Abstract
Purpose::
Previous studies have indicated that a gelsolin-like protein (called C/L-gelsolin) comprises about 50% of the water-soluble protein of the zebrafish cornea and consequently is called a corneal crystallin. In view of the genome duplication that occurred in zebrafish, we have re-investigated the gelsolin family in zebrafish in order to establish which member was recruited as a corneal crystallin and how the various other members vary in expression profile, and to initiate molecular analyses on promoter activity of the appropriate gelsolin-like genes in the superfamily.
Methods::
Multiple alignments for phylogenetic analysis were constructed using the MUSCLE* program. RT-PCR, 2D-gel and MALDI-TOF were performed by conventional methods. A 4 kb fragment upstream of the ATG translation start site was amplified and cloned into an EGFP expression vector containing a I-SceI recognition site. The plasmid and I-SceI were co injected into 1 cell stage embryos, and EGFP expression was examined by fluorescence microscopy.
Results::
Six very similar gelsolin family genes were found in the zebrafish genome: two (gsna and gsnb) group with mammalian gelsolin, two (scina and scinb) group with mammalian scinderin (adseverin), and two (scinla and scinlb) form a sister group to mammalian scinderin. The scinderin-like scinla and scinlb genes have not been reported among mammals. RT-PCR and protein analyses established that scinla is the abundant corneal crystallin, and that scinlb is corneal-preferred but expressed to a lesser extent in the cornea. scinla is also expressed in lens; scinlb is more widely expressed, including brain, heart and lens. gsna and scina and scinb are expressed at low levels in many tissues. Surprisingly, gsnb is expressed specifically at low level in cornea. A 4 kb promoter fragment (2.5 kb upstream, exon 1, intron 1 and part of exon 2) of scinla showed corneal activity followed by brain and olfactory placode activity during the first 3 days of embryogenesis in transgenic zebrafish.
Conclusions::
Zebrafish have at least 6 closely related gelsolin genes, with the corneal crystallin belonging to a unique sister group of mammalian scinderin. Corneal preferred expression occurs in 3 of the 6 gelsolin family genes with variable levels of intensity. Regulatory elements for the differential expression pattern of scinla during development are present in the 4 kb promoter fragment.
Keywords: cornea: basic science • gene/expression