Local retinoic acid signaling directs emergence of the extraocular muscle functional unit.

Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments.

ISWI ATPase Smarca5 Regulates Differentiation of Thymocytes Undergoing ß-Selection.

Development of lymphoid progenitors requires a coordinated regulation of gene expression, DNA replication, and gene rearrangement. Chromatin-remodeling activities directed by SWI/SNF2 superfamily complexes play important roles in these processes. In this study, we used a conditional knockout mouse model to investigate the role of Smarca5, a member of the ISWI subfamily of such complexes, in early lymphocyte development. Smarca5 deficiency results in a developmental block at the DN3 stage of αß thymocytes and pro-B stage of early B cells at which the rearrangement of Ag receptor loci occurs. It also disturbs the development of committed (CD73+) γδ thymocytes. The αß thymocyte block is accompanied by massive apoptotic depletion of ß-selected double-negative DN3 cells and premitotic arrest of CD4/CD8 double-positive cells. Although Smarca5-deficient αß T cell precursors that survived apoptosis were able to undergo a successful TCRß rearrangement, they exhibited a highly abnormal mRNA profile, including the persistent expression of CD44 and CD25 markers characteristic of immature cells. We also observed that the p53 pathway became activated in these cells and that a deficiency of p53 partially rescued the defect in thymus cellularity (in contrast to early B cells) of Smarca5-deficient mice. However, the activation of p53 was not primarily responsible for the thymocyte developmental defects observed in the Smarca5 mutants. Our results indicate that Smarca5 plays a key role in the development of thymocytes undergoing ß-selection, γδ thymocytes, and also B cell progenitors by regulating the transcription of early differentiation programs.

Complete Ring Artifacts Reduction Procedure for Lab-Based X-ray Nano CT Systems.

In this article, we introduce a new ring artifacts reduction procedure that combines several ideas from existing methods into one complex and robust approach with a goal to overcome their individual weaknesses and limitations. The procedure differentiates two types of ring artifacts according to their cause and character in computed tomography (CT) data. Each type is then addressed separately in the sinogram domain. The novel iterative schemes based on relative total variations (RTV) were integrated to detect the artifacts. The correction process uses the image inpainting, and the intensity deviations smoothing method. The procedure was implemented in scope of lab-based X-ray nano CT with detection systems based on charge-coupled device (CCD) and scientific complementary metal-oxide-semiconductor (sCMOS) technologies. The procedure was then further tested and optimized on the simulated data and the real CT data of selected samples with different compositions. The performance of the procedure was quantitatively evaluated in terms of the artifacts' detection accuracy, the comparison with existing methods, and the ability to preserve spatial resolution. The results show a high efficiency of ring removal and the preservation of the original sample's structure.

Developmental mechanisms driving complex tooth shape in reptiles.

BACKGROUND: In mammals, odontogenesis is regulated by transient signaling centers known as enamel knots (EKs), which drive the dental epithelium shaping. However, the developmental mechanisms contributing to formation of complex tooth shape in reptiles are not fully understood. Here, we aim to elucidate whether signaling organizers similar to EKs appear during reptilian odontogenesis and how enamel ridges are formed. RESULTS: Morphological structures resembling the mammalian EK were found during reptile odontogenesis. Similar to mammalian primary EKs, they exhibit the presence of apoptotic cells and no proliferating cells. Moreover, expression of mammalian EK-specific molecules (SHH, FGF4, and ST14) and GLI2-negative cells were found in reptilian EK-like areas. 3D analysis of the nucleus shape revealed distinct rearrangement of the cells associated with enamel groove formation. This process was associated with ultrastructural changes and lipid droplet accumulation in the cells directly above the forming ridge, accompanied by alteration of membranous molecule expression (Na/K-ATPase) and cytoskeletal rearrangement (F-actin). CONCLUSIONS: The final complex shape of reptilian teeth is orchestrated by a combination of changes in cell signaling, cell shape, and cell rearrangement. All these factors contribute to asymmetry in the inner enamel epithelium development, enamel deposition, ultimately leading to the formation of characteristic enamel ridges.

Characterization of the Morphological Nature of Hollow Spray Dried Dispersion Particles Using X-ray Submicron-Computed Tomography.

Graphical Abstract.

The effect of oral and nasal breathing on the deposition of inhaled particles in upper and tracheobronchial airways.

The inhalation route has a substantial influence on the fate of inhaled particles. An outbreak of infectious diseases such as COVID-19, influenza or tuberculosis depends on the site of deposition of the inhaled pathogens. But the knowledge of respiratory deposition is important also for occupational safety or targeted delivery of inhaled pharmaceuticals. Simulations utilizing computational fluid dynamics are becoming available to a wide spectrum of users and they can undoubtedly bring detailed predictions of regional deposition of particles. However, if those simulations are to be trusted, they must be validated by experimental data. This article presents simulations and experiments performed on a geometry of airways which is available to other users and thus those results can be used for intercomparison between different research groups. In particular, three hypotheses were tested. First: Oral breathing and combined breathing are equivalent in terms of particle deposition in TB airways, as the pressure resistance of the nasal cavity is so high that the inhaled aerosol flows mostly through the oral cavity in both cases. Second: The influence of the inhalation route (nasal, oral or combined) on the regional distribution of the deposited particles downstream of the trachea is negligible. Third: Simulations can accurately and credibly predict deposition hotspots. The maximum spatial resolution of predicted deposition achievable by current methods was searched for. The simulations were performed using large-eddy simulation, the flow measurements were done by laser Doppler anemometry and the deposition has been measured by positron emission tomography in a realistic replica of human airways. Limitations and sources of uncertainties of the experimental methods were identified. The results confirmed that the high-pressure resistance of the nasal cavity leads to practically identical velocity profiles, even above the glottis for the mouth, and combined mouth and nose breathing. The distribution of deposited particles downstream of the trachea was not influenced by the inhalation route. The carina of the first bifurcation was not among the main deposition hotspots regardless of the inhalation route or flow rate. On the other hand, the deposition hotspots were identified by both CFD and experiments in the second bifurcation in both lungs, and to a lesser extent also in both the third bifurcations in the left lung.

Loss of ISWI ATPase SMARCA5 (SNF2H) in Acute Myeloid Leukemia Cells Inhibits Proliferation and Chromatid Cohesion.

ISWI chromatin remodeling ATPase SMARCA5 (SNF2H) is a well-known factor for its role in regulation of DNA access via nucleosome sliding and assembly. SMARCA5 transcriptionally inhibits the myeloid master regulator PU.1. Upregulation of SMARCA5 was previously observed in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Since high levels of SMARCA5 are necessary for intensive cell proliferation and cell cycle progression of developing hematopoietic stem and progenitor cells in mice, we reasoned that removal of SMARCA5 enzymatic activity could affect the cycling or undifferentiated state of leukemic progenitor-like clones. Indeed, we observed that CRISPR/cas9-mediated SMARCA5 knockout in AML cell lines (S5KO) inhibited the cell cycle progression. We also observed that the SMARCA5 deletion induced karyorrhexis and nuclear budding as well as increased the ploidy, indicating its role in mitotic division of AML cells. The cytogenetic analysis of S5KO cells revealed the premature chromatid separation. We conclude that deleting SMARCA5 in AML blocks leukemic proliferation and chromatid cohesion.

Error in the estimation of periosteal and endosteal contours from micro-CT scans for nonadult tibiae and humeri.

OBJECTIVES: This article summarized errors obtained by diverse techniques used for the derivation of cross-sectional contours in nonadult humeri and tibiae. MATERIALS AND METHODS: We analyzed cross-sectional contours in a total sample of 62 humeral and 75 tibial diaphyses in the age between birth and 12 years divided into three age groups. Long bone 35% (humeri) and midshaft (tibiae) cross-sections were taken on micro-CT images and analyzed by EPJMacro in FIJI. Properties were extracted from contours derived by manual, automatic, spline, and ellipse techniques. Agreement between techniques was assessed using manually extracted properties such as the true value using percent prediction error (%PE), reduced major axis regression, and ±95% limits of agreement. RESULTS: The lowest measurement errors were obtained for total areas, moderate errors for cortical areas and section moduli, and the highest errors for medullary areas for both bones. Derivation of humeral nonadult cross-sectional properties is less sensitive to the technique used for derivation of periosteal and endosteal contours, reaching mean %PEs below 5%. In contrast, tibial nonadult cross-sectional properties are more sensitive to the technique used and exceed 5% for some combinations. DISCUSSION: Automatic techniques provide reasonably high agreement with manually extracted contours for nonadult humeri but low agreement for tibiae. Semiautomatic approaches-spline and ellipse techniques-may reduce the error for all studied properties in tibiae, especially when combined with manually traced periosteal contours. The positive effect of the semiautomatic technique on measurement error is low for humeri.

Effect of deriving periosteal and endosteal contours from microCT scans on computation of cross-sectional properties in non-adults: the femur.

Derivation of periosteal and endosteal contours taken from transversal long bone cross-sections limits the accuracy of calculated biomechanical properties. Although several techniques are available for deriving both contours, the effect of these techniques on accuracy of calculated cross-sectional properties in non-adults is unknown. We examine a sample of 86 non-adult femora from birth to 12 years of age to estimate the effect of error in deriving periosteal and endosteal contours on cross-sectional properties. Midshaft cross-sections were taken from microCT scans and contours were derived using manual, fully automatic, spline, and ellipse techniques. Agreement between techniques was assessed against manually traced periosteal and endosteal contours using percent prediction error (%PE), reduced major axis analysis, and limits of agreement. The %PEs were highest in the medullary area and lowest in the total area. Mean %PEs were sufficiently below the 5% level of acceptable error, except for medullary areas, but individual values can greatly exceed this 5% boundary given the high standard deviation of %PE means and wide minimum-maximum range of %PEs. Automatic processing produces greater errors than does combination with manual, spline, and ellipse processing. Although periosteal contour is estimated with stronger agreement compared with endosteal contour, error in deriving periosteal contour has a substantially greater effect on calculated section moduli than does error in deriving endosteal contours. We observed no size effect on the resulting bias. Nevertheless, cross-sectional properties in a younger age category may be estimated with greater error compared with in an older age category. We conclude that non-adult midshaft cross-sectional properties can be derived from microCT scans of femoral diaphyses with mean error of < 5% and that derivation of endosteal contour can be simplified by the ellipse technique because fully automatic derivation of endosteal contour may increase the resulting error, especially in small samples.

The ISWI ATPase Smarca5 (Snf2h) Is Required for Proliferation and Differentiation of Hematopoietic Stem and Progenitor Cells.

The imitation switch nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair, and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1-iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells accumulated but their maturation toward erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S15Ph °s ) second with CBP/p300 (K376Ac ), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor-iCre) and observed that animals were anemic postnatally. Furthermore, 4-hydroxytamoxifen-mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis. Stem Cells 2017;35:1614-1623.

Superficial cells are self-renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice.

Articular cartilage has little regenerative capacity. Recently, genetic lineage tracing experiments have revealed chondrocyte progenitors at the articular surface. We further characterized these progenitors by using in vivo genetic approaches. Histone H2B-green fluorescent protein retention revealed that superficial cells divide more slowly than underlying articular chondrocytes. Clonal genetic tracing combined with immunohistochemistry revealed that superficial cells renew their number by symmetric division, express mesenchymal stem cell markers, and generate chondrocytes via both asymmetric and symmetric differentiation. Quantitative analysis of cellular kinetics, in combination with phosphotungstic acid-enhanced micro-computed tomography, showed that superficial cells generate chondrocytes and contribute to the growth and reshaping of articular cartilage. Furthermore, we found that cartilage renewal occurs as the progeny of superficial cells fully replace fetal chondrocytes during early postnatal life. Thus, superficial cells are self-renewing progenitors that are capable of maintaining their own population and fulfilling criteria of unipotent adult stem cells. Furthermore, the progeny of these cells reconstitute adult articular cartilage de novo, entirely substituting fetal chondrocytes.-Li, L., Newton, P. T., Bouderlique, T., Sejnohova, M., Zikmund, T., Kozhemyakina, E., Xie, M., Krivanek, J., Kaiser, J., Qian, H., Dyachuk, V., Lassar, A. B., Warman, M. L., Barenius, B., Adameyko, I., Chagin, A. S. Superficial cells are self-renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice.

Age-related changes in the tooth-bone interface area of acrodont dentition in the chameleon.

Chameleon teeth develop as individual structures at a distance from the developing jaw bone during the pre-hatching period and also partially during the post-hatching period. However, in the adult, all teeth are fused together and tightly attached to the jaw bone by mineralized attachment tissue to form one functional unit. Tooth to bone as well as tooth to tooth attachments are so firm that if injury to the oral cavity occurs, several neighbouring teeth and pieces of jaw can be broken off. We analysed age-related changes in chameleon acrodont dentition, where ankylosis represents a physiological condition, whereas in mammals, ankylosis only occurs in a pathological context. The changes in hard-tissue morphology and mineral composition leading to this fusion were analysed. For this purpose, the lower jaws of chameleons were investigated using X-ray micro-computed tomography, laser-induced breakdown spectroscopy and microprobe analysis. For a long time, the dental pulp cavity remained connected with neighbouring teeth and also to the underlying bone marrow cavity. Then, a progressive filling of the dental pulp cavity by a mineralized matrix occurred, and a complex network of non-mineralized channels remained. The size of these unmineralized channels progressively decreased until they completely disappeared, and the dental pulp cavity was filled by a mineralized matrix over time. Moreover, the distribution of calcium, phosphorus and magnesium showed distinct patterns in the different regions of the tooth-bone interface, with a significant progression of mineralization in dentin as well as in the supporting bone. In conclusion, tooth-bone fusion in chameleons results from an enhanced production of mineralized tissue during post-hatching development. Uncovering the developmental processes underlying these outcomes and performing comparative studies is necessary to better understand physiological ankylosis; for that purpose, the chameleon can serve as a useful model species.

Accurate micro-computed tomography imaging of pore spaces in collagen-based scaffold.

In this work we have used X-ray micro-computed tomography (µCT) as a method to observe the morphology of 3D porous pure collagen and collagen-composite scaffolds useful in tissue engineering. Two aspects of visualizations were taken into consideration: improvement of the scan and investigation of its sensitivity to the scan parameters. Due to the low material density some parts of collagen scaffolds are invisible in a µCT scan. Therefore, here we present different contrast agents, which increase the contrast of the scanned biopolymeric sample for µCT visualization. The increase of contrast of collagenous scaffolds was performed with ceramic hydroxyapatite microparticles (HAp), silver ions (Ag(+)) and silver nanoparticles (Ag-NPs). Since a relatively small change in imaging parameters (e.g. in 3D volume rendering, threshold value and µCT acquisition conditions) leads to a completely different visualized pattern, we have optimized these parameters to obtain the most realistic picture for visual and qualitative evaluation of the biopolymeric scaffold. Moreover, scaffold images were stereoscopically visualized in order to better see the 3D biopolymer composite scaffold morphology. However, the optimized visualization has some discontinuities in zoomed view, which can be problematic for further analysis of interconnected pores by commonly used numerical methods. Therefore, we applied the locally adaptive method to solve discontinuities issue. The combination of contrast agent and imaging techniques presented in this paper help us to better understand the structure and morphology of the biopolymeric scaffold that is crucial in the design of new biomaterials useful in tissue engineering.

3D atlas of the human fetal chondrocranium in the middle trimester.

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.

Raman microspectroscopy and laser-induced breakdown spectroscopy for the analysis of polyethylene microplastics in human soft tissues.

People are exposed to microplastics (MPs) on a large scale in everyday life. However, it is not clear whether MPs can also be distributed and retained in certain tissues. Therefore, the development of analytical methods capable of detecting MPs in specific human organs/tissues is of utmost importance. In this study, the use and combination of spectroscopic techniques, namely Raman microspectroscopy and laser-induced breakdown spectroscopy (LIBS), was tested for the detection of polyethylene (PE) MPs in human tonsils. Preliminary results showed that Raman microspectroscopy was able to detect MPs down to 1 µm in size and LIBS down to 10 µm. In the next step, human tonsils were spiked with PE MPs, and digested. The filtered particles were analyzed using Raman microspectroscopy and LIBS, and complemented by X-ray fluorescence (XRF). The results showed that Raman microspectroscopy could reliably detect PE MPs in spiked human tonsils, while LIBS and XRF served as a reference analytical method to characterize particles that could not be classified by Raman microspectroscopy for their non-organic origin. The results of this study, supported by a current feasibility study conducted on clinical samples, demonstrated the reliability and feasibility of this approach for monitoring MPs in biotic samples.

Unveiling vertebrate development dynamics in frog Xenopus laevis using micro-CT imaging.

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.

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.

Differential requirements for Smarca5 expression during hematopoietic stem cell commitment.

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.

Technology and provenience of the oldest pottery in the northern Pannonian Basin indicates its affiliation to hunter-gatherers.

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.

Region-specific gene expression profiling of early mouse mandible uncovered SATB2 as a key molecule for teeth patterning.

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.