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Mammalian dentition exhibits distinct heterodonty, with more simple teeth located in the anterior area of the jaw and more complex teeth situated posteriorly. While some region-specific differences in signalling have been described previously, here we performed a comprehensive analysis of gene expression at the early stages of odontogenesis to obtain complete knowledge of the signalling pathways involved in early jaw patterning. Gene expression was analysed separately on anterior and posterior areas of the lower jaw at two early stages (E11.5 and E12.5) of odontogenesis. Gene expression profiling revealed distinct region-specific expression patterns in mouse mandibles, including several known BMP and FGF signalling members and we also identified several new molecules exhibiting significant differences in expression along the anterior-posterior axis, which potentially can play the role during incisor and molar specification. Next, we followed one of the anterior molecules, SATB2, which was expressed not only in the anterior mesenchyme where incisor germs are initiated, however, we uncovered a distinct SATB2-positive region in the mesenchyme closely surrounding molars. Satb2-deficient animals demonstrated defective incisor development confirming a crucial role of SATB2 in formation of anterior teeth. On the other hand, ectopic tooth germs were observed in the molar area indicating differential effect of Satb2-deficiency in individual jaw regions. In conclusion, our data provide a rich source of fundamental information, which can be used to determine molecular regulation driving early embryonic jaw patterning and serve for a deeper understanding of molecular signalling directed towards incisor and molar development.
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Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Mandíbula , Proteínas de Unión a la Región de Fijación a la Matriz , Odontogénesis , Factores de Transcripción , Animales , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Ratones , Mandíbula/metabolismo , Mandíbula/embriología , Odontogénesis/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Diente/metabolismo , Diente/embriología , Diente/crecimiento & desarrollo , Incisivo/metabolismo , Incisivo/embriología , Incisivo/crecimiento & desarrollo , Tipificación del Cuerpo/genética , Transducción de SeñalRESUMEN
Consensus holds that pottery technology came to Central Europe from the Northern Balkans with independent pottery traditions existing concurrently in Eastern Europe. An unusual grass-tempered pottery dating back to around 5800 cal BC found in lake sediments at Santovka, Slovakia, predated the earliest known Neolithic pottery in the region (~ 5500 cal BC), suggesting unexplored narratives of pottery introduction. Analyses of the pottery's technology, origin, and grass temper shedding light on ceramic traditions' spread can unveil mobility patterns and community lifestyles. Our findings indicate a non-local provenance, low temperature firing, Festugc sp. grass temper and unique rectangular or cylindrical vessel shapes which align with Eastern European hunter-gatherer practices. Moreover, the pottery style and technology have no analogies in the contemporary Danubian pottery traditions and have more similarities to those of the Eastern traditions. The pottery's raw materials likely originated from distant areas, indicating extensive territorial access for its creators. Our findings imply late Mesolithic hunter-gatherers as the probable artisans and with implications for the site's significance in the late Mesolithic landscape.
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BACKGROUND: Xenopus laevis, the African clawed frog, is a versatile vertebrate model organism in various biological disciplines, prominently in developmental biology to study body plan reorganization during metamorphosis. However, a notable gap exists in the availability of comprehensive datasets encompassing Xenopus' late developmental stages. FINDINGS: This study utilized micro-computed tomography (micro-CT), a noninvasive 3-dimensional (3D) imaging technique with micrometer-scale resolution, to explore the developmental dynamics and morphological changes in Xenopus laevis. Our approach involved generating high-resolution images and computed 3D models of developing Xenopus specimens, spanning from premetamorphosis tadpoles to fully mature adults. This dataset enhances our understanding of vertebrate development and supports various analyses. We conducted a careful examination, analyzing body size, shape, and morphological features, focusing on skeletogenesis, teeth, and organs like the brain and gut at different stages. Our analysis yielded valuable insights into 3D morphological changes during Xenopus' development, documenting details previously unrecorded. These datasets hold the solid potential for further morphological and morphometric analyses, including segmentation of hard and soft tissues. CONCLUSIONS: Our repository of micro-CT scans represents a significant resource that can enhance our understanding of Xenopus' development and the associated morphological changes in the future. The widespread utility of this amphibian species, coupled with the exceptional quality of our scans, which encompass a comprehensive series of developmental stages, opens up extensive opportunities for their broader research application. Moreover, these scans can be used in virtual reality, 3D printing, and educational contexts, further expanding their value and impact.
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Imagenología Tridimensional , Microtomografía por Rayos X , Xenopus laevis , Animales , Xenopus laevis/crecimiento & desarrollo , Microtomografía por Rayos X/métodos , Imagenología Tridimensional/métodos , Metamorfosis Biológica , Larva/crecimiento & desarrolloRESUMEN
The chondrocranium provides the key initial support for the fetal brain, jaws and cranial sensory organs in all vertebrates. The patterns of shaping and growth of the chondrocranium set up species-specific development of the entire craniofacial complex. The 3D development of chondrocranium have been studied primarily in animal model organisms, such as mice or zebrafish. In comparison, very little is known about the full 3D human chondrocranium, except from drawings made by anatomists many decades ago. The knowledge of human-specific aspects of chondrocranial development are essential for understanding congenital craniofacial defects and human evolution. Here advanced microCT scanning was used that includes contrast enhancement to generate the first 3D atlas of the human fetal chondrocranium during the middle trimester (13 to 19 weeks). In addition, since cartilage and bone are both visible with the techniques used, the endochondral ossification of cranial base was mapped since this region is so critical for brain and jaw growth. The human 3D models are published as a scientific resource for human development.
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Imagenología Tridimensional , Humanos , Feto/diagnóstico por imagen , Femenino , Microtomografía por Rayos X , Cráneo/diagnóstico por imagen , Cráneo/embriología , Embarazo , Cartílago/diagnóstico por imagen , Cartílago/embriologíaRESUMEN
The development of craniofacial skeletal structures is fascinatingly complex and elucidation of the underlying mechanisms will not only provide novel scientific insights, but also help develop more effective clinical approaches to the treatment and/or prevention of the numerous congenital craniofacial malformations. To this end, we performed a genome-wide analysis of RNA transcription from non-coding regulatory elements by CAGE-sequencing of the facial mesenchyme of human embryos and cross-checked the active enhancers thus identified against genes, identified by GWAS for the normal range human facial appearance. Among the identified active cis-enhancers, several belonged to the components of the PI3/AKT/mTORC1/autophagy pathway. To assess the functional role of this pathway, we manipulated it both genetically and pharmacologically in mice and zebrafish. These experiments revealed that mTORC1 signaling modulates craniofacial shaping at the stage of skeletal mesenchymal condensations, with subsequent fine-tuning during clonal intercalation. This ability of mTORC1 pathway to modulate facial shaping, along with its evolutionary conservation and ability to sense external stimuli, in particular dietary amino acids, indicate that the mTORC1 pathway may play a role in facial phenotypic plasticity. Indeed, the level of protein in the diet of pregnant female mice influenced the activity of mTORC1 in fetal craniofacial structures and altered the size of skeletogenic clones, thus exerting an impact on the local geometry and craniofacial shaping. Overall, our findings indicate that the mTORC1 signaling pathway is involved in the effect of environmental conditions on the shaping of craniofacial structures.
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Transducción de Señal , Pez Cebra , Embarazo , Ratones , Animales , Femenino , Humanos , Proteínas , Diana Mecanicista del Complejo 1 de la Rapamicina , DietaRESUMEN
The formation of hematopoietic cells relies on the chromatin remodeling activities of ISWI ATPase SMARCA5 (SNF2H) and its complexes. The Smarca5 null and conditional alleles have been used to study its functions in embryonic and organ development in mice. These mouse model phenotypes vary from embryonic lethality of constitutive knockout to less severe phenotypes observed in tissue-specific Smarca5 deletions, e.g., in the hematopoietic system. Here we show that, in a gene dosage-dependent manner, the hypomorphic allele of SMARCA5 (S5tg) can rescue not only the developmental arrest in hematopoiesis in the hCD2iCre model but also the lethal phenotypes associated with constitutive Smarca5 deletion or Vav1iCre-driven conditional knockout in hematopoietic progenitor cells. Interestingly, the latter model also provided evidence for the role of SMARCA5 expression level in hematopoietic stem cells, as the Vav1iCre S5tg animals accumulate stem and progenitor cells. Furthermore, their hematopoietic stem cells exhibited impaired lymphoid lineage entry and differentiation. This observation contrasts with the myeloid lineage which is developing without significant disturbances. Our findings indicate that animals with low expression of SMARCA5 exhibit normal embryonic development with altered lymphoid entry within the hematopoietic stem cell compartment.
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Hematopoyesis , Células Madre Hematopoyéticas , Ratones , Animales , Células Madre Hematopoyéticas/metabolismo , Hematopoyesis/genética , Diferenciación Celular/genética , Adenosina Trifosfatasas/metabolismoRESUMEN
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.
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Ventrículos Cerebrales , Plexo Coroideo , Animales , Ratones , Plexo Coroideo/diagnóstico por imagen , Microtomografía por Rayos X , Rombencéfalo/diagnóstico por imagen , EncéfaloRESUMEN
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.
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Trastornos de la Motilidad Ciliar , Enfermedades Renales Poliquísticas , Retinitis Pigmentosa , Femenino , Embarazo , Humanos , Ratones , Animales , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Retina/metabolismo , Enfermedades Renales Poliquísticas/genética , Retinitis Pigmentosa/metabolismo , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/metabolismoRESUMEN
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.
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Diente , Pez Cebra , Ratones , Animales , Diente/fisiología , Raíz del Diente , Huesos , Desarrollo ÓseoRESUMEN
In this study we use comparative genomics to uncover a gene with uncharacterized function (1700011H14Rik/C14orf105/CCDC198), which we hereby name FAME (Factor Associated with Metabolism and Energy). We observe that FAME shows an unusually high evolutionary divergence in birds and mammals. Through the comparison of single nucleotide polymorphisms, we identify gene flow of FAME from Neandertals into modern humans. We conduct knockout experiments on animals and observe altered body weight and decreased energy expenditure in Fame knockout animals, corresponding to genome-wide association studies linking FAME with higher body mass index in humans. Gene expression and subcellular localization analyses reveal that FAME is a membrane-bound protein enriched in the kidneys. Although the gene knockout results in structurally normal kidneys, we detect higher albumin in urine and lowered ferritin in the blood. Through experimental validation, we confirm interactions between FAME and ferritin and show co-localization in vesicular and plasma membranes.
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Metabolismo Energético , Estudio de Asociación del Genoma Completo , Animales , Humanos , Peso Corporal , Metabolismo Energético/genética , Ferritinas/genética , Riñón , Hombre de NeandertalRESUMEN
Formation of oriented myofibrils is a key event in musculoskeletal development. However, the mechanisms that drive myocyte orientation and fusion to control muscle directionality in adults remain enigmatic. Here, we demonstrate that the developing skeleton instructs the directional outgrowth of skeletal muscle and other soft tissues during limb and facial morphogenesis in zebrafish and mouse. Time-lapse live imaging reveals that during early craniofacial development, myoblasts condense into round clusters corresponding to future muscle groups. These clusters undergo oriented stretch and alignment during embryonic growth. Genetic perturbation of cartilage patterning or size disrupts the directionality and number of myofibrils in vivo. Laser ablation of musculoskeletal attachment points reveals tension imposed by cartilage expansion on the forming myofibers. Application of continuous tension using artificial attachment points, or stretchable membrane substrates, is sufficient to drive polarization of myocyte populations in vitro. Overall, this work outlines a biomechanical guidance mechanism that is potentially useful for engineering functional skeletal muscle.
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Músculo Esquelético , Pez Cebra , Animales , Ratones , Pez Cebra/genética , Músculo Esquelético/fisiología , Miofibrillas/fisiología , Morfogénesis , Mioblastos/fisiologíaRESUMEN
We report on the formation of silver nanoparticles by gas aggregation in a reaction chamber at room temperature. The size distribution of nanoparticles deposited on a silicon substrate for various lengths of an aggregation (high-pressure) chamber was investigated by atomic force microscopy. Nanoparticles were characterized by scanning and transmission electron microscopy and spectral ellipsometry. The physical shape of the nanoparticles and its distribution was correlated with their optical properties. Metal-dielectric nanocomposites were deposited employing simultaneous deposition of Ag NPs via high-pressure magnetron sputtering and the dielectric matrix was deposited via thermal evaporation. Pure and Eu-, Er-, and Yb-doped lithium fluoride was used as the dielectric host matrix. Optical transmittance of lithium fluoride containing silver nanoparticles was measured and their theoretical absorption cross-section calculated. The nanoparticles were also embedded in Eu3+-doped downshifting and Er3+- and Yb3+-doped up-conversion materials to study their influence on emission spectra. Spectra of identical layers with and without nanoparticles were compared. Their transmittance at various annealing temperatures is also presented.
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There are major differences in duration and scale at which limb development and regeneration proceed, raising the question to what extent regeneration is a recapitulation of development. We address this by analyzing skeletal elements using a combination of micro-CT imaging, molecular profiling and clonal cell tracing. We find that, in contrast to development, regenerative skeletal growth is accomplished based entirely on cartilage expansion prior to ossification, not limiting the transversal cartilage expansion and resulting in bulkier skeletal parts. The oriented extension of salamander cartilage and bone appear similar to the development of basicranial synchondroses in mammals, as we found no evidence for cartilage stem cell niches or growth plate-like structures during neither development nor regeneration. Both regenerative and developmental ossification in salamanders start from the cortical bone and proceeds inwards, showing the diversity of schemes for the synchrony of cortical and endochondral ossification among vertebrates.
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Osteogénesis , Urodelos , Animales , Huesos , Cartílago , División Celular , MamíferosRESUMEN
Spontaneous bleeds are a leading cause of death in the pediatric JAG1-related liver disease Alagille syndrome (ALGS). We asked whether there are sex differences in bleeding events in patients, whether Jag1Ndr/Ndr mice display bleeds or vascular defects, and whether discovered vascular pathology can be confirmed in patients non-invasively. We performed a systematic review of patients with ALGS and vascular events following PRISMA guidelines, in the context of patient sex, and found significantly more girls than boys reported with spontaneous intracranial hemorrhage. We investigated vascular development, homeostasis, and bleeding in Jag1Ndr/Ndr mice, using retina as a model. Jag1Ndr/Ndr mice displayed sporadic brain bleeds, a thin skull, tortuous blood vessels, sparse arterial smooth muscle cell coverage in multiple organs, which could be aggravated by hypertension, and sex-specific venous defects. Importantly, we demonstrated that retinographs from patients display similar characteristics with significantly increased vascular tortuosity. In conclusion, there are clinically important sex differences in vascular disease in ALGS, and retinography allows non-invasive vascular analysis in patients. Finally, Jag1Ndr/Ndr mice represent a new model for vascular compromise in ALGS.
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Síndrome de Alagille , Femenino , Masculino , Animales , Ratones , Síndrome de Alagille/complicaciones , Caracteres Sexuales , Retina , Factores de RiesgoRESUMEN
Scanning electron microscopy (SEM) is a common method for the analysis of painting micro-samples. The high resolution of this technique offers precise surface analysis and can be coupled with an energy-dispersive spectrometer for the acquisition of the elemental composition. For light microscopy and SEM analysis, the painting micro-samples are commonly prepared as cross-sections, where the micro-sample positioned on the side is embedded in a resin. Therefore, the sequence of its layers is exposed after the cross-section is polished. In common cases outside of cultural heritage, a conductive layer is applied on the polished side, but in this field, the measurements are mostly done in low-vacuum SEM (LV-SEM). Although the charging effect is reduced in LV-SEM, it can still occur, and can hardly be prevented even with carbon tape or paint. This work presents two conductive cross-section preparation methods for non-conductive samples, which reduce charging effects without impairing the sample integrity.
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Cross-section preparation of painting micro-samples is part of their routine analysis. This type of preparation can be used for several analytical techniques, such as scanning electron microscopy, Fourier-transform infrared spectroscopy, Raman spectroscopy, and optical microscopy. These techniques offer high-resolution imaging and/or elemental information, providing access to technical and material data important for the interpretation, preservation, and restoration of painted artworks. However, it also means that the material from the sample embedded in the resin becomes unreachable for further analysis, except for the polished surface of the cross-section. Degradation of the embedding medium can also occur over time, which can lead to misinterpretation, loss of information, or even complete destruction of the embedded sample. In the field of cultural heritage, cyclododecane (CDD) is commonly used for the consolidation and protection of objects, and is used in the preparation of cross-sections to prevent contamination of the sample by the embedding medium. This study enhanced the existing preparation process by shaping the CDD layer to enable extraction of the micro-sample from the resin if needed, without compromising the integrity of the sample. Moreover, the purity, the sublimation rate in a normal environment and a vacuum, and the impact of CDD on three different types of samples (historical painting on a canvas, wall painting fragment, model sample) were examined.
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Characterization of brain infarct lesions in rodent models of stroke is crucial to assess stroke pathophysiology and therapy outcome. Until recently, the analysis of brain lesions was performed using two techniques: (1) histological methods, such as TTC (Triphenyltetrazolium chloride), a time-consuming and inaccurate process; or (2) MRI imaging, a faster, 3D imaging method, that comes at a high cost. In the last decade, high-resolution micro-CT for 3D sample analysis turned into a simple, fast, and cheaper solution. Here, we successfully describe the application of brain contrasting agents (Osmium tetroxide and inorganic iodine) for high-resolution micro-CT imaging for fine location and quantification of ischemic lesion and edema in mouse preclinical stroke models. We used the intraluminal transient MCAO (Middle Cerebral Artery Occlusion) mouse stroke model to identify and quantify ischemic lesion and edema, and segment core and penumbra regions at different time points after ischemia, by manual and automatic methods. In the transient-ischemic-attack (TIA) mouse model, we can quantify striatal myelinated fibers degeneration. Of note, whole brain 3D reconstructions allow brain atlas co-registration, to identify the affected brain areas, and correlate them with functional impairment. This methodology proves to be a breakthrough in the field, by providing a precise and detailed assessment of stroke outcomes in preclinical animal studies.
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Yodo , Accidente Cerebrovascular , Animales , Ratones , Tetróxido de Osmio , Microtomografía por Rayos X , Accidente Cerebrovascular/diagnóstico por imagen , Accidente Cerebrovascular/patología , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Infarto de la Arteria Cerebral Media/patología , Modelos Animales de EnfermedadRESUMEN
Selective laser melting (SLM) is an additive manufacturing technology suitable for producing cellular lattice structures using fine metal powder and a laser beam. However, the shape and dimensional deviations occur on the thin struts during manufacturing, influencing the mechanical properties of the structure. There are attempts in the literature to describe the actual shape of the struts' geometry, however, on a smaller data sample only, and there is a lack of a universal FEA material model applicable to a wider range of lattice structure diameters. To describe the actual dimensions of the struts, a set of lattice structures, with diameters ranging from 0.6 to 3.0 mm, were manufactured using SLM. These samples were digitized using micro-computed tomography (µCT) and fully analyzed for shape and dimensions. The results show large deviations in diameters of inscribed and circumscribed cylinders, indicating an elliptical shape of the struts. With increasing lattice structure diameter, the deviations decreased. In terms of the effect of the shape and dimensions on the mechanical properties, the Gaussian cylinder was found to describe struts in the diameter range of 1.5 to 3.0 mm sufficiently well. For smaller diameters, it is appropriate to represent the actual cross-section by an ellipse. The use of substitute ellipses, in combination with the compression test results, has resulted in FEA material model that can be used for the 0.6 to 3.0 mm struts' diameter range. The model has fixed Young's and tangential modules for these diameters and is controlled only by the yield strength parameter (YST).