Figure 8

EryCs deliver TGFβ1 and BMP2 into the growing blastema. (a) Representative image showing TGFβ1 expression in EryCs in a growing blastema (n = 3). The section was obtained from a regenerating limb in a transitional stage from stage II to stage III. Intense TGFβ1 immunoreactivity was observed in a large number of dots located along the equatorial plane of most PcNobs, though the cytoplasm of PcNobs exhibited weak immunoreactivity. A sample indicated by the arrowhead was enlarged in the lower panel. Topro-3: nucleus. Scale bars, 50 μm (upper) and 10 μm (lower). (b) Representative image showing TGFβ1 expression in PcNobs circulating along the thick blood vessels near the elbow (n = 3). The section was obtained from the same limb in (a). Dotted lines show the vessel wall. TGFβ1 immunoreactivity in PcNobs was substantially the same as that observed in blood cell suspensions (Fig. 7a), though it was difficult to recognize differences in immunoreactivity between EryCs and free Newtic1-negative PcNobs. Inset: enlarged image of the equatorial plane of PcNob (arrowhead). Scale bars, 50 μm and 10 μm (inset). (c) Representative image showing BMP2 expression in EryCs in a growing blastema (n = 3). The section was obtained from a regenerating limb in the transitional stage from stage II to stage III. BMP2 immunoreactivity in the cytoplasm of PcNobs obviously declined while intense immunoreactivity was observed in small dots distributed around the PcNobs as well as along the wall of regenerating capillaries/vessels. Arrows: neutrophil (NP) which exhibited BMP2 immunoreactivity in its cytoplasm. Inset: enlargement of the neutrophil (asterisk). Scale bars, 50 μm and 20 μm (inset). (d) Representative image showing BMP2 expression in PcNobs circulating along thick blood vessels near the elbow (n = 3). The section was obtained from the same limb in (c). Dotted lines show the wall of the vessel. BMP2 immunoreactivity in PcNobs was substantially the same as that observed in blood cell suspensions (Fig. 7b). Scale bars, 50 μm. (e) Example showing control immunoreactivity with Newtic1 (red) and RFP (green) antibodies (n = 3). The image shows tissues near the elbow. Dotted lines show the vessel wall. The section was obtained from the same limb in (c,d). Scale bars, 50 μm.