Aberrant overexpression of TGF alpha in stroma disturbs cornea development

Abstract

Purpose : Transforming growth factor alpha (TGFα) plays important roles in morphogenesis of multiple tissue/organ systems during the embryonic development and maintenance of homeostasis in adult. In this study, we examined the hypothesis that conditionally overexpression of TGFα in mouse corneal stroma can perturb corneal morphogenesis during development.

Methods : The new driver mouse line Kera^Rkl containing a IRES2-rtTA3 minigene was inserted behind the stop codon in exon 3 of Keratocan (Kera) gene was generated by gene targeting techniques using ES cells (Zhang Y, ARVO, 2015). Kera^Rki/tet-O-TGFα bitransgenic mice (TG) were obtained by crossing Kera^Rkl with the effector mice tet-O-TGFα and were administrated with doxycycline (Dox) for TGFα overexpression from embryonic day 12 (E12) to various developing stages including postnatal day 1 (P1), P7, P14, and P21. Stereomicroscopy, histology, western blot and immunofluorescence staining (IFS) were applied to evaluate ocular surface conditions. Single transgenic littermates were used as controls (WT).

Results : TGFα overexpression by the corneal stroma of TG mice after Dox induction was confirmed by Western blot. The cell density of the corneal stroma keratocytes in the induced TG mice was 2-3 fold increased than those of WT mice at P1 and P7. We found that unlike WT littermates which consist of 1-2 cell layers of corneal epithelium, TGFα overexpression resulted in 13-17 epithelial layers at P1. Interestingly, TGFα overexpression also caused cell degeneration with vacuoles formation in the TGs at P7. IFS with antibody against corneal epithelium-specific Krt12 showed that peripheral corneal epithelium progressively lost corneal phenotype starting from P1. On the other hand, conjunctiva epithelial-specific Krt13 expression extended into the corneal surface at the same time. Furthermore, small cell clumps located at corneal epithelium at P7 of the TG mice were negative of Krt14 expression.

Conclusions : These data suggest that physiological level of TGFα is critical for the corneal morphogenesis during mouse embryonic development.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.