Table 1.
ACh bead implantation.
Table 2.
ChAT bead implantation.
Table 3.
BW284c51 bead implantation.
Table 4.
MAB304 bead implantation.
Fig 1.
AChE activity in limbs of whole-mounted chick (a-i) and duck (j) embryos. a) weak AChE in HH16 hind limb bud (arrow); b) AChE in HH22 embryo. Limb buds have extended distally; note AChE is strongest at their rostral corners (arrows). c) pair of hind limb buds at HH19. In ventral aspect (upper), AChE is strong at rim and along a longitudinal centre stripe (arrow); AChE is higher on dorsal (lower limb) than on ventral side (upper); d) stained cryosection at HH19; note internal AChE+ cells, and ectodermal AChE, being strongest at AER (arrow); e) pair of front limb buds at HH25; note outer rim and inner stripe of AChE activity (arrow); f) AChE on cryosection at HH22; note AChE at AER; g) at HH27, AChE outlines ends of future digits 2–4 ("2–4"); note interdigital space is free of AChE (stippled arrow); h) by HH30, foot structure has become complex, indicating individual phalanges; note that interdigital areas begin to present AChE (stippled arrow); i) foot by HH32 presents four digits, individual phalanges clearly outlined by AChE; note interdigital space shows AChE at rim (stippled arrow); j) AChE expression in foot of E14 duck embryo; note interdigital space is free of AChE (stippled arrow). al, allantois; dt, digit; ey, eye; lg, leg; ht, heart; mt, metatarsus; ot, optic tectum; ph, phalange; so, somites; tl, telencephalon; wg, wing.
Fig 2.
ChAT expression by ISH in whole-mounted embryos from HH17 to HH25.
a) onset of ChAT expression in head and heart; b) first ChAT expression at proximal basis of HH18 hindlimb; c) ChAT in HH 21 embryo (head at upper end missing); note strong ChAT in caudal corners of hindlimbs (arrows), and high expression in heart and allantois; d) by HH22, caudal ChAT expression expands rostrally; e) by HH22+, in both hind and front limbs ChAT has further expanded; note highest expression on caudal and distal parts of limb (cf. with f); f) for comparison, AChE expression in HH22 is concentrated rostrally; g) distinct ChAT in wing, and h) in leg of HH25 embryo; i) ChAT in ventral hind trunk of a HH25+ embryo. al, allantois; ht, heart; lwg, left wing; rwg, right wing; llg, left leg; rlg, right leg.
Fig 3.
AChE (a-f) and BChE expression (g) in maturing chicken foot. (a) AChE in HH34 foot. Note strong expression near joints, but also in interdigital spaces (e.g., white star); metatarsi are numbered 1–4; some phalanges (ph) are visible. (b) Detail of a HH34 digit seen from side. AChE was very high at end of distal phalange and at distal tip of digits (star; cf. AER in Fig 3); (c) three-banded AChE in joint region (arrows); (d) AChE in HH34 foot viewed from side. Note ectodermal AChE at degenerating rim of interdigital space (star); (e) by HH41, digits are fully separated, AChE is very distinct in perichondria near distal end of bones (arrow); (f, g) AChE and BChE expressions in digit of a HH37 embryo; f) strong AChE at distal ends of bones, whereby most proximal bone is farthest developed, as indicated by its sharp distal border staining for AChE (right arrow); length of individual bone is indicated by two arrows. Inner end structures of bones (growth plate, epiphysis) are void of AChE, while minor staining in centres of bones (diaphysis; star) indicates bone differentiation (see also in a, e). g) BChE is strong in bone centres (arrow, g), likely associated with hematopoietic stem cells (bone marrow). Further see text; ph, phalanges; tl, talon.
Fig 4.
Implantation of BW284c51- (a-c) or MAB304-soaked beads (d, e) decelerates mineralization. a) AChE-stained HH21 embryo with a BW284c51-soaked bead implanted into left hind limb at HH17; note that at HH21 AChE activity was almost completely inhibited. b), and details in b´) BW284c51-bead implanted into right wing from HH17-37. Note severe inhibition of mineralization in treated wing (upper; blue, AB staining, red AR staining); c) BW284c51-bead implanted into right wing from HH25-32. Note smaller size of treated wing (lower; blue, AB staining). d) bead soaked in MAB304, an AChE-specific antibody, implanted into left wing (arrow) from HH17-26. Note smaller size and much retarded chondrogenesis, as revealed by AB staining (blue); e) dto, from HH17-38. Note retarded AR staining (red) in treated wing (arrow), particularly, see radius. "con", untreated control limbs.
Fig 5.
Implantation of ACh-soaked beads accelerates skeletogenesis.
a) increased growth and chondrogenesis (blue, AB staining) in treated right wing (arrow) of HH18-30 embryo; b) dto. effects on treated right leg (arrow) of another HH18-30 embryo; c), details in c´) accelerated mineralization (red, AR staining) in left wing of a HH18-38 embryo. "con", untreated control limbs.
Fig 6.
Implantation of ChAT-soaked beads accelerates skeletogenesis.
a) huge developmental advance by ChAT treatment of right wing from HH17-24; b) and details in b´) accelerated growth in right wing from HH18-32; c) and details in c´) accelerated mineralization in right wing of HH18-37; d), details in d´) dto. effects in right wing of a HH19-39 embryo are slightly detectable. "con", untreated control limbs.