PLoS One. 2017 Jan 24;12(1):e0170252. doi: 10.1371/journal.pone.0170252. eCollection 2017.

Endochondral Ossification Is Accelerated in Cholinesterase-Deficient Mice and in Avian Mesenchymal Micromass Cultures.

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Balancing nicotinic acceleration of vertebrate skeletogenesis by cholinesterases

Janine Spieker, Johannes Frieß, Thomas Mudersbach, Astrid Vogel-Höpker and Paul G. Layer.

Developmental Biology and Neurogenetics, Technische Universität Darmstadt, Schnittspahnstrasse 13, D-64287 Darmstadt, Germany



A wealth of epidemiologic and other studies documented deleterious effects of exposure to anticholinesterases (cholinesterase inhibitors, ChEIs) on bone growth and physiology. However, a molecular understanding, in particular based on in vivo experiments is missing [1-3]. Using four independent in vivo and in vitro approaches in chick and mouse embryos, we asked: what are cholinergically sensitive molecular and histological steps during bone formation? What roles do acetyl- and/or butyrylcholinesterase (AChE, BChE, respectively) play to balance cholinergic bone stimulation? The following answers were detailed in two previous articles [4, 5]: i) In the chicken limb anlage, highly dynamic expressions of AChE and choline acetyltransferase (ChAT) are elevated in skeletogenic fronts and organizing centers. ii) Skeletogenesis in chick is regulated by an ACh-dependent cholinergic system, but also to some extent by an ACh-independent function of the AChE protein. iii) The in vivo findings were further substantiated by chicken micromass cultures, and, iv) in fetal knockout mice in absence of either one, or both cholinesterases. Thus, both in vivo and in vitro, an increase in cholinergic signalling, either due to nicotinic stimulation or AChE inhibition, led to accelerated chondroblast production and premature mineralization at the expense of incomplete chondrogenic differentiation.


Results and Discussion

Cholinesterases (AChE and BChE) can function conventionally as transmitter-degrading enzymes at synapses, but also as components of non-neuronal cholinergic systems (NNCS; [1]). Previous in vitro studies revealed that acetylcholine (ACh) can stimulate proliferation of both chondro- and osteoblasts, and several types of acetylcholine receptors (AChRs) are present in the growth plate; in which AChE and BChE are also distinctively expressed [2]). As an excellent study model of a NNCS, we have focused on endochondral osteogenesis [3]) of long bones in vertebrate limbs, using four different approaches:

i) Expression studies of acetylcholinesterase (AChE) were performed by IHC and of choline acetyltransferase (ChAT) by ISH in developing embryonic chicken limbs (stages HH17-37; Fig. 1; see details in [4]). Briefly, AChE outlined formation of bones, being strongest at their distal tips, and later also marked interdigital 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 indicating a leading role of AChE in limb formation.

ii) Loss-of-function experiments were performed via unilateral implantation of beads into chicken limb anlagen, which were soaked in cholinergic components (Fig. 2; see details in [4]). After varying periods, the formation of cartilage matrix and of mineralizing bones was followed by Alcian blue (AB) and Alizarin red (AR) stainings, respectively. Beads soaked in an inhibitor of AChE, eitherBW284c51, or the AChE-specific monoclonal antibody MAB304 delayed cartilage formation (Fig. 2A). Beads either soaked in acetylcholine (ACh), or in ChAT accelerated bone formation in ovo (Fig. 2B). Since bead inhibition of BChE was ineffective, an ACh-independent action during BW284c51 and MAB304 inhibition was indicated also.

iii) An improved in vitro micromass model from chicken limb buds allowed histological distinction between areas of cartilage, apoptosis and mineralization (Fig. 3C-E; [5]). Corresponding to our previous bead implantation studies, treatment with the AChE inhibitor BW284c51, or with nicotine, resulted in a concentration-dependent decrease in cartilage and accelerated mineralization. The effect of nicotine was counter-acted by MLA, a specific inhibitor of the α7-nAChR. 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 (since a BChE inhibitor was ineffective; for details, see [5]).

iv) In prenatal ChE knockout mice (provided by Dr. O. Lockridge, Omaha, USA; abbrev. AB+; A+B and AB; whereby A=AChE, B=BChE;) profound changes of endochondral ossification were detected (Fig. 3A, B; details see in [5]). In all mutant embryos bone growth and cartilage remodeling into mineralizing bone occurred a few days earlier than in wild type and AB+ mice (Fig. 3A). In all mutants between E18.5 to birth AB staining disappeared from epiphyses prematurely (Fig. 3B) and the skeletogenic genes Col-II, Col-X, Ihh, Mmp-13 and ALP were changed (details see, [5]). Instead, AB+ cells were dislocated into diaphyses, most pronounced so in AB mutants, indicating additive effects of both missing ChEs in AB mutant mice.



Due to inhibition of AChE, or, to direct cholinergic stimulation in chicken limb anlagen in ovo, or in chicken micromass cultures, or absence of either one or both cholinesterases in KO mice, there is increase in cholinergic signalling, which leads to increased chondroblast production and premature mineralization, at the expense of incomplete chondrogenic differentiation (Fig. 4). With its high relevance for skeletogenesis and bone physiology, skeletal cholinergic signalling deserves enhanced biomedical attention.




Fig. 1.AChE (A, B) and ChAT (C) expression at skeletogenic frontiers of chicken limbs (cf. with duck in j). A. AChE marks differentiation in whole-mounted embryos and limbs. a) HH16: weak AChE in hind limb bud (arrow); b) HH22: strong AChE at rostral limb bud corners (arrows). c) HH19: strong AChE at dorsal rim and along a longitudinal centre stripe (arrow) of hind limbs (upper); d) HH19: AChE strongest at AER (arrow); e) HH25: outer rim and inner stripe of AChE (arrow); f) HH22: AChE at AER; g) at HH27: AChE outlines ends of future digits 2-4; interdigital space free of AChE (stippled arrow); h) HH30: AChE in foot announces phalanges; AChE in interdigital areas indicates cell death (stippled arrow); i) foot at HH32 presents four digits, phalanges outlined by AChE; j) AChE in E14 duck foot: interdigital space is void of AChE (stippled arrow; cf. with arrow in h, white star in k). B.AChE (k-m) and BChE expression (n) in maturing chicken foot. (k) AChE in HH34 foot: strong expression near joints, and in interdigital spaces (star); (l) three-banded AChE in joint region (arrows); m) HH37: end structures of bones (growth plates) void of AChE; minor staining in bone centres (diaphysis; star) indicates bone differentiation. n) at HH37: BChE strong in bone centres (arrow). C. As compared with AChE, ChAT expression is delayed (by ISH in whole-mounted embryos). o) HH17: onset of ChAT in head and heart; p) HH18: first ChAT at proximal hindlimb base; q) HH 21: strong ChAT in caudal hindlimb corners (arrows), high expression in heart and allantois; r) HH22: caudal ChAT expands rostrally; s) HH22+: in all limbs ChAT is highest on caudal and distal limbs (cf. with f); t) HH22: for comparison, AChE concentrated rostrally; u) HH25: distinct ChAT in wing, and v) in leg; w) HH25+: ChAT in ventral hind trunk.



Fig. 2. Cholinergic bead implantations in vivo.A. AChE inhibition by BW284c51- (a-c) or MAB304-soaked beads (d, e) decelerates mineralization. a) a BW284c51-soaked bead implanted into hind limb at HH17: by HH21, AChE activity was inhibited, b) dto, at HH26: chondrogenesis is retarded; c) HH17-37 with BW284c51-bead: severe inhibition of mineralization in treated wing (upper; blue, A-blu staining, red, A-red staining); d) bead soaked in MAB304, an AChE-specific antibody, implanted into left wing (arrow) from HH17-26. Note smaller size and retarded chondrogenesis (blue); e) dto, from HH17-36. Note retarded mineralisation (red) in treated wing. B.ACh-soaked beads accelerate skeletogenesis. a) AChE activity in control and ACh-treated limb; b) increased growth and chondrogenesis (blue) in treated right wing (arrow) of HH18-30 embryo; c) dto. accelerated mineralization (red) in left wing of a HH17-38 embryo. “con”, untreated control limbs.



Fig 3. A. Cartilage matrix (blue) and mineralization (red) are changed in legs of ChE knockout mice. Note larger size of all mutants at E13.5, earlier onset of mineralization in A+B and in AB mutant legs (A=AChE, B=BChE), and earlier degradation of cartilage matrix (A-blu) in AB+ and AB mutants, along with appearance of A-blu+ cells in their diaphyses. B. Cartilage remodeling is accelerated in perinatal ChE mutants. (a-h) Cryosections of WT and ChE mutant femurs at P0 stained by A-blu at pH 1.0 (upper; revealing glucosaminoglycans), and at pH 2.5 (lower; revealing proteoglycans). Compare strong staining of epiphyses of WT with step-wise advanced degradation of A-blu in epiphyses of mutants (stippled line and arrow), and its appearance in diaphyses. Note onset of secondary ossification in (g, star). (i-l) Whole-mounted femurs double-stained by A-blu/A-red at pH2.5. C. Micromass cultures from chicken limb buds as skeletogenic in vitro models. (a) Cultures incubated for 13 days in presence of 2% chicken serum (CS, right) show improved spatial order. Note cartilage forms in an inner tissue core (upper), followed by a ring of ALP (lower) and mineralization (middle), as shown by A-blu, A-red and ALP stainings. (b) Combined FDA/EtBr staining to detect living cells (green) and dead cells (red). (c) Combined A-blu/A-red (left), merged with ALP staining (right). A-blu indicates chondrocytes; zone of high cell death and ALP activity marks zone of hypertrophic chondrocytes; A-red staining marks zone of chondrocyte-derived osteoprogenitor cells. D. AChE inhibition, or stimulation of nAChRs reduces proteoglycan content and ALP activity, and increases mineralization in vitro. Micromass cultures of chicken limb-bud cells of HH stage 22-24 were stained with A-blu, A-red and for ALP activity. Cultures were incubated for 13 days (a) in presence of increasing doses of BW284c51, or control conditioned medium. (b) dto, in presence of increasing doses of nicotine. E. Combined cultivation of chick limb micromass cultures with 20µM nicotine plus increasing doses of MLA. Note protection by MLA, a specific inhibitor of the α7-nAChR.




Fig 4. Models of cholinergic balancing of chondro- vs. osteogenesis.A. Scheme of the growth plate of developing long bones, including four different states of chondrocytes (rc, pc, ph, hc), before cells reach the mineralizing zone (MZ, red). Epiphyseal and diaphyseal spaces are separated by a chondro-osseous junction (COJ; also called degeneration zone, DZ). Osteoblastic precursors (obp, orange circles) migrate from GP into MZ. (A, right) represents spatial distributions of ACh, of α7-nAChR and of AChE. B. depicts two effective pathways of AChE actions. (B, left) whenever systemic AChE is low (e.g. in ChE KO mice), systemic ACh will be high, leading to increased/advanced proliferation of both proliferative chondrocytes (pc) and osteoblastic precursors (obp). (B, right) At normal levels of ChEs, as in diaphyses of wt, a lower level of ACh in GP will allow chondrocytes to normal terminal differentiation, remodeling and mineralization.


Abbreviations: A-blu, Alcian blue; AER, apical ectodermal ridge; al, allantois; ALP, alkaline phosphatase; A-red, Alizarin red; dt, digit; ey, eye; hc, hypertrophic chondrocytes; HH, Hamburger-Hamilton chicken stages; ht, heart; IHC, immunohistochemistry; ISH, in situ hybridisation; lg, leg; lwg, left wing; llg, left leg; rlg, right leg; mt, metatarsus; MZ, mineralizing zone; ot, optic tectum; pc, proliferating chondrocytes; ph, pre-hypertrophic chondrocytes; ph, phalange; rc, resting chondrocytes; rwg, right wing; so, somites; tel, telencephalon; tl, talon; wg, wing; wt, wild type; ZPA, zone of polarizing activity.



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