Visualizing Normal and Defective Bone Development in Zebrafish Embryos Using the Fluorescent Chromophore Calcein

Abstract

Zebrafish have recently become a model of choice among developmental biologists. This unique model enables both modern molecular and genetic studies to be carried out to identify genes involved in a wide variety of developmental processes. The success of the genetic approach depends largely on the application of an easy and effective screening method to identify interesting mutants. In order to develop a method for visualizing skeletal structures in zebrafish embryos that would be suitable for screening skeletal mutants, we investigated the use of the fluorescent chromophore calcein, which binds specifically to calcified skeletal structures. By using this method, we followed the development of the skeletal structures in zebrafish embryos from day 1 to day 21 postfertilization, and analyzed the effect of bone morphogenetic protein-2 (BMP2) on axial skeleton development. We found the development of the calcified skeletal structure to appear in a progressive fashion from head to tail. Calcified structures in the head (i.e., the jaw) developed first, which were then followed by the axial skeleton in the trunk. Interesting to note was that there appeared to be two domains in the calcification of vertebrae within the axial skeleton. The first three vertebrae were in the first domain; the rest being in the second domain. Compared with Alcian blue staining, we found that calcein staining indeed labels calcified skeletal structures, and, moreover, it is a more sensitive and inclusive method for visualizing skeletal structures. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed BMP2 in zebrafish notochord cells. We demonstrated that ectopic expression of BMP2 in notochord cells inhibited the development of the axial skeleton. Together, these results clearly demonstrated the sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants that have bone structure defects.

Postembryonic staging of wild‐type goldfish, with brief reference to skeletal systems

“…22, 23); we describe here the developmental process of goldfish skeletal system formation based on earlier descriptions of the zebrafish skeletal system (Cubbage and Mabee, 1996; Du et al, 2001; Bird and Mabee, 2003; Parichy et al, 2009). Skeletal features which are not well‐described in zebrafish are also depicted based on early studies of adult goldfish and other related teleost species (Koh, 1931; Gregory, 1933; Fink and Fink, 1981; Fujita, 1990).…”

“…Of the internal skeletal structures of the pectoral girdle, the cleithrum can be observed at early stages (Kimmel et al, 1995; Grandel and Schulte‐Merker, 1998; Du et al, 2001; Mabee et al, 2002). By the prot stage at the latest, this skeleton can be observed as calcified tissues (Fig.…”

Hydrogel-based Delivery of rhBMP-2 Improves Healing of Large Bone Defects Compared With Autograft

“…Two animals were administered a series of bone labels: calcein green (10 mg/kg; Sigma C0875) at 4 weeks, xylenol orange (100 mg/kg; Sigma 398187) at 8 weeks, and calcein blue (10 mg/kg; Sigma M1255) at 12 weeks postsurgery as subcutaneous injections to identify temporal mineralization patterns [15,29,33,42]. Fixed samples were processed into cryoblocks, and nondecalcified sections (5 lm) were obtained to qualitatively observe repair, the mineral deposition pattern visualized by bone labels, and alkaline phosphatase (AP) staining for osteoblastic activity [21,22,41,44].…”

Japanese medaka (Oryzias latipes): developmental model for the study of alcohol teratology

“…Calcein (Sigma-Aldrich) is a fluorescent chromophore that specifically binds to calcium and fluorescently stains the calcified skeletal structure in live fish (Du et al, 2001). Calcein (0.2%) was dissolved in hatching solution and adjusted to pH 7.4 using 0.5 M NaOH.…”