Osteoblast maturation occurs in overlapping proximal-distal compartments during fin regeneration in zebrafish

Abstract

During fin regeneration, osteoblasts must continually differentiate for outgrowth of the bony fin rays. Bone maturity increases in a distal-proximal manner, and osteoblast maturation can be detected similarly when following gene expression. We find that early markers for osteoblast differentiation are expressed in a discrete domain at the distal end of the fin, just proximal to the adjacent germinal compartment of dividing cells. Matrix genes, required at later stages developmentally, are expressed in a population of cells proximally to the early genes. A marker for mature osteoblasts is expressed in cells further proximal. These domains of gene expression are partially overlapping, perhaps revealing additional levels of osteoblast maturity. We suggest a model for growth where new cells are continually added to the distal-most osteoblast compartment, while osteoblasts in more proximal locations differentiate, thus translating developmental time to location on the proximal-distal axis.

Figure 1

Differences in the maturity of bone matrix can be detected using alizarin red. (A) Ontogenetic fin stained for both alizarin red and ZNS5. Square bracket identifies transition from the alizarin red positive staining (proximally) to the alizarin red negative region (A’ shows this in higher magnification). (B) Regenerating fin (5 dpa) stained for both alizarin red and ZNS5. Arrow points to amputation plane. Square bracket identifies transition from the alizarin red positive staining (proximally) to the alizarin red negative region (B’ shows this in higher magnification). Curved brackets in (A) and (B) identifies the ZNS5-negative region (the remainder of the fin ray is ZNS5-positive). (C) and (D) Sequential images of a single longitudinal cryosection through a ZNS5-stained fin ray (i.e. staining was completed on whole mount fins prior to sectioning). Arrow points to unstained bone matrix. Asterisk identifies a landmark melanocyte. Arrowhead in (D) points to ZNS5-positive signal on the lateral surface of the bone matrix (signal can also be observed medial to the bone matrix). Scale bar for C and D is 50 μm.

Figure 2

Expression of osteoblast markers during ontogeny and regeneration is in the lateral osteoblast compartment. Ontogeny is on the left, 5 dpa regenerating fin is in the middle, cryosection of 5 dpa regenerating fins is on the right. (A,B,C) runx2a; (D,E,F) runx2b; (G,H,I) osx; (J,K,L) spp-1. Cells expressing all genes are located medial to the deposited bone matrix (arrows).

Figure 3

Proliferating cells leave the blastema within 6 hours of dividing. Regenerating fins were treated with BrdU and harvested immediately (A,B,C), following a 2 hour chase in fresh water (D,E,F), or following a 6 hour chase in fresh water (G,H,I). Harvested fins were processed for cx43 in situ hybridization (left panels) and BrdU detection (middle panels). Rightward panels show overlap of bright field and fluorescence. Brackets indicate the cx43-positive domain, revealing that the majority of BrdU-labeled cells overlap with cx43. By the 6 hour chase, very few BrdU positive cells remain in the cx43-positive region.

Figure 4

Model for osteoblast maturation in the fin ray. Early (runx2a, runx2b, osx), intermediate (spp-1), and mature (ZNS5) osteoblast domains are represented on the left. These genes are expressed in partially overlapping domains in the lateral osteoblast compartment (between the basal layer of the epidermis,e, and the actintrichia, yellow). The region of cx43-positive cells is indicated in purple in the cartoon of the fin ray. We suggest that the cx43-positive proliferating cells cross actinotrichia and contribute to the cells in zone 1.