Abstract
Purpose: :
50% of the water–soluble corneal protein in adult zebrafish belongs to the extensive gelsolin family (called C/L–gelsolin or gelsolin–1). Subsequently, we cloned a second gelsolin–like gene (called U–gelsolin or gelsolin–2) from zebrafish with 56% sequence identity to the gelsolin–1 gene. The 2 genes have different exon–intron structures. Here we explore their structural and functional relationships.
Methods: :
Multiple alignments for phylogenetic analysis were constructed using the MUSCLE* program or Clustal W. Digoxigenin–UTP labeled antisense RNA probes (∼500 bp) were generated by in vitro transcription and used for whole mount in situ hybridization. Morpholino (MO) antisense oligonucleotides designed against 25 bases including the AUG of the mRNAs were microinjected into the yolk in 1–2 cell embryos.
Results: :
Phylogenetic analysis using amino acid sequences of gelsolins identified previously in different species showed that zebrafish gelsolin–1 clusters with adseverin rather than authentic gelsolin; gelsolin–2 clusters with gelsolins of other species. A similar result was found for the gelsolin–like corneal protein of the ‘4–eyed’ fish, Anableps. The zebrafish genome contains two adseverin genes and one gelsolin gene. 48 hour embryos showed that the gelsolin gene is expressed in many tissues during zebrafish development in contrast to the corneal–preferred adseverin gene. As reported earlier (Kanungo et al. Proc. Natl. Acad. Sci. USA 100: 3287–3292, 2003), reduction of adseverin expression by microinjection of a specific MO into fertilized eggs ventralized the embryo. By contrast, microinjection of a specific gelsolin MO did not affect development. Gelsolin expression was reduced about 50% as judged by in vitro transcription and translation tests using a S–tagged reporter construct.
Conclusions: :
We conclude that the abundant corneal protein (previously called gelsolin–1) is more closely related to adseverin than gelsolin, that gelsolin–2 is the zebrafish homologue to mammalian gelsolin, and that the two genes have vastly different developmental expression patterns and functions.
Keywords: cornea: basic science • gene/expression