Abstract
Purpose::
To examine the expression of a number of corneal genes, including the paired-like homeobox gene Vsx1 in mice during eye lid opening and to use X ray diffraction techniques to study the changes in structure that occur during this critical stage in development.
Methods::
Eight corneas at 5, 12 and 17 days of development were excised and extracted RNA was quantified and transcribed to cDNA for quantitative PCR. cDNA was amplified with SYBR Green for real-time quantitative PCR using RNA specific primers for the following genes: Vsx1, Aqp1, Aqp5, and Col8A2. Rpl19 was used as a housekeeping gene for normalization. To further analyse this time period high angle x ray scatter patterns were obtained at 0.2mm intervals across whole murine corneas aged between 7 and 21 days. The X ray scatter patterns were analysed to produce quantitative information about the relative collagen mass distribution and preferred orientation of collagen fibrils in each cornea. This data is illustrated in the form of contour and vector plot maps.
Results::
Vsx1 is expressed at all time points in the cornea and shows a 2.43 fold increase in expression at P12 with slight decrease at P17. Expression of Aqp5 has a ~30 fold higher expression and Aqp1 has a ~2 fold increase in expression from P5 to P17. Col8A2 shows highest expression at P5 with a subsequent ~3 fold decrease at P12 and ~2 fold increase at P17. High angle XRD shows the formation of an annulus of collagen around the edge of the cornea at day 10 of development that continues until fully formed. Preferential alignment of collagen in the cornea can be seen to develop after the annulus starts to form.
Conclusions::
The period of eyelid opening in murine development sees many changes especially in terms of collagen structural organisation. Vsx1 is expressed during corneal development and expression increases from day 5 to day 12 just prior to eye opening. Recent findings have suggested that Vsx1 plays a role in corneal wound healing as expression is detected in activated fibroblasts in the corneal stroma during wound healing. Mutations in Vsx1 have been implicated in keratoconus, a corneal thinning ectasia and the gene Col8A2 has been linked with PPCD. This suggests that Vsx1 could be involved in the collagen production pathways and that these pathways are not just confined to development but maintenance and repair as well.
Keywords: development • gene/expression • cornea: basic science