_:b10244431 "were caused by changing cell cycle distributions in the \u223C4000\u20138000 cell embryo at the MBT, since S-phase is when dNTP levels would be expected to be highest if these levels were cell cycle regulated at later stages of development [>>23<<], , but the first cell cycle phase to be extended at the X.laevis MBT is the S-phase [27]." . _:b10244412 . _:b10244444 "Newport and Dasso were unable to repeat the results of Lovtrup and colleagues, who claimed that injecting dNTPs could increase the number of sychronous cleavage cycles in X.laevis [28], [>>30<<]. When we attempted this experiment, we consistently found that injecting dNTPs to 100\u2013300 \u00B5M, or \u223C2.5\u20137.5 fold their endogenous concentration, slowed the cleavage cycles relative to control siblings. The unknown toxic effects of dNTP" . _:b10244447 "As frog eggs have been shown to quickly form nuclei around injected plasmids [33], and frog extracts as well as frog eggs replicate injected plasmid DNA [34], [>>35<<], the limiting dNTP model is compatible with experiments showing that injected plasmids can cause a precocious MBT [36]." . _:b10244453 . . _:b10244437 . . _:b10244435 . _:b10244438 . . _:b10244407 "At least in cultured cells, this anabolic state appears to be sustained primarily by the consumption of glucose and glutamine [>>4<<]." . _:b10244453 "As the orthologs of Chk1 and ATR are required to slow the rapid replication cycles of syncytial Drosophila embryos and to coordinate cell size and cell cycle time in early C.elegans embryos [39], [>>40<<], limiting dNTPs could be a conserved feature of early animal development, possibly including mammals [5]." . _:b455779300 . _:b10244439 . . _:b10244407 . _:b455779274 . _:b10244435 "The 13th cycle is one cycle later than was reported in two studies of cells from dissociated embryos [>>7<<], [8], but is consistent with a previous report in which intact embryos were imaged [6], [28]." . . _:b10244436 "The 13th cycle is one cycle later than was reported in two studies of cells from dissociated embryos [7], [>>8<<], but is consistent with a previous report in which intact embryos were imaged [6], [28]." . _:b10244427 "By this stoichiometry, the nitrogen is roughly accounted for and free ammonium levels would not rise, which is known to be the case [>>19<<]. In this view, the egg is stockpiled with aspartate, which acts as a source of nitrogen for generating glutamine for the embryo. Although we are unsure what the benefit of these changes to the embryo are, as aspartate and glutamine are" . _:b10244420 "Phosphorylation of tyrosine-15 (pY15) in Cdc2 increased 8 hr after fertilization, which marks the onset of the MBT (Figure 1B) [>>12<<], [13]. The time course ends prior to gastrulation (\u223C13 hr post-fertilization)." . _:b10244440 . . _:b10244448 "nuclei around injected plasmids [33], and frog extracts as well as frog eggs replicate injected plasmid DNA [34], [35], the limiting dNTP model is compatible with experiments showing that injected plasmids can cause a precocious MBT [>>36<<]. It has been demonstrated that the ATR-Chk1 replication checkpoint is transiently activated during the MBT [13]." . _:b10244446 "As frog eggs have been shown to quickly form nuclei around injected plasmids [33], and frog extracts as well as frog eggs replicate injected plasmid DNA [>>34<<], [35], the limiting dNTP model is compatible with experiments showing that injected plasmids can cause a precocious MBT [36]." . _:b455779259 . _:b10244452 . . _:b455779265 . . . _:b10244445 . _:b10244403 "introduction" . . . _:b10244403 . . . _:b455779286 . _:b455779280 . _:b10244414 . _:b10244443 . . . _:b10244454 "cycles of syncytial Drosophila embryos and to coordinate cell size and cell cycle time in early C.elegans embryos [39], [40], limiting dNTPs could be a conserved feature of early animal development, possibly including mammals [>>5<<]. An attractive feature of the limiting dNTP model is that it provides a mechanistic explanation for how the MBT occurs so reproducibly from embryo-to-embryo." . _:b10244404 "Recently it has been demonstrated that even mouse embryonic stem cells have a specialized metabolic state [>>1<<], [2]." . . _:b10244434 . _:b10244411 "For the remainder of the blastula period, cells continue to cycle but the length of the cell cycles increases, in inverse proportion to cell size [>>8<<], [9]. Although the MBT is also associated with an increased level of zygotic transcription and the appearance of cell motility, for the purposes of this paper we consider the MBT solely from the perspective of the cell cycle. It is" . _:b10244433 . . _:b10244432 . . _:b10244409 "Once embryos reach a threshold ratio of DNA:cytoplasm after 12\u201313 cleavage cycles [6], [>>7<<], [8], the cleavage cycles slow and lose synchrony, an event termed the mid-blastula transition (MBT)." . . . . _:b455779305 . _:b10244430 . _:b455779299 . . _:b455779306 . _:b10244430 "For instance, in mammalian cells, dATP pools are far more sensitive to inhibition of RNR by the small molecule inhibitor hydroxyurea (HU) than are the other dNTP pools [21], [>>22<<]." . _:b455779273 . . _:b455779262 . . _:b10244413 . . _:b10244427 . _:b10244434 "The number of genomes increases exponentially during the cleavage cycles but the length of S-phase remains constant (\u223C20 min) until the MBT [>>27<<]. Therefore, for the embryo as a whole, the rate of dNTP fixation into DNA also increases exponentially. We used time lapse imaging of the animal poles of intact embryos to re-examine the timing of the MBT. We found that cells at the" . _:b10244442 "Although dATP does not decrease dramatically in abundance (\u223C2-fold, Figure 4A), replication forks in yeast arrest when dNTP concentrations are only 20% lower than normal [>>31<<]. One significant problem with the limiting dNTP model is how difficult it has been to definitively test. Newport and Dasso were unable to repeat the results of Lovtrup and colleagues, who claimed that injecting dNTPs could increase the" . . _:b10244456 . "10.1371%2Fjournal.pone.0016881" . . . . . . . _:b455779298 . _:b10244425 "It has been proposed that prior to gastrulation, amino acids are the primary source of energy for X.laevis embryos [>>17<<]. The cognate amino acid(s) was never identified, although very early work had suggested that aspartate levels fall prior to gastrulation in several amphibian species [18]." . _:b455779269 . . . . _:b10244452 "As the orthologs of Chk1 and ATR are required to slow the rapid replication cycles of syncytial Drosophila embryos and to coordinate cell size and cell cycle time in early C.elegans embryos [>>39<<], [40], limiting dNTPs could be a conserved feature of early animal development, possibly including mammals [5]." . _:b455779281 . _:b10244450 . . . _:b455779283 . _:b455779277 . _:b10244416 "Injection solutions were adjusted to pH 7.7\u20137.9, the measured pH of X.laevis early embryo cytoplasm [>>46<<], and filter sterilized." . _:b10244405 "Recently it has been demonstrated that even mouse embryonic stem cells have a specialized metabolic state [1], [>>2<<]." . . _:b10244408 . . _:b10244439 "\u223C4096 cells after 12 synchronous divisions, these experimental findings are fairly consistent with the finding that unfertilized X.laevis eggs contain \u223C10 pmol of each dNTP which would suffice to synthesize \u223C2500 X.laevis genomes [>>29<<]." . . . _:b455779260 . . _:b455779301 . _:b10244415 . . _:b10244404 . _:b455779268 . _:b10244405 . . _:b10244457 . _:b10244413 "It is generally thought that some maternal protein stored in the egg, potentially a replication origin factor [>>10<<], becomes limiting at the MBT, thereby reporting the DNA:" . _:b10244406 . _:b455779264 . . _:b10244407 . _:b455779290 . _:b455779302 . . _:b10244418 _:b10244456 . _:b10244418 _:b10244457 . _:b10244418 _:b10244452 . . _:b10244418 _:b10244453 . _:b10244418 _:b10244454 . _:b10244418 _:b10244455 . _:b455779293 . _:b10244418 _:b10244448 . _:b10244444 . _:b10244418 _:b10244449 . _:b10244418 _:b10244450 . _:b10244418 _:b10244451 . _:b10244403 . _:b10244412 . _:b455779270 . _:b10244443 "Newport and Dasso were unable to repeat the results of Lovtrup and colleagues, who claimed that injecting dNTPs could increase the number of sychronous cleavage cycles in X.laevis [>>28<<], [30]. When we attempted this experiment, we consistently found that injecting dNTPs to 100\u2013300 \u00B5M, or \u223C2.5\u20137.5 fold their endogenous concentration, slowed the cleavage cycles relative to control siblings. The unknown toxic effects of" . _:b10244455 "after 13 cleavage divisions has \u223C60 pL of non-yolk volume and is much too large to be affected by the cell size requirement for the G1/S transition that has been surmised to exist in typical somatic cells (e.g. a HeLa cell is \u223C2 pL) [>>41<<], [42]. The limiting dNTP model suggests that diminishing cell size first limits the speed of the cell cycle by decreasing the absolute rate of the metabolic fluxes required for DNA synthesis." . _:b10244413 . . _:b10244419 "This platform has been previously demonstrated to provide reproducible and linear measurements of more than 100 common metabolites [>>11<<]. We were able to detect a core set of 48 metabolites in single eggs or embryos in nearly all of our experiments (Figure 1A). We focused our analyses on this robust set of 48 metabolites, which represents a broad cross-section of central" . _:b10244414 . . _:b10244415 . _:b10244418 _:b10244428 . _:b10244441 . _:b10244418 _:b10244429 . _:b10244418 _:b10244430 . _:b10244408 . _:b10244418 _:b10244431 . _:b455779279 . . _:b10244418 _:b10244424 . _:b10244418 _:b10244425 . _:b10244418 _:b10244426 . _:b10244409 . _:b10244418 _:b10244427 . _:b10244418 _:b10244420 . _:b455779289 . _:b10244418 _:b10244421 . _:b10244418 _:b10244422 . _:b10244410 . _:b10244418 _:b10244423 . _:b10244411 . _:b10244421 "Phosphorylation of tyrosine-15 (pY15) in Cdc2 increased 8 hr after fertilization, which marks the onset of the MBT (Figure 1B) [12], [>>13<<]. The time course ends prior to gastrulation (\u223C13 hr post-fertilization)." . _:b455779284 . _:b10244418 _:b10244419 . _:b10244418 _:b10244444 . _:b10244418 _:b10244445 . . _:b10244418 _:b10244446 . _:b10244420 . _:b10244418 _:b10244447 . _:b455779261 . _:b10244418 _:b10244440 . . _:b10244418 _:b10244441 . _:b10244418 _:b10244442 . _:b10244421 . _:b10244418 _:b10244443 . _:b10244448 . _:b455779260 . . _:b10244418 _:b10244436 . _:b10244418 _:b10244437 . _:b455779288 . _:b10244418 _:b10244438 . _:b10244422 . _:b10244418 _:b10244439 . _:b455779263 . _:b10244418 _:b10244432 . _:b10244450 "ATR-Chk1 is known to be the primary signaling route for stalled replication forks in animal cells and, in X.laevis egg extracts, ATR signaling has been shown to suppress origin firing [>>37<<], [38]." . _:b10244449 . _:b10244418 _:b10244433 . _:b10244418 _:b10244434 . _:b10244423 . _:b455779262 . _:b10244418 _:b10244435 . _:b455779258 . . _:b10244417 . _:b10244416 . _:b455779257 . _:b10244420 . . _:b10244423 "Amino acid incorporation into proteins has been estimated to increase from \u223C50 pmol hr\u22121 amino acid\u22121 embryo\u22121 just following fertilization to \u223C150 pmol hr\u22121 amino acid\u22121 embryo\u22121 in late blastulae [>>15<<],[16]. Based on our AAA, these low rates would still be sufficient to drain the pools of most essential amino acids over the 11 hr time course. As most of the 9 essential amino acid pools that we could measure remained constant, it is" . . _:b10244417 . _:b10244418 . _:b455779259 . . _:b10244431 . _:b10244419 . _:b455779258 . . _:b455779291 . _:b10244428 . _:b455779269 . _:b10244429 . _:b455779268 . _:b455779261 "3"^^ . _:b10244404 . _:b10244430 . _:b455779271 . _:b455779260 "3"^^ . _:b10244431 . _:b455779270 . _:b455779275 . _:b455779263 "3"^^ . _:b10244424 . _:b455779265 . . _:b455779262 "3"^^ . _:b10244425 . _:b455779264 . _:b455779266 . . . _:b455779257 "4"^^ . _:b10244426 . _:b455779267 . _:b455779307 . _:b10244406 . _:b10244427 . _:b455779266 . _:b455779259 "3"^^ . _:b10244436 . _:b455779277 . _:b455779258 "3"^^ . _:b10244437 . _:b455779276 . _:b455779269 "2"^^ . _:b10244438 . _:b455779279 . _:b455779272 . _:b455779268 "3"^^ . _:b455779263 . . _:b10244439 . _:b455779278 . _:b10244411 . . _:b455779271 "2"^^ . _:b10244432 . _:b455779273 . _:b10244410 . . _:b455779270 "2"^^ . _:b10244426 . _:b10244433 . _:b455779272 . _:b455779265 "3"^^ . . _:b10244434 . _:b455779275 . . _:b455779264 "3"^^ . . _:b10244435 . _:b10244406 "There is an increasingly deep understanding of the metabolic attributes of rapidly growing cells, particularly cancerous cells, and how these attributes are programmed by growth factor signaling in mouse and human cells [>>3<<]. Continual cell proliferation requires not only that cells duplicate and divide their genetic material but also that they double in size. To grow in size, rapidly proliferating cells require a constant supply of building blocks, such as" . _:b455779274 . _:b10244415 "Metabolites were shipped overnight at \u221220\u00B0C from Boston, MA to Princeton, NJ for LC-MS/MS metabolomic analysis the following day. Metabolites were quantified as described [>>45<<]." . _:b10244403 _:b10244408 . _:b455779292 . _:b10244403 _:b10244409 . _:b455779267 "3"^^ . _:b10244403 _:b10244410 . _:b10244444 . _:b455779285 . _:b10244403 _:b10244411 . _:b455779294 . _:b10244403 _:b10244412 . _:b455779266 "3"^^ . _:b10244403 _:b10244413 . _:b10244445 . _:b455779284 . _:b10244438 "Also as previously reported, incubating embryos with HU led to few subsequent divisions after the 12th cleavage cycle, and embryo death (Figure 4D) [>>28<<]. As HU will prevent the synthesis of additional dNTPs by RNR, this result strongly suggests that embryonic pools of at least one dNTP are functionally exhausted around the time of the MBT, leading to cell cycle arrest in S-phase and cell" . _:b455779277 "2"^^ . _:b10244412 "For the remainder of the blastula period, cells continue to cycle but the length of the cell cycles increases, in inverse proportion to cell size [8], [>>9<<]. Although the MBT is also associated with an increased level of zygotic transcription and the appearance of cell motility, for the purposes of this paper we consider the MBT solely from the perspective of the cell cycle. It is generally" . _:b10244429 "For instance, in mammalian cells, dATP pools are far more sensitive to inhibition of RNR by the small molecule inhibitor hydroxyurea (HU) than are the other dNTP pools [>>21<<], [22]." . _:b10244451 . _:b10244446 . _:b455779287 . _:b455779257 . _:b10244403 _:b10244404 . _:b455779276 "2"^^ . _:b455779258 . _:b10244440 "between the time at which maternal pools of dNTPs are exhausted and the MBT, as well as our novel result that dATP pools are diminished around the time of the MBT, support an old idea that the MBT is triggered by declining dNTP pools [>>7<<],[30]. In this model, the MBT occurs when dNTP pools and the biosynthetic capacity of RNR are outpaced by the exponentially increasing demands for dNTPs at replication forks." . _:b10244403 _:b10244405 . . _:b10244447 . _:b10244403 _:b10244406 . _:b455779259 . _:b455779260 . _:b455779304 . _:b10244403 _:b10244407 . _:b455779286 . _:b455779261 . _:b455779279 "2"^^ . _:b10244418 "results and discussion" . _:b455779262 . _:b455779263 . _:b10244440 . _:b455779281 . _:b10244414 _:b10244415 . _:b455779278 "2"^^ . . . _:b455779297 . _:b10244441 . _:b455779280 . . _:b455779273 "2"^^ . _:b455779285 . _:b10244442 . _:b10244414 "materials and methods" . _:b455779283 . . _:b10244421 . _:b455779272 "2"^^ . _:b10244445 "The MBT occurs when the DNA:cytoplasm ratio increases above a critical threshold [>>7<<], [32]. In the limiting dNTP model, the amount of DNA is represented by the number of replication forks that are fixing dNTPs into polymer, while the cytoplasm is represented by dNTP pools and RNR enzyme. As frog eggs have been shown to" . _:b10244443 . _:b455779282 . _:b455779275 "2"^^ . _:b10244452 . _:b455779280 . _:b455779293 . . _:b10244446 . _:b455779274 "2"^^ . _:b455779281 . _:b455779282 . _:b10244425 . _:b455779283 . _:b10244453 . _:b455779284 . _:b455779292 . _:b455779285 . _:b10244405 . _:b455779285 "2"^^ . _:b455779286 . _:b10244419 . _:b455779287 . _:b10244454 . _:b455779288 . _:b455779295 . _:b455779289 . _:b455779284 "2"^^ . . _:b455779290 . _:b10244441 "the time at which maternal pools of dNTPs are exhausted and the MBT, as well as our novel result that dATP pools are diminished around the time of the MBT, support an old idea that the MBT is triggered by declining dNTP pools [7],[>>30<<]. In this model, the MBT occurs when dNTP pools and the biosynthetic capacity of RNR are outpaced by the exponentially increasing demands for dNTPs at replication forks." . _:b455779291 . _:b10244455 . . _:b455779292 . _:b455779294 . _:b455779293 . _:b455779287 "2"^^ . _:b455779294 . _:b10244447 . _:b455779295 . _:b10244448 . _:b455779264 . _:b455779289 . _:b455779265 . _:b455779286 "2"^^ . _:b455779266 . _:b455779267 . _:b10244449 . _:b455779295 . _:b455779268 . _:b455779288 . _:b455779269 . _:b455779281 "2"^^ . _:b455779270 . _:b455779271 . _:b10244450 . _:b455779272 . _:b455779257 . _:b455779291 . _:b455779273 . _:b455779280 "2"^^ . _:b455779274 . _:b455779275 . _:b10244457 "Even for the largest dNTP pool in mouse fibroblasts (dTTP) [>>22<<], the amount of dTTP that needs to be polymerized to duplicate the genome is greater than 40 times the pool size." . _:b10244451 . _:b10244422 "Of the 20 amino acids, 11 cannot be synthesized de novo by animals and are often called essential (we include cysteine and tyrosine in the essential group because their synthesis requires the breakdown of other essential amino acids) [>>14<<]. While phenyalanine and to a lesser extent histidine increased during early development, the other 7 measured, essential amino acids did not change in abundance." . _:b455779276 . _:b455779290 . . _:b455779283 "2"^^ . _:b455779277 . _:b455779278 . _:b455779279 . _:b455779301 . _:b455779282 "2"^^ . . _:b10244429 . _:b455779300 . _:b455779293 "2"^^ . _:b455779303 . _:b455779292 "2"^^ . _:b10244426 "The cognate amino acid(s) was never identified, although very early work had suggested that aspartate levels fall prior to gastrulation in several amphibian species [>>18<<]. Here, we have definitively identified alanine and aspartate as amino acids that are consumed during the early development of X.laevis (Figure 1A, 1E, Figure S1, S3)." . _:b10244414 _:b10244416 . _:b10244414 _:b10244417 . _:b455779302 . _:b10244417 "200 \u00B5L of each supernatant was then dried under liquid nitrogen and subjected to metabolomic analysis as described [>>47<<]." . _:b455779295 "2"^^ . _:b10244408 "As in many, if not most, animal species, fertilization of the X.laevis egg initiates a series of rapid and synchronous cell division cycles [>>5<<]. These cleavage cycles represent the minimal cell cycle, alternating between genome replication (S-phase) and genome segregation (M-phase) and lacking gap phases (G1 and G2-phase) and cell cycle checkpoints. Little to no cell growth" . _:b10244456 . _:b455779296 . _:b455779297 . _:b455779297 . _:b455779294 "2"^^ . . _:b455779298 . _:b455779299 . _:b10244457 . _:b455779300 . _:b455779296 . _:b455779301 . _:b455779289 "2"^^ . . _:b455779302 . . _:b10244410 "Once embryos reach a threshold ratio of DNA:cytoplasm after 12\u201313 cleavage cycles [6], [7], [>>8<<], the cleavage cycles slow and lose synchrony, an event termed the mid-blastula transition (MBT)." . _:b455779303 . _:b455779304 . _:b455779299 . _:b455779305 . _:b455779288 "2"^^ . _:b455779306 . _:b455779307 . _:b455779298 . _:b10244424 "Amino acid incorporation into proteins has been estimated to increase from \u223C50 pmol hr\u22121 amino acid\u22121 embryo\u22121 just following fertilization to \u223C150 pmol hr\u22121 amino acid\u22121 embryo\u22121 in late blastulae [15],[>>16<<]. Based on our AAA, these low rates would still be sufficient to drain the pools of most essential amino acids over the 11 hr time course. As most of the 9 essential amino acid pools that we could measure remained constant, it is likely" . _:b455779291 "2"^^ . _:b455779290 "2"^^ . . _:b10244449 "It has been demonstrated that the ATR-Chk1 replication checkpoint is transiently activated during the MBT [>>13<<]. In the limiting dNTP model, depleted dNTP pools would cause replication forks to stall after \u223C12\u201313 replication cycles, activating the ATR-Chk1 checkpoint pathway to retard origin firing and extending S-phase. ATR-Chk1 is known to be the" . _:b455779301 "2"^^ . . _:b10244436 . . _:b455779300 "2"^^ . _:b455779303 "2"^^ . _:b455779305 . "PMC0" . _:b10244455 . _:b455779302 "2"^^ . _:b455779304 . _:b455779297 "2"^^ . . _:b455779261 . _:b455779307 . _:b455779296 "2"^^ . _:b455779287 . _:b455779306 . _:b10244428 . _:b455779299 "2"^^ . _:b10244409 . _:b455779298 "2"^^ . _:b10244428 "While not as dramatic as many of the changes we observed, these changes were nevertheless somewhat surprising, as it is thought that cells tightly control dNTP concentrations [>>20<<]. All four dNTPs are synthesized through the action of ribonucleotide reductase (RNR). A complex set of allosteric feedback loops are thought to allow the four dNTP pools to achieve the desired concentrations and stoichiometry, in large" . _:b10244424 . . _:b10244454 . _:b10244456 "13 cleavage divisions has \u223C60 pL of non-yolk volume and is much too large to be affected by the cell size requirement for the G1/S transition that has been surmised to exist in typical somatic cells (e.g. a HeLa cell is \u223C2 pL) [41], [>>42<<]. The limiting dNTP model suggests that diminishing cell size first limits the speed of the cell cycle by decreasing the absolute rate of the metabolic fluxes required for DNA synthesis." . . . _:b10244437 "The 13th cycle is one cycle later than was reported in two studies of cells from dissociated embryos [7], [8], but is consistent with a previous report in which intact embryos were imaged [6], [>>28<<]. Also as previously reported, incubating embryos with HU led to few subsequent divisions after the 12th cleavage cycle, and embryo death (Figure 4D) [28]. As HU will prevent the synthesis of additional dNTPs by RNR, this result strongly" . _:b455779305 "2"^^ . . _:b455779278 . _:b455779282 . _:b455779304 "2"^^ . _:b455779307 "2"^^ . _:b455779306 "2"^^ . _:b10244418 . _:b10244423 . _:b10244422 . _:b455779276 . _:b10244432 "MBT, since S-phase is when dNTP levels would be expected to be highest if these levels were cell cycle regulated at later stages of development [23], , but the first cell cycle phase to be extended at the X.laevis MBT is the S-phase [>>27<<]. In fact, the proportion of each cell cycle devoted to S-phase does not decrease as cells traverse the MBT and cell cycles elongate in late blastulae [27]." . . _:b10244416 . _:b455779267 . . . _:b455779303 . _:b455779271 . . _:b455779296 . . _:b10244442 . _:b10244433 "In fact, the proportion of each cell cycle devoted to S-phase does not decrease as cells traverse the MBT and cell cycles elongate in late blastulae [>>27<<]." . . _:b10244451 "ATR-Chk1 is known to be the primary signaling route for stalled replication forks in animal cells and, in X.laevis egg extracts, ATR signaling has been shown to suppress origin firing [37], [>>38<<]." . . . .