Lens Placode Formation and Its Regulation by BMP Receptor Signaling

Abstract

Purpose: : To identify the mechanism of lens placode formation and its regulation by signaling through type–I BMP receptors, Alk2 and Alk3.

Methods: : Lens placodes lacking Alk2, Alk3 or both were generated by mating Alk2 and/or Alk3 floxed mice with LeCre mice, which express Cre recombinase in the lens. Conditional knockout (CKO) placodes were compared to wild type (WT) littermate placodes. Pregnant females were injected with BrdU (50 mg /kg) and sacrificed one hr later. Embryos were fixed, embedded in 5% agar for orientation and in paraffin for sectioning. Sections were labeled with an antibody to BrdU or with the TUNEL assay.

Results: : The lens placode, a thickening of the head ectoderm, forms after contact with the optic vesicle at embryonic day 9 (E9.0). At E9.5, the placodal and adjacent non–placodal ectoderm had comparable rates of proliferation, measured by BrdU labeling index (42 and 45%, respectively; p = 0.5). However, there was higher TUNEL labeling in the placodal (18%) than the peri–placodal ectoderm (7%)(p < 0.0001). Alk3^CKO and Alk3^WT placodes had the same rate of proliferation (BrdU index 42%). TUNEL labeling was much higher in the Alk3^CKO placodes (46%) than Alk3^WT (18%; p < 0.0001). Alk3^CKO placodes gave rise to smaller lenses with disorganized and disintegrating fibers and attenuated epithelial cells that often failed to separate from the corneal epithelium. Alk2^CKO;Alk3^CKO placodes failed to form lenses. The double CKO placodes also had increased apoptosis (39%), compared to WT (19%; p < 0.05). Unlike the Alk3^CKO placodes, the double CKO placodes showed a small decrease in BrdU index (32%) compared to Alk3^WT placodes (39%)(p < 0.05).

Conclusions: : Paradoxically, lens placode thickening is not associated with a higher rate of proliferation, but with higher cell death. This is consistent with the hypothesis of Hendrix and Zwaan, who postulated that adhesion to the optic vesicle limits spreading of placode cells, leading to crowding and cell elongation. Increased apoptosis does not account for failure of lens formation, since both Alk3^CKO and Alk2^CKO;Alk3^CKO placodes have high rates of apoptosis, but only Alk2^CKO;Alk3^CKO placodes do not form lenses. BMP signaling through Alk2 and Alk3 regulates the survival and proliferation of placode cells and is essential for lens formation.

Keywords: antiviral drugs • transgenics/knock-outs • development