Structurally normal corneas in aldehyde dehydrogenase 3a1-deficient mice

Abstract

We have constructed an ALDH3a1 null mouse to investigate the role of this enzyme that comprises nearly one-half of the total water-soluble protein in the mouse corneal epithelium. ALDH3a1-deficient mice are viable and fertile, have a corneal epithelium with a water-soluble protein content approximately half that of wild-type mice, and contain no ALDH3a1 as determined by zymograms and immunoblots. Despite the loss of protein content and ALDH3a1 activity, the ALDH3a1(-/-) mouse corneas appear indistinguishable from wild-type corneas when examined by histological analysis and electron microscopy and are transparent as determined by light and slit lamp microscopy. There is no evidence for a compensating protein or enzyme. Even though the function of ALDH3a1 in the mouse cornea remains unknown, our data indicate that its enzymatic activity is unnecessary for corneal clarity and maintenance, at least under laboratory conditions.

FIG. 1.

Generation of ALDH3a1-deficient mice. (A) The mouse ALDH3a1 gene locus is represented by gray lines, with exons identified by the number above the black boxes. Restriction maps are shown that were used for cloning, Southern blot analysis, and linearization of the vector at the polylinker. Double-ended arrows delineate the genomic fragments from AflII digests for Southern blot analysis; the hatched boxes indicate regions used as probes. The SstII site is in parentheses to indicate that it was present only in the polylinker of pBS SK(−). The small arrows followed by dotted lines delineate amplified regions in PCR analyses with predicted sizes shown below arrows. The neomycin cassette is labeled, and the arrow indicates the direction of its transcription. The box marked S indicates sequences that contained translation termination codons and a putative transcriptional terminator (see the text). (B) Southern blot analysis of wild-type (+/+), heterozygote (+/−), and homozygote (−/−) animals for the ALDH3a1 allele. For wild-type and homozygote lanes, 1 μg of DNA was loaded, and for the heterzygote lane, 2 μg of DNA was loaded. (C) Regions for PCR analysis of wild-type and knockout ALDH3a1 alleles in progeny from matings between heterozygotes. (D) Results of PCR analyses for wild-type (+/+), heterozygote (+/−), and knockout (−/−) ALDH3a1 alleles in mice. (E) Distribution of ALDH3a1 genotypes for 206 progeny from matings of ALDH3a1 heterozygotes.

FIG. 2.

ALDH3a1 expression. (A) SDS-PAGE (10% polyacrylamide) of 10 μg of water-soluble protein from the corneal epithelium of ALDH3a1 wild-type (+/+), heterozygote (+/−), and knockout (−/−) mice. The size and position of markers are shown on the left. The major 68-kDa band is transketolase (38), and the major 51-kDa band is ALDH3a1. (B) Immunoblot probed with anti-ALDH3 antibody. SDS-PAGE was performed as in panel A, except that 1 μg of protein was loaded in each lane. (C) Zymogram for ALDH3a1 activity using benzaldehyde and NADP⁺ as substrates. SDS-PAGE was performed as in Panel A.

FIG. 3.

Water-soluble protein content in corneal epithelial cells. (A) The water-soluble protein was normalized to the DNA content of corneal epithelial cells from ALDH3a1 knockout (−/−) and wild-type (+/+) mice. (B) The corneal epithelial water-soluble protein was normalized against detergent-soluble protein from ALDH3a1 knockout (−/−) and wild-type (+/+) mice.

FIG. 4.

Immunoblot analysis to test for compensation by ALDH1. (A) For SDS-PAGE, 10 μg of water-soluble protein from wild-type (+/+) and knockout (−/−) corneal epithelia were loaded onto each lane; after SDS-PAGE, the blots were stained with SyproRuby to reveal proteins. (B) Immunoblot probed with anti-ALDH1 antibody.

FIG. 5.

Hematoxylin-and-eosin-stained cross sections of cornea from a 42-day-old ALDH3a1 knockout mouse (KO) and a 129/Sv wild-type mouse (WT) showing normal tissue stratification. Micrographs reveal intact epithelial, stromal, and endothelial layers in both corneas.

FIG. 6.

Transmission electron microscopy of a transected cornea from a 51-day-old ALDH3a1 knockout mouse showing normal structure. (A) Superficial epithelium consisting mainly of nucleus-free squamous epithelium (se) but occasionally showing a nucleated cell (nu); (B) basal portion of the basal epithelum (be) and adjacent anterior stroma (st) with normal structures for a mouse, including basal lamina (arrowheads) and adjacent hemidesmosomes (arrows), but no Bowman’s membrane; (C) normal keratocyte (kr) in the central stroma (st); (D) normal-appearing posterior stroma (st). Descemet’s membrane (dm), and endothelium (en). The corneas of heterozygote and homozygote wild-type mice were similarly normal in structure. Magnification, ×15,000. Calibration bar, 1.0 μm for all micrographs.

FIG. 7.

Wild-type (WT) and ALDH3a1 knockout (KO) eyes are shown. The eyes were imaged immediately after dissection. The brown iris is observable underneath the cornea of both eyes. Each lens is observable through the pupil.