Fig. 3

Stump Osteoblasts Proliferate in an FGF-Dependent Manner

(A) osteocalcin:GFP+ osteoblasts are capable of dividing after amputation. Frames from a movie obtained from an osteocalcin:GFP; Histone 2a-mCherry transgenic fish fin ray at 1 dpa. Times are indicated (hours:minutes). Scale bar, 20 μm.

(B–D) GFP+ and GFP- stump osteoblasts incorporate BrdU (1 hr pulse) in osteocalcin:GFP fish at 1 dpa.

(B) Overview.

(C) A BrdU+ osteoblast that is Zns 5+ but negative for GFP (arrow) (n = 4 fish, 37/44 sections). Also note the GFP+ cell located distally to the amputation plane (asterisk).

(D) A BrdU+ osteoblast that is positive for Zns 5 and GFP (arrowhead) (n = 4 fish, 5/17 sections). Scale bars, 50 μm (B) and 10 μm (C and D).

(E) Proliferation of Zns 5+ cells in the distal stump (0–350 μm proximal to the amputation plane at 2 dpa or equivalent area in uncut fins) is highly upregulated at 2 dpa as indicated by BrdU incorporation (5 mM for 9 hr). Error bars represent SEM. Student′s t test, p = 0.021 (n_uncut = 3 fish with 31 sections; n_{2 dpa} = 3 fish with 43 sections).

(F) Quantification of the proliferative response of stump osteoblasts at 3 dpa. Average number +SEM of BrdU+ Zns 5+ cells (45 min BrdU pulse) per section is shown in bins of 175 μm along the proximal-distal axis (n = 5 fish each, uncut: 214 sections; 3 dpa: 284 sections). Student′s t test, ^*p < 0.05, ^***p < 0.001.

(G) Inhibition of FGF signaling by the FGFR1 inhibitor SU5402 significantly downregulates proliferation in distal stump osteoblasts at 2 dpa, as determined by BrdU incorporation (5 mM for 9 hr). Error bars represent SEM. Student′s t test, ^**p = 0.0013 (n_DMSO = 4 fish with 43 sections; n_SU5402 = 4 fish with 40 sections). See also Figure S2.