Proteomic Analysis of Differences in Ectoderm and Mesoderm

Article pubs.acs.org/jpr

Proteomic Analysis of Differences in Ectoderm and Mesoderm Membranes by DiGE Renee Wang, Xiaoyong Liu, Elke Küster-Schöck, and François Fagotto* Department of Biology, McGill University, Montreal, Canada S Supporting Information *

ABSTRACT: Ectoderm and mesoderm can be considered as prototypes for epithelial and mesenchymal cell types. These two embryonic tissues display clear differences in adhesive and motility properties, which are phenomenologically well characterized but remain largely unexplored at the molecular level. Because the key downstream regulations must occur at the plasma membrane and in the underlying actin cortical structures, we have set out to compare the protein content of membrane fractions from Xenopus ectoderm and mesoderm tissues using 2-dimensional difference gel electrophoresis (DiGE). We have thus identified several proteins that are enriched in one or the other tissues, including regulators of the cytoskeleton and of cell signaling. This study represents to our knowledge the first attempt to use proteomics specifically targeted to the membrane-cortex compartment of embryonic tissues. The identified components should help unraveling a variety of tissue-specific functions in the embryo.

KEYWORDS: Gastrulation, Morphogenesis, Cell migration, Cell adhesion, Cytoskeleton, Xenopus

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In a first attempt toward addressing this question, we have used cell fractionation and difference gel electrophoresis (DiGE)9,10 on Xenopus embryo tissues. Xenopus is a particularly well suited model for such investigation, since the specific tissues can be unambiguously identified and easily dissected in large amounts. We have set to prepare fractions enriched in membrane and cytoskeletal proteins from microdissected ectoderm and mesoderm tissues and to compare them by DiGE, and we have identified several proteins reproducibly enriched in one or the other tissue. In the second part of this study, we expanded this approach to investigate tissues artificially induced to a mesoderm fate by expression of FGF or Activin in the prospective ectoderm. The goal of this experiment was to eliminate parameters depending on the position of the tissues, in particular the influence of maternally inherited prelocalized components, and to isolate changes specifically due to mesoderm-inducing signals. Note that with this particular experimental design, all dissected tissues, whether normal ectoderm or induced mesoderm, included the polarized outer cell layer, and thus this feature should not contribute to proteomic differences. A second interest of this approach was to detect common and distinct effects of FGF and TGFβ signals on cell properties. These two pathways are known to produce qualitatively different mesoderm tissues: the original findings indicated that FGF could only induce a subset of markers considered to correspond to ventral-type mesoderm, while Activin could induce the whole range of mesoderm (and endoderm) markers and

INTRODUCTION

Primary embryonic germ layers display strikingly different behaviors: ectoderm cells have strong cell−cell adhesion and the capacity to set a basolateral polarity, but low motility, whereas mesoderm cells bind more efficiently to the extracellular matrix but less to other cells, migrate very actively, and remain usually nonpolarized.1−8 These represent the very same characteristics that distinguish the classical “epithelial” and “mesenchymal” cell types in all animals and in human. While there is a large body of knowledge about the molecular composition and cell organization of differentiated epithelia and mesenchymal cells, it is difficult to distinguish those traits that reflect the fundamental properties defining epithelial and mesenchymal cells from those that result from final differentiated states of particular cell types. We believe that much could be learnt from the early embryo, where cells have just undergone these basic changes in cell behavior but are still otherwise undifferentiated. We indeed postulate that early ectoderm and mesoderm tissues are largely identical in molecular composition and cellular properties, except for the ones immediately involved in the critical functions of these two tissues, i.e., the signaling/transcriptional circuitry that controls cell fate determination and the above-mentioned differences in morphogenetic functions. Such structural, adhesive, and migratory differences must be reflected at the molecular level in the composition and organization of the plasma membrane and of the underlying cytoskeletal cortex. They may not necessarily correspond to changes in expression levels but rather may rely on local recruitment and/or posttranslational modifications. © 2012 American Chemical Society

Received: April 20, 2012 Published: August 1, 2012 4575

dx.doi.org/10.1021/pr300379m | J. Proteome Res. 2012, 11, 4575−4593

Journal of Proteome Research

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Figure 1. Source and nature of embryonic tissues used in this study. (A) Diagram of early Xenopus gastrula indicating the dissected ectoderm and mesoderm tissues. (B) Schematic representation of induction of various mesoderm/endoderm markers by increasing levels of Activin (arbitrary scales). (C) Diagram of the experimental scheme for preparation of induced tissues. Animal explants were dissected at blastula stage to ensure that the exact same cell population was analyzed in all conditions, since at the onset of gastrulation induced cells that have acquired mesoderm/endoderm fate may change position within the embryo. Explants were then cultured until early gastrula stage. (D) RT-PCR analysis of animal explants. Embryos injected with Activin (A) or FGF (F) mRNA, which induced, respectively, mesendoderm (expressing Xgsc, Sox17, and low levels of Xbra) and ventral mesoderm (expressing only Xbra). C = control animal explants. Asterix indicates nonspecific bands.

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structures, roughly in a dose-dependent manner, from ventralposterior mesoderm at low doses, to trunk mesoderm, then anterior mesoderm and endoderm with increasing doses (Figure 1B,D).11−21 However, the in vivo picture is more complex, and the classical “dorsal-ventral” organization of the mesoderm has been revised into a more subtle ensemble of regionalized subtypes of mesoderm that seem to correspond to regions under the control of FGFs, TGFβs, and both,22−26 consistent with the fact that these activities have distinct but overlapping spatial patterns.27 Some mesoderm genes appear to depend uniquely or mostly on FGF signaling, others on Activin/TGFβ signaling, and still others require both.11,16,17,28−32 In addition, there are multiple positive and negative cross-interactions between these pathways at various levels.16,17,32,33 Little is known about the cellular mechanisms that differentiate the behavior of the different types of mesoderm. Some typical properties of the mesoderm, such as formation of lamellipodia, bipolarity, and migration, can be induced by both FGFs and TGFβs, while others, such as strong adhesion to fibronectin, appears to be a specific effect of TGFβ signaling.34 Our experimental design has thus the potential to explore at the proteomic level possible common and specific features of cells reacting to these two factors and teach us about the cellular mechanisms underlying the morphogenetic properties of the mesoderm.

EXPERIMENTAL PROCEDURES

Antibodies

Antibodies used were rabbit α-C-Cadherin and mouse α-Ccadherin 5G5 (both gifts from B. M. Gumbiner), rabbit α-βCatenin P14L,27 mouse anti-eGFP (Invitrogen), mouse antitubulin E7 (DSHB), mouse anti-actin (Sigma.), mouse anticytokeratin (DSHB), rabbit anti-cofilin and P-cofilin (gift from J.Bamburg), rabbit anti-FatVg (gift from M.Kloc35), rabbit antiGDI (Santa-Cruz Technologies), anti-RBB7 (gift from Dr. Wang36), EP45 (gift from Drs. Kirschner and Jorgensen37), and rat anti-RPSA/LAMR (gift from V. B. Loktev and G. G. Karpova38). DNA Constructs

myc-tagged GFP,39 myc-tagged-GAP43-GFP,8 mito-GFP,40 and C-cadherin-GFP were in pCS2+. Xenopus Embryo Manipulations

mRNA was microinjected in the animal side at the 2-cell stage (5 nL/blastomere). Embryos were collected at stage 10.5−11.41 Cell Fractionation

All steps were carried out at 4 °C. For each experimental replicate, 40 whole embryos, 40 ectoderm explants, and 40 dorsal mesoderm explants were dissected from stage 10.5 embryos obtained from one female. The tissues were homogenized in 100 μL of homogeneization buffer (250 mM sucrose, 10 mM Hepes-NaOH pH 7.4, 2 mM MgCl2, 2 mM 4576



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Na-vanadate, 1 mM Na-EDTA, 1 mM Na-molybdate, and 0.2 μM okadaic acid, supplemented with a cocktail of protease inhibitors) using a 1 mL Dounce homogenizer with 10 strokes on ice. Crude homogenates were carefully laid on top of a 200 μL sucrose cushion (1 M sucrose, 10 mM Hepes-NaOH pH 7.4, 2 mM MgCl2, 2 mM Na-vanadate, 1 mM Na-EDTA, 1 mM molybdate, and 0.2 μM okadaic acid) in 1.5 mL microcentrifuge tubes and centrifuged for 10 min at 1000 rpm (100g). Four fractions were collected and analyzed (Figure 2). The top layer (fraction 1, ∼90 μL) was transferred to a clean microcentrifuge tube, and centrifuged for 10 min at 13,200 rpm (16,100 g). The resulting supernatant was carefully removed, leaving the bottom part of the tube (∼20 μL), including the pellet and some

supernatant, undisturbed. This fraction was used for the DiGE analysis: The pellet was then resuspended with 20 μL 2x Laemmli SDS-PAGE buffer for Western blot) or 2D ureacontaining lysis buffer (30 mM TrisHCl pH 8.5, 8 M urea, 4% CHAPS, 10 mM DTT) for DiGE. In experiment 2 (Figure 6B), a variant of this protocol was used, aimed at further cleaning the enriched fraction: The supernatant (fractions 1−2) was laid again on a sucrose cushion and spun for 10 min at 1000 rpm. The same step was repeated a third time. The resulting supernatant was centrifuged for 10 min at 13,200 rpm (as in the original), the pellet was then resuspended in 100 μL of homogeneization buffer, centrifuged, resuspended and centrifuged again a third time, and finally resuspended in 2D lysis buffer. A 4 μL portion was taken for protein concentration determination (Bradford). The typical protein concentrations in membrane fractions were 4 mg/mL for whole embryo sample, 2−3 mg/mL for ectoderm, and 1.5−2 mg/mL for mesoderm. Samples were stored at −80 °C. Immunoblots Analysis

The equivalent of 3 embryos, 20 ectoderms, and 30 mesoderms were loaded per lane on 10% SDS−polyacrylamide gels and electrophoretically separated and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk in PBS + 0.1% Triton X100 and probed with appropriate antibodies. Detection was done with enhanced chemiluminesence (ECL plus Immunoblotting Detection System, Amersham Bioscience/GE Healthcare). Difference Gel Electrophoresis (DiGE)

The mixed internal standard methodology10 was used according to the manufacturer’s instructions (GE Healthcare) with the following modifications. Each experiment contained two or three sample groups (ectoderm vs mesoderm, uninjected vs FGF- or Activin-injected) repeated in triplicates/quadruplicates, yielding six to twelve experimental samples. Twenty-five micrograms (experiment 1a+b) or 40 μg (experiment 2) of each sample was labeled with either Cy3 or Cy5, using a dye-swap approach that avoids labeling all samples in one group with the same dye. Equal aliquots of each sample in one experiment were mixed to give a pooled internal standard. For each gel, 25 μg/40 μg of the standard was labeled with Cy2 and combined with one Cy3- and one Cy5-labeled experimental sample. The mixtures were brought up to final volume of 250 μL (experiments 1a+b) or 450 μL (experiment 2) with 1X sample buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.002% bromophenol blue, 50 mg/mL DTT, 2% IPG buffer pH3−11NL (GE Healthcare) and loaded by passive rehydration onto 13 cm (experiments 1a+b) or 24 cm (experiment 2) pH3−11NL Immobiline IPG strips (GE Healthcare). Isoelectric focusing was performed according to the manufacturer’s recommendations. Following IEF, strips were equilibrated for 15 min each, first into equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue) with 10 mg/mL DTT, then with equilibration buffer supplemented with 25 mg/mL iodoacetamide. Second dimension SDS-PAGE was performed on hand-cast 10% SDS-PAGE gels using low fluorescence glass plates. The differentially labeled co-resolved proteome maps within each gel were imaged at 100 μm resolution separately by dye-specific excitation and emission wavelengths using a Typhoon trio+ Variable Mode Imager (GE Healthcare). After imaging for CyDye components, the low fluorescence glass plates were

Figure 2. Preparation of membrane enriched fractions from embryonic tissues. (A) Analysis of the cell fractions. Four fractions were collected from a low speed centrifugation through a sucrose layer. The top fraction 1 was further centrifuged at higher speed to separate soluble from insoluble components. The pellet 1P was used for the DiGE experiments. The distribution of GFP-fusions targeted to various subcellular compartments was analyzed by immunoblot. The markers were GFP (cytosol), C-cadherin-GFP and membrane-GFP (plasma membrane), C-cadherin and β-catenin (endogenous cell membrane), mitoGFP, i.e., GFP fused to a signal sequence targeting to the outer mitochondrial membrane (mitochondria), actin, tubulin, and cytokeratin (cytoskeletal markers). (B) Amounts of yolk protein were determined on Coomassie-stained SDS-PAGE gels, and DAPI-stained nuclei were counted under a dissecting microscope. 4577



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removed, the gels were fixed in 40% methanol, 5% acetic acid for 60 min, and then silver stained for spot picking.

points of phosphorylated proteins were calculated using the Scansite software (http://scansite.mit.edu/calc_mw_pi.html).

DiGE Analysis and Quantification

RT-PCR Analysis

The gel images were analyzed using the software DeCyder v6.5 (GE Healthcare). The Differential In-gel Analysis (DIA) module was used to define the spot positions and normalize the value from a Cy3- or Cy5-labeled spot by forming the ratio over the corresponding Cy2 signal from the pooled internal standard. On the basis of the assumption that the majority of spots will not change between experimental samples, all Cy3/ Cy5 ratios were then standardized by fitting to a Gaussian curve that was centered around no change. The DIA data sets for all gels of one experiment were then collectively analyzed using the Biological Variation Analysis (BVA) module, which allowed for the matching of protein spot patterns across gels, and statistical analysis of matched spots. Statistical significance was assigned using Student’s t test and analysis of variance (ANOVA) analysis that compare the variation of expression within a group to the magnitude of change between groups. Principal component analysis (PCA) was performed using the Extended Data Analysis (EDA) DeCyder module.

Total RNA was prepared from embryos using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription (RT) was performed with 1 μg of RNA using Revert Aid H Minus M-MuLV Reverse Transcriptase (MBI Fermentas). One-twentieth of the RT mixture was used as templates in 25 μL PCR reactions (Taq-Polymerase, Invitrogen). Sequences of primers used for PCR analysis with the number of cycles in brackets (upstream primer listed first): XSox17α, 5′-GGACGAGTGCCAGATGATG-3′ and 5′CTGGCAAGTACATCTGTCC-3′ (25 cycles); goosecoid, 5′ACAACTGGAAGCACTGA-3′ and 5′-TCTTATTCCAGAGGAACC-3′ (27 cycles); Xbra, 5′-CACAGTTCATGCAGTGACCG-3′ and 5′-TTCTGTGAGTGTACGGACTGG-3′ (27 cycles). The annealing temperatures were 55 °C, except for ODC and Xbra (65 °C). The products were then analyzed on 1% agarose gels. Immunofluorescence

Immunofluorescence was performed on cryosections from paraformaldehyde (PFA)-fixed, gelatin-embedded embryos as previously described.27,45 For RGI staining, samples were fixed with 1% PFA, and for the other stainings with 4% PFA.

In-Gel Digestion, Mass Spectrometry, and Data Analysis

Twenty-four centimeter preparative 2D gels were loaded with 100 μg of protein from membrane fractions. They were silver stained42 to locate the spots of interest. Sample handling and mass spectrometry were carried out at the Proteomics Platform of the McGill University/Genome Quebec Innovation Centre. Proteins of interest were excised and digested with trypsin on a Mass Prep Robotic Workstation (PerkinElmer). Proteins were subjected to in-gel digestion with trypsin (Promega) using an automated MassPrep Workstation (Micromass). Extracted peptides were injected onto a Zorbax C18 (Agilent) desalting column and subsequently chromatographically separated on a Biobasic C18 Integrafrit column (New Objective), using a nano high-performance liquid chromatography system (1100 Series unit; Agilent). Eluted peptides were analyzed on a Q-ToF mass spectrometer (Waters). Individual sample tandem mass spectrometry spectra were peak listed using Distiller version 2.3.2.0 (Matrix Science) software with peak picking parameters set at 5 SRN and 0.4 CT. The peak-listed data was then searched against a copy of the Uniprot database by using Mascot version 2.3.01 (Matrix Science) and X! Tandem (http://www.thegpm.org version 2007.01.01.1). Mascot and X!Tandem were set up to search the Xenopus laevis (Taxonomy ID: 8355) database (release of September 17, 2010; 15,643 protein entries), assuming the digestion enzyme trypsin, a fragment ion mass tolerance of 0.8 Da, and a parent ion mass tolerance of 1.5 Da. Iodoacetamide derivative of cysteine was specified in both search engines as a fixed modification. Oxidation of methionine was specified in Mascot as a variable modification, whereas Pyro-glu from Q of glutamine, deamidation of asparagine, and oxidation of methionine were specified in X! Tandem as variable modifications. Scaffold (Proteome Software Inc.) was used to validate MS/ MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm.43 Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 1 identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm.44 Theoretical isoelectric

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RESULTS

Analysis of Differences between Endogenous Ectoderm and Mesoderm and MS Identification

Because we wanted to focus our analysis on membrane and cytoskeletal components, we developed a simple protocol for the enrichment of these elements from small amounts of Xenopus embryonic tissues. Crude tissue homogenates were layered on top of a sucrose cushion. After a short low speed centrifugation, nuclei and yolk platelets had sedimented through the cushion. The supernatant was then centrifuged at higher speed to pellet heavy/large membranes (Figure 2A). The enrichment of this pellet was estimated using several markers, including endogenous proteins and GFP fusions targeted to specific compartments (Figure 2B). Cadherin and myristylated-GFP, which specifically inserts into the plasma membrane,8 were strongly enriched in the pellet. This fraction also contained most of the mitochondria and intermediate filaments (cytokeratin), together with a smaller fraction of actin and tubulin. GFP, used as a marker for soluble proteins, was efficiently excluded. Using this protocol, we prepared a membrane-enriched fraction from dissected ectoderm and mesoderm tissues (Figure 1A) and compared their protein composition by DiGE (Figure 3A). We performed two independent DiGE experiments, called experiments 1a and 1b for the rest of the text, each of them analyzing ectoderm and mesoderm membrane fractions obtained from three embryo batches. We ranked the spots of both experiments according to the statistical significance of the observed changes between ectoderm and mesoderm. In selecting candidates for further analysis and protein identification, we eliminated spots that were very faint, only found in two out of three gels, or located in poorly resolved areas of the gels. The 19 spots that were eventually selected for MS on the basis of their low Student’s t test p-value of ±1.2 in one of the two experiments (10 spots), the second value was always similar if not virtually identical. The variability was particularly low in experiment 1b, with all matches but one showing a pvalue 1.3 times enriched (see Table 1), included AnnexinIV (a calcium and lipid binding protein55,56), the WD repeat domain 77 protein (a component of the methylosome57), chitobiase (a secreted glycosidase present in cortical granules58), peroxiredoxins (redox reactions), and GDI (a GDP-GDP dissociation inhibitor involved in the release of Rab proteins from membranes59). We also identified FatVg/perilipin2 and perilipin-like protein, a family of proteins known to accumulate at the periphery of fat lipid droplets and regulate lipid storage homeostasis.60 The two proteins have virtually identical sequences and are likely “pseudo-alleles” as often found in Xenopus laevis.61 Finally, a series of spots corresponded to the protease inhibitor EP45/ Seryp, a secreted protein that accumulates in yolk platelets.37 Multiple EP45 isoforms had been previously reported.62 Analysis of Differences between Ectoderm and Activin/FGF-Induced Mesoderm

We similarly analyzed membrane fractions from “ectodermal” animal caps dissected from uninjected embryos and from embryos injected with FGF or Activin mRNA (Figure 1C). The mRNA were titrated and the tissues analyzed by RT-PCR for classical markers (Figure 1D), in order to obtain the expected patterns for, respectively, FGF-induced ventral-posterior mesoderm (high Xbra, no Goosecoid, no Sox17) or for 4581



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Figure 5. Scattered plot of spot intensities for FGF/Activin-induced explants. Besides the spots found to be significantly different by DiGE, additional neighboring spots (B2b, B2c) corresponding to related isoforms (probably differing in terms of posttranslational modifications) are also included for comparison’s sake. Circles correspond to individual values, crosses to average values. Lines connect average values to illustrate trends.

Validation of Subcellular Localization and Tissue Distribution by Immunofluorescence

The strongest signal was observed around the blastopore lip, i.e., the mesoderm undergoing involution. P-cofilin, on the contrary, was strongly enriched in the ectoderm and could barely be detected in the involuted mesoderm (Figure 7B). FatVg has been reported to localize to cortical granules in the oocyte.35 Using the antibody used by Chan et al. in their original report, we did not detect in the gastrula more than a few spots that might correspond to such granules, but rather a strong nuclear signal and a sharp cell peripheral staining closely coinciding with the plasma membrane (Figure 8). This cell peripheral staining was almost exclusively observed in the mesoderm and was strongest in the involuted mesoderm. The nuclear signal was widespread throughout the embryo and may be due to nonspecific binding to this protein-dense organelle, as observed for many antibodies (unpublished observations).

To validate the tissue enrichment of a few candidates, we used immunofluorescence as a completely independent technique. We obtained antibodies recognizing the Xenopus forms of six proteins, cofilin, FatVg, GDI, RBB7, EP45, and RPSA/LAMR. We failed to detect Annexin IV71 and hspa8.72 All antibodies recognized on immunoblots single bands or doublets with the expected apparent size (Supplementary Figure S5). We used two antibodies for cofilin, one detecting total cofilin and one specific for the phosphorylated form.73,74 Both labeled cell peripheries, consistent with a cortical localization (Figure 7). The two antibodies produced two perfectly complementary patterns: total cofilin was found in all tissues but was more abundant in the mesoderm than in the ectoderm (Figure 7A). 4582



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Table 2. Summary Table of DiGE Experiment 2 (FGF/Activin)a

a

Each protein is presented with its most common names and abbreviations, the average intensity ratios between FGF-induced or Activin-induced and uninjected control, the reported general subcellular localization and cellular functions, as well as localization and loss-of-function phenotype in Xenopus embryos whenever available. The color code indicates proteins significantly enriched (orange to red) or depleted (light to dark green) in induced tissues. Numbers correspond to citation of published references. Additional information can be found in the Supporting Information.

Immunodetection of GDI (Figure 9) gave a characteristic subcellular pattern of bright cytoplasmic spots (arrowheads). Some finer signal was observed along the plasma membrane (arrows). This pattern was consistent with the known function of GDI in regulating vesicular trafficking. We thus thought to compare it with markers of the endocytic compartment. In the absence of antibodies raised in different species, we could not perform double staining, but we did observe very similar intracellular puncta by staining contiguous sections from the same embryos for the lysosomal marker Lamp1 (Supplementary Figure S2). Lamp1 spots were less abundant in the cell periphery, a region where one rather expects early endosomes. We have not yet found adequate antibodies to detect this compartment in Xenopus. Interestingly, in terms of tissue distribution, Lamp1-positive puncta did not show any obvious enrichment between the three germ layers, whereas GDIpositive puncta were strikingly asymmetrically distributed, rare in the ectoderm, numerous in the mesoderm, and most

abundant in the anterior mesendoderm (Figure 9) and endoderm (Supplementary Figure S2). GDI staining increased gradually around the blastopore lip (data not shown), suggesting that it progressively appeared as mesoderm was passing through the lip. The antibody against RSPA/LAMR 38 recognized an accumulation of spots mostly confined to the cell periphery (Figure 10), but in a broader pattern than would be expected for a localization purely to the plasma membrane. The signal was strongest in a restricted area of the ectoderm corresponding to the prospective neuroderm (Figure 10E). Some weak staining was observed in the mesoderm (Figure 10F), but none in the animal ectoderm (Figure 10D) nor on the ventral side (not shown). Because this pattern was reminiscent of the region where FGF signaling is active in the early gastrula,27,32 we also examined a pregrastrula (late blastula) stage. The signal at this stage was generally stronger and peaked all around a circumferal supra-equatorial region 4583



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Figure 6. Global analysis of ectoderm/mesoderm differences. (A) Summary of subcellular distribution of all identified proteins. See text for details. (B,C) Principal component analysis. (B) PCA plot of experiment 2 spot maps, base set (all spots that are found in at least 75% of spot maps), 897 proteins. Clusters reflect sample groups (frog mothers). PC1 and PC2 account for only 54.7% of variance. (C) PCA plot of experiment 2 spot maps, “ANOVA 0.1” set (all spots of the base set that have an ANOVA score of ≤0.1), 17 proteins. Clusters reflect experimental groups. PC1 and PC2 account for 84.8% of variance. (D) Summary of the number of spots with statistical differences detected between mesoderm and ectoderm and between FGF/Activin-induced and control tissues. See Supplementary Table S5 for more details. The Venn diagrams show the number of spots with p-values less than 0.03 calculated on the basis of Student’s t test (one tailed), either considering all samples as independent or paired based on the embryo batch, as well as the number of spots that showed significance in both tests, and for experiment 2, the number of cases where significant differences where found for both FGF- and Activin-induced tissues. Also indicated are the subgroups of spots identified by mass spectroscopy (MS).

involved “sedimentable” components (plasma membrane, organelles or cortical structures).

(Figure 10B), again coinciding with the pattern of active FGF signaling,27,32 fully consistent with enrichment of RPSA/LAMR in FGF-injected tissue (Table 2 and Figure 5). In agreement with the original report,37 the anti-EP45 antibody decorated all yolk platelets with a regular ring (Supplementary Figure S4). The mesoderm enrichment detected by DiGE could be readily accounted for by the larger size and number of platelets. The anti-RBB7/RbAp46 antibody gave a strong nuclear staining in all tissues (data not shown), consistent with the expected nuclear function of this protein. We did not find evidence for a cytoplasmic/plasma membrane pool. In summary, in all cases but RBB7, the immunofluorescence data largely matched the tissue distribution inferred from the DiGE experiments and confirmed that these differences indeed

General Analysis of Tissue Proteomic Differences: Principal Component and Pairwise Analyses

To extract global information about the properties of different embryonic tissues, we performed multivariate statistics on our DiGE data. Principal component analysis (PCA) allows looking at relationships between points within a data set in an unbiased way. It “rotates” the ensemble of data points to produce a description of the data as “principal components” that contain the maximal variance between the points. It is graphically represented as a 2D projection where one axis, the first component or PC1, contains the maximal variance, and the second orthogonal axis PC2 contains the second highest variance that is fully uncorrelated with PC1. This type of 4584



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Figure 8. Immunofluorescence validation of FatVg tissue distribution and cellular localization. (A−C) Representative fields in the dorsal region of a gastrula embryo. (A) Anterior mesoderm. (A′) Enlarged view of mesodermal cells showing the details of subcellular localization. (B) Dorsal ectoderm (prospective neuroderm). (C) Lower part of the boundary and lip region. The FatVg antibody showed a strong, likely nonspecific, reaction with all nuclei. Sparse punctuate cytoplasmic staining was observed (arrowheads), and prominent accumulation at the cell periphery (arrows). This peripheral staining closely coincides with the weak continuous cadherin staining, suggesting that FatVg may interact with the plasma membrane or the submembrane cortex, but not with the cadherin spots corresponding to adhesive clusters. Dotted line: ectoderm-mesoderm boundary.

Figure 7. Immunofluorescence validation of cofilin tissue distribution and cellular localization. (A,B) General view of the dorsal region of a gastrula embryo. Cryosections of wild type embryos were immunostained with antibodies recognizing total (A) and phosphorylated cofilin (B). Plasma membranes were stained with anti-cadherin antibody (green), and nuclei with Hoechst (blue). (A) Total cofilin signal is strongest in the blastopore lip region of the mesoderm (meso), corresponding to the region of most active cell migration (asterisk). (B) P-cofilin signal is much stronger in the ectoderm (ecto) compared to the mesoderm and endoderm (endo). A gradual decrease is observed along the non-involuted mesoderm (non-inv meso), where mesodermal properties are thought to be progressively acquired. (A′, B′) Enlarged views of the lower part of the ectoderm/mesoderm boundary, just above the lip. (A′) Cofilin staining spans through the whole cell with some enrichment at cell peripheries. Accumulation is also observed along membranes in contact with the ectoderm (arrows). (B′) P-cofilin strongly accumulates along the plasma membrane, with prominent concentrations at the apical adherens junctions (arrowheads). It is very weak along the basal side in contact with the mesoderm (arrows).

Activin treatments. We performed two series of PCA analysis, one on the whole set of spots that were matched across the experiment’s gels (Figure 6B), and the second on the subset of 17 spots selected for their statistically significant different enrichments (Figure 6C). PCA for the whole data sets revealed an interesting trend: although the two principal components taken together would account for only about half of the variance, the samples in all three experiments were clustered remarkably into their respective embryo batches, suggesting

analysis is particularly informative when several conditions are compared, such as in experiment 2 between controls, FGF and 4585



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Figure 9. Immunofluorescence validation of GDI tissue distribution and cellular localization. (A) Representative field in the dorsal region of a gastrula embryo. (B) Enlargement of part of this field. Arrowheads and arrows point to the same structures in all panels. Arrowheads indicate bright cytoplasmic spots, arrows weaker plasma membrane signal. Bright GDI spots are more abundant in the endoderm and in the mesoderm than in the ectoderm.

that on the whole resolved proteome the major variable is the individual variability between mother frogs. On the other hand, when the 17 spots with ANOVA values

Proteomic Analysis of Differences in Ectoderm and Mesoderm

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