Immunolabeling for p-Smad 1/5/8 (green, A–D) and p-c-Jun (green, E), to show the zones of active BMP signaling in the course of digit formation.
Note in A, the absence of labeling in the tendon blastema (tb) at 7,5 days of incubation. In addition, zones of intense labeling paralleling the expression of BMPER of are observed, (arrows, compared with Fig. 1K). B is a higher magnification image of A to show the labeling in the differentiating cartilage except in the most peripheral region subjacent to the perichondrium (p). C shows positive labeling in a proximal region of the tendons corresponding with the myotendinous junction (day 8 of incubation). D is a longitudinal section of digit 3 at day 6 of incubation showing intense labeling in the growing tip (dt) and reduced labeling in the zones of joint formation (arrow heads). E, is a longitudinal section of digit 3, showing intense labeling for p-c-Jun in the joint forming region (arrow heads). In A and B the actin cytoskeleton was labeled in red with Phalloidin-TRITC. F–M, are longitudinal (F, G, I, K, and M) and transverse (H, J, and L) sections through digit 3 showing the expression of type 1 BMP receptors in the autopod. Bmpr1b (F–H) is highly expressed at RNA (F–H) and protein (G) levels in the early chondrogenic digit ray with zones of reduced expression in the joint forming region. In the non-chondrogenic domains of the autopod, the expression of Bmpr1b is predominant in the subectodermal mesenchyme and interdigital regions. Expression of Bmpr1a (I–J) and Alk2 (K–L) is poor in the chondrogenic rays, but transcripts are abundant in the tissues around the digit cartilage including the perichondrium, the interdigital mesenchyme. Transcripts of Alk2, are also appreciated in the developing joints (arrow in K). Alk 1 was only appreciated in the peridigital blood vessels (M).