Acetylcholinesterase Regulates Skeletal In Ovo Development of Chicken Limbs by ACh-Dependent and -Independent Mechanisms

Abstract

Formation of the vertebrate limb presents an excellent model to analyze a non-neuronal cholinergic system (NNCS). Here, we first analyzed the expression of acetylcholinesterase (AChE) by IHC and of choline acetyltransferase (ChAT) by ISH in developing embryonic chicken limbs (stages HH17-37). AChE outlined formation of bones, being strongest at their distal tips, and later also marked areas of cell death. At onset, AChE and ChAT were elevated in two organizing centers of the limb anlage, the apical ectodermal ridge (AER) and zone of polarizing activity (ZPA), respectively. Thereby ChAT was expressed shortly after AChE, thus strongly supporting a leading role of AChE in limb formation. Then, we conducted loss-of-function studies via unilateral implantation of beads into chicken limb anlagen, which were soaked in cholinergic components. After varying periods, the formation of cartilage matrix and of mineralizing bones was followed by Alcian blue (AB) and Alizarin red (AR) stainings, respectively. Both acetylcholine (ACh)- and ChAT-soaked beads accelerated bone formation in ovo. Notably, inhibition of AChE by BW284c51, or by the monoclonal antibody MAB304 delayed cartilage formation. Since bead inhibition of BChE was mostly ineffective, an ACh-independent action during BW284c51 and MAB304 inhibition was indicated, which possibly could be due to an enzymatic side activity of AChE. In conclusion, skeletogenesis in chick is regulated by an ACh-dependent cholinergic system, but to some extent also by an ACh-independent aspect of the AChE protein.

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.