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.

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.

Imaging the elemental distribution within human malignant melanomas using Laser-Induced Breakdown Spectroscopy.

The diagnosis of malignant melanoma, often an inconspicuous but highly aggressive tumor, is most commonly done by histological examination, while additional diagnostic methods on the level of elements and molecules are constantly being developed. Several studies confirmed differences in the chemical composition of healthy and tumor tissue. Our study presents the potential of the LIBS (Laser-Induced-Breakdown Spectroscopy) technique as a diagnostic tool in malignant melanoma (MM) based on the quantitative changes in elemental composition in cancerous tissue. Our patient group included 17 samples of various types of malignant melanoma and one sample of healthy skin tissue as a control. To achieve a clear perception of results, we have selected two biogenic elements (calcium and magnesium), which showed a dissimilar distribution in cancerous tissue from its healthy surroundings. Moreover, we observed indications of different concentrations of these elements in different subtypes of malignant melanoma, a hypothesis that requires confirmation in a more extensive sample set. The information provided by the LIBS Imaging method could potentially be helpful not only in the diagnostics of tumor tissue but also be beneficial in broadening the knowledge about the tumor itself.

The level of protein in the maternal murine diet modulates the facial appearance of the offspring via mTORC1 signaling.

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.

Comparison of single and double pulse laser-induced breakdown spectroscopy for the detection of biomolecules tagged with photon-upconversion nanoparticles.

BACKGROUND: Laser-induced breakdown spectroscopy (LIBS) is a well-recognized analytical technique used for elemental analysis. This method is gaining considerable attention also in biological applications thanks to its ability for spatial mapping and elemental imaging. The implementation of LIBS in the biomedical field is based on the detection of metals or other elements that either naturally occur in the samples or are present artificially. The artificial implementation of nanoparticle labels (Tag-LIBS) enables the use of LIBS as a readout technique for immunochemical assays. However, one of the biggest challenges for LIBS to meet immunoassay readout standards is its sensitivity. RESULTS: This paper focuses on the improvement of LIBS sensitivity for the readout of nanoparticle-based immunoassays. First, the LIBS setup was optimized on photon-upconversion nanoparticle (UCNP) droplets deposited on the microtiter plate wells. Two collection optics systems were compared, with single pulse (SP) and collinear double pulse (DP) LIBS arrangements. By deploying the second laser pulse, the sensitivity was improved up to 30 times. The optimized SP and DP setups were then employed for the indirect detection of human serum albumin based on immunoassay with UCNP-based labels. Compared to our previous LIBS study, the detection limit was enhanced by two orders of magnitude, from 10 ng mL-1 to 0.29 ng mL-1. In addition, two other immunochemical methods were used for reference, based on the readout of upconversion luminescence of UCNPs and absorbance measurement with enzyme labels. Finally, the selectivity of the assay was tested and the practical potential of Tag-LIBS was demonstrated by the successful analysis of urine samples. SIGNIFICANCE AND NOVELTY: In this work, we improved the sensitivity of the Tag-LIBS method by combining new labels based on UCNPs with the improved collection optics and collinear DP configuration. In the instrumental setup optimization, the DP LIBS showed better sensitivity and signal-to-noise ratio than SP. The optimizations allowed the LIBS readout to surpass the sensitivity of enzyme immunoassay, approaching the qualities of upconversion luminescence readout, which is nowadays a state-of-the-art readout technique.

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.

Directionality of developing skeletal muscles is set by mechanical forces.

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.

A previously uncharacterized Factor Associated with Metabolism and Energy (FAME/C14orf105/CCDC198/1700011H14Rik) is related to evolutionary adaptation, energy balance, and kidney physiology.

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.

Laser-based techniques: Novel tools for the identification and characterization of aged microplastics with developed biofilm.

Microplastics found in the environment are often covered with a biofilm, which makes their analysis difficult. Therefore, the biofilm is usually removed before analysis, which may affect the microplastic particles or lead to their loss during the procedure. In this work, we used laser-based analytical techniques and evaluated their performance in detecting, characterizing, and classifying pristine and aged microplastics with a developed biofilm. Five types of microplastics from different polymers were selected (polyamide, polyethylene, polyethylene terephthalate, polypropylene, and polyvinyl chloride) and aged under controlled conditions in freshwater and wastewater. The development of biofilm and the changes in the properties of the microplastic were evaluated. The pristine and aged microplastics were characterized by standard methods (e.g., optical and scanning electron microscopy, and Raman spectroscopy), and then laser-induced breakdown spectroscopy (LIBS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) were used. The results show that LIBS could identify different types of plastics regardless of the ageing and major biotic elements of the biofilm layer. LA-ICP-MS showed a high sensitivity to metals, which can be used as markers for various plastics. In addition, LA-ICP-MS can be employed in studies to monitor the adsorption and desorption (leaching) of metals during the ageing of microplastics. The use of these laser-based analytical techniques was found to be beneficial in the study of environmentally relevant microplastics.

Sex differences and risk factors for bleeding in Alagille syndrome.

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.

Altered developmental programs and oriented cell divisions lead to bulky bones during salamander limb regeneration.

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.

Conductive cross-section preparation of non-conductive painting micro-samples for SEM analysis.

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.

Cyclododecane shaping, sublimation rate and residue analysis for the extraction of painting micro-samples from resin cross-sections.

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.

High-resolution micro-CT for 3D infarct characterization and segmentation in mice stroke models.

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.

Surface flow for colonial integration in reef-building corals.

Reef-building corals are endangered animals with a complex colonial organization. Physiological mechanisms connecting multiple polyps and integrating them into a coral colony are still enigmatic. Using live imaging, particle tracking, and mathematical modeling, we reveal how corals connect individual polyps and form integrated polyp groups via species-specific, complex, and stable networks of currents at their surface. These currents involve surface mucus of different concentrations, which regulate joint feeding of the colony. Inside the coral, within the gastrovascular system, we expose the complexity of bidirectional branching streams that connect individual polyps. This system of canals extends the surface area by 4-fold and might improve communication, nutrient supply, and symbiont transfer. Thus, individual polyps integrate via complex liquid dynamics on the surface and inside the colony.

Serotonin limits generation of chromaffin cells during adrenal organ development.

Adrenal glands are the major organs releasing catecholamines and regulating our stress response. The mechanisms balancing generation of adrenergic chromaffin cells and protecting against neuroblastoma tumors are still enigmatic. Here we revealed that serotonin (5HT) controls the numbers of chromaffin cells by acting upon their immediate progenitor "bridge" cells via 5-hydroxytryptamine receptor 3A (HTR3A), and the aggressive HTR3Ahigh human neuroblastoma cell lines reduce proliferation in response to HTR3A-specific agonists. In embryos (in vivo), the physiological increase of 5HT caused a prolongation of the cell cycle in "bridge" progenitors leading to a smaller chromaffin population and changing the balance of hormones and behavioral patterns in adulthood. These behavioral effects and smaller adrenals were mirrored in the progeny of pregnant female mice subjected to experimental stress, suggesting a maternal-fetal link that controls developmental adaptations. Finally, these results corresponded to a size-distribution of adrenals found in wild rodents with different coping strategies.