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 identify the ZNS5-negative region (the remainder of the fin ray is ZNS5-positive). C, 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 = 50 μm.

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-C: runx2a. D-F: runx2b. G-I: osx. J-L: spp-1. Cells expressing all genes are located medial to the deposited bone matrix (arrows).

Proliferating cells leave the blastema within 6 hr of dividing. Regenerating fins were treated with BrdU and harvested immediately (A-C), following a 2-hr chase in fresh water (D-F), or following a 6-hr chase in fresh water (G-I). Harvested fins were processed for cx43 in situ hybridization (left) and BrdU detection (middle). Right: 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-hr chase, very few BrdU-positive cells remain in the cx43-positive region.

Acknowledgments
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