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R. Rajagopal, V. Kaartinen, D.C. Beebe; Role of Alk2 Receptor Signaling in Lens Development . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1884.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previous studies showed that bone morphogenetic protein (BMP) signaling is important for lens induction and fiber cell formation. We examined the function of the type–I BMP and activin receptor, Alk2, in lens development. Methods:Lenses lacking Alk2 were generated by mating Alk2 floxed mice with mice expressing Cre recombinase in the lens (LeCre). Post–natal day 3 (P3) mice or pregnant females were injected with BrdU (50mg /kg) and sacrificed 1 hour later. Whole embryos or heads were fixed in 10% formalin. Lenses were embedded in paraffin, sectioned, treated for antigen retrieval and labeled with antibody to BrdU or using the TUNEL assay. Other P3 lenses were embedded in agar, sliced in 100 µm sections, permeabilized, labeled with antibodies to molecules downstream of Alk2, such as phosphorylated SMAD1 (pSMAD1), pSMAD2, pSMAD3, or SMAD4 and viewed by confocal microscopy. Results:Cre–positive lenses were compared to lenses from Cre–negative littermates. At embryonic day 12.5 (E12.5), the knockout and wildtype lenses appeared similar and their epithelial cells had comparable rates of proliferation, as measured by the BrdU labeling index. At P3, the knockout lenses were smaller in size and their fiber cells appeared disorganized. There was variability in the reduced size of the knockout lenses; the smallest lenses developed cataracts later in postnatal development. The epithelia of the knockout lenses were thinner than wildtype. In wildtype lenses, the BrdU labeling index decreased from 37.2% at E12.5 to 17.9% at P3. However, in knockout lenses, the BrdU labeling index remained high at P3 (31.7%). Some of the fiber nuclei in the knockout lenses were BrdU positive, indicating their failure to withdraw from the cell cycle. TUNEL labeling revealed large increases in apoptosis in knockout epithelial and fiber cells (P < 0.01 for both). The nuclear levels of pSMAD1 and pSMAD2 were similar in the knockout and wild type lenses. However, preliminary results show that the nuclear levels of pSMAD3 and SMAD4 were lower in knockout epithelial and fiber cells, compared to wild–type lenses. Conclusions:Signaling through Alk2 is required for normal lens development. Between E12.5 and P3, Alk2 signaling causes a decrease in the rate of lens epithelial cell proliferation. In spite of increased proliferation in lenses lacking Alk2, a concomitant increase in cell death results in a smaller lens. Alk2 signaling is required for fiber cells to withdraw from the cell cycle. These phenotypes may be caused by decreased signaling through pSmad3 and Smad4.
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