Abstract
Purpose: :
To determine the global apoptosis gene expression profile in post–natal mouse lenses using arrays.
Methods: :
RNA was isolated from pooled lenses from individual litters of mouse strain 129Sv/Ev using TRIzol® reagent (Invitrogen Corps) at postnatal days 7 (P7), and 14 (P14). Target cDNA was generated from the mouse lens RNA with incorporation of 33P–dCTP (Amersham Biosciences) and hybridised to the array (PanoramaTM mouse apoptosis gene array, Sigma Genosys). Apoptosis genes significantly differentially expressed between lenses at various stages of maturation (P7, P14) were identified. Lenses were also collected from White Leghorn chickens at embryonic days 6, 8, 10, 12, 14 and 16 and the RNA extracted using the TRIzol® reagent. Immunocytochemistry and Western blotting in mouse and chick lenses for the protein products of selected genes was carried out using standard procedures.
Results: :
Over 100 apoptosis genes were expressed above background in at least one of the stages examined. Both highly and differentially expressed genes were identified from the arrays. The expression of these genes was confirmed by semi–quantitative RT–PCR using cDNA from mouse lenses. The chick homologues of these genes have also been examined and, to date, the expression of 95% of them has been confirmed in embryonic chick lenses using RT–PCR. Fifteen mouse genes identified using the arrays were shown to be differentially regulated by greater than two–fold when P14 results were compared to those at P7 (P value < 0.05). Two genes were selected for further detailed analysis using RT–PCR, immunocytochemistry and Western blotting in both the mouse and chick systems; defender against death 1 (Dad–1) was shown to be highly expressed at both P7 and P14, while death associated protein 1 (Dap1) was shown to be differentially expressed, with highest expression at P14.
Conclusions: :
These studies are the first in which a comprehensive analysis of apoptosis gene expression in the maturing mouse lens has been carried out. A number of these genes are also expressed in the chick lens during development. The results reveal expression of apoptosis genes that may have important roles in lens differentiation in both mouse and chick and pave the way for further gene expression and functional analyses of apoptosis genes in the lens.
Keywords: apoptosis/cell death • gene microarray • development