Fig 1.
Newborn cholinesterase double-KO mice present strong skeletal phenotypes.
Whole-mount staining by Alcian blue (A-blu) for cartilage (blue) and Alizarin red (A-red) for mineralizing bone elements (red) of P0 wild type (a) and A-B- double-KO mouse (b). Note, in mutants A-blu staining is stronger in areas where mineralization is decreased, e.g. in distal long bones (b, stippled arrow) and dorsal ribs; A-blu is absent in ventral ribs (b, star) and hind limb joints (b, stippled circle). (c-f) details of mice in (a, b) of chest and vertebral column in wt (c, e) and A-B- mutant (d, f). Further see text; see supplemental S1 Fig for A-blu staining of whole-mount mice only.
Fig 2.
Cartilage matrix (blue) and mineralization (red) are changed in ChE mutants from E13.5 until birth.
Note larger size of all mutants at E13.5 (cf. S1 and S2 Figs), and earlier onset of mineralization in A+B- and in A-B- mutant legs. Note earlier and stronger degradation of cartilage matrix (A-blu) in A-B+ and more so in A-B- mutants, along with appearance of A-blu+ cells in their diaphyses. Size relations between legs are approximated.
Fig 3.
Cartilage remodeling is accelerated in perinatal ChE mutants.
(a-h) Cryosections of WT and three ChE mutant femurs at P0 were stained by Alcian blue (A-blu) at pH 1.0 (upper; revealing sulfated proteo- and glucosaminoglycans, GAGs) and at pH 2.5 (lower; revealing carboxylated and weakly sulfated glycoproteins and mucopolysaccarides, PGs). Compare strong staining of epiphyses of WT with step-wise advanced degradation of A-blu in epiphyses of mutants (yellow stippled line and arrow), and its appearance in diaphyses. Note onset of secondary ossification in (g, star). (i-l) Whole-mounted femurs were double-stained by A-blue and A-red at pH2.5. rc, resting chondrocytes; GP, growth plate; MZ, mineralizing zone; pc, proliferating chondrocytes; ph, pre-hypertrophic chondrocytes; hc hypertrophic chondrocytes. Cryosections were counterstained by nuclear fast red. n = 5 per each sample. Further see text.
Fig 4.
(a, b) Expression of AChE and BChE in diaphysis of WT. Note ChEs were also expressed on parts of perichondrium and periosteum, but were completely absent in epiphyses. (c -l) Major matrix components and GP structure are changed in P0 A-B- mutant mice. (c-f) DAPI staining to detect specifically cell nuclei in GP of A-B- KO and WT mice at P0 at low (c, d) and higher magnification (e, f). Note many brightly stained, mostly apoptotic cells in mutant. White arrows indicate high numbers of abnormally positioned of cells in mutant growth plate. (g-l) Serial longitudinal sections of distal femoral growth plates from newborn WT (g-i) and mutant hind limbs (j-l) were hybridized with Col-II (g, j), Col-X (h, k) and MMP-13 (i, l) riboprobes. Note that in mutant both Col-X and MMMP-13 were strongly reduced, while Col-II was still quite high in epiphysis and in periosteal cells on diaphysis of mutant mouse. n = 5 per each sample.
Fig 5.
Changes of major gene expressions in A-B- mutant tibias between E18.5 and P0.
(a, b, g, h) ALP as expressed in hc and diaphysis of WT (a, b), disappeared completely by P0 in mutant (g, h). (c-f, i-l) Expressions of Ihh and Runx2 disappear in mutant tibias. In WT, ISH staining for Ihh expression shifted from a small zone in epiphysis into the diaphysis (c, d), while Runx2 was expressed at both stages in diaphyses (e, f). Note that in mutant at P0 both markers were not detectable anymore (j, l); however, in the E18.5 mutant, Ihh was nearly absent (i), while Runx2 was weakly expressed in diaphysis (k). n = 3 per each sample.
Fig 6.
Micromass cultures from chicken limb buds as skeletogenic in vitro models: chicken serum reorganizes improves their spatial order.
(a) Micromass cultures of chicken limb bud cells were incubated in absence (left) and presence (right) of 2% chicken serum (CS) for 13 days. Addition of CS leads to a stepwise chondrogenic differentiation, whereby cartilage forms in an inner tissue core (upper; equiv. to a GP-like area), followed by a ring of ALP (lower; equiv. to a DZ-like area) and mineralization (middle; equiv. to a MZ-like area) near the periphery, as shown by A-blu, A-red and Alkaline phosphatase (ALP) stainings. (b) Combined FDA/EtBr staining to detect living cells (green) and dead cells (red). (c) Combined A-blu/A-red staining (left), and merged with ALP staining (right). Zone of A-blu indicates proliferative and matured chondrocytes; zone of high cell death and ALP activity without proteoglycan matrix secretion marks the zone of hypertrophic and “terminal” hypertrophic chondrocytes. A-red staining identified calcification and marks the zone of chondrocyte-derived osteoprogenitor cells. n = 5 per each series. DZ, in vitro zone corresponding to DZ; GP, iv zone corresp. to GP; MZ, iv zone corresp. to MZ.
Fig 7.
AChE inhibition or stimulation of nAChRs reduces proteoglycan content and ALP activity, and increases mineralization in vitro.
(a-d) Micromass cultures of chicken limb-bud cells of HH stage 22–24 were stained with A-blu, A-red and for ALP activity. Mesenchymal micromass cultures were incubated for 13 days (a) or 9 days (c) in presence of increasing doses of BW284c51, or control conditioned medium. (b) Chick limb micromass cultures were incubated for 13 days in the presence of increased doses of nicotine or control conditioned medium. (d) Combined cultivation of chick limb micromass cultures with 20μM nicotine plus increasing doses of MLA. ALP staining of div9 micromass cultures (c) indicated dose-dependent premature chondrogenesis at early stages leading to a suppressed chondrogenic differentiation at later cultivating times (a). All concentrations are indicated as μM; n = 7 per each treatment.
Fig 8.
Inhibition of AChE accelerates progression into cell death in chondrogenic zone, as shown by a FDA/EtBr dual staining of micromass cultures on div13.
Cell viability and cell death were revealed by combined FDA/EtBr staining for living and dead cells (green, red, respectively). Note with increasing BW284c51 concentrations dead cells are increased in inner compartment of micromass culture, corresponding with chondrogenic zone of GP (GP; below stippled lines), but decrease in outer compartment, corresponding with MZ. n = 3 per each sample. DZ, in vitro zone corresp. to DZ; GP, iv zone corresp. to GP; MZ, iv zone corresp. to MZ.
Fig 9.
Models of cholinergic balancing of chondro- vs. osteogenesis.
(a, left) 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). Note that osteoblastic precursors (obp, orange circles), a second source of proliferative cells besides pc, will migrate from GP into MZ [31]. (a, right) represents spatial distributions of ACh, of α7-nAChR and of AChE. Note that in regions of ChE activity in MZ, ACh concentration and thus, cholinergic signaling will be low (cf. Fig 4A and 4B). (b) depicts two effective pathways of AChE actions. (left in b) whenever systemic AChE is low (e.g. in ChE mutants), 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. Further see Discussion. rc, resting chondrocytes; pc, proliferating chondrocytes; ph, pre-hypertrophic chondrocytes; hc, hypertrophic chondrocytes; obp, osteopblastic precursors; COJ, chondro-osseous junction; MZ, mineralizing zone.