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

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

Cholinesterases in development: AChE as a firewall to inhibit cell proliferation and support differentiation.

Acetylcholinesterase (AChE) is a most remarkable protein, not only because it is one of the fastest enzymes in nature, but also since it appears in many molecular forms and is regulated by elaborate genetic networks. AChE is expressed in many tissues during development and in mature organisms, as well as in healthy and diseased states. In search for alternative, "non-classical" functions of cholinesterases (ChEs), AChE could either work within the frame of classic cholinergic systems, but in non-neural tissues ("non-synaptic function"), or act non-enzymatically. Here, we review briefly some of the major ideas and advances of this field, and report on some recent progress from our own experimental work, e.g. that i) non-neural ChEs have pronounced, predominantly enzymatic effects on early embryonic (limb) development in chick and mouse, that ii) retinal R28 cells of the rat overexpressing synaptic AChE present a significantly decreased cell proliferation, and that iii) in developing chick retina ACh-synthesizing and ACh-degrading cells originate from the same postmitotic precursor cells, which later form two locally opposing cell populations. We suggest that such distinct distributions of ChAT(+) vs. AChE(+) cells in the inner half retina provide graded distributions of ACh, which can direct cell differentiation and network formation. Thus, as corroborated by works from many labs, AChE can be considered a highly co-opting protein, which can combine enzymatic and non-enzymatic functions within one molecule

BW284c51 bead implantation.

<p>BW284c51 bead implantation.</p

MAB304 bead implantation.

<p>MAB304 bead implantation.</p

ChAT expression by ISH in whole-mounted embryos from HH17 to HH25.

<p>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.</p

ChAT bead implantation.

<p>ChAT bead implantation.</p

Implantation of ChAT-soaked beads accelerates skeletogenesis.

<p>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.</p

ACh bead implantation.

<p>ACh bead implantation.</p

Copy number variation of two separate regulatory regions upstream of SOX9 causes isolated 46,XY or 46,XX disorder of sex development

Background: SOX9 mutations cause the skeletal malformation syndrome campomelic dysplasia in combination with XY sex reversal. Studies in mice indicate that SOX9 acts as a testis-inducing transcription factor downstream of SRY, triggering Sertoli cell and testis differentiation. An SRY-dependent testis-specific enhancer for Sox9 has been identified only in mice. A previous study has implicated copy number variations (CNVs) of a 78 kb region 517–595 kb upstream of SOX9 in the aetiology of both 46,XY and 46,XX disorders of sex development (DSD). We wanted to better define this region for both disorders. Results: By CNV analysis, we identified SOX9 upstream duplications in three cases of SRY-negative 46,XX DSD, which together with previously reported duplications define a 68 kb region, 516–584 kb upstream of SOX9, designated XXSR (XX sex reversal region). More importantly, we identified heterozygous deletions in four families with SRY-positive 46,XY DSD without skeletal phenotype, which define a 32.5 kb interval 607.1–639.6 kb upstream of SOX9, designated XY sex reversal region (XYSR). To localise the suspected testis-specific enhancer, XYSR subfragments were tested in cell transfection and transgenic experiments. While transgenic experiments remained inconclusive, a 1.9 kb SRY-responsive subfragment drove expression specifically in Sertoli-like cells. Conclusions: Our results indicate that isolated 46,XY and 46,XX DSD can be assigned to two separate regulatory regions, XYSR and XXSR, far upstream of SOX9. The 1.9 kb SRY-responsive subfragment from the XYSR might constitute the core of the Sertoli-cell enhancer of human SOX9, representing the so far missing link in the genetic cascade of male sex determination