BMP signaling induces digit regeneration in neonatal mice

Abstract

The regenerating digit tip of mice is a novel epimorphic response in mammals that is similar to fingertip regeneration in humans. Both display restricted regenerative capabilities that are amputation-level dependent. Using this endogenous regeneration model in neonatal mice, we have found that noggin treatment inhibits regeneration, thus suggesting a bone morphogenetic protein (BMP) requirement. Using non-regenerating amputation wounds, we show that BMP7 or BMP2 can induce a regenerative response. BMP-induced regeneration involves the formation of a mammalian digit blastema. Unlike the endogenous regeneration response that involves redifferentiation by direct ossification (evolved regeneration), the BMP-induced response involves endochondral ossification (redevelopment). Our evidence suggests that BMP treatment triggers a reprogramming event that re-initiates digit tip development at the amputation wound. These studies demonstrate for the first time that the postnatal mammalian digit has latent regenerative capabilities that can be induced by growth factor treatment.

Fig. 1.

BMP signaling and endogenous digit tip regeneration. (A-C) Wholemount stain with Alizarin Red 14 days after BSA (A), noggin (B) or BMP4 (C) bead (asterisk) implantation following amputation at a distal level (solid line in A). (D-G) In situ hybridization of Bmp7 (D), Bmp2 (E), Bmpr1a (F) and Bmpr1b (G) 7 days after distal amputation. Bmp2- and Bmpr1b-expressing cells in the blastema are indicated (arrows). ne, nail epithelium; gp, growth plate; m, bone marrow. (H) RT-PCR comparison of expression of Bmp2, Bmp7, Bmpr1a, Bmpr1b and Bmpr2 in the blastema (Blast) and unamputated control tissue (Digit).

Fig. 2.

BMP induces bone regeneration from the proximally amputated digit tip. (A) Diagram showing the level of proximal amputation and the position of bead placement. (B-D,I) Control digits with BSA bead. (E-H,J) Experimental digits with BMP bead. (B,E,F) Wholemount Alizarin Red-stained samples 14 DPI treated with BSA (B), BMP7 (E) or BMP2 (F). (C,G) Wholemount stained samples processed for the histological analysis using Mallory triple stain. (D,J) 14 DPI digits stained with calcein to identify regions of ossification. (D) BSA-treated control digit showing the formation of ossification caps (white arrows). (J) BMP7 bead-implanted digit showing a robust distal ossification center. (H,I) 7 DPI samples processed for histological staining with Mallory triple stain. (H) In BMP7-treated digits, chondrocytes (inset) are observed in the regenerate newly forming bone. (I) BSA control digits do not form chondrocytes in the distal stump. Solid line indicates the amputation plane, asterisk indicates implanted bead.

Fig. 3.

Comparison of the proximal-distal length of the terminal phalangeal bone at 14 DPI in BSA- and BMP-treated digits. Student's t-test ± s.e.m. ^***, P< 0.001.

Fig. 4.

Final anatomy of regenerated digits at 35 DPI. (A-C) Final anatomy after BMP7 (A) and BSA (B) treatment compared with a stage-matched unamputated digit (C). A near-normal digit tip is regenerated following BMP7 treatment. (D,E) Wholemount bone stain (Alizarin Red S) of BMP7-(D) or BSA (E)-treated digits. (F) Calcein labeling of the BMP7-treated digit at 35 DPI showing the persistence of a distal ossification center (arrow). Solid line indicates the amputation plane, asterisk indicates implanted bead. Scale bars: 200 μm.

Fig. 5.

Cell proliferation during BMP7-induced regeneration. (A-F) BrdU incorporation at 4 (A) and 7 (B) DPA (PN7 and PN10, respectively) after proximal amputation. BrdU incorporation at 3 (C) and 7 (D) DPI (PN10 and PN14, respectively) after treatment with BSA. BrdU incorporation at 3 (E) and 7 (F) DPI (PN10 and PN14, respectively) after treatment with BMP7. Increased zones of proliferation are induced by BMP7 in the connective tissue (arrowheads) and in the bone stump (arrows). Asterisks indicates implanted beads. (G) BrdU labeling was quantified by cell counts in comparable regions of BSA and BMP7-treated digits at 3 and 7 DPI. The data show BMP7-enhanced proliferation in the connective tissue at 3 DPI, and in both connective tissue and the stump at 7 DPI. Student's t-test ± s.d.; ^*, P<0.05, ^***, P<0.001.

Fig. 6.

Gene expression in the digit blastema. (A-L) In situ hybridization of Msx1 (A-D), Sfrp2 (E-H) and Pedf (I-L) in BMP7-induced regenerates and BSA controls at 3 and 7 DPI. Msx1 (B) and Sfrp2 (F) show BMP7-induced upregulation (arrows) at 3 DPI as compared with stage-matched controls (A,E). At 7 DPI, Msx1 (D) and Sfrp2 (H) expression is absent from the blastema and is restricted to the dorsal-proximal mesenchyme (arrowheads), similar to in control digits (C,G). Cells expressing Pedf are enhanced at 3 DPI in BMP7-treated digits (J, arrow). At 7 DPI, Pedf expression is prominent in the BMP7-induced digit blastema and in the marrow region (L, arrows), but largely absent in the stage-matched BSA controls (K). Asterisks indicate implanted beads.

Fig. 7.

Endochondral marker genes are induced by BMP7 treatment. (A-H) At 7 DPI, the expression of endochondral marker genes (Col2a1, Ihh and Col10a1) and osteogenic marker genes (osteocalcin) was examined by in situ hybridization in control (A-D) and BMP7-induced regenerates (E-H). BSA control digits display expression of endochondral marker genes at the proximal base of the digit (A-C), with osteocalcin expression capping the distal digit stump (D). In BMP7-treated digits, ectopic expression domains of the endochondral marker genes (arrows in E-G) are induced in the regenerate, and enhanced osteocalcin expression is observed throughout the distal stump (H). Asterisks indicate implanted beads.