The Pax6 transcription factor functions early during embryogenesis to control key steps in brain, pancreas, olfactory and ocular system development. A requirement for Pax6 in proper formation of lens, iris and retina is well documented. By examining the corneas of heterozygous Small eye (SEY) mice, this report shows that Pax6 is also necessary for normal corneal morphogenesis. In particular, the epithelial component of the postnatal and adult SEY (+/-) cornea is thinner owing to a reduction in the number of cell layers, despite a tenfold increase in the proliferative index and no change in TUNEL labeling. Ultrastructural views revealed large gaps between corneal epithelial cells and a change in the appearance of desmosomes, suggesting that adhesion abnormalities contribute to the corneal phenotype of SEY (+/-) mice. Western blot analysis and immunofluorescence showed equivalent amounts and normal localization of E-cadherin in SEY (+/-) corneas, and the actin cytoskeleton appeared normal as judged by phalloidin staining. By contrast, the levels of desmoglein, β-catenin and γ-catenin were reduced in the SEY (+/-) cornea. In addition, the amount of keratin-12 mRNA and protein, the major intermediate filament, was reduced in SEY (+/-) corneal epithelium as shown by in situ hybridization and immunohistochemistry. Finally, the SEY (+/-) corneal epithelium adheres less well than wild-type when challenged with gentle rubbing using a microsponge. In conclusion, our results indicate that cellular adhesion is compromised in the SEY (+/-) corneal epithelium and suggests a role for Pax6 in the proper generation and maintenance of the adult cornea.

A twofold thickening of capillary basement membranes of rat retinas resulting from dietary galactose was prevented by sorbinil, an inhibitor of aldose reductase. Since the basement membrane thickening was ultrastructurally similar to that typical of diabetic retinopathy, it may indicate changes in vessel permeability and susceptibility to hemorrhage. Galactosemic rats should be useful models for studying basement membrane-related complications of diabetes and for examining the potential biochemical regulation of basement membrane synthesis by aldose reductase inhibitors.

Development of the functional secretory epithelium in the mammary gland of the female mouse requires the elongation of the anlage through the mammary fat pad to form the primary/secondary ductal network from which tertiary ductal side-branches and lobuloalveoli develop. In this study we examined the hormonal requirements for the spatial development of the primary/secondary epithelial network and tertiary side-branches by quantifying ductal growth and epithelial cell proliferation in normal and hormone-treated BALB/c mice between 21 and 39 days of age. In normal mice, an allometric increase in ductal length commenced at 31 days of age and resulted in completion of the primary/secondary ductal network by 39 days of age. Concurrent with this allometric growth was a significant increase in cellular proliferation in the terminal end-buds (TEBs) of the ductal epithelium from 29 days of age, as determined by 5-bromo-2 -deoxyuridine (BrdU) incorporation. A level of cellular proliferation similar to that in the TEBs of 33-day-old control mice could be induced in the TEBs of 25-day-old mice following treatment for 1 day with estrogen (E), or progesterone (P) or both (E/P), indicating that both E and P were mitogenic for epithelial cells of the peripubertal TEBs. However, the period of allometric ductal growth in untreated mice did not correspond to an increase in serum E or P (which might have been expected during the estrous cycle). In addition, epithelial growth was not observed in mammary glands from 24-day-old mice that were cultured in vitro with E, P or E/P. In contrast to treatment with E, treatment with P promoted a dramatic increase, relative to control mice, in the number of tertiary branch points upon the primary/secondary ductal network. BrdU labeling of mammary glands from 24-33-day-old mice pelleted with cholesterol (C), E, P or E/P confirmed the greater mitogenicity of P on the epithelial cells of the secondary/tertiary ducts as compared with C or E. Concurrent with these changes, localized progesterone receptor (PR) expression in clusters of cells in the ductal epithelium was associated with structures that histologically resembled early branch points from ductules. In conclusion, our results suggest that additional endocrine growth factor(s) other than E and P contribute to the development of the primary/secondary ductal network, and that P is responsible for the formation of tertiary side-branches in the mammary glands of mice during puberty.

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