ZFIN Image: Knopf et al., 2011, Fig. S3

Image

Figure Caption

Fig. S3 Dedifferentiated osteoblasts migrate to the blastema where they re-differentiate

(A) Zns 5 can be detected in the forming blastema from 1 dpa on (red arrows). Cryosections of fin rays at different stages of regeneration are shown. The white dotted line demarcates the border of the blastema to the wound epidermis. n_0hpa = 3 fish, 43/43 sections, n_1dpa = 3 fish, 27/38 sections, n_1.5dpa = 1 fish, 17/18 sections, n_2dpa = 4 fish, 40/48 sections. Scalebar 20 μm.

(B) The number of osteocalcin:GFP+ osteoblasts in the distal stump decreases after amputation. The fraction of Zns 5+ cells expressing GFP in osteocalcin:GFP transgenic fins in the distal stump segment is shown at 0 hpa (49 ± 2 %) and 2 dpa (19 ± 4 %). At 2 dpa, 12 ± 2 % of the Zns 5+ cells in the blastema are also GFP positive. n_{0 hpa} = 4 fish, 18 sections, n_{2 dpa} = 3 fish, 34 sections. Error bars represent s.e.m. 6

(C) osterix:CreERT2-p2a-mCherry fish express CreERT2 specifically in osteoblasts. Fin rays of osterix:CreERT2-p2a-mCherry fish shown as wholemounts (left side) and on cryosections co-stained with Zns 5 (right side). The transgenic line reproduces the pattern of osterix:nlsGFP and osterix:mCherry expression: In uncut fin rays fluorescence is especially high at the growing tips and it is upregulated in the regenerating fin. mCherry expression co-localizes with Zns 5 demonstrating that the expression of the line is restricted to osteoblasts only. As in osterix:nlsGFP and osterix:mCherry fish expression is not detected in cells located directly at the segment joints (asterisk). Scalebar (wholemount) 200 μm, scalebar (section) 20 μm. BF = Brightfield.

(D) Genetically labeled GFP+ stump osteoblasts in osterix:CreERT2-p2a-mCherry; hs:R to nG double transgenic fish treated with 4-HT more than a week prior to fin amputation migrate towards the amputation plane and cross a segment boundary (white asterisk) on their way towards the regenerate. The same whole mount fin ray is shown at 6 h intervals in brightfield and GFP fluorescence plus the overlay. The amputation plane is indicated by the red dashed line. Scalebar 100 μm.

(E) gfp expression can be detected by whole mount in situ hybridization in the forming regenerate of osterix:nlsGFP fish at 2 dpa. n = 5/5 fins.

Acknowledgments

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Reprinted from Developmental Cell, 20(5), Knopf, F., Hammond, C., Chekuru, A., Kurth, T., Hans, S., Weber, C.W., Mahatma, G., Fisher, S., Brand, M., Schulte-Merker, S., and Weidinger, G., Bone Regenerates via Dedifferentiation of Osteoblasts in the Zebrafish Fin, 713-724, Copyright (2011) with permission from Elsevier. Full text @ Dev. Cell