Three-dimensional imaging of whole midgestation murine embryos shows an intravascular localization for all hematopoietic clusters.
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ABSTRACT:
SUBMITTER: Yokomizo T
PROVIDER: S-EPMC3122938 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Hematopoietic cell clusters in the aorta of vertebrate embryos play a pivotal role in the formation of the adult blood system. Despite their importance, hematopoietic clusters have not been systematically quantitated or mapped because of technical limitations posed by the opaqueness of whole mouse embryos. Here, we combine an approach to make whole mouse embryos transparent, with multicolor marking, to allow observation of hematopoietic clusters using high-resolution 3-dimensional confocal microscopy. Our method provides the first complete map and temporal quantitation of all hematopoietic clusters in the mouse embryonic vasculature. We show that clusters peak in number at embryonic day 10.5, localize to specific vascular subregions and are heterogeneous, indicating a basal endothelial to non-basal (outer cluster) hematopoietic cell transition. Clusters enriched with the c-Kit(+)CD31(+)SSEA1(-) cell population contain functional hematopoietic progenitors and stem cells. Thus, three-dimensional cartography of transparent mouse embryos provides novel insight into the vascular subregions instrumental in hematopoietic progenitor/stem cell development, and represents an important technological advancement for comprehensive in situ hematopoietic cluster analysis.
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Project description:The de novo generation of hematopoietic cells occurs during midgestation when a population of endothelial cells called hemogenic endothelium transitions into hematopoietic progenitors and stem cells. In mammalian embryos, the newly formed hematopoietic cells form clusters in the lumens of the major arteries in the embryo proper and in the vascular plexus of the yolk sac. Small clusters of hematopoietic cells that are independent of the vasculature (referred to here as extravascular islands) were shown to form in the mesentery during vascular remodeling of the vitelline artery. Using three-dimensional imaging of whole mouse embryos we demonstrate that extravascular budding of hematopoietic clusters is a more widespread phenomenon that occurs from the vitelline and the umbilical arteries both proximal to the embryo proper and distal in the extraembryonic yolk sac and placenta. Furthermore, we show that there are several mechanisms by which hematopoietic clusters leave the arteries, including vascular remodeling and extrusion. Lastly, we provide static images suggesting that extravascular islands contribute to the formation of new blood vessels. Thus, extravascular islands may represent a novel mechanism of vasculogenesis whereby established vessels contribute endothelial and hematopoietic cells to developing vascular beds.
| S-EPMC5810923 | biostudies-literature
Project description:When a single molecule is detected in a wide-field microscope, the image approximates the point spread function of the system. However, as the distribution of molecules becomes denser and their images begin to overlap, existing solutions to determine the number of molecules present and their precise three-dimensional locations can tolerate little to no overlap. We propose a localization scheme that can identify several overlapping molecule images while maintaining high localization precision. A solution to this problem involving matched optical and digital techniques, as here proposed, can substantially increase the allowable labeling density and accelerate the data collection time of single-molecule localization microscopy by more than one order of magnitude.
| S-EPMC4066253 | biostudies-literature
Project description:We describe a three-dimensional (3D) confocal imaging technique to characterize and enumerate rare, newly emerging hematopoietic cells located within the vasculature of whole-mount preparations of mouse embryos. However, the methodology is broadly applicable for examining the development and 3D architecture of other tissues. Previously, direct whole-mount imaging has been limited to external tissue layers owing to poor laser penetration of dense, opaque tissue. Our whole-embryo imaging method enables detailed quantitative and qualitative analysis of cells within the dorsal aorta of embryonic day (E) 10.5-11.5 embryos after the removal of only the head and body walls. In this protocol we describe the whole-mount fixation and multimarker staining procedure, the tissue transparency treatment, microscopy and the analysis of resulting images. A typical two-color staining experiment can be performed and analyzed in ?6 d.
| S-EPMC3629302 | biostudies-literature
Project description:The embryonic site of definitive hematopoietic stem cell (dHSC) origination has been debated for decades. Although an intra-embryonic origin is well supported, the yolk sac (YS) contribution to adult hematopoiesis remains controversial. The same developmental origin makes it difficult to identify specific markers that discern between an intraembryonic versus YS-origin using a lineage trace approach. Additionally, the highly migratory nature of blood cells and the inability of pre-circulatory embryonic cells (i.e., 5-7 somite pairs (sp)) to robustly engraft in transplantation, even after culture, has precluded scientists from properly answering these questions. Here we report robust, multi-lineage and serially transplantable dHSC activity from cultured 2-7sp murine embryonic explants (Em-Ex). dHSC are undetectable in 2-7sp YS explants. Additionally, the engraftment from Em-Ex is confined to an emerging CD31+CD45+c-Kit+CD41- population. In sum, our work supports a model in which the embryo, not the YS, is the major source of lifelong definitive hematopoiesis.
| S-EPMC6302089 | biostudies-other
Project description:BackgroundHematopoietic progenitors are generated in the yolk sac and aorta-gonad-mesonephros region during early mouse development. At embryonic day 10.5 the first hematopoietic stem cells emerge in the aorta-gonad-mesonephros. Subsequently, hematopoietic stem cells and progenitors are found in the fetal liver. The fetal liver is a potent hematopoietic site, playing an important role in the expansion and differentiation of hematopoietic progenitors and hematopoietic stem cells. However, little is known concerning the regulation of fetal liver hematopoietic stem cells. In particular, the role of cytokines such as interleukin-1 in the regulation of hematopoietic stem cells in the embryo has been largely unexplored. Recently, we observed that the adult pro-inflammatory cytokine interleukin-1 is involved in regulating aorta-gonad-mesonephros hematopoietic progenitor and hematopoietic stem cell activity. Therefore, we set out to investigate whether interleukin-1 also plays a role in regulating fetal liver progenitor cells and hematopoietic stem cells.Design and methodsWe examined the interleukin-1 ligand and receptor expression pattern in the fetal liver. The effects of interleukin-1 on hematopoietic progenitor cells and hematopoietic stem cells were studied by FACS and transplantation analyses of fetal liver explants, and in vivo effects on hematopoietic stem cell and progenitors were studied in Il1r1(-/-) embryos.ResultsWe show that fetal liver hematopoietic progenitor cells express the IL-1RI and that interleukin-1 increases fetal liver hematopoiesis, progenitor cell activity and promotes hematopoietic cell survival. Moreover, we show that in Il1r1(-/-) embryos, hematopoietic stem cell activity is impaired and myeloid progenitor activity is increased.ConclusionsThe IL-1 ligand and receptor are expressed in the midgestation liver and act in the physiological regulation of fetal liver hematopoietic progenitor cells and hematopoietic stem cells.
| S-EPMC2663609 | biostudies-literature
Project description:BackgroundWhole-mount in situ hybridization (WISH) is a fundamental tool for studying the spatio-temporal expression pattern of RNA molecules in intact embryos and tissues. The available methodologies for detecting mRNAs in embryos rely on enzymatic activities and chemical reactions that generate diffusible products, which are not fixed to the detected RNA, thereby reducing the spatial resolution of the technique. In addition, current WISH techniques are time-consuming and are usually not combined with methods reporting the expression of protein molecules.ResultsThe protocol we have developed and present here is based on the RNAscope technology that is currently employed on formalin-fixed, paraffin-embedded and frozen tissue sections for research and clinical applications. By using zebrafish embryos as an example, we provide a robust and rapid method that allows the simultaneous visualization of multiple transcripts, demonstrated here for three different RNA molecules. The optimized procedure allows the preservation of embryo integrity, while exhibiting excellent signal-to-noise ratios. Employing this method thus allows the determination of the spatial expression pattern and subcellular localization of multiple RNA molecules relative to each other at high resolution, in the three-dimensional context of the developing embryo or tissue under investigation. Lastly, we show that this method preserves the function of fluorescent proteins that are expressed in specific cells or cellular organelles and conserves antigenicity, allowing protein detection using antibodies.ConclusionsBy fine-tuning the RNAscope technology, we have successfully redesigned the protocol to be compatible with whole-mount embryo samples. Using this robust method for zebrafish and extending it to other organisms would have a strong impact on research in developmental, molecular and cell biology. Of similar significance would be the adaptation of the method to whole-mount clinical samples. Such a protocol would contribute to biomedical research and clinical diagnostics by providing information regarding the three-dimensional expression pattern of clinical markers.
| S-EPMC4172952 | biostudies-literature
Project description:Alveolar architecture plays a fundamental role in the processes of ventilation and perfusion in the lung. Alterations in the alveolar surface area and alveolar cavity volume constitute the pathophysiological basis of chronic respiratory diseases such as pulmonary emphysema. Previous studies based on micro-computed tomography (micro-CT) of lung samples have allowed the geometrical study of acinar units. However, our current knowledge is based on the study of a few tissue samples in random locations of the lung that do not give an account of the spatial distributions of the alveolar architecture in the whole lung. In this work, we combine micro-CT imaging and computational geometry algorithms to study the regional distribution of key morphological parameters throughout the whole lung. To this end, 3D whole-lung images of Sprague-Dawley rats are acquired using high-resolution micro-CT imaging and analyzed to estimate porosity, alveolar surface density, and surface-to-volume ratio. We assess the effect of current gold-standard dehydration methods in the preparation of lung samples and propose a fixation protocol that includes the application of a methanol-PBS solution before dehydration. Our results show that regional porosity, alveolar surface density, and surface-to-volume ratio have a uniform distribution in normal lungs, which do not seem to be affected by gravitational effects. We further show that sample fixation based on ethanol baths for dehydration introduces shrinking and affects the acinar architecture in the subpleural regions. In contrast, preparations based on the proposed dehydration protocol effectively preserve the alveolar morphology.
| S-EPMC8692792 | biostudies-literature
Project description:BackgroundValproic acid (VPA) is prescribed therapeutically for multiple conditions, including epilepsy. When taken during pregnancy, VPA is teratogenic, increasing the risk of several birth and developmental defects including neural tube defects (NTDs). The mechanism by which VPA causes NTDs remains controversial and how VPA interacts with folic acid (FA), a vitamin commonly recommended for the prevention of NTDs, remains uncertain. We sought to address both questions by applying untargeted metabolite profiling analysis to neural tube closure (NTC) stage mouse embryos.MethodsPregnant SWV dams on either a 2 ppm or 10 ppm FA supplemented diet were injected with a single dose of VPA on gestational day E8.5. On day E9.5, the mouse embryos were collected and evaluated for NTC status. Liquid chromatography coupled to mass spectrometry metabolomics analysis was performed to compare metabolite profiles of NTD-affected VPA-exposed whole mouse embryos with profiles from embryos that underwent normal NTC from control dams.ResultsNTDs were observed in all embryos from VPA-treated dams and penetrance was not diminished by dietary FA supplementation. The most profound metabolic perturbations were found in the 10ppm FA VPA-exposed mouse embryos, compared with the other three treatment groups. Affected metabolites included amino acids, nucleobases and related phosphorylated nucleotides, lipids, and carnitines.ConclusionMaternal VPA treatment markedly perturbed purine and pyrimidine metabolism in E9.5 embryos. In combination with a high FA diet, VPA treatment resulted in gross metabolic changes, likely caused by a multiplicity of mechanisms, including an apparent disruption of mitochondrial beta-oxidation. Birth Defects Research 109:106-119, 2017. © 2016 Wiley Periodicals, Inc.
| S-EPMC5388579 | biostudies-literature
Project description:BackgroundHyperthyroidism during pregnancy is treated with the antithyroid drugs (ATD) propylthiouracil (PTU) and methimazole (MMI). PTU currently is recommended as the drug of choice during early pregnancy. Yet, despite widespread ATD use in pregnancy, formal studies of ATD teratogenic effects have not been performed.MethodsWe examined the teratogenic effects of PTU and MMI during embryogenesis in mice. To span different periods of embryogenesis, dams were treated with compounds or vehicle daily from embryonic day (E) 7.5 to 9.5 or from E3.5 to E7.5. Embryos were examined for gross malformations at E10.5 or E18.5 followed by histological and micro-CT analysis. Influences of PTU on gene expression levels were examined by RNA microarray analysis.ResultsWhen dams were treated from E7.5 to E9.5 with PTU, neural tube and cardiac abnormalities were observed at E10.5. Cranial neural tube defects were significantly more common among the PTU-exposed embryos than those exposed to MMI or vehicle. Blood in the pericardial sac, which is a feature indicative of abnormal cardiac function and/or abnormal vasculature, was observed more frequently in PTU-treated than MMI-treated or vehicle-treated embryos. Following PTU treatment, a total of 134 differentially expressed genes were identified. Disrupted genetic pathways were those associated with cytoskeleton remodeling and keratin filaments. At E 18.5, no gross malformations were evident in either ATD group, but the number of viable PTU embryos per dam at E18.5 was significantly lower from those at E10.5, indicating loss of malformed embryos. These data show that PTU exposure during embryogenesis is associated with delayed neural tube closure and cardiac abnormalities. In contrast, we did not observe structural or cardiac defects associated with MMI exposure except at the higher dose. We find that PTU exposure during embryogenesis is associated with fetal loss. These observations suggest that PTU has teratogenic potential.
| S-EPMC3329451 | biostudies-literature