Scrib is Required for Lens Morphogenesis in the Mouse

Abstract

Purpose: : PDZ proteins such as Scrib, are known to play roles in cell cycle regulation, adhesion, polarity, and differentiation in invertebrates. Using MLR39cre mediated knockouts, we have reported a lens role for Scrib in multiple aspects of fiber cell differentiation including roles in fiber cell shape, denucleation, apoptosis, adhesion and cytoskeletal organization. To address the possibility that Scrib is required for lens morphogenesis, we generated mice in which Scrib was deleted at the lens placode stage and characterized the effect of this mutation on lens cell proliferation and differentiation.

Methods: : Mice carrying a conditional null allele of Scrib were crossed to Lens-Cre transgenic mice. Paraffin sections of Day E11.5, E13.5, E15.5, and E17.5 Scrib^f/f (control)and Scrib^f/f;cre (CKO) embryos or eyes were stained with hematoxylin and eosin (H&E) and propidium iodide (PI) to assess lens morphology. BrdU and TUNEL analyses were carried out to assess cell proliferation and apoptosis, respectively. To examine differentiation, sections were stained with antibodies against p57, ,β, and γ-crystallin, cMAF, and pERK.

Results: : At E11.5, Scrib^f/f;cre lenses exhibited a failure of the lens vesicle to separate from the ectoderm. We immunostained for - crystallin and found crystallin staining throughout the lens vesicle except in the region still associated with the ectoderm. By E13.5, Scrib^f/f;cre lenses showed defects in fiber cell elongation and nuclear organization and fibers were vacuolated. By E15.5, the epithelium was disorganized and multilayered. BrdU analyses on E13.5 and E15.5 lenses showed that the percent BrdU positive cells in the epithelium was similar in CKO and control lenses; however, total number of cells in the CKO was reduced as compared to control. Furthermore, BrdU positive cells were found in the transition zone and fibers of E13.5. TUNEL analyses showed apoptotic cells throughout the lens as early as E11.5. Analysis of differentiation markers in E13.5 lenses showed defects in CKO lenses including a mosaic staining pattern for p57 in the transition zone and reduced pERK staining. No differences in cMAF, β or γ-crystallin expression patterns were detected.

Keywords: development • cell adhesions/cell junctions