Single blastomere expression profiling of Xenopus laevis embryos of 8 to 32-cells reveals developmental asymmetry

Flachsová, Monika; Šindelka, Radek; Kubista, Mikael

doi:10.1038/srep02278

Cited by 30 publications

(31 citation statements)

References 32 publications

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“…We find that D11, V11, and V21 blastomeres exhibit characteristic smallmolecular activity that reproducibly contributes to their commitment to neuronal, epidermal, and hindgut tissues in the adult organism, as validated by microinjection and cell-fate tracing experiments. The metabolomic differences uncovered in this work complement recent transcriptomic asymmetry (9) in the animalvegetal axis of the early embryo and provide previously unidentified findings for asymmetry in the dorso-ventral axis, underscoring the importance of performing metabolic measurements on the level of the single cell. These small-molecular differences between single embryonic cells and different regions of the embryo's body have not been technologically discernable by classical, cell-averaging measurements thus far.…”

Section: Discussionsupporting

confidence: 60%

Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo

Onjiko

Moody

Nemes

2015

Proc. Natl. Acad. Sci. U.S.A.

178

239

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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.single cell | mass spectrometry | metabolomics | embryo development | Xenopus T he ability to understand the basic mechanisms that regulate embryonic development requires knowledge of the complete suite of biomolecules expressed by each cell in the organism. Although technological advances in single-cell isolation, genome sequencing, and transcriptome analyses have made it possible to determine spatial and temporal changes for large molecules (1, 2), little is known about small-molecular events that unfold in the individual cells (blastomeres) of the embryo. In many animals, mRNAs and proteins synthesized during oogenesis are sequestered to different cytoplasmic domains, which after fertilization then specify the germ-cell lineage and determine the anteriorposterior and dorsal-ventral axes of the embryo. For example, in the South African clawed frog (Xenopus laevis), several mRNAs are localized to the animal pole region (Fig. 1), which later gives rise to the embryonic ectoderm and the nervous system (3), whereas VegT mRNA localization to the vegetal pole specifies endoderm formation (4), and region-specific relocalization of the Wnt and Dsh maternal proteins govern the dorsal-ventral patterning of the embryo (5). However, there is abundant evidence that in developing systems not all transcripts are translated into proteins, and not all proteins are active; therefore, analyses of the mRNAs and proteins within single cells may not reveal...

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Section: Discussionsupporting

confidence: 60%

Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo

Onjiko

Moody

Nemes

2015

Proc. Natl. Acad. Sci. U.S.A.

178

239

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“…The full molecular composition of the cytoplasmic determinant and the mechanism by which β-catenin is stabilized remain unknown although the participation of maternal Wnt11 and Dvl proteins, perhaps in combination with the formation of multivesicular bodies, has been proposed (22,38). It is likely that the elusive cytoplasmic determinant is protein in nature because recent RNA-seq analyses of early cleavage stages (eight-cell) of X. laevis or X. tropicalis embryos have not detected any asymmetrically expressed mRNAs along the D-V axis (39,40). The early β-catenin stabilization, in combination with zygotic Siamois and Nodal signaling, induces Spemann organizer formation at the gastrula stage (11).…”

Section: Discussionmentioning

confidence: 99%

Spemann organizer transcriptome induction by early beta-catenin, Wnt, Nodal, and Siamois signals in Xenopus laevis

Ding

Ploper

Sosa

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The earliest event in Xenopus development is the dorsal accumulation of nuclear β-catenin under the influence of cytoplasmic determinants displaced by fertilization. In this study, a genome-wide approach was used to examine transcription of the 43,673 genes annotated in the Xenopus laevis genome under a variety of conditions that inhibit or promote formation of the Spemann organizer signaling center. Loss of function of β-catenin with antisense morpholinos reproducibly reduced the expression of 247 mRNAs at gastrula stage. Interestingly, only 123 β-catenin targets were enriched on the dorsal side and defined an early dorsal β-catenin gene signature. These genes included several previously unrecognized Spemann organizer components. Surprisingly, only 3 of these 123 genes overlapped with the late Wnt signature recently defined by two other groups using inhibition by Dkk1 mRNA or Wnt8 morpholinos, which indicates that the effects of β-catenin/ Wnt signaling in early development are exquisitely regulated by stage-dependent mechanisms. We analyzed transcriptome responses to a number of treatments in a total of 46 RNA-seq libraries. These treatments included, in addition to β-catenin depletion, regenerating dorsal and ventral half-embryos, lithium chloride treatment, and the overexpression of Wnt8, Siamois, and Cerberus mRNAs. Only some of the early dorsal β-catenin signature genes were activated at blastula whereas others required the induction of endomesoderm, as indicated by their inhibition by Cerberus overexpression. These comprehensive data provide a rich resource for analyzing how the dorsal and ventral regions of the embryo communicate with each other in a self-organizing vertebrate model embryo.

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“…The ‘inputs’ in this GRN represent the TFs and signaling molecules (transduced via intracellular TFs) important for mesendoderm formation and early endoderm patterning, many of which have been elucidated [8,9]. Recently, genome-wide approaches have identified additional localized maternal and zygotic transcripts encoding transcription factors [10-16]. Based on a comprehensive catalogue of the X. tropicalis TFs [14], 130 TFs are found to be enriched vegetally (in comparison to the animal pole), and we have focused on the ∼50 TFs expressed at relatively high abundance in the vegetal tissue (Transcripts Per Million values ≥ 50).…”

Section: Generation Of the Mesendoderm Gene Regulatory Networkmentioning

confidence: 99%

A gene regulatory program controlling early Xenopus mesendoderm formation: Network conservation and motifs

Charney

Paraiso

Blitz

et al. 2017

Seminars in Cell & Developmental Biology

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Germ layer formation is among the earliest differentiation events in metazoan embryos. In triploblasts, three germ layers are formed, among which the endoderm gives rise to the epithelial lining of the gut tube and associated organs including the liver, pancreas and lungs. In frogs (Xenopus), where early germ layer formation has been studied intensively, the process of endoderm specification involves the interplay of dozens of transcription factors. Here, we review the interactions between these factors, summarized in a transcriptional gene regulatory network (GRN). We highlight regulatory connections conserved between Xenopus, zebrafish, mouse, and human endodermal lineages. Especially prominent is the conserved role and regulatory targets of the Nodal signaling pathway and the T-box transcription factors, Vegt and Eomes. Additionally, we highlight some network topologies and motifs, and speculate on their possible roles in development.

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Single blastomere expression profiling of Xenopus laevis embryos of 8 to 32-cells reveals developmental asymmetry

Cited by 30 publications

References 32 publications

Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo

Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo

Spemann organizer transcriptome induction by early beta-catenin, Wnt, Nodal, and Siamois signals in Xenopus laevis

A gene regulatory program controlling early Xenopus mesendoderm formation: Network conservation and motifs

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