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J. Huang, Y. Liu, D. Beebe; The Role of the Extracellular Matrix in Lens Placode Formation. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2358.
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© ARVO (1962-2015); The Authors (2016-present)
The mechanism responsible for the formation of the lens placode is not known. We tested the hypothesis, proposed by Hendrix and Zwaan in 1975, that adhesion between the optic vesicle and the surface ectoderm, due to assembly of extracellular matrix (ECM) between them, drives the formation and subsequent invagination of the lens placode.
Pregnant dams carrying ERCre-Tam; Fn1fx/fx or Fn1fx/fx embryos were injected i.p. with Tamoxifen (Tam; 0.2mg/kg) at E8.75 and E9.25. Embryos were collected at E9.5 or E10.25, when the lens placode is formed and invaginating, respectively. Antibody staining for placode-specific molecular markers and with Alexa488-phalloidin was examined by confocal and wide field fluorescence microscopy.
We previously reported (ARVO 2009) that deletion of Pax6 in the surface ectoderm prevented placode formation and decreased the expression of transcripts encoding several components of the ECM, including fibronectin (FN). Since FN is required for assembly of the ECM, we deleted Fn1 globally prior to placode formation using Tam-inducible Cre recombinase. ERCre-Tam; Fn1fx/fx embryos treated with Tam were alive, but developed pericardial edema by E10.25, consistent with the known requirement for FN in the formation of heart valves. In these embryos, the prospective placodal ectoderm showed no evidence of thickening and the lens pit did not form on E10.25, although morphogenesis occurred in other regions of the embryo. Placodes and lenses formed normally in Tam-treated, Fn1fx/fx embryos that did not carry the ERCre-Tam transgene. Antibody staining confirmed that FN was not detected between the optic vesicle and the ectoderm in the knockout embryos. Staining with antibodies to Pax6 or with phalloidin showed that placode cell specification, cytoskeletal arrangement and cell polarity were normal in Fn1CKO embryos. By labeling the Pax6-expressing surface ectoderm, it appeared that ectoderm cells spread, rather than clustering into the placode. We are analyzing the phenotype of Fn1CKO embryos generated with two Cre transgenes, targeted to the prospective lens ectoderm and optic vesicle.
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