family of nuclear receptors
.
Nature
330
,
444
450
.
Rabacchi
,
S. A.
,
Neve
,
R. L.
and
Dräger
,
U. C.
(
1990
).
A positional marker for the dorsal retina is homologous to the 68kD-laminin receptor
.
Development
109
,
521
531
.
Russo
,
J. E.
and
Hilton
,
J.
(
1988
).
Characterization of cytosolic aldehyde dehydrogenase from cyclophosphamide resistant L1210 cells
.
Cancer Res
.
48
,
2963
2968
.
Sladek
,
N. E.
,
Manthey
,
C. L.
,
Maki
,
P. A.
,
Zhang
,
Z.
and
Landkamer
,
G. J.
(
1989
).
Xenobiotic oxidation catalyzed by aldehyde dehydrogenases
.
Drug Metabol. Rev
.
20
(
2-4
),
697
720
.
Sperry
,
R. W.
(
1963
).
Chemoaffinity in the orderly growth of nerve fiber patterns and connections
.
Proc. natn. Acad. Sci. U.S.A
.
50
,
703
710
.
Stahl
,
B.
,
Müller
,
B.
,
Von Boxberg
,
Y.
,
Cox
,
E. C.
and
Bonhoeffer
,
F.
(
1990
).
Biochemical characterization of a putative axonal guidance molecule of the chick visual system
.
Neuron
5
,
735
743
.
Tempst
,
P.
,
Link
,
A. J.
,
Riviere
,
L. R.
,
Fleming
,
M.
and
Elicone
,
C.
(
1990
).
Internal sequence analysis of proteins separated on polyacrylamide gels at the submicrogram level: improved methods, applications and gene cloning strategies
.
Electrophoresis
11
,
537
553
.
Thaller
,
C.
and
Eichele
,
G.
(
1987
).
Identification and spatial distribution of retinoids in the developing chick limb bud
.
Nature
327
,
625
628
.
Theiler
,
K.
(
1972
).
The House Mouse. Development and Normal Stages from Fertilization to 4 Weeks of Age
.
Berlin, Heidelberg, New York
:
Springer-Verlag
.
Tickle
,
C.
,
Alberts
,
B.
,
Wolpert
,
L.
and
Lee
,
J.
(
1982
).
Local application of retinoic acid to the limb bud mimics the action of the polarizing region
.
Nature
296
,
564
566
.
Trisler
,
G.
,
Schneider
,
M. D.
and
Nirenberg
,
M.
(
1981
).
A topographic gradient of molecules in retina can be used to identify neuron position
.
Proc. natn. Acad. Sci. U.S.A
.
78
,
2145
2149
.
Vaessen
,
M.-J.
,
Meders
,
J. H. C.
,
Bootsma
,
D.
and
Geurts Van Kessel
,
A
. (
1990
).
The cellular retinoic-acid-binding protein is expressed in tissues associated with retinoic-acid-induced malformations
.
Development
110
,
371
378
.
Walter
,
J.
,
Kern-Veits
,
B.
,
Hue
,
J.
,
Stolze
,
B.
and
Bonhoeffer
,
F.
(
1987
).
Recognition of position-specific properties of tectal cell membranes by retinal axons in vitro
.
Development
101
,
685
696
.
Warkany
,
J.
and
Schraffenberger
,
E.
(
1946
).
Congenital malformations induced in rats by maternal vitamin A deficiency. I. Defects of the eye
.
Arch. Ophthal
.
35
,
150
169
.
Wilson
,
J. G.
,
Roth
,
C. B.
and
Warkany
,
J.
(
1953
).
An analysis of the syndrome of malformations induced by maternal vitamin A deficiency. Effects of restoration of vitamin A at various times during gestation
.
Am. J. Anat
.
92
,
189
217
.