Excessive Hedgehog Signaling Results in Misexpression of Cell Cycle Proteins and Abnormal Cell Cycle Behaviour During Lens Development

Abstract

Purpose: : We have previously shown in an activated smoothened (smo) mouse model mimicking excessive Hedgehog (Hh) signaling, that lenses display disrupted patterns of lens cell proliferation and apoptosis. In the current study, we investigated which aspects of the cell cycle are affected in this mutant mouse model.

Methods: : An ectodermal cre-recombinase (cre-ect) was used to create a conditional activation of smo (gene encoding for the smo receptor in Hh signaling) in the head ectoderm, including the lens placode, of mice to study the effects of constitutive Hh signaling. Eyes and lenses were examined at embryonic day (E) 12.5, 15.5, 16.5 and 18.5 using histological and immunofluorescent techniques.

Results: : Previous proliferation and apoptosis studies with PCNA and TUNEL have shown aberrant patterns of cellular proliferation and cell death respectively, beginning after E12.5, indicating possible cell cycle defects in the activated smo mutants. CyclinD1 promotes cell cycle entry, and is normally expressed in the developing anterior lens epithelium and cells transitioning into fiber cells at the lens equator, but not in differentiated fiber cells. However, at E16.5 in our mutants, CyclinD1 is expressed throughout the entire lens. The cyclin dependant kinase inhibitors (CDKIs) p27^kip1 and p57^kip2 which work to inhibit cyclinD/cdk activity in post mitotic fiber cells are also misexpressed in the activated smo mutant lens. Beginning at E15.5, p27^kip1 and p57^kip2, normally expressed in the transitional zone of the lens, display expanded expression in the lens epithelium and entire posterior fiber cell region of the mutants. Furthermore, Prox1 and c-maf, required for proper lens fiber cell elongation and differentiation display ectopic expression throughout the epithelium and fiber cell compartments of the mutants at E15.5 and E18.5.

Conclusions: : Constitutive activation of smo promotes the continual activation of a known Hh target gene cyclinD1 which is incorrectly expressed in the fiber cell region of the mutant lenses. The fact that p27^kip1 and p57^kip2 expression is also misexpressed in the fiber cell population suggests that these CDKIs may be attempting to remove these aberrantly expressing cyclinD1 cells from the cell cycle. Interestingly, Prox1 and c-maf have been shown to regulate p27^kip1 and p57^kip2 expression, and disruptions in proper expression of these proteins in our mutants may offer insights into the abnormal cell cycle behaviour in our mutant model.

Keywords: development • proliferation • apoptosis/cell death