Barbora Veselá

Embryonic Toxicity of Nanoparticles

Applications of nanoparticles (NP) in medicine, industry and other branches of human activities undoubtedly contribute to technology development and well-being. However, as NP are very small units in a wide range of materials, there is a lack of information related to possible side effects potentially affecting the health of organisms. There is increasing experimental interest in the determination of environmental effects on humans exposed to NP. Most such experimental studies focus on adult populations or adult experimental animals. However, embryos can be more sensitive to pollutants and environmental impacts in some species. Therefore, some investigations dealing particularly with the effects of NP on embryonic development have appeared recently and this issue is becoming of great concern. The aim of this review is to summarize the knowledge on the effects of various nanomaterials on embryonic development. A comprehensive collection of significant experimental nanotoxicity data is presented, which also indicate how the toxic effect of NP can be mediated and modulated with respect to possible effective protection strategies.

Expression of Fas, FasL, caspase-8 and other factors of the extrinsic apoptotic pathway during the onset of interdigital tissue elimination

Elimination of the interdigital web is considered to be the classical model for assessing apoptosis. So far, most of the molecules described in the process have been connected to the intrinsic (mitochondrial) pathway. The extrinsic (receptor mediated) apoptotic pathway has been rather neglected, although it is important in development, immunomodulation and cancer therapy. This work aimed to investigate factors of the extrinsic apoptotic machinery during interdigital regression with a focus on three crucial initiators: Fas, Fas ligand and caspase-8. Immunofluorescent analysis of mouse forelimb histological sections revealed abundant expression of these molecules prior to digit separation. Subsequent PCR Array analyses indicated the expression of several markers engaged in the extrinsic pathway. Between embryonic days 11 and 13, statistically significant increases in the expression of Fas and caspase-8 were observed, along with other molecules involved in the extrinsic apoptotic pathway such as Dapk1, Traf3, Tnsf12, Tnfrsf1A and Ripk1. These results demonstrate for the first time the presence of extrinsic apoptotic components in mouse limb development and indicate novel candidates in the molecular network accompanying the regression of interdigital tissue during digitalisation.

Osteogenic Profile of Mesenchymal Cell Populations Contributing to Alveolar Bone Formation.

Teeth develop within the surrounding periodontal tissues, involving the alveolar bone, periodontal ligament and cementum. The alveolar bone originates through the process of intramembranous ossification involving mesenchymal cells from the tooth germ. As most available data are related to endochondral ossification, we examined the molecular background of alveolar bone development. We investigated the osteogenic profile of mesenchymal cells dissected from mouse mandible slices at the stage of early alveolar bone formation. Relative monitoring of gene expression was undertaken using PCR Arrays; this included the profiles of 84 genes associated with osteogenesis. To examine the tooth-bone interface, stages with detectable changes in bone remodelling during development (E13.0, E14.0 and E15.0) were chosen and compared with each other. These results showed a statistically significant increase in the expression of the genes Fgf3, Ctsk, Icam-1, Mmp9, Itga3 and Tuft1, and of a wide range of collagens (Col1a2, Col3a1, Col7a1, Col12a1, Col14a1). Decreased expression was detected in the case of Col2a1, Sox9, Smad2 and Vegfb. To confirm these changes in gene expression, immunofluorescence analyses of Mmp9 and Sox9 proteins were performed in situ. Our research has identified several candidate genes that may be crucial for the initiation of alveolar bone formation and is the basis for further functional studies.

Non-apoptotic role for caspase-7 in hair follicles and the surrounding tissue

Hair follicles are unique organs undergoing regular cycles of proliferation, differentiation, and apoptosis. The final step of apoptosis is, in general, mediated by executioner caspases comprising caspase-3, -6 and -7. Despite their commonly accepted apoptotic function, executioner caspases also participate in non-apoptotic processes. In the present study, we investigated activation (cleavage) of caspase-7 in mouse hair follicles and surrounding tissue during embryonic development into adulthood. Casp7−/− mice were examined to understand the effect of caspase-7 deficiency in the skin. The activated form of caspase-7 was observed during embryonic hair follicle development, as well as in the first hair cycle. In general, activation of caspase-7 did not correlate with apoptosis and activation of caspase-3, except during physiological hair follicle regression. Notably, cleaved caspase-7 was observed in mast cells and its deficiency in the adult skin resulted in increased mast cell number. Our study shows for the first time activated caspase-7 in hair follicles and mast cells and indicates its non-apoptotic roles in the skin.

Expression of apoptosis-related genes in the mouse skin during the first postnatal catagen stage, focused on localization of Bnip3L and caspase-12

Abstract Hair follicles undergo repetitive stages of cell proliferation and programmed cell death. The catagen stage of physiological apoptosis is connected with dynamic changes in morphology and alterations in gene expression. However, hair follicle apoptosis must be in balance with events in surrounding tissues, such as keratinocyte cornification, to maintain complex skin homeostasis. Several pro- and anti-apoptotic molecules in the skin have been reported but mainly in pathological states. In this investigation, apoptosis-related gene expression was examined during the first catagen stage of mouse hair follicle development by PCR arrays under physiological conditions. Postnatal stages P15 and P17, representing early and late catagen stages, were evaluated relatively to stage P6, representing the hair follicle growing phase, to demonstrate dynamics of gene activation during the catagen. Several statistically significant alterations were observed at P15 and particularly at P17. Bnip3L and caspase-12 identified by the PCR arrays at both catagen stages were additionally localized using immunofluorescence and were reported in physiological hair development for the first time.

Activation of pro-apoptotic caspases in non-apoptotic cells during odontogenesis and related osteogenesis

Caspases are well known proteases in the context of inflammation and apoptosis. Recently, novel roles of pro-apoptotic caspases have been reported, including findings related to the development of hard tissues. To further investigate these emerging functions of pro-apoptotic caspases, the in vivo localisation of key pro-apoptotic caspases (-3,-6,-7,-8, and -9) was assessed, concentrating on the development of two neighbouring hard tissues, cells participating in odontogenesis (represented by the first mouse molar) and intramembranous osteogenesis (mandibular/alveolar bone). The expression of the different caspases within the developing tissues was correlated with the apoptotic status of the cells, to produce a picture of whether different caspases have potentially distinct, or overlapping non-apoptotic functions. The in vivo investigation was additionally supported by examination of caspases in an osteoblast-like cell line in vitro. Caspases-3,-7, and -9 were activated in apoptotic cells of the primary enamel knot of the first molar; however, caspase-7 and -8 activation was also associated with the non-apoptotic enamel epithelium at the same stage and later with differentiating/differentiated odontoblasts and ameloblasts. In the adjacent bone, active caspases-7 and -8 were present abundantly in the prenatal period, while the appearance of caspases-3,-6, and -9 was marginal. Perinatally, caspases-3 and -7 were evident in some osteoclasts and osteoblastic cells, and caspase-8 was abundant mostly in osteoclasts. In addition, postnatal activation of caspases-7 and -8 was retained in osteocytes. The results provide a comprehensive temporo-spatial pattern of pro-apoptotic caspase activation, and demonstrate both unique and overlapping activation in non-apoptotic cells during development of the molar tooth and mandibular/alveolar bone. The importance of caspases in osteogenic pathways is highlighted by caspase inhibition in osteoblast-like cells, which led to a significant decrease in osteocalcin expression, supporting a role in hard tissue cell differentiation.

FasL Modulates Expression of Mmp2 in Osteoblasts

FasL is a well-known actor in the apoptotic pathways but recent reports have pointed to its important novel roles beyond cell death, as observed also for bone cells. This is supported by non-apoptotic appearance of FasL during osteogenesis and by significant bone alterations unrelated to apoptosis in FasL deficient (gld) mice. The molecular mechanism behind this novel role has not yet been revealed. In this report, intramembranous bone, where osteoblasts differentiate directly from mesenchymal precursors without intermediary chondrogenic step, was investigated. Mouse mandibular bone surrounding the first lower molar was used as a model. The stage where a complex set of bone cells (osteoblasts, osteocytes, osteoclasts) is first present during development was selected for an initial examination. Immunohistochemical staining detected FasL in non-apoptotic cells at this stage. Further, FasL deficient vs. wild type samples subjected to osteogenic PCR Array analysis displayed a significantly decreased expression of Mmp2 in gld bone. To examine the possibility of this novel FasL–Mmp2 relationship, intramembranous bone-derived osteoblastic cells (MC3T3-E1) were treated with anti-FasL antibody or rmFasL. Indeed, the FasL neutralization caused a decreased expression of Mmp2 and rmFasL added to the cells resulted in the opposite effect. Since Mmp2-/- mice display age-dependent alterations in the intramembranous bone, early stages of gld mandibular bone were examined and age-dependent phenotype was confirmed also in gld mice. Taken together, the present in vivo and in vitro findings point to a new non-apoptotic function of FasL in bone development associated with Mmp2 expression.

Detection of Bim and Puma in mouse hair follicles using immunofluorescence and TUNEL assay double staining

Apoptosis in hair follicles often is studied under pathological conditions; little is known about apoptotic mechanisms during normal hair follicle formation and maintenance. We investigated proteins of intrinsic apoptotic pathway, Bim and Puma, during hair follicle development and the first catagen stage using immunofluorescence to describe their expression patterns and to correlate them with apoptosis as determined by TUNEL assay. Both proteins were found in developing follicles. Bim and Puma overlapped apoptosis only partially during physiological apoptotic stage and they were present in non-apoptotic parts of the follicles. Our findings suggest that these primary apoptotic molecules participate in postnatal development and maintenance of hair follicles.

Mouse Incisor Stem Cell Niche and Myb Transcription Factors

Dental hard tissues are formed particularly by odontoblasts (dentin) and ameloblasts (enamel). Whereas the reparation of dentin is often observed, enamel does not regenerate in most species. However, in mouse incisor, a population of somatic stem cells in the cervical loop is responsible for the incisor regeneration. Understanding of the specificities of these cells is therefore of an interest in basic research as well as regenerative therapies. The Myb transcription factors are involved in essential cellular processes. B‐Myb is often linked to the stem cell phenotype, and c‐Myb expression marks undifferentiated and proliferating cells such as the stem cells. In the presented study, temporo‐spatial expression of B‐Myb and c‐Myb proteins was correlated with localisation of putative somatic stem cells in the mouse incisor cervical loop by immunohistochemistry. B‐Myb expression was localised mostly in the zone of transit‐amplifying cells, and c‐Myb was found in the inner enamel epithelium, the surrounding mesenchyme and in differentiated cells. Taken together, neither B‐Myb nor c‐Myb was exclusively present or abundant in the area of the incisor stem cell niche. Their distribution, however, supports recently reported novel functions of c‐Myb in differentiation of hard tissue cells.

Mybs in mouse hair follicle development.

The Myb transcription factors are involved in essential cellular processes, such as cell proliferation, differentiation and cell death. Biological functions carried out by specific Myb proteins are distinct. Hair follicles are ectodermal-derived organs with cycling character of the growth resulting from the presence of somatic stem cells. In this study, we followed the expression of the Myb proteins in developing hair follicles and in the hair follicle stem cell niche by immunofluorescence staining. During hair follicle development, B-Myb was present in a few cells located in the area of cell division; c-Myb was abundant postanally in dividing cells but also in keratinizing zone. In addition, c-Myb was also detected in cells under the hair follicle bulge. These findings indicate possible involvement of c-Myb in regulation of activated stem cells leaving the niche.