Craniofacial studies in chicken embryos confirm the pathogenicity of human FZD2 variants associated with Robinow syndrome.

Robinow syndrome is a rare disease caused by variants of seven WNT pathway genes. Craniofacial features include widening of the nasal bridge and jaw hypoplasia. We used the chicken embryo to test whether two missense human FZD2 variants (1301G>T, p.Gly434Val; 425C>T, p.Pro142Lys) were sufficient to change frontonasal mass development. In vivo, the overexpression of retroviruses with wild-type or variant human FZD2 inhibited upper beak ossification. In primary cultures, wild-type and variant human FZD2 significantly inhibited chondrogenesis, with the 425C>T variant significantly decreasing activity of a SOX9 luciferase reporter compared to that for the wild type or 1301G>T. Both variants also increased nuclear shuttling of ß-catenin (CTNNB1) and increased the expression of TWIST1, which are inhibitory to chondrogenesis. In canonical WNT luciferase assays using frontonasal mass cells, the variants had dominant-negative effects on wild-type FZD2. In non-canonical assays, the 425C>T variant failed to activate the reporter above control levels and was unresponsive to exogenous WNT5A. This is the first single amino acid change to selectively alter ligand binding in a FZD receptor. Therefore, FZD2 missense variants are pathogenic and could lead to the altered craniofacial morphogenesis seen in Robinow syndrome.

DiaPASEF proteotype analysis indicates changes in cell growth and metabolic switch induced by caspase-9 inhibition in chondrogenic cells.

Caspase-9 is the major apical caspase responsible for triggering the intrinsic apoptotic pathway. Our previous study indicated that specific inhibition of caspase-9 caused microscopically evident alterations in appearance of the primary chondrogenic cultures which cannot be explained by decrease in apoptosis. To describe a complex molecular background of this effect, proteomics analysis of control and caspase-9 inhibitor-treated chondrogenic cultures were performed. Proteins were extracted, identified and quantified using LC-MS in both data dependent and data independent acquisition (DIA) mode. While directDIA analysis of diaPASEF data obtained using timsTOF Pro LC-MS system revealed 7849 protein groups (Q-value <0.01), a parallel analysis of iTRAQ-2DLC-MS3 and conventional DIA-MS data identified only 5146 and 4098 protein groups, respectively, showing diaPASEF a superior method for the study. The detailed analysis of diaPASEF data disclosed 236/551 significantly down-/up-regulated protein groups after caspase-9 inhibition, respectively (|log2FC|>0.58, Q value <0.05). Classification of downregulated proteins revealed changes in extracellular matrix organization, collagen metabolism, and muscle system processes. Moreover, deregulations suggest a switch from glycolytic to lipid based metabolism in the inhibited cells. No essential changes were found in the proteins involved in apoptosis. The data indicate new non-apoptotic participation of caspases in chondrocyte homeostasis with potential applications in cartilage pathophysiology.

Cholinergic control of bone development and beyond.

There is ample evidence that cholinergic actions affect the health status of bones in vertebrates including man. Nicotine smoking, but also exposure to pesticides or medical drugs point to the significance of cholinergic effects on bone status, as reviewed here in Introduction. Then, we outline processes of endochondral ossification, and review respective cholinergic actions. In Results, we briefly summarize our in vivo and in vitro studies on bone development of chick and mouse [1,2], including (i) expressions of cholinergic components (AChE, BChE, ChAT) in chick embryo, (ii) characterisation of defects during skeletogenesis in prenatal ChE knockout mice, (iii) loss-of-function experiments with beads soaked in cholinergic components and implanted into chicken limb buds, and finally (iv) we use an in vitro mesenchymal 3D-micromass model that mimics cartilage and bone formation, which also had revealed complex crosstalks between cholinergic, radiation and inflammatory mechanisms [3]. In Discussion, we evaluate non-cholinergic actions of cholinesterases during bone formation by considering: (i) how cholinesterases could function in adhesive mechanisms; (ii) whether and how cholinesterases can form bone-regulatory complexes with alkaline phosphatase (ALP) and/or ECM components, which could regulate cell division, migration and adhesion. We conclude that cholinergic actions in bone development are driven mainly by classic cholinergic, but non-neural cycles (e.g., by acetylcholine); in addition, both cholinesterases can exert distinct ACh-independent roles. Considering their tremendous medical impact, these results bring forward novel research directions that deserve to be pursued.

Osteogenic Potential of Caspases Related to Endochondral Ossification.

Caspases have functions particularly in apoptosis and inflammation. Increasing evidence indicates novel roles of these proteases in cell differentiation, including those involved in osteogenesis. This investigation provides a complex screening of osteogenic markers affected by pan caspase inhibition in micromass cultures derived from mouse forelimbs. PCR Array analysis showed significant alterations in expression of 49 osteogenic genes after 7 days of inhibition. The largest change was a decrease in CD36 expression, which was confirmed at organ level by caspase inhibition in cultured mouse ulnae followed by CD36 immunohistochemical analysis. So far, available data point to osteogenic potential of pro-apoptotic caspases. Therefore, the expression of pro-apoptotic caspases (-3, -6, -7, -8, -9) within the growth plate of mouse forelimbs at the stage where the individual zones are clearly apparent was studied. Caspase-9 was reported in the growth plate for the first time as well as caspase-6 and -7 in the resting zone, caspase-7 in the proliferation, and caspase-6 and -8 in the ossification zone. For all caspases, there was a gradient increase in activation toward the ossification zone. The distribution of staining varied significantly from that of apoptotic cells, and thus, the results further support non-apoptotic participation of caspases in osteogenesis.

Osteogenic Potential of the Transcription Factor c-MYB.

The transcription factor c-MYB is a well-known marker of undifferentiated cells such as haematopoietic cell precursors, but recently it has also been observed in differentiated cells that produce hard tissues. Our previous findings showed the presence of c-MYB in intramembranous bones and its involvement in the chondrogenic steps of endochondral ossification, where the up-regulation of early chondrogenic markers after c-myb overexpression was observed. Since we previously detected c-MYB in osteoblasts, we aimed to analyse the localisation of c-MYB during later stages of endochondral bone formation and address its function during bone matrix production. c-MYB-positive cells were found in the chondro-osseous junction zone in osteoblasts of trabecular bone as well as deeper in the zone of ossification in cells of spongy bone. To experimentally evaluate the osteogenic potential of c-MYB during endochondral bone formation, micromasses derived from embryonic mouse limb buds were established. Nuclear c-MYB protein expression was observed in long-term micromasses, especially in the areas around nodules. c-myb overexpression induced the expression of osteogenic-related genes such as Bmp2, Comp, Csf2 and Itgb1. Moreover, alizarin red staining and osteocalcin labelling promoted mineralised matrix production in c-myb-overexpressing cultures, whereas downregulation of c-myb by siRNA reduced mineralised matrix production. In conclusion, c-Myb plays a role in the osteogenesis of long bones by inducing osteogenic genes and causing the enhancement of mineral matrix production. This action of the transcription factor c-Myb might be of interest in the future for the establishment of novel approaches to tissue regeneration.

Role of c-Myb in chondrogenesis.

The Myb locus encodes the c-Myb transcription factor involved in controlling a broad variety of cellular processes. Recently, it has been shown that c-Myb may play a specific role in hard tissue formation; however, all of these results were gathered from an analysis of intramembranous ossification. To investigate a possible role of c-Myb in endochondral ossification, we carried out our study on the long bones of mouse limbs during embryonic development. Firstly, the c-myb expression pattern was analyzed by in situ hybridization during endochondral ossification of long bones. c-myb positive areas were found in proliferating as well as hypertrophic zones of the growth plate. At early embryonic stages, localized expression was also observed in the perichondrium and interdigital areas. The c-Myb protein was found in proliferating chondrocytes and in the perichondrium of the forelimb bones (E14.5-E17.5). Furthermore, protein was detected in pre-hypertrophic as well as hypertrophic chondrocytes. Gain-of-function and loss-of-function approaches were used to test the effect of altered c-myb expression on chondrogenesis in micromass cultures established from forelimb buds of mouse embryos. A loss-of-function approach using c-myb specific siRNA decreased nodule formation, as well as downregulated the level of Sox9 expression, a major marker of chondrogenesis. Transient c-myb overexpression markedly increased the formation of cartilage nodules and the production of extracellular matrix as detected by intense staining with Alcian blue. Moreover, the expression of early chondrogenic genes such as Sox9, Col2a1 and activity of a Col2-LUC reporter were increased in the cells overexpressing c-myb while late chondrogenic markers such as Col10a1 and Mmp13 were not significantly changed or were downregulated. Taken together, the results of this study demonstrate that the c-Myb transcription factor is involved in the regulation and promotion of endochondral bone formation.

Cell communication and tissue engineering.

Gap junction intercellular communication (GJIC) is ubiquitous in the majority of cells and is indispensable for proper development and function of most tissues. The loss of gap junction mediated cell to cell communication leads to compromised development in many tissues and organs, and also facilitates tumorigenesis and autonomous cell behavior in cancerous cells. Because cells embedded in an extracellular matrix constantly interact through gap junctions to coordinate normal tissue functions and homeostasis, our group hypothesized that increasing cell to cell communication, via genetically engineering cells to overexpress gap junction proteins, could improve cell signaling and increase differentiation in interior regions of engineered tissue equivalents. In a recent paper,1 we presented a platform to regenerate full 3D equivalents of engineered tissue, providing a strategy to overcome a barrier in regenerative medicine. These findings suggest that both targeted delivery and cell-based strategies can be used as treatments to enhance communication in 3D living tissue.2 In this addendum, we address the effects of extracellular calcium (Ca(2+) (e)) on intracellular calcium (Ca(2+) (i)), GJIC and osteogenic differentiation under conditions in which bone marrow stromal cells (BMSCs) also exhibit higher cell-to-cell communication. As a key secondary messenger in many biological processes, the levels of Ca(2+) (e) and Ca(2+) (i) play a role in cell differentiation and may be a tunable signal in tissue regeneration. Higher cell-to-cell communication was achieved by both genetically engineering cells to overexpress connexin 43 (Cx43) and by a high density cell seeding technique, denoted micromass seeding (MM). The results presented in this addendum show that the intensity and duration of a second messenger, like calcium, can be augmented in a platform that enables higher cell-to-cell communication. The ability to modulate calcium signaling, combined with our previous approaches to modulate GJIC, may have an impact on tissue regeneration and therapies for communication incompetent cells, such as those associated with heart disease and certain types of cancer.

Developmental toxicity of Ochratoxin A in rat embryo midbrain micromass cultures.

Embryonic midbrain micromass cultures were exposed for five days to ochratoxin A (OTA) at seven concentrations (ranging from 0.16 to 10 microg/mL). Cell viability was assessed in neutral red uptake test (NRU), and differentiation - by immunoenzymatic determination of structural proteins (beta(III)-tubulin, MAP2, GFAP) expression level as well as by computer image analysis. Dose dependent decrease in cell number and differentiation was observed. Concentration-response curves were analysed and the mean inhibition concentrations (microg/mL) for cytotoxicity (IC(50)) and differentiation (ID(50)) were calculated. There were no significant differences in the sensitivity of neurons in early and late stage of differentiation and astrocytes to the toxic activity of this compound. For all endpoints ID(50) value was very low (< 10 microg/mL) so OTA was classified as a strong teratogen. IC(50)/ ID(50) ratios <2 pointed out that with harmful action of OTA the basic cytotoxicity should be connected.