We advance mass spectrometry from a cell population‐averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary ...electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh‐resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE‐μESI‐HRMS enabled the identification of 500–800 nonredundant protein groups by measuring 20 ng, or <0.2% of the total protein content in single blastomeres that were isolated from the 16‐cell frog (Xenopus laevis) embryo, amounting to a total of 1709 protein groups identified between n=3 biological replicates. By quantifying ≈150 nonredundant protein groups between all blastomeres and replicate measurements, we found significant translational cell heterogeneity along multiple axes of the embryo at this very early stage of development when the transcriptional program of the embryo has yet to begin.
Back to the beginning: the cell. The encoded proteome offers basic insight into normal development. Capillary electrophoresis, electrospray ionization, high‐resolution mass spectrometry, and bottom‐up proteomics were integrated to enable the identification of 1709 proteins in single embryonic Xenopus cells. Quantification of hundreds of proteins revealed translational differences between cells that give rise to different tissues during development.
Spatial and temporal changes in molecular expression are essential to embryonic development, and their characterization is critical to understand mechanisms by which cells acquire different ...phenotypes. Although technological advances have made it possible to quantify expression of large molecules during embryogenesis, little information is available on metabolites, the ultimate indicator of physiological activity of the cell. Here, we demonstrate that single-cell capillary electrophoresis-electrospray ionization mass spectrometry is able to test whether differential expression of the genome translates to the domain of metabolites between single embryonic cells. Dissection of three different cell types with distinct tissue fates from 16-cell embryos of the South African clawed frog ( Xenopus laevis ) and microextraction of their metabolomes enabled the identification of 40 metabolites that anchored interconnected central metabolic networks. Relative quantitation revealed that several metabolites were differentially active between the cell types in the wild-type, unperturbed embryos. Altering postfertilization cytoplasmic movements that perturb dorsal development confirmed that these three cells have characteristic small-molecular activity already at cleavage stages as a result of cell type and not differences in pigmentation, yolk content, cell size, or position in the embryo. Changing the metabolite concentration caused changes in cell movements at gastrulation that also altered the tissue fates of these cells, demonstrating that the metabolome affects cell phenotypes in the embryo.
Significance Spatiotemporal characterization of molecular expression during embryonic development is critical for understanding how cells become different and give rise to distinct tissues and organs. Technological advances enabled the measurement of RNAs and proteins in single cells of embryos, but there is very little information on small molecules, metabolites, that are the ultimate indication of the physiological state. To fill this knowledge gap, we developed and used a single-cell technology to find that embryonic cells that give rise to different tissues have characteristically different metabolic signatures that are not simply a reflection of cell pigmentation, yolk content, size, or position in the embryo, but also affect cell fate. This approach is likely to provide new mechanistic insights into early embryo development.
Label-free single-cell proteomics by mass spectrometry (MS) is currently incompatible with complex tissues without requiring cell culturing, single-cell dissection, or tissue dissociation. We here ...report the first example of label-free single-cell MS-based proteomics directly in single cells in live vertebrate embryos. Our approach integrates optically guided in situ subcellular capillary microsampling, one-pot extraction–digestion of the collected proteins, peptide separation by capillary electrophoresis, ionization by an ultrasensitive electrokinetically pumped nanoelectrospray, and detection by high-resolution MS (Orbitrap). With a 700 zmol (420 000 copies) lower limit of detection, this trace-sensitive technology confidently identified and quantified ∼750–800 protein groups (<1% false-discovery rate) by analyzing just ∼5 ng of protein digest, viz. <0.05% of the total protein content from individual cells in a 16-cell Xenopus laevis (frog) embryo. After validating the approach by recovering animal–vegetal-pole proteomic asymmetry in the frog zygote, the technology was applied to uncover proteomic reorganization as the animal–dorsal (D11) cell of the 16-cell embryo gave rise to its neural-tissue-fated clone in the embryo developing to the 32-, 64-, and 128-cell stages. In addition to enabling proteomics on smaller cells in X. laevis, we also demonstrated this technology to be scalable to single cells in live zebrafish embryos. Microsampling single-cell MS-based proteomics raises exciting opportunities to study cell and developmental processes directly in complex tissues and whole organisms at the level of the building block of life: the cell.
Knowledge of single-cell metabolism would provide a powerful look into cell activity changes as cells differentiate to all the tissues of the vertebrate embryo. However, single-cell mass spectrometry ...technologies have not yet been made compatible with complex three-dimensional changes and rapidly decreasing cell sizes during early development of the embryo. Here, we bridge this technological gap by integrating capillary microsampling, microscale metabolite extraction, and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) to enable direct metabolic analysis of identified cells in the live frog embryo (Xenopus laevis). Microprobe CE-ESI-MS of <0.02% of the single-cell content allowed us to detect ∼230 different molecular features (positive ion mode), including 70 known metabolites, in single dorsal and ventral cells in 8-to-32-cell embryos. Relative quantification followed by multivariate and statistical analysis of the data found that microsampling enhanced detection sensitivity compared to whole-cell dissection by minimizing chemical interferences and ion suppression effects from the culture media. In addition, higher glutathione/oxidized glutathione ratios suggested that microprobed cells exhibited significantly lower oxidative stress than those dissected from the embryo. Fast (5 s/cell) and scalable microsampling with minimal damage to cells in the 8-cell embryo enabled duplicate and triplicate metabolic analysis of the same cell, which surprisingly continued to divide to the 16-cell stage. Last, we used microprobe single-cell CE-ESI-MS to uncover previously unknown reorganization of the single-cell metabolome as the dorsal progenitor cell from the 8-cell embryo formed the neural tissue fated clone through divisions to the 32-cell embryo, peering, for the first time, into the formation of metabolic single-cell heterogeneity during early development of a vertebrate embryo.
Xenopus Sally A. Moody; Abraham Fainsod
2022, 20220419
eBook
Odprti dostop
This book focuses on the amphibian, Xenopus, one of the most commonly used model animals in the biological sciences. Over the past 50 years, the use of Xenopus has made possible many fundamental ...contributions to our knowledge in cell biology, developmental biology, molecular biology, and neurobiology. In recent years, with the completion of the genome sequence of the main two species and the application of genome editing techniques, Xenopus has emerged as a powerful system to study fundamental disease mechanisms and test treatment possibilities. Xenopus has proven an essential vertebrate model system for understanding fundamental cell and developmental biological mechanisms, for applying fundamental knowledge to pathological processes, for deciphering the function of human disease genes, and for understanding genome evolution. Key Features Provides historical context of the contributions of the model system Includes contributions from an international team of leading scholars Presents topics spanning cell biology, developmental biology, genomics, and disease model Describes recent experimental advances Incorporates richly illustrated diagrams and color images Related Titles Green, S. L. The Laboratory Xenopus sp. (ISBN 978-1-4200-9109-0) Faber, J. & P. D. Nieuwkoop. Normal Table of Xenopus laevis (Daudin): A Systematical & Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis (ISBN 978-0-8153-1896-5) Jarret, R. L. & K. McCluskey. The Biological Resources of Model Organisms (ISBN 978-1-0320-9095-5)
Normal tables of development are essential for studies of embryogenesis, serving as an important resource for model organisms, including the frog Xenopus laevis. Xenopus has long been used to study ...developmental and cell biology, and is an increasingly important model for human birth defects and disease, genomics, proteomics and toxicology. Scientists utilize Nieuwkoop and Faber's classic 'Normal Table of Xenopus laevis (Daudin)' and accompanying illustrations to enable experimental reproducibility and reuse the illustrations in new publications and teaching. However, it is no longer possible to obtain permission for these copyrighted illustrations. We present 133 new, high-quality illustrations of X. laevis development from fertilization to metamorphosis, with additional views that were not available in the original collection. All the images are available on Xenbase, the Xenopus knowledgebase (http://www.xenbase.org/entry/zahn.do), for download and reuse under an attributable, non-commercial creative commons license. Additionally, we have compiled a 'Landmarks Table' of key morphological features and marker gene expression that can be used to distinguish stages quickly and reliably (https://www.xenbase.org/entry/landmarks-table.do). This new open-access resource will facilitate Xenopus research and teaching in the decades to come.
Model animals are crucial to biomedical research. Among the commonly used model animals, the amphibian, Xenopus, has had tremendous impact because of its unique experimental advantages, cost ...effectiveness, and close evolutionary relationship with mammals as a tetrapod. Over the past 50 years, the use of Xenopus has made possible many fundamental contributions to biomedicine, and it is a cornerstone of research in cell biology, developmental biology, evolutionary biology, immunology, molecular biology, neurobiology, and physiology. The prospects for Xenopus as an experimental system are excellent: Xenopus is uniquely well‐suited for many contemporary approaches used to study fundamental biological and disease mechanisms. Moreover, recent advances in high throughput DNA sequencing, genome editing, proteomics, and pharmacological screening are easily applicable in Xenopus, enabling rapid functional genomics and human disease modeling at a systems level.
Yes-associated protein 65 (YAP) contains multiple protein-protein interaction domains and functions as both a transcriptional co-activator and as a scaffolding protein. Mouse embryos lacking YAP did ...not survive past embryonic day 8.5 and showed signs of defective yolk sac vasculogenesis, chorioallantoic fusion, and anterior-posterior (A-P) axis elongation. Given that the YAP knockout mouse defects might be due in part to nutritional deficiencies, we sought to better characterize a role for YAP during early development using embryos that develop externally. YAP morpholino (MO)-mediated loss-of-function in both frog and fish resulted in incomplete epiboly at gastrulation and impaired axis formation, similar to the mouse phenotype. In frog, germ layer specific genes were expressed, but they were temporally delayed. YAP MO-mediated partial knockdown in frog allowed a shortened axis to form. YAP gain-of-function in Xenopus expanded the progenitor populations in the neural plate (sox2(+)) and neural plate border zone (pax3(+)), while inhibiting the expression of later markers of tissues derived from the neural plate border zone (neural crest, pre-placodal ectoderm, hatching gland), as well as epidermis and somitic muscle. YAP directly regulates pax3 expression via association with TEAD1 (N-TEF) at a highly conserved, previously undescribed, TEAD-binding site within the 5' regulatory region of pax3. Structure/function analyses revealed that the PDZ-binding motif of YAP contributes to the inhibition of epidermal and somitic muscle differentiation, but a complete, intact YAP protein is required for expansion of the neural plate and neural plate border zone progenitor pools. These results provide a thorough analysis of YAP mediated gene expression changes in loss- and gain-of-function experiments. Furthermore, this is the first report to use YAP structure-function analyzes to determine which portion of YAP is involved in specific gene expression changes and the first to show direct in vivo evidence of YAP's role in regulating pax3 neural crest expression.
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