Early embryogenesis in CHDFIDD mouse model reveals facial clefts and altered cranial neurogenesis.

Congenital heart defects, facial dysmorphism and intellectual development disorder (CHDFIDD) is associated with mutations in CDK13 gene, which encodes a transcription regulating Cyclin-dependent kinase 13 (CDK13). Here, we focused on development of craniofacial structures and analyzed early embryonic stages of CHDFIDD mouse models with hypomorphic mutation in Cdk13 gene, which exhibits cleft lip/palate and knockout of Cdk13 with stronger phenotype including midfacial cleft. Cdk13 was found to be physiologically strongly expressed in the mouse embryonic craniofacial structures, namely in the forebrain, nasal epithelium and maxillary mesenchyme. We also uncovered that Cdk13-deficiency leads to development of hypoplastic branches of the trigeminal nerve including maxillary branch and additionally, we detected significant gene expression changes of molecules involved in neurogenesis (Ache, Dcx, Mef2c, Neurog1, Ntn1, Pou4f1) within the developing palatal shelves. These results, together with changes of gene expression of other key face-specific molecules (Fgf8, Foxd1, Msx1, Meis2 and Shh) at early stages in Cdk13 mutant embryos, demonstrate a key role of CDK13 in regulation of craniofacial morphogenesis.

ChOP-CT: quantitative morphometrical analysis of the Hindbrain Choroid Plexus by X-ray micro-computed tomography.

The Hindbrain Choroid Plexus is a complex, cerebrospinal fluid-secreting tissue that projects into the 4th vertebrate brain ventricle. Despite its irreplaceability in the development and homeostasis of the entire central nervous system, the research of Hindbrain Choroid Plexus and other Choroid Plexuses has been neglected by neuroscientists for decades. One of the obstacles is the lack of tools that describe the complex shape of the Hindbrain Choroid Plexus in the context of brain ventricles. Here we introduce an effective tool, termed ChOP-CT, for the noninvasive, X-ray micro-computed tomography-based, three-dimensional visualization and subsequent quantitative spatial morphological analysis of developing mouse Hindbrain Choroid Plexus. ChOP-CT can reliably quantify Hindbrain Choroid Plexus volume, surface area, length, outgrowth angle, the proportion of the ventricular space occupied, asymmetries and general shape alterations in mouse embryos from embryonic day 13.5 onwards. We provide evidence that ChOP-CT is suitable for the unbiased evaluation and detection of the Hindbrain Choroid Plexus alterations within various mutant embryos. We believe, that thanks to its versatility, quantitative nature and the possibility of automation, ChOP-CT will facilitate the analysis of the Hindbrain Choroid Plexus in the mouse models. This will ultimately accelerate the screening of the candidate genes and mechanisms involved in the onset of various Hindbrain Choroid Plexus-related diseases.

Role of ciliopathy protein TMEM107 in eye development: insights from a mouse model and retinal organoid.

Primary cilia are cellular surface projections enriched in receptors and signaling molecules, acting as signaling hubs that respond to stimuli. Malfunctions in primary cilia have been linked to human diseases, including retinopathies and ocular defects. Here, we focus on TMEM107, a protein localized to the transition zone of primary cilia. TMEM107 mutations were found in patients with Joubert and Meckel-Gruber syndromes. A mouse model lacking Tmem107 exhibited eye defects such as anophthalmia and microphthalmia, affecting retina differentiation. Tmem107 expression during prenatal mouse development correlated with phenotype occurrence, with enhanced expression in differentiating retina and optic stalk. TMEM107 deficiency in retinal organoids resulted in the loss of primary cilia, down-regulation of retina-specific genes, and cyst formation. Knocking out TMEM107 in human ARPE-19 cells prevented primary cilia formation and impaired response to Smoothened agonist treatment because of ectopic activation of the SHH pathway. Our data suggest TMEM107 plays a crucial role in early vertebrate eye development and ciliogenesis in the differentiating retina.

Spatiotemporal monitoring of hard tissue development reveals unknown features of tooth and bone development.

Mineralized tissues, such as bones or teeth, are essential structures of all vertebrates. They enable rapid movement, protection, and food processing, in addition to providing physiological functions. Although the development, regeneration, and pathogenesis of teeth and bones have been intensely studied, there is currently no tool to accurately follow the dynamics of growth and healing of these vital tissues in space and time. Here, we present the BEE-ST (Bones and tEEth Spatio-Temporal growth monitoring) approach, which allows precise quantification of development, regeneration, remodeling, and healing in any type of calcified tissue across different species. Using mouse teeth as model the turnover rate of continuously growing incisors was quantified, and role of hard/soft diet on molar root growth was shown. Furthermore, the dynamics of bones and teeth growth in lizards, frogs, birds, and zebrafish was uncovered. This approach represents an effective, highly reproducible, and versatile tool that opens up diverse possibilities in developmental biology, bone and tooth healing, tissue engineering, and disease modeling.

Metastasising ameloblastoma or ameloblastic carcinoma? A case report with mutation analyses.

BACKGROUND: Ameloblastic carcinoma and metastasising ameloblastoma are rare epithelial odontogenic tumours with aggressive features. Distinguishing between these two lesions is often clinically difficult but necessary to predict tumour behaviour or to plan future therapy. Here, we provide a brief review of the literature available on these two types of lesions and present a new case report of a young man with an ameloblastoma displaying metastatic features. We also use this case to illustrate the similarities and differences between these two types of tumours and the difficulties of their differential diagnosis. CASE PRESENTATION: Our histopathological analyses uncovered a metastasising tumour with features of ameloblastic carcinoma, which developed from the ameloblastoma. We profiled the gene expression of Wnt pathway members in ameloblastoma sample of this patient, because multiple molecules of this pathway are involved in the establishing of cell polarity, cell migration or for epithelial-mesenchymal transition during tumour metastasis to evaluate features of tumor behaviour. Indeed, we found upregulation of several cell migration-related genes in our patient. Moreover, we uncovered somatic mutation BRAF p.V600E with known pathological role in cancerogenesis and germline heterozygous FANCA p.S858R mutation, whose interpretation in this context has not been discussed yet. CONCLUSIONS: In conclusion, we have uncovered a unique case of ameloblastic carcinoma associated with an alteration of Wnt signalling and the presence of BRAF mutation. Development of harmful state of our patient might be also supported by the germline mutation in one FANCA allele, however this has to be confirmed by further analyses.

Primary cilia and hypoxia-associated signaling in developmental odontogenic cysts in relation to autosomal dominant polycystic kidney disease - A novel insight.

Developmental cysts are pathological epithelial-lined cavities arising in various organs as a result of systemic or hereditary diseases. Molecular mechanisms involved in the formation of developmental odontogenic cysts (OCs) are not fully understood yet; the cystogenesis of renal cysts originating from the autosomal dominant polycystic kidney disease (ADPKD) has been, however, explored in much greater detail. This narrative review aimed i) to summarize molecular and cellular processes involved in the formation and growth of developmental OCs, especially dentigerous cysts (DCs) and odontogenic keratocysts (OKCs), ii) to find if there are any similarities in their cystogenesis to ADPKD cysts, and, based on that, iii) to suggest potential factors, candidate molecules, and mechanisms that could be involved in the DC formation, thus proposing further research directions. Here we suggest a possible association of developmental OCs with primary cilia disruption and with hypoxia, which have been previously linked with cyst formation in ADPKD patients. This is illustrated on the imagery of tissues from an ADPKD patient (renal cyst) and from developmental OCs, supporting the similarities in cell proliferation, apoptosis, and primary cilia distribution in DC/OKC/ADPKD tissues. Based on all that, we propose a novel hypothesis of OCs formation suggesting a crucial role of mutations associated with the signaling pathways of primary cilia (in particular, Sonic Hedgehog). These can lead to excessive proliferation and formation of cell agglomerates, which is followed by hypoxia-driven apoptosis in the centers of such agglomerates (controlled by molecules such as Hypoxia-inducible factor-1 alpha), leading to cavity formation and, finally, the OCs development. Based on this, we propose future perspectives in the investigation of OC pathogenesis.

Molecular profiling of the vestibular lamina highlights a key role for Hedgehog signalling.

The vestibular lamina (VL) forms the oral vestibule, creating a gap between the teeth, lips and cheeks. In a number of ciliopathies, formation of the vestibule is defective, leading to the creation of multiple frenula. In contrast to the neighbouring dental lamina, which forms the teeth, little is known about the genes that pattern the VL. Here, we establish a molecular signature for the usually non-odontogenic VL in mice and highlight several genes and signalling pathways that may play a role in its development. For one of these, the Sonic hedgehog (Shh) pathway, we show that co-receptors Gas1, Cdon and Boc are highly expressed in the VL and act to enhance the Shh signal from the forming incisor region. In Gas1 mutant mice, expression of Gli1 was disrupted and the VL epithelium failed to extend due to a loss of proliferation. This defect was exacerbated in Boc/Gas1 double mutants and could be phenocopied using cyclopamine in culture. Signals from the forming teeth, therefore, control development of the VL, coordinating the development of the dentition and the oral cavity.

Titanium Dioxide Nanoparticles Modulate Systemic Immune Response and Increase Levels of Reduced Glutathione in Mice after Seven-Week Inhalation.

Titanium dioxide nanoparticles (TiO2 NPs) are used in a wide range of applications. Although inhalation of NPs is one of the most important toxicologically relevant routes, experimental studies on potential harmful effects of TiO2 NPs using a whole-body inhalation chamber model are rare. In this study, the profile of lymphocyte markers, functional immunoassays, and antioxidant defense markers were analyzed to evaluate the potential adverse effects of seven-week inhalation exposure to two different concentrations of TiO2 NPs (0.00167 and 0.1308 mg TiO2/m3) in mice. A dose-dependent effect of TiO2 NPs on innate immunity was evident in the form of stimulated phagocytic activity of monocytes in low-dose mice and suppressed secretory function of monocytes (IL-18) in high-dose animals. The effect of TiO2 NPs on adaptive immunity, manifested in the spleen by a decrease in the percentage of T-cells, a reduction in T-helper cells, and a dose-dependent decrease in lymphocyte cytokine production, may indicate immunosuppression in exposed mice. The dose-dependent increase in GSH concentration and GSH/GSSG ratio in whole blood demonstrated stimulated antioxidant defense against oxidative stress induced by TiO2 NP exposure.

Enhancing oral squamous cell carcinoma prediction: the prognostic power of the worst pattern of invasion and the limited impact of molecular resection margins.

Objective: Oral squamous cell carcinoma (OSCC) originates from the mucosal lining of the oral cavity. Almost half of newly diagnosed cases are classified as advanced stage IV disease, which makes resection difficult. In this study, we investigated the pathological features and mutation profiles of tumor margins in OSCC. Methods: We performed hierarchical clustering of principal components to identify distinct patterns of tumor growth and their association with patient prognosis. We also used next-generation sequencing to analyze somatic mutations in tumor and marginal tissue samples. Results: Our analyses uncovered that the grade of worst pattern of invasion (WPOI) is strongly associated with depth of invasion and patient survival in multivariable analysis. Mutations were primarily detected in the DNA isolated from tumors, but several mutations were also identified in marginal tissue. In total, we uncovered 29 mutated genes, mainly tumor suppressor genes involved in DNA repair including BRCA genes; however none of these mutations significantly correlated with a higher chance of relapse in our medium-size cohort. Some resection margins that appeared histologically normal harbored tumorigenic mutations in TP53 and CDKN2A genes. Conclusion: Even histologically normal margins may contain molecular alterations that are not detectable by conventional histopathological methods, but NCCN classification system still outperforms other methods in the prediction of the probability of disease relapse.

Role of LGR5-positive mesenchymal cells in craniofacial development.

Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5), a Wnt pathway member, has been previously recognised as a stem cell marker in numerous epithelial tissues. In this study, we used Lgr5-EGFP-CreERT2 mice to analyse the distribution of LGR5-positive cells during craniofacial development. LGR5 expressing cells were primarily located in the mesenchyme adjacent to the craniofacial epithelial structures undergoing folding, such as the nasopharyngeal duct, lingual groove, and vomeronasal organ. To follow the fate of LGR5-positive cells, we performed lineage tracing using an inducible Cre knock-in allele in combination with Rosa26-tdTomato reporter mice. The slight expansion of LGR5-positive cells was found around the vomeronasal organ, in the nasal cavity, and around the epithelium in the lingual groove. However, most LGR5 expressing cells remained in their original location, possibly supporting their signalling function for adjacent epithelium rather than exerting their role as progenitor cells for the craniofacial structures. Moreover, Lgr5 knockout mice displayed distinct defects in LGR5-positive areas, especially in the reduction of the nasopharyngeal duct, the alteration of the palatal shelves shape, abnormal epithelial folding in the lingual groove area, and the disruption of salivary gland development. The latter defect manifested as an atypical number and localisation of the glandular ducts. The gene expression of several Wnt pathway members (Rspo1-3, Axin2) was altered in Lgr5-deficient animals. However, the difference was not found in sorted EGFP-positive cells obtained from Lgr5 +/+ and Lgr5 -/- animals. Expression profiling of LGR5-positive cells revealed the expression of several markers of mesenchymal cells, antagonists, as well as agonists, of Wnt signalling, and molecules associated with the basal membrane. Therefore, LGR5-positive cells in the craniofacial area represent a very specific population of mesenchymal cells adjacent to the epithelium undergoing folding or groove formation. Our results indicate a possible novel role of LGR5 in the regulation of morphogenetic processes during the formation of complex epithelial structures in the craniofacial areas, a role which is not related to the stem cell properties of LGR5-positive cells as was previously defined for various epithelial tissues.

Macrophage-mediated tissue response evoked by subchronic inhalation of lead oxide nanoparticles is associated with the alteration of phospholipases C and cholesterol transporters.

BACKGROUND: Inhalation of lead oxide nanoparticles (PbO NPs), which are emitted to the environment by high-temperature technological processes, heavily impairs target organs. These nanoparticles pass through the lung barrier and are distributed via the blood into secondary target organs, where they cause numerous pathological alterations. Here, we studied in detail, macrophages as specialized cells involved in the innate and adaptive immune response in selected target organs to unravel their potential involvement in reaction to subchronic PbO NP inhalation. In this context, we also tackled possible alterations in lipid uptake in the lungs and liver, which is usually associated with foam macrophage formation. RESULTS: The histopathological analysis of PbO NP exposed lung revealed serious chronic inflammation of lung tissues. The number of total and foam macrophages was significantly increased in lung, and they contained numerous cholesterol crystals. PbO NP inhalation induced changes in expression of phospholipases C (PLC) as enzymes linked to macrophage-mediated inflammation in lungs. In the liver, the subchronic inhalation of PbO NPs caused predominantly hyperemia, microsteatosis or remodeling of the liver parenchyma, and the number of liver macrophages also significantly was increased. The gene and protein expression of a cholesterol transporter CD36, which is associated with lipid metabolism, was altered in the liver. The amount of selected cholesteryl esters (CE 16:0, CE 18:1, CE 20:4, CE 22:6) in liver tissue was decreased after subchronic PbO NP inhalation, while total and free cholesterol in liver tissue was slightly increased. Gene and protein expression of phospholipase PLCß1 and receptor CD36 in human hepatocytes were affected also in in vitro experiments after acute PbO NP exposure. No microscopic or serious functional kidney alterations were detected after subchronic PbO NP exposure and CD68 positive cells were present in the physiological mode in its interstitial tissues. CONCLUSION: Our study revealed the association of increased cholesterol and lipid storage in targeted tissues with the alteration of scavenger receptors and phospholipases C after subchronic inhalation of PbO NPs and yet uncovered processes, which can contribute to steatosis in liver after metal nanoparticles exposure.

Alteration of primary cilia morphology and associated signalling in ameloblastoma.

OBJECTIVES: Primary cilium is a cellular organelle with growing significance confirmed in tumour biology. Primary cilia have been associated with fine tuning of numerous cell signalling pathways and the role of this structure in cancer initiation and progression is recently at the forefront of attention. Here, we investigated possible alterations in the occurrence of primary cilia and changes of associated signalling in ameloblastoma, which represents the most common odontogenic tumour. METHODS: We performed immunohistochemistry to assess the number and morphology of primary cilia in ameloblastoma tissues. The gene expression of key SHH pathway members was analysed by qPCR. As a functional experiment, we treated a primary ameloblastoma cell line by a SHH pathway inhibitor Sonidegib (LDE225). RESULTS: We uncovered differences in primary cilia distribution and appearance in histological subtypes of ameloblastoma with the highest number of ciliated cells in plexiform and follicular subtypes. SHH protein was located close to primary cilia in ameloblastoma epithelial cells and the expression of molecules downstream of SHH signalling was upregulated. Moreover, the inhibition of SHH pathway by Sonidegib caused downregulation of SHH effector gene GLI1 and cell cycle regulator CCND1 in ameloblastoma primary cell line. The inhibition of SHH signalling also altered the expression of molecules involved in intraflagellar transport. CONCLUSIONS: In conclusion, our study uncovered alterations in number of ciliated cells and associated signalling in ameloblastoma, which indicate SHH inhibitors as potential therapeutic target to treat this disease.

Epigenetic Regulations of Perineural Invasion in Head and Neck Squamous Cell Carcinoma.

Carcinomas of the oral cavity and oropharynx belong among the ten most common malignancies in the human population. The prognosis of head and neck squamous cell carcinoma (HNSCC) is determined by the degree of invasiveness of the primary tumor and by the extent of metastatic spread into regional and distant lymph nodes. Moreover, the level of the perineural invasion itself associates with tumor localization, invasion's extent, and the presence of nodal metastases. Here, we summarize the current knowledge about different aspects of epigenetic changes, which can be associated with HNSCC while focusing on perineural invasion (PNI). We review epigenetic modifications of the genes involved in the PNI process in HNSCC from the omics perspective and specific epigenetic modifications in OSCC or other neurotropic cancers associated with perineural invasion. Moreover, we summarize DNA methylation status of tumor-suppressor genes, methylation and demethylation enzymes and histone post-translational modifications associated with PNI. The influence of other epigenetic factors on the HNSCC incidence and perineural invasion such as tobacco, alcohol and oral microbiome is overviewed and HPV infection is discussed as an epigenetic factor associated with OSCC and related perineural invasion. Understanding epigenetic regulations of axon growth that lead to tumorous spread or uncovering the molecular control of axon interaction with cancer tissue can help to discover new therapeutic targets for these tumors.

Copper Oxide Nanoparticles Stimulate the Immune Response and Decrease Antioxidant Defense in Mice After Six-Week Inhalation.

Copper oxide nanoparticles (CuO NPs) are increasingly used in various industry sectors. Moreover, medical application of CuO NPs as antimicrobials also contributes to human exposure. Their toxicity, including toxicity to the immune system and blood, raises concerns, while information on their immunotoxicity is still very limited. The aim of our work was to evaluate the effects of CuO NPs (number concentration 1.40×106 particles/cm3, geometric mean diameter 20.4 nm) on immune/inflammatory response and antioxidant defense in mice exposed to 32.5 µg CuO/m3 continuously for 6 weeks. After six weeks of CuO NP inhalation, the content of copper in lungs and liver was significantly increased, while in kidneys, spleen, brain, and blood it was similar in exposed and control mice. Inhalation of CuO NPs caused a significant increase in proliferative response of T-lymphocytes after mitogenic stimulation and basal proliferative activity of splenocytes. CuO NPs significantly induced the production of IL-12p70, Th1-cytokine IFN-γ and Th2-cytokines IL-4, IL-5. Levels of TNF-α and IL-6 remained unchanged. Immune assays showed significantly suppressed phagocytic activity of granulocytes and slightly decreased respiratory burst. No significant differences in phagocytosis of monocytes were recorded. The percentage of CD3+, CD3+CD4+, CD3+CD8+, and CD3-CD19+ cell subsets in spleen, thymus, and lymph nodes did not differ between exposed and control animals. No changes in hematological parameters were found between the CuO NP exposed and control groups. The overall antioxidant protection status of the organism was expressed by evaluation of GSH and GSSG concentrations in blood samples. The experimental group exposed to CuO NPs showed a significant decrease in GSH concentration in comparison to the control group. In summary, our results indicate that sub-chronic inhalation of CuO NPs can cause undesired modulation of the immune response. Stimulation of adaptive immunity was indicated by activation of proliferation and secretion functions of lymphocytes. CuO NPs elicited pro-activation state of Th1 and Th2 lymphocytes in exposed mice. Innate immunity was affected by impaired phagocytic activity of granulocytes. Reduced glutathione was significantly decreased in mice exposed to CuO NPs.

X-ray microtomography imaging of craniofacial hard tissues in selected reptile species with different types of dentition.

BACKGROUND: Reptiles exhibit a large heterogeneity in teeth morphology. The main variability comprises the different tooth shape, the type of tooth attachment to the underlying bone, or the ability to replace the teeth. FINDINGS: Here, we provide full datasets of microtomography scans and 3D models of reptilian dentitions and skulls. We selected representative species for each of 9 reptilian families on the basis of their characteristic dental features. Because there are ≥4 different types of tooth-bone attachments, ranging from the mammalian-like thecodont attachment found in crocodilians to the simple acrodont implantation observed in some lizards, we aimed to evaluate species with different types of tooth-bone attachments. Moreover, another interesting feature varying in reptilian species is the complexity of tooth shape or the number of tooth generations, which can be associated with the type of tooth attachment to the jawbone. Therefore, selected model species also include animals with distinct tooth morphology along the jaw or different number of tooth generations. The development of tooth attachment and relationship of the tooth to the jaw can be further analysed in detail on a large collection of pre-hatching stages of chameleon. Next, we introduce different possibilities for how these datasets can be further used to study tooth-bone relationships or tooth morphology in 3D space. Moreover, these datasets can be valuable for additional morphological and morphometric analyses of reptilian skulls or their individually segmented skeletal elements. CONCLUSIONS: Our collection of microcomputed tomography scans can bring new insight into dental or skeletal research. The broad selection of reptilian species, together with their unique dental features and high quality of these scans including complete series of developmental stages of our model species and provide large opportunities for their reuse. Scans can be further used for virtual reality, 3D printing, or in education.

Loss of Sprouty Produces a Ciliopathic Skeletal Phenotype in Mice Through Upregulation of Hedgehog Signaling.

The Sprouty family is a highly conserved group of intracellular modulators of receptor tyrosine kinase (RTK)-signaling pathways, which have been recently linked to primary cilia. Disruptions in the structure and function of primary cilia cause inherited disorders called ciliopathies. We aimed to evaluate Sprouty2 and Sprouty4 gene-dependent alterations of ciliary structure and to focus on the determination of its association with Hedgehog signaling defects in chondrocytes. Analysis of the transgenic mice phenotype with Sprouty2 and Sprouty4 deficiency revealed several defects, including improper endochondral bone formation and digit patterning, or craniofacial and dental abnormalities. Moreover, reduced bone thickness and trabecular bone mass, skull deformities, or chondroma-like lesions were revealed. All these pathologies might be attributed to ciliopathies. Elongation of the ciliary axonemes in embryonic and postnatal growth plate chondrocytes was observed in Sprouty2-/- and Sprouty2+/- /Sprouty4-/- mutants compared with corresponding littermate controls. Also, cilia-dependent Hedgehog signaling was upregulated in Sprouty2/4 mutant animals. Ptch1 and Ihh expression were upregulated in the autopodium and the proximal tibia of Sprouty2-/- /Sprouty4-/- mutants. Increased levels of the GLI3 repressor (GLI3R) form were detected in Sprouty2/4 mutant primary fibroblast embryonic cell cultures and tissues. These findings demonstrate that mouse lines deficient in Sprouty proteins manifest phenotypic features resembling ciliopathic phenotypes in multiple aspects and may serve as valuable models to study the association between overactivation of RTK and dysfunction of primary cilia during skeletogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Role of Cell Death in Cellular Processes During Odontogenesis.

The development of a tooth germ in a precise size, shape, and position in the jaw, involves meticulous regulation of cell proliferation and cell death. Apoptosis, as the most common type of programmed cell death during embryonic development, plays a number of key roles during odontogenesis, ranging from the budding of the oral epithelium during tooth initiation, to later tooth germ morphogenesis and removal of enamel knot signaling center. Here, we summarize recent knowledge about the distribution and function of apoptotic cells during odontogenesis in several vertebrate lineages, with a special focus on amniotes (mammals and reptiles). We discuss the regulatory roles that apoptosis plays on various cellular processes during odontogenesis. We also review apoptosis-associated molecular signaling during tooth development, including its relationship with the autophagic pathway. Lastly, we cover apoptotic pathway disruption, and alterations in apoptotic cell distribution in transgenic mouse models. These studies foster a deeper understanding how apoptotic cells affect cellular processes during normal odontogenesis, and how they contribute to dental disorders, which could lead to new avenues of treatment in the future.

The Expression of Selected Wnt Pathway Members (FZD6, AXIN2 and ß-Catenin) in Canine Oral Squamous Cell Carcinoma and Acanthomatous Ameloblastoma.

The Wnt signaling pathway is well known to be involved in many types of human cancer; however, in veterinary medicine, the investigation of individual Wnt members' expression, and their role in or association with oral tumor pathogenesis, is still underevaluated. We aim to determine the expression pattern of Frizzled-6 (FZD6) as one of the Wnt receptors in two of the most common canine oral neoplastic lesions-canine oral squamous cell carcinoma (COSCC) and canine acanthomatous ameloblastoma (CAA). While COSCC is a malignant tumor with aggressive biological behavior and a tendency to metastasize, CAA is a benign tumor with high local invasiveness. In CAA, the expression of FZD6 was mostly located in the center of the epithelial tumorous tissue, and cells exhibiting features of squamous metaplasia were strongly positive. In well-differentiated COSCC, FZD6 was expressed in the tumorous epithelium as well as the tumorous stroma. There was a negative correlation between cytokeratin expression and FZD6 expression in COSCC, where the central parts of the epithelial tumorous tissue were often FZD6-negative. The non-differentiated COSCC with low expression of cytokeratin exhibited a diffuse FZD6 signal. The invasive front with areas of tumor budding exhibited high FZD6 expression with a loss of cytokeratin expression. Moreover, the expression of ß-catenin and AXIN2 was increased in comparison to gingiva. In conclusion, our study revealed significant differences in the expression patterns and the levels of FZD6 between COSCC and CAA, indicating the differential engagement of the Wnt pathway in these tumors.

An RNA aptamer restores defective bone growth in FGFR3-related skeletal dysplasia in mice.

Achondroplasia is the most prevalent genetic form of dwarfism in humans and is caused by activating mutations in FGFR3 tyrosine kinase. The clinical need for a safe and effective inhibitor of FGFR3 is unmet, leaving achondroplasia currently incurable. Here, we evaluated RBM-007, an RNA aptamer previously developed to neutralize the FGFR3 ligand FGF2, for its activity against FGFR3. In cultured rat chondrocytes or mouse embryonal tibia organ culture, RBM-007 rescued the proliferation arrest, degradation of cartilaginous extracellular matrix, premature senescence, and impaired hypertrophic differentiation induced by FGFR3 signaling. In cartilage xenografts derived from induced pluripotent stem cells from individuals with achondroplasia, RBM-007 rescued impaired chondrocyte differentiation and maturation. When delivered by subcutaneous injection, RBM-007 restored defective skeletal growth in a mouse model of achondroplasia. We thus demonstrate a ligand-trap concept of targeting the cartilage FGFR3 and delineate a potential therapeutic approach for achondroplasia and other FGFR3-related skeletal dysplasias.