Identification and characterization of a second fibronectin gene in zebrafish

Fibronectin (FN) is a highly conserved extracellular matrix protein that plays crucial roles in vertebrate embryogenesis. Previously, it was reported that zebrafish possess a single FN gene (fn1a). Here we report the presence of a second zebrafish FN gene (fn1b) that encodes a protein with a predicted primary structure that is similar to FNs identified in other vertebrates possessing 12 type I, 2 type II and 17 type III repeats including two alternative splice sites (EIIIA and EIIIB) and a variable region (V). Zebrafish FN1b exhibits 62.0% amino acid identity with zebrafish FN1a, 54% with human and 55% with Xenopus laevis FNs respectively. Employing RT-PCR analysis, we demonstrate that EIIIB- and V- isoforms are produced by alternative splicing of a single fn1b transcript. The FN1b EIIIA- isoform was not detected in zebrafish embryos or adult tissues nor were EIIIA, EIIIB or V region splice variants of fn1a found. FN1b mRNA was detected by RT-PCR in embryos at the gastrula-stage (8hpf) through 72hpf and in various adult tissues. EIIIB- and V+ are the predominant forms of FN1b present in the zebrafish embryo. Unlike FN1a, which is present at a relatively high amount at the embryonic stages before gastrulation, the FN1b isoforms are present at very low amounts at the early cleavage stage. The presence of multiple isoforms of FN1b along with a different pattern of expression compared to FN1a indicates that the two fn genes have separate roles in zebrafish development.