Remodeling of the Metabolome during Early Frog Development

Vastag, Livia; Jorgensen, Paul; Peshkin, Leonid; Wei, Ru; Rabinowitz, Joshua D.; Kirschner, Marc W.

doi:10.1371/journal.pone.0016881

Cited by 62 publications

(79 citation statements)

References 46 publications

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“…Several of the small molecules were shared among the pathways, creating nodes in the network. In agreement, recent experiments on whole Xenopus embryos eloquently demonstrated alanine to be the energy source that rapidly converts to aspartate and glutamate, which then fluxes to the synthesis of multiple amino acids including valine, proline, and glutamine (12). Thus, our measurements captured an information-rich snapshot of small-molecule activity in the blastomeres, providing data to complement already available transcriptomic and some proteomic information in single blastomeres and metabolic and proteomic expression in the whole embryo of Xenopus (11,12).…”

Section: Resultssupporting

confidence: 60%

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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.

175

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: Resultssupporting

confidence: 60%

“…S3, encompassing nearly all essential and nonessential amino acids, osmolites, and classical neurotransmitters. This level of coverage of the metabolome from a single blastomere in the 16-cell embryo compares favorably with ∼50 metabolites that were recently identified in a whole Xenopus egg and embryo using liquid chromatography MS (12).…”

Section: Resultsmentioning

confidence: 51%

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.

175

239

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“…frog, a vertebrate embryo model amenable to both metabolomic analyses (10) 1 C and D). Notably, a comparison of NADP levels showed that embryos injected with 6xhis-Xtnadk mRNA possessed nearly four times more NADP than eyfp mRNA-injected embryos (Fig.…”

Section: Significancementioning

confidence: 99%

NAD kinase controls animal NADP biosynthesis and is modulated via evolutionarily divergent calmodulin-dependent mechanisms

Love

Pollak

Dölle

et al. 2015

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

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Nicotinamide adenine dinucleotide phosphate (NADP) is a critical cofactor during metabolism, calcium signaling, and oxidative defense, yet how animals regulate their NADP pools in vivo and how NADP-synthesizing enzymes are regulated have long remained unknown. Here we show that expression of Nadk, an NAD + kinase-encoding gene, governs NADP biosynthesis in vivo and is essential for development in Xenopus frog embryos. Unexpectedly, we found that embryonic Nadk expression is dynamic, showing cell type-specific up-regulation during both frog and sea urchin embryogenesis. We analyzed the NAD kinases (NADKs) of a variety of deuterostome animals, finding two conserved internal domains forming a catalytic core but a highly divergent N terminus. One type of N terminus (found in basal species such as the sea urchin) mediates direct catalytic activation of NADK by Ca 2+ /calmodulin (CaM), whereas the other (typical for vertebrates) is phosphorylated by a CaM kinase-dependent mechanism. This work indicates that animal NADKs govern NADP biosynthesis in vivo and are regulated by evolutionarily divergent and conserved CaM-dependent mechanisms. + and reduced form NADPH) is one of the most fundamental metabolic molecules inside cells. NADPH serves essential functions in reductive biosyntheses, detoxification processes (mediated by cytochrome P450), and the regeneration of oxidative defense systems. Moreover, it acts as an electron donor for NADPH oxidases, which generate reactive oxygen species for immune cell respiratory bursts and for redox signaling (1). NADP + can also be converted to NAADP, a potent intracellular calcium messenger (2). However, despite its importance for cellular function, surprisingly little is known about how animals regulate their cellular NADP pools.Earlier work has shown that animals use vitamin B3 to produce NAD, and in vitro studies have established that NAD can be phosphorylated to yield NADP by NAD kinase (NADK) enzymes using ATP as a phosphoryl donor (3-5) (Fig. 1A). However, there have been no in vivo studies examining the function of animal NADK genes, and thus whether NADK is required for sustaining NADP levels in vivo has remained unknown.In addition, the mechanisms governing the regulation of the NADK enzyme are not well-understood. Previous studies, however, have demonstrated a species-dependent differential response of NADK activity following exposure to calcium/calmodulin (Ca 2+ /CaM). For instance, NADK activity in plant extracts is increased by the addition of Ca 2+ /CaM (6). However, none of the three recombinantly expressed NADK isoforms from Arabidopsis thaliana were responsive to Ca 2+ /CaM (7,8). In animals, a Ca 2+ / CaM-dependent rise of NADP following sea urchin egg fertilization was observed more than 30 y ago, although the molecular mechanisms underlying this phenomenon have not been elucidated.We previously identified and characterized the human NADK encoded by the gene Nadk (1, 9), finding its enzymatic activity insensitive to Ca 2+ /CaM. Thus, the interaction between the N...

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“…Since metabolite pools are known to become crucial before gastrulation 21 , we subjected t e × l s hybrid embryos to metabolic profiling at 7 hours post-fertilization (hpf), just prior to the characteristic deformation preceding lysis. Levels of 17 out of 179 metabolites detected were significantly altered (Fig.…”

mentioning

confidence: 99%

Paternal chromosome loss and metabolic crisis contribute to hybrid inviability in Xenopus

Gibeaux

Acker

Kitaoka

et al. 2018

Nature

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Remodeling of the Metabolome during Early Frog Development

Cited by 62 publications

References 46 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

NAD kinase controls animal NADP biosynthesis and is modulated via evolutionarily divergent calmodulin-dependent mechanisms

Paternal chromosome loss and metabolic crisis contribute to hybrid inviability in Xenopus

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