Abstract
Purpose: :
Lens transparency depends on the production of large amounts of "fiber cell-specific" proteins. The control of gene and protein expression in fiber cell differentiation is incompletely understood.
Methods: :
Laser microdissection was used to isolate E9.5 lens placode cells from wild type (WT), Pax6 (Pax6fx/fx; Le-Cre) conditional knockout (CKO) and BMP receptor CKO (Bmpr1a/Acvr1CKO) embryos and cortical fiber cells from one-month-old mice. Epithelia from 1- or 8-month-old WT lenses were separated into central and peripheral regions. RNA was amplified (NuGEN Ovation kit) and used to probe Illumina whole genome microarrays. Transcripts were detected and quantified by PCR and localized by in situ hybridization. Proteins were detected by western blotting and confocal microscopy.
Results: :
αB is the only crystallin previously identified in the mammalian lens placode. Microarray analysis showed that WT placode cells accumulate transcripts encoding a subset of β/γ crystallins (Cryba1, b3, Crygb, c and e) and the "fiber-specific" protein, MIP. These RNAs were greatly reduced in Pax6 or BMP receptor CKO placodes. Adult lens central epithelial cells had abundant transcripts encoding most "fiber-specific" proteins, including Cryba1, 2 and 4, Cryb1-3, Crygc, d, e, n and s, Mip, Prox1, Lim2 and Bfsp2. Crygb, c and d transcripts were more abundant in central than in peripheral epithelial cells. Cryga, e, and f transcripts decreased with age in the epithelium, but increased markedly in fiber cells. Most other "fiber-specific" mRNAs were present at similar levels in epithelial and fiber cells. MIP transcripts were among the most abundant in epithelial cells, although MIP protein was not detected in these cells. Similarly, Prox1 mRNA was abundant in central epithelial cells, but Prox1 protein was detected only in transition zone epithelial cells and in fiber cells. Prox1 staining was prominent in the nuclei of all epithelial cells from lenses lacking the BMP receptor, Acvr1.
Conclusions: :
It is generally thought that fiber-specific genes are expressed at the onset of fiber cell differentiation. Instead, Mip and some β/γ-crystallin genes are transcribed under the control of Pax6 and BMP signaling in the placode. Most "fiber-specific" transcripts are already abundant in adult epithelial cells. Synthesis of MIP and Prox1 proteins is controlled by differential translation, a previously unrecognized mechanism regulating fiber cell differentiation.
Keywords: differentiation • gene/expression • growth factors/growth factor receptors