Zebrafish sp7:EGFP: A transgenic for studying otic vesicle formation, skeletogenesis, and bone regeneration

Mechanisms underlying the morphogenesis of skeletal shape are largely not understood, mainly because the ability to study the dynamic processes of cellular behavior giving rise to cartilage and bone in living embryos has been limited. In recent years, techniques have emerged that make the study of tissue formation possible in vivo (Beis and Stainier, 2006; Field et al., 2003; Koster and Fraser, 2004), suggesting that skeletal morphogenesis can be similarly studied. The zebrafish is an ideal model organism for studying skeletal development because embryos and larvae are small and transparent, enabling the study of organogenesis in the living organism. Zebrafish cartilage and bone elements develop early and have distinct morphologies, and the genetic mechanisms underlying skeletal formation are shared with other vertebrates (Yelick and Schilling, 2002). Transgenic lines are especially valuable for analysis of living embryos, including time-lapse confocal microscopy (Cooper et al., 2005; Glickman et al., 2003; Smith et al., 2008). Transgenic fish lines have already been produced that express GFP in cells that give rise to, amongst other tissues, cartilage elements in the head including Tg(−1252sox10:GFP)ba5 (Dutton et al., 2008) and Tg(foxp2-enhancerA:EGFP)zc42 (Bonkowsky et al., 2008). A transgenic line expressing an observable marker of osteoblasts would help us explore osteoblast behavior specifically during the formation of intramembranous bony elements, which have no cartilaginous precursor, and for studying the induction of osteoblasts in perichondrium during endochondral ossification. Sp7 is a zinc-finger-containing transcription factor expressed in osteoblasts and not chondrocytes, making it an excellent marker for studying osteoblasts (Nakashima et al., 2002). Recently, the promoter of sp7 has been shown to drive mCherry in osteoblasts of medaka fish allowing for the analysis of osteoblast behavior in the forming skeleton of this species (Renn and Winkler, 2009). This medaka sp7 regulatory sequence has been used to drive fluorescent marker expression in zebrafish (Hammond and Schulte-Merker, 2009; Spoorendonk et al., 2008), however, there exists no transgenic line using the regulatory region of sp7 in zebrafish to drive a fluorescent marker in zebrafish. We used BAC-mediated transgenesis to drive EGFP under the control of sequence upstream of sp7 in a zebrafish BAC. In the case of zebrafish sp7, we do not know the regulatory elements necessary for gene transcription. Therefore, an advantage of using BACs for transgenesis is that they often contain large genomic clones that include the essential regulatory elements of a gene of interest. The presence of large inserts of a zebrafish-specific sequence makes it likely that the BAC contains sequence essential to drive a transgene in an expression pattern consistent with the endogenous gene. We injected the BAC into embryos to generate stable transgenic lines expressing GFP in osteoblasts. We show here the native expression pattern of sp7 by in situ hybridization in whole-mounts and sections, and compare it to the expression of GFP in the Tg(sp7:EGFP)b1212 transgenic line. We found that GFP expression reproduces endogenous sp7 gene expression, indicating that this line will be an excellent tool for future study of the dynamic behavior of cells that create the skeleton.