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.

Most components of the cholinergic system are detected in skeletogenic cell types in vitro, yet the function of this system in skeletogenesis remains unclear. Here, we analyzed endochondral ossification in mutant murine fetuses, in which genes of the rate-limiting cholinergic enzymes acetyl- (AChE), or butyrylcholinesterase (BChE), or both were deleted (called here A-B+, A+B-, A-B-, respectively). In all mutant embryos bone growth and cartilage remodeling into mineralizing bone were accelerated, as revealed by Alcian blue (A-blu) and Alizarin red (A-red) staining. In A+B- and A-B- onset of mineralization was observed before E13.5, about 2 days earlier than in wild type and A-B+ mice. In all mutants between E18.5 to birth A-blu staining disappeared from epiphyses prematurely. Instead, A-blu+ cells were dislocated into diaphyses, most pronounced so in A-B- mutants, indicating additive effects of both missing ChEs in A-B- mutant mice. The remodeling effects were supported by in situ hybridization (ISH) experiments performed on cryosections from A-B- mice, in which Ihh, Runx2, MMP-13, ALP, Col-II and Col-X were considerably decreased, or had disappeared between E18.5 and P0. With a second approach, we applied an improved in vitro micromass model from chicken limb buds that allowed histological distinction between areas of cartilage, apoptosis and mineralization. When treated with the AChE inhibitor BW284c51, or with nicotine, there was decrease in cartilage and accelerated mineralization, suggesting that these effects were mediated through nicotinic receptors (α7-nAChR). We conclude that due to absence of either one or both cholinesterases in KO mice, or inhibition of AChE in chicken micromass cultures, there is increase in cholinergic signalling, which leads to increased chondroblast production and premature mineralization, at the expense of incomplete chondrogenic differentiation. This emphasizes the importance of cholinergic signalling in cartilage and bone formation.

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Zusammenfassung Sowohl die frühe phylogenetische und ontogenetische Existenz von cholinergen Systemen, als auch das Vorkommen in nicht‐neuronalen Geweben legen cholinerge und nicht‐cholinerge Funktionalitäten nahe, die weit über deren klassische Funktion an Synapsen hinausgehen. Die Fähigkeit von Cholinesterasen zur Bildung riesiger Proteinkomplexe eröffneten ihnen vielseitige Funktionsfelder. Schon in Stammzellen vertreten, begünstigen Cholinesterasen im Verbund mit Komponenten der Zellmatrix die Zelldifferenzierung; dabei erscheint ihre Enzymaktivität (teilweise) als nicht notwendig. Dies wurde durch Effekte inaktiver AChE in nicht‐neuronalen Zellen einerseits, und davon unabhängig durch die Entdeckung der CLAM‐Proteinfamilie beeindruckend untermauert. Vieles spricht somit dafür, dass die ursprünglichen Funktionsfelder der Cholinesterasen, wie auch von cholinergen Systemen insgesamt, in allgemeinen Zell‐Zell‐Wechselwirkungen zu suchen sind. Diese Einsichten wurden hier an einigen Zellkulturstudien und ausgewählten Beispielen der Normalentwicklung dargestellt. In der Wirbeltierretina beeinflussen die als erste differenzierenden cholinergen Amakrinzellen die Netzwerkbildung. Nicht weniger bedeutend ist das cholinerge System bei der Bildung von Röhrenknochen. Acetylcholin beschleunigt die Knochenbildung, und die Cholinesterasen regulieren dabei nicht nur dessen Konzentration, sondern spielen beide zudem strukturelle Rollen. Ebenso überzeugend ist eine Studie an Froschlarven, die zeigt, dass bei der Darmbildung von Xenopus laevis sehr wohl das AChE‐Protein, aber nicht dessen Enzymaktivität beteiligt ist. Die volle Aufklärung der Wirkungsweise der Cholinesterasen ist notwendig, denn eine Vielzahl von spezifischen Anticholinesterasen finden breite Anwendungen in wichtigen gesellschaftlichen Bereichen (Landwirtschaft, Gesundheit, Sicherheit). Die Forschung hat dies erkannt und widmet sich verstärkt nicht nur den Cholinesterasen, sondern insgesamt der Aufklärung nicht‐neuronaler cholinerger Systeme (NNCS).

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