Ectopic Fgf signaling induces the intercalary response in developing chicken limb buds

Abstract

Background: Intercalary pattern formation is an important regulatory step in amphibian limb regeneration. Amphibian limb regeneration is composed of multiple steps, including wounding, blastema formation, and intercalary pattern formation. Attempts have been made to transfer insights from regeneration-competent animals to regeneration-incompetent animalsat each step in the regeneration process. In the present study, we focused on the intercalary mechanism in chick limb buds. In amphibian limb regeneration, a proximodistal axis is organized as soon as a regenerating blastema is induced. Intermediate structures are subsequently induced (intercalated) between the established proximal and distal identities. Intercalary tissues are derived from proximal tissues. Fgf signaling mediates the intercalary response in amphibian limb regeneration.

Results: We attempted to transfer insights into intercalary regeneration from amphibian models to the chick limb bud. The zeugopodial part was dissected out, and the distal and proximal parts were conjunct at st. 24. Delivering ectopic Fgf2 + Fgf8 between the distal and proximal parts resulted in induction of zeugopodial elements. Examination of HoxA11 expression, apoptosis, and cell proliferation provides insights to compare with those in the intercalary mechanism of amphibian limb regeneration. Furthermore, the cellular contribution was investigated in both the chicken intercalary response and that of axolotl limb regeneration.

Conclusions: We developed new insights into cellular contribution in amphibian intercalary regeneration, and found consistency between axolotl and chicken intercalary responses. Our findings demonstrate that the same principal of limb regeneration functions between regeneration-competent and -incompetent animals. In this context, we propose the feasibility of the induction of the regeneration response in amniotes.

Keywords: Axolotl; Chick; Fgf signaling; HoxA11; Intercalation; Limb development; Limb regeneration.

Fig. 1

Summary of the experimental procedures and gene expression pattern. a Chicken embryo at stage 24, which was used for the surgery in the present study. b–e Summary of surgical procedures. b The schematic diagram of the surgical procedure. The distal one-quarter was isolated and stabbed with a gelatin bead. c The distal one-quarter of the limb bud was dissected out and the isolated part was used for the graft. d The next two quarters, the intermediate part, was removed. e The remaining one-quarter of the stump was stuck with the distal graft and the bead. f–m Gene expression pattern in non-amputated limb buds (f–i) and operated limb buds (j–m). Pg-h (Aggrecan) expression (f, j), Meis2 expression (g, k), Shox2 expression (h, l), and HoxA13 expression (i, m). The circles indicate the position of the grafted bead (J–M). The scale bars in a, b, c, and F are 2, 1, 1, and 0.5 mm, respectively

Fig. 2

The skeletal phenotypes. a Time schedule of the experiment. b The intact chicken wing. c The skeletal pattern derived from the amputated limb bud three-quarters from the distal tip at st. 24. d The skeletal pattern of the limb bud from which the middle part was dissected out. An intercalary induced structure could be observed. e An Fgf2 + Fgf8-soaked bead induced the intermediate (zeugopodial) structures between the stump and the graft. The scale bar in B is 2 mm

Fig. 3

HoxA11 expression pattern in the operated limbs. HoxA11 expression in the control (PBS) samples (a, c, e) and Fgf2 + Fgf8 bead-grafted limb buds (b, d, f). a, b HoxA11 expression remained at the distal parts on day 6 (2 days after surgery). HoxA11 expression disappeared in the control samples at later time points, however (c, e). d, f HoxA11 expression was observed in the proximal region in the Fgf2 + Fgf8 bead-grafted limbs. Asterisks indicate the grafted beads. The scale bar in A is 500 μm

Fig. 4

Mitogenic activity was measured by pH 3 immunofluorescence. The left column shows the control samples with the PBS-soaked bead. The right column shows the limb buds with the Fgf2 + Fgf8-soaked bead. Asterisks and arrowheads indicate the beads and the ischemic region, respectively. The scale bar in A is 250 μm. (M) The summary of the result of A–L and the statistical analysis. n.s. = no significant difference

Fig. 5

Apoptosis was investigated by TUNEL analysis. The left column shows the control samples with the PBS-soaked bead. The right column shows the limb buds with the Fgf2 + Fgf8-soaked bead. Asterisks and arrowheads indicate the beads and the ischemic region, respectively. The scale bar in A is 250 μm. (M) The summary of the result of A–L and the statistical analysis. n.s. = no significant difference.* = P < 0.05

Fig. 6

Re-evaluation of cellular contribution in the axolotl intercalary response. a Schematic diagram of the experimental design. b Hand grafting 10 days after surgery. The line indicates the approximate border of the graft. The scale bar is 3 mm. c Day 30. The elongated portion is recognizable between the grafted hand and the stump (double-headed arrows). d, e Skeletal pattern was revealed by Alcian blue staining. d Fgf2 + Fgf8 bead-grafted sample. Arrowheads indicate the two induced cartilages between the graft and the stump. (D’) Higher magnification view of D. e PBS-soaked bead grafting resulted in no intercalary responses (E’). f Histological observation of the Fgf2 + Fgf8 bead-grafted limb. Cartilage formation is readily evident between the hand graft and the stump. The scale bar indicates 1 mm. g–o The identical section underwent in situ hybridization and immunofluorescence. (G–I) Type II collagen (Col2A) expression pattern in the intercalary induced cartilage. (J–L) GFP expression pattern in the intercalary induced cartilage. The host was a GFP transgenic axolotl. Proximally derived cells were GFP-positive. m–o Hoechst staining for nuclei. g, j, and m are the distal regions of the induced zeugopod, and i, l, and o are the proximal. H, K, and N are the higher magnification views of g, j, and m, respectively. The scale bars in G and H are 500 and 200 μm, respectively. p–s The same experiment was performed with a normal host animal and a graft derived from a GFP transgenic axolotl. p, r) Type II collagen (Col2A) expression pattern in the intercalary induced cartilage. q, s GFP expression pattern in the intercalary induced cartilage. R and S are higher magnification views

Fig. 7

Cellular contribution of the intercalary induced cartilage in chick limb buds. The grafted hand part was derived from a quail limb bud. a, c, e The control sample. b, d, f Fgf2 + Fgf8 bead-grafted limb. a, b Histological observation. (c–f) Immunofluorescent analysis using the antibody specific for chick cells (8F3; c, d) and for quail cells (QCPN; e, f). C’ and D’ are the higher magnification views of the boxed regions in c and d, respectively. Asterisks indicate the beads. Carp. = carpal cartilage. (G–I) The relationship between the HoxA11 expression domain and the 8F3 expression domain was investigated in the adjacent sections. g HoxA11 expression. h 8F3 antigen expression on the adjacent section. I is Hoechst staining for nuclei and is the identical section to H. The scale bars in A, C, C’, and G are 500, 500, 100, and 100 μm, respectively