Name: cell-lineage guided mass spectrometry proteomics in the developing (frog) embryo
Uploaded: 2022-04-21T13:00:00.000+00:00
Duration: 9 min 18 s
Description: Watch this Scientific Journal Video about Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo at JoVE.com

We are grateful to Jie Li (University of Maryland, College Park) for valuable discussions on embryonic dissociation and FACS. We thank Vi M. Quach and Camille Lombard-Banek for assistance with sample preparation and data collection in previous studies exemplifying the proteomic applications that are highlighted in this protocol. Parts of this work were supported by the National Science Foundation under award number IOS-1832968 CAREER (to P.N.), the National Institutes of Health under award number R35GM124755 (to P.N.), the University of Maryland-National Cancer Institute Partnership Program (to P.N.), and COSMOS Club Foundation research awards (to A.B.B. and L.R.P.).

The authors declare no competing interests.

This protocol enables the characterization of protein expression in identified cell lineages in embryos of the Xenopus species. Stemming from HRMS, the methodology combines exquisite specificity in molecular identification, capability for multi-protein detection without molecular probes (usually hundreds to thousands of different proteins), and a capability for quantification. Adaptability to classical tools and workflows in cell and developmental (neuro)biology expand HRMS proteomics to exciting applications, including holistic characterization of stem cell differentiation in the vertebrate X. laevis embryo.
The steps describe cell lineage-guided proteomics in the X. laevis embryo. As examples, we demonstrate the analysis of neural-fated single cells and their lineages and provide the corresponding CE- and nanoLC HRMS datasets through a public data repository (see PRIDE). This approach is readily adaptable to studies on the spatiotemporal regulation of proteins in multiple cell types (and lineages). Spatiotemporally guided proteomics in the developing embryo can also be extended to transcriptomic and metabolomic studies to promote the molecular systems biology understanding of cell differentiation and development of key organs, such as the nervous system.
This protocol adds new dimensions to bioanalysis. Cell cultures, such as the induced pluripotent stem cells (iPSCs)1,2, facilitate the measurement of temporal proteome dynamics during cell differentiation. The approach detailed in this study peers into cell fate induction in the 3-dimensional live embryo, where complex morphogen gradients, parallel signaling pathways, and convergent extension processes collaborate to bring about tissue induction and morphogenesis. Studying proteome dynamics in the context of an embryo can provide information on parallel mechanisms that guide cell differentiation, an exciting direction to deepen understanding of differentiation and development.
The approach described here borrows the experimental benefits of the Xenopus species to this end, X. laevis in particular. Each cell of the early 16- and 32- cell X. laevis embryo is mapped to reproducible tissue fates in the adult organism, essentially a spatial projection of cells in the cleavage stage embryos. Reproducibility for cell-guided proteomics builds on accurate cell typing. We aid success by credentialing embryos for stereotypical pigmentation and cleavage before proceeding to experiments describing cell dissection and lineage tracing16,20. It is important to note that cells of cleavage stage embryos often contribute to the formation of different tissue types to various degrees; the details of their level contribution to each tissue type is available on Xenbase23. The embryonic stage and precursor cells to be lineage traced should therefore be chosen based on the biological question at hand. When culturing embryos over longer periods of time (e.g., &#62;1 d), care ought to be taken to remove damaged/dead embryos, which may, in turn, diminish viability for the other embryos in the population.
For successful HRMS proteomics, careful attention should be paid to the analytical foundations of cell/tissue collection, protein extraction, and HRMS measurement. Because Xenopus embryos are cultured in media containing high concentrations of nonvolatile salts that are known to reduce HRMS sensitivity, it is recommended to reduce the aspirated media when collecting the cells and tissues for proteomic studies. It is a good practice to explore desalting to advance the identification and quantification of the proteins. Prior to HRMS measurement, the analytical performance of the CE- and LC-ESI-HRMS instruments should be evaluated, including separation, ionization, reproducibility, and linear dynamic range of quantification. With good analytical metrics, this protocol helps reduce animal usage in biological research, aids sensitivity for obtaining more powerful sets of biochemical data, and enhances the power of statistical data analysis to interpret results. By default, we analyze each biological replicate in at least 3 technical replicates using CE or LC-HRMS, which are used to evaluate technical reproducibility and deepen the coverage of the detectable proteome. Power analysis helps the estimation of statistical power and design of the biological replicate size. The use of different sets of parent frogs is advised to account for naturally occurring biological variability among embryos.
SUPPLEMENTARY INFORMATION 
The HRMS-MS/MS proteomics data and related processing files were deposited to the ProteomeExchange Consortium via the PRIDE60 partner repository with the data set identifier PXD030059.

Our understanding of cell differentiation and the genesis of tissues and organs is the result of decades of elaborate targeted screens of genes and their products. Increasing our knowledge of all the biomolecules and their quantities during important cellular events would help unravel molecular mechanisms that control the spatial and temporal patterning of the vertebrate body plan. Technologies enabling molecular amplification and sequencing are now able to routinely report on large numbers of genes and transcripts, supporting hypothesis-driven studies in basic biological and translational research. To understand developing systems, a complex relationship between transcription and translation advocates for direct analysis of multiple proteins and their post-translational modifications. Global proteomics using in vitro biological systems, such as induced pluripotent stem cells, began to delineate mechanisms of tissue induction1,2. In complex organisms, such as the vertebrate embryo, development relies on morphogen gradients in the context of space and time3. It follows that gaining knowledge of proteomic changes as cells differentiate to form specialized tissues, such as neural tissues, offers a key to unlock molecular programs controlling normal and defective development and guide next-generation therapeutics.
The vertebrate South African clawed frog (Xenopus laevis) is a well-established model in cell and developmental, neuro-, and regenerative biology. Sir John Gurdon&#39;s 2012 Nobel Prize in Physiology or Medicine4,5 for the discovery of pluripotency of the somatic nucleus highlighted the importance of this model for discoveries in basic and translational studies. Xenopus embryos develop externally to the mother, thus facilitating direct manipulation of cells, cell clones, and gene expression over various stages of development. Asymmetrical pigmentation and stereotypical cell divisions enabled the charting of reproducible fate maps from the 16-6 and 32-cell7,8 stage embryo. For high-resolution mass spectrometry (HRMS) based proteomics, additional advantages of the model include relatively large size (~1 mm in diameter), which yields abundant protein content for analysis (~130 &#181;g in early cleavage-stage embryos, ~10 &#181;g of protein content in single cells of the 16-cell embryo)9,10.
At present, HRMS is the leading technology of choice for detecting proteins. This technology enables direct, sensitive, and specific detection and quantification of multiple, usually hundreds-to-thousands of different proteins11. Bottom-up proteomics by HRMS involves a series of interconnected steps. Following extraction from the cell/tissue sample, proteins are digested with a proteolytic enzyme, such as trypsin (bottom-up proteomics). The resulting peptides are separated based on their different physicochemical properties, including hydrophobicity (reversed-phase liquid chromatography, LC), net charge (ion-exchange chromatography), size (size exclusion chromatography), or electrophoretic mobility (capillary electrophoresis, CE). Peptides are then charged (ionized), typically using electrospray ionization (ESI), and peptide ions are detected and sequenced via gas-phase fragmentation by tandem HRMS. The resulting peptide data are mapped to the proteome of the organism being studied. With protein-specific (proteotypic) peptide ion signal intensity correlating with concentration, protein quantification can be performed label-free or label-based (multiplexing quantitation). HRMS proteomics yields a rich resource of information on the molecular state of the system under study, allowing for the generation of hypotheses and follow-up functional studies.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63586/63586fig01v2.jpg" /> 
Figure 1: Spatiotemporally scalable proteomics enabling cell-lineage guided HRMS proteomics in the developing (frog) embryo. (A) Visualization of the specimen (1) using a stereomicroscope (2) for injection of an identified cell (inset), using a fabricated micropipette (3) under control by a translation-stage (4). (B) Subcellular sampling of the identified left D11 cell in a 16-cell embryo. (C)&#160;Dissection of a whole D11 cell from a 16-cell embryo. (D) Fluorescent (green) tracing of the left and right D111 progenies from a 32-cell embryo to guide dissection of the neural ectoderm (NE) in the gastrula (stage 10) and isolation of the descendent tissue from the tadpole using FACS. Scale bars: 200 &#181;m for embryos, 1.25 mm for the vial. Figures were adapted with permission from references15,19,21,59. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63586/63586fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The protocol presented here enables HRMS-based quantification of large numbers of proteins in identified cells/tissues in developing X. laevis embryos. The approach builds on accurate cell identification, reproducible cell fate maps, and established methodologies to track cell lineages in this biological model6,7,8. As shown in Figure 1, we study proteomes from single cells by employing whole-cell dissection or capillary microsampling to aspirate cellular content. Monitoring the lineage of a cell permits us to study the spatiotemporal evolution of the proteome as cells form tissues during gastrulation. The cell progeny is fluorescently marked by injecting a fluorophore conjugated to inert dextran or mRNA for fluorescent protein (e.g., green fluorescent protein, or GFP). The labeled progeny is isolated at desired developmental time points. During gastrulation, cell clones that are tightly clustered may be isolated by dissection. After gastrulation, cell clones may be distributed within the embryo owing to migratory movements and can be isolated from dissociated tissues by fluorescence-activated cell sorting (FACS). Proteins in these cells and tissues are measured via bottom-up proteomics employing HPLC or CE for separation and ESI tandem HRMS for identification. Cell-lineage-guided HRMS proteomics is scalable to different cell sizes and lineages within the embryo and is specific, sensitive, and quantitative. Through select examples shown here, we also demonstrate this protocol to be scalable and broadly adaptable to different types of cells and cell lineages.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63586/63586fig02.jpg" /> 
Figure 2: The bioanalytical workflow. Micro-dissection and capillary aspiration, or FACS facilitated sampling of cellular and clonal protein content. Depletion of abundant yolk proteins and separation by capillary electrophoresis (CE) or nano-flow liquid chromatography (LC) enhanced identification (ID) sensitivity using electrospray ionization (ESI) high-resolution mass spectrometry (HRMS). Quantification revealed dysregulation, supplying new information for hypothesis-driven studies in conjunction with information available from gene ontology (GO). Figures were adapted with permission from reference15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63586/63586fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Acetonitrile (LC-MS-grade)</td><td>Fisher Scientific</td><td>A955</td><td></td></tr><tr><td>Agarose</td><td>ThermoFisher Scientific</td><td>R0492</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Fisher Scientific</td><td>A643-500</td><td></td></tr><tr><td>Analytical Column</td><td>Thermo Scientific</td><td>164941</td><td></td></tr><tr><td>Analytical microbalance</td><td>Mettler-Toledo</td><td>XSE105DU</td><td></td></tr><tr><td>Automatic peptide fractionation platform</td><td>Agilent</td><td>1260 Infinity II</td><td></td></tr><tr><td>Borosilicate Capillaries</td><td>Sutter Instruments Co.</td><td>B100-50-10</td><td></td></tr><tr><td>Borosilicate Capillaries (for making Emmitters)</td><td>Sutter Instruments</td><td>B100-75-10</td><td></td></tr><tr><td>C18 spin columns (for desalting)</td><td>ThermoFisher Scientific</td><td>89870</td><td></td></tr><tr><td>Camera ro monitor electrospray</td><td>Edmund Optics Inc.</td><td>EO-2018C</td><td></td></tr><tr><td>Combretastatin A4</td><td>Millipore Sigma</td><td>C7744</td><td></td></tr><tr><td>Commercial CESI system</td><td>AB SCIEX</td><td>CESI</td><td></td></tr><tr><td>(Cyclohexylamino)-1-propanesulfonic acid (CAPS)</td><td>VWR</td><td>97061-492</td><td></td></tr><tr><td>Cytochalasin D</td><td>Millipore Sigma</td><td>C8273</td><td></td></tr><tr><td>Dextran, Alexa Fluor 488; 10,000 MW, Anionic, Fixable</td><td>ThermoFisher Scientific</td><td>D22910</td><td></td></tr><tr><td>Diothiothreitol</td><td>Fisher Scientific</td><td>FERR0861</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11252-30</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>AAJ62786AP</td><td></td></tr><tr><td>Epifluorescence light source</td><td>Lumencore</td><td>AURA III</td><td></td></tr><tr><td>Eppendorf LoBing microcentrifuge&amp;nbsp; tubes: protein</td><td>Fisher Scientific</td><td>13-698-793</td><td></td></tr><tr><td>Formic acid (LC-MS-grade)</td><td>Fisher Scientific</td><td>A117-50</td><td></td></tr><tr><td>Freezer (-20 &amp;deg;C)</td><td>Fisher Scientific</td><td>97-926-1&amp;nbsp;</td><td></td></tr><tr><td>Freezer (-80 &amp;deg;C)</td><td>Thermo Scientific</td><td>TSX40086A</td><td></td></tr><tr><td>Fused silica capillary</td><td>Molex</td><td>1088150596</td><td></td></tr><tr><td>Heat Block</td><td>Benchmark</td><td>BSH300</td><td></td></tr><tr><td>High pressure liquid Chromatography System</td><td>ThermoFisher Scientific</td><td>Dionex Ultimate 3000 RSLC nanosystem</td><td></td></tr><tr><td>High voltage power supply</td><td>Spellman</td><td>CZE1000R</td><td></td></tr><tr><td>High-resolution Mass Spectrometer</td><td>ThermoFisher Scientific</td><td>Orbitrap Fusion Lumos Tribrid Mass Spectrometer</td><td></td></tr><tr><td>HPLC caps</td><td>Thermo Scientific</td><td>C4013-40A</td><td></td></tr><tr><td>HPLC Vials</td><td>Thermo Scientific</td><td>C4013-11</td><td></td></tr><tr><td>Illuminator e.g. Goosenecks</td><td>Nikon</td><td>C-FLED2</td><td></td></tr><tr><td>Ingenuity Pathway Analysis</td><td>Qiagen</td><td></td><td></td></tr><tr><td>Iodoacetamide</td><td>Fisher Scientific</td><td>AC122275000</td><td></td></tr><tr><td>Methanol (LC-MS-grade)</td><td>Fisher Scientific</td><td>A456</td><td></td></tr><tr><td>Methanol (LC-MS-grade)</td><td>Fisher Scientific</td><td>A456-4</td><td></td></tr><tr><td>Microcapillary puller</td><td>Suttor Instruments</td><td>P-2000</td><td></td></tr><tr><td>Microinjector</td><td>Warner Instrument, Handem, CT</td><td>PLI-100A</td><td></td></tr><tr><td>Micropippette puller</td><td>Sutter Instruments Co.</td><td>P-1000</td><td></td></tr><tr><td>MS data analysis software, commercial</td><td>ProteomeDiscoverer</td><td></td><td></td></tr><tr><td>MS data analysis software, opensource</td><td>MaxQuant</td><td></td><td></td></tr><tr><td>non-idet 40 substitute</td><td>Millipore Sigma</td><td>11754599001</td><td></td></tr><tr><td>Petri dish 60 mm and 80 mm</td><td>Fisher Scientific</td><td>S08184</td><td></td></tr><tr><td>Pierce 10 &amp;micro;L bed Zip-tips (for desalting)</td><td>ThermoFisher Scientific</td><td>87782</td><td></td></tr><tr><td>Pierce bicinchoninic acid protein assay kit</td><td>ThermoFisher Scientific</td><td>23225</td><td></td></tr><tr><td>Pierce quantitative colorimetric peptide assay</td><td>ThermoFisher Scientific</td><td>23275</td><td></td></tr><tr><td>Pierce Trypsin Protease (MS Grade)</td><td>Fisher Scientific</td><td>PI90058</td><td></td></tr><tr><td>Protein LoBind vials</td><td>Eppendorf</td><td>0030108434&lt;br/&gt; , 0030108442</td><td></td></tr><tr><td>Refrigerated Centrifuge</td><td>Eppendorf</td><td>5430R</td><td></td></tr><tr><td>Refrigerated Incubator</td><td>Thermo Scientific</td><td>PR505755R/3721</td><td></td></tr><tr><td>sodium isethionate</td><td>Millipore Sigma</td><td>220078</td><td></td></tr><tr><td>sodium pyrophosphate</td><td>Sigma Aldrich</td><td>221368-100G</td><td></td></tr><tr><td>Stainless steel BGE vial</td><td>Custom-Built</td><td></td><td></td></tr><tr><td>Stainless steel sample vials</td><td>Custom-Built</td><td></td><td></td></tr><tr><td>Stereomicroscope (objective 10x)</td><td>Nikon</td><td>SMZ 1270, SZX18</td><td></td></tr><tr><td>Sucrose</td><td>VWR</td><td>97063-790</td><td></td></tr><tr><td>Syringe pumps (2)</td><td>Harvard Apparatus</td><td>704506</td><td></td></tr><tr><td>Syringes (gas-tight): 500&amp;ndash;1000 &amp;micro;L</td><td>Hamilton</td><td>1750TTL</td><td></td></tr><tr><td>Transfer pipettes (Plastic, disposable)</td><td>Fisher Scientific</td><td>13-711-7M</td><td></td></tr><tr><td>Trap Column</td><td>Thermo Scientific</td><td>164750</td><td></td></tr><tr><td>Tris-HCl (1 M solution)</td><td>Fisher Scientific</td><td>AAJ22638AP</td><td></td></tr><tr><td>Vacuum concentrator capable of operation at 4&amp;ndash;10 &amp;deg;C</td><td>Labconco</td><td>7310022</td><td></td></tr><tr><td>Vortex-mixer</td><td>Benchmark</td><td>BS-VM-1000</td><td></td></tr><tr><td>Water (LC-MS-grade)</td><td>Fisher Scientific</td><td>W6</td><td></td></tr><tr><td>Water (LC-MS-grade)</td><td>Fisher Scientific</td><td>W6</td><td></td></tr><tr><td>XYZ translation stage</td><td>Thorlabs</td><td>PT3</td><td></td></tr><tr><td>XYZ translation stage</td><td>Custom-Built</td><td></td><td></td></tr></tbody></table>

cell-lineage guided mass spectrometry proteomics in the developing (frog) embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

This protocol enabled the study of proteins in single cells and their lineages as they establish tissues in X. laevis embryos. Figure 1 illustrates one such application of the approach to study proteins in neural-tissue-fated cells and the newly induced neural ectoderm in the embryo. As shown in Figure 1A, the bioanalytical workflow integrated traditional tools of cell and developmental biology to identify, inject/aspirate cells, and collect specimens. Figure 1B shows microprobe sampling of the left dorsal-animal (D11) cell in the 16-cell embryo in vivo using a microinjector; after the experiment, embryos successfully developed to tadpoles with normal anatomy56. Large embryonic cells (~100-250 &#181;m in diameter) were conducive to manual microdissection as well. Dissection of a right dorsal-animal midline (D11) cell is illustrated from the 16-cell embryo in Figure 1C. The setup also permitted tracing a clonal trajectory by injecting a fluorescent tracer into the identified precursor. As shown in Figure 1D, the approach allowed to isolate clones arising from the left and right D111 cells via tissue dissection or by fluorescence-activated cell sorting (FACS). The sample collection strategies described here are sufficiently scalable in space and time to study embryonic development in new details.
The bioanalytical workflow integrated HRMS technologies to improve sensitivity and quantification (Figure 2). The collected proteins were measured via a bottom-up proteomic approach. Optional fractionation of samples based on orthogonal chemistry (e.g., high-pH then low-pH reversed-phase LC) aided detection sensitivity. To separate peptides, CE was selected for trace amounts of samples (&#60;&#60;~100 ng)) and nanoLC for limited amounts of materials (&#62;&#62;150 ng). Peptides were sequenced using ESI-HRMS. The detected proteins were quantified using label-free and label-based strategies. Simplification of sample processing for single-cell analysis, such as elimination of the typical steps of reduction and alkylation followed by CE analysis, facilitated the identification of ~400-800 different proteins. Among the reported proteins were many with important functional roles, such as chaperonin containing TCP1 subunit 3 (Cct3), voltage-dependent ion channel (Vdac2), and creatine kinase-brain (Ckb). Multivariate and statistical data models helped us10,57 and others9,58 find previously unknown differences in the proteomic composition of select cells and tissues using the approaches summarized in this protocol. Notably, these HRMS measurements required no functioning probes or knowledge about the composition of the specimens ahead of time, supporting discoveries.
In a series of studies, the proteomic state of identified cells in developing embryos of X. laevis (Fig. 3) was quantified21. Figure 3A shows the detection of gene translational differences between D11 cells that were dissected from different embryos10. Microsampling CE-ESI-HRMS allowed us to identify and quantify up to ~700 proteins from single cells21. The representative primary HRMS-MS/MS data on identifying ~400 cumulative proteins from technical duplicate measurements on a D11 cell in the embryo was deposited to PRIDE. The approach was scalable to smaller cells and embryos from other model organisms, such as zebrafish21. Scalability to smaller cell sizes allowed us to explore the spatiotemporal reorganization of the D11 progeny from a live embryo as it developed in time. Figure 3B presents the use of this protocol to perform subcellular quantitative proteomics of identified cells in the 16-, 32-, 64-, and 128-cell embryos. Proteins were separated into four groups that displayed distinct abundance profiles over clonal development21.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63586/63586fig03.jpg" /> 
Figure 3: Protocol scalability from the subcellular space to clonal tissues in X. laevis embryo. (A) Measurement of proteomic differences between identified whole D11 cells, revealing cell-cell heterogeneity. Gene names shown for select proteins. (B) Reorganization of the cellular proteome in the developing D11 cell clone. Fuzzy C-means cluster analysis (GProx) of protein dynamics, grouping proteins based on similar expression patterns. Gray numbers show the number of different proteins that were quantified in each cluster. Figures were adapted with permission from references21,57. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63586/63586fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This protocol allowed us to conduct spatial and temporal proteomics in identified, developing cell clones (Figure 4). Figure 4A demonstrates the application of the approach for labeling and isolating cell clones constituting two tissues with important roles in neural tissue development and patterning of the embryo. The majority of the Spemann organizer (SO) was traced by labeling the left and right D112 and D212 cells via injection of fluorescent dextran. In parallel, the neighboring dorsal-animal (D111) cells were labeled to mark the majority of the neural ectoderm (NE). The tissues were dissected, and their protein content was analyzed following this protocol. nanoLC-ESI-HRMS returned up to 2,000 different proteins from the tissues, including signaling, such as the Wnt, Fgf, and Tgf&#946; pathways. The representative primary HRMS-MS/MS data on identifying ~1,800 cumulative proteins from technical duplicate measurements on a pool of dissected SO tissues was deposited to PRIDE. The Wnt pathway ligand Wnt10a and Wntless (Wls), a membrane protein dedicated to the secretion of Wnt ligands, were detected only in the NE proteome. The Wnt pathway was found suppressed in the SO and may explain a lack of Wnt-interacting proteins detected in the SO. These results showcase the applicability of this protocol for studying lineage-specific differences within the embryo.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63586/63586fig04.jpg" /> 
Figure 4: Example of data analysis from spatial tissue proteomics in the X. laevis embryos. (A) Differential labeling of the Spemann&#39;s Organizer (SO) and the neural ectoderm (NE) tissues by injection of fluorescent protein mRNA in the predecessor D112 and D212 in the 32-cell embryo, respectively. (B) Top 5 overrepresented biological processes in the SO (red) and NE (green) proteome showing detectable differences. Pathway overrepresentation analysis shows biological processes using Bonferroni correction. (C) Protein domain enrichment analysis (SMART), revealing enrichment of DNA and RNA binding motif-containing proteins in the SO. (D) STRING analysis predicting canonical protein-protein interactions based on the detected SO proteome. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63586/63586fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The diverse proteomic data serve as valuable information for the assessment of function. The proteomic data can be evaluated using canonical knowledge bases. As shown in Figure 4B, pathway analysis of dysregulated proteins showed overrepresented translation and energy metabolism in both the SO and the NE datasets in our recent studies. The NE proteome was enriched in proteins associated with nuclear transport of protein cargo in the cell, likely indicating downstream events following signaling in the newly established NE (Figure 4C). The enrichment analysis in Figure 4D found upregulation in translation initiation, RNA-binding, binding in the proteasome complex, suggesting a role for dynamic protein turnover developing SO. Cell lineage-guided HRMS proteomics is scalable in space and time and sufficiently sensitive to help better understand the molecular organization of cells during normal and impaired development.
Supplementary File. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63586/supplemental file.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo at JoVE.com

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

cell-lineage-guided-mass-spectrometry-proteomics-developing-frog

Nanyang Technological University

Research

JoVE Journal

Chemistry

1.6K Views.  University of Maryland. Deze afstammingsgeleide benadering maakt proteomische analyse van belangrijke ontwikkelingsgebeurtenissen met ruimtelijke en temporele resolutie binnen het gewervelde embryo mogelijk, waardoor informatie wordt verstrekt die niet toegankelijk zou zijn via metingen van het hele embryo. Deze aanpak kan gemakkelijk worden uitgebreid om eiwitten, metabolieten en transcripties van verschillende embryonale cellen te bestuderen terwijl ze differentiëren tijdens de ontwikkeling. Deze methode kan ons ...

Video: Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq&#8212;the most popular approach to investigate chromatin features at a global level&#8212;is outlined here for Xenopus embryos.

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

Developmental Biology

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Medicine

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Biology

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

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