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    Tissue-specific regulation of the number of cell division rounds by inductive cell interaction and transcription factors during ascidian embryogenesis

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    AbstractMechanisms that regulate the number of cells constituting the body have remained largely elusive. We approached this issue in the ascidian, Halocynthia roretzi, which develops into a tadpole larva with a small number of cells. The embryonic cells divide 11 times on average from fertilization to hatching. The number of cell division rounds varies among tissue types. For example, notochord cells divide 9 times and give rise to large postmitotic cells in the tadpole. The number of cell division rounds in partial embryos derived from tissue-precursor blastomeres isolated at the 64-cell stage also varied between tissues and coincided with their counterparts in the intact whole embryos to some extent, suggesting tissue-autonomous regulation of cell division. Manipulation of cell fates in notochord, nerve cord, muscle, and mesenchyme lineage cells by inhibition or ectopic activation of the inductive FGF signal changed the number of cell divisions according to the altered fate. Knockdown and missexpression of Brachyury (Bra), an FGF-induced notochord-specific key transcription factor for notochord differentiation, indicated that Bra is also responsible for regulation of the number of cell division rounds, suggesting that Bra activates a putative mechanism to halt cell division at a specific stage. The outcome of precocious expression of Bra suggests that the mechanism involves a putative developmental clock that is likely shared in blastomeres other than those of notochord and functions to terminate cell division at three rounds after the 64-cell stage. Precocious expression of Bra has no effect on progression of the developmental clock itself

    Regulation of the Number of Cell Division Rounds by Tissue-Specific Transcription Factors and Cdk Inhibitor during Ascidian Embryogenesis

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    <div><p>Mechanisms that regulate the number of cell division rounds during embryogenesis have remained largely elusive. To investigate this issue, we used the ascidian, which develops into a tadpole larva with a small number of cells. The embryonic cells divide 11.45 times on average from fertilization to hatching. The number of cell division rounds varies depending on embryonic lineages. Notochord and muscle consist of large postmitotic cells and stop dividing early in developing embryos. Here we show that conversion of mesenchyme to muscle cell fates by inhibition of inductive FGF signaling or mis-expression of a muscle-specific key transcription factor for muscle differentiation, Tbx6, changed the number of cell divisions in accordance with the altered fate. Tbx6 likely activates a putative mechanism to halt cell division at a specific stage. However, precocious expression of Tbx6 has no effect on progression of the developmental clock itself. Zygotic expression of a <i>cyclin-dependent kinase inhibitor, CKI-b</i>, is initiated in muscle and then in notochord precursors. CKI-b is possibly downstream of tissue-specific key transcription factors of notochord and muscle. In the two distinct muscle lineages, postmitotic muscle cells are generated after 9 and 8 rounds of cell division depending on lineage, but the final cell divisions occur at a similar developmental stage. <i>CKI-b</i> gene expression starts simultaneously in both muscle lineages at the 110-cell stage, suggesting that CKI-b protein accumulation halts cell division at a similar stage. The difference in the number of cell divisions would be due to the cumulative difference in cell cycle length. These results suggest that muscle cells do not count the number of cell division rounds, and that accumulation of CKI-b protein triggered by tissue-specific key transcription factors after cell fate determination might act as a kind of timer that measures elapsed time before cell division termination.</p></div

    <i>CKI-b</i> gene expression.

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    <p>(A) (upper row) <i>In situ</i> hybridization with the <i>Hr-CKI-b</i> probe. The expression becomes evident at the 110-cell stage in the B7.4 and B7.8 muscle lineage precursors simultaneously. At the 110-cell stage plus one hour, there are 4 B7.4 descendants and 2 B7.8 descendants. At 2 hours after the 110-cell stage, the expression has become evident in notochord precursors. The stages from the 110-cell stage through the gastrula and up to the neural plate stage are shown. (bottom) Expression of <i>CKI-b</i> in embryos whose cleavages were arrested at the 110-cell stage. Asterisks indicate <i>de novo</i> expression at the 110-cell stage plus 4 hours, which probably corresponds to the heavily stained cells in the above photo. (B) Schematic representation of the arrested 110-cell embryos, showing the position of each tissue precursor cell. Vegetal view. (C) Timetable of <i>CKI-b</i> expression and cell division progression after the 64-cell stage. Mus, muscle. Not, notochord.</p

    Numbers of cell divisions of mesenchyme cells in Tbx6-mis-expressing embryos.

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    <p>B7.3 and B7.7 mesenchyme blastomeres were isolated from 64-cell embryos that had been injected with control <i>H2B:mCherry</i> mRNA (gray bars) and <i>Tbx6</i> mRNA (green bars). The numbers of descendant cells were counted, and the numbers of cell divisions were then calculated. On the abscissa, e.g., 3 cell divisions represents partial embryos that divided 2.50 to 3.49 times. Numbers of partial embryos observed are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090188#pone-0090188-g002" target="_blank">Fig. 2B</a>. Proportions of major specimens are given above the columns.</p

    Expression of <i>CKI-b</i> occurs downstream of inductive cell interaction and the key transcription factor.

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    <p>(A) Schematic representation of arrested 110-cell embryos, showing the position of each tissue precursor cell. Vegetal view. (B) <i>CKI-b</i> expression in a cleavage-arrested 110-cell embryo treated with DMSO as a control. (C) That treated with MEK inhibitor. Expression in the notochord was lost, and ectopic expression was evident in mesenchyme cells. (D) <i>CKI-b</i> expression in a cleavage-arrested 110-cell embryo injected with control MO. (E) That in an embryo injected with Brachyury MO. The expression in notochord cells was lost. Numbers of embryos that showed the expression in each tissue per those of observed embryos are shown. Mch, mesenchyme. Mus, muscle. Not, notochord.</p

    Cell counts of partial embryos treated with MEK inhibitor.

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    <p>DMSO, the solvent of MEK inhibitor, was used as a control.</p><p>Cell counts and numbers of cell divisions are presented as mean ± s.e.m.</p><p>Conditions are highlighted in bold and italic letters when cell fate changes are expected.</p><p>Numbers of cell divisions are highlighted in bold and italic letters when a change in the number was observed.</p><p>Mch, mesenchyme. Mu, muscle.</p

    Numbers of descendant cells derived from precursor blastomeres of various tissues.

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    <p>(A) Vegetal view of 64-cell embryos color-coded for precursor blastomeres of each tissue. Anterior is up. Names of blastomeres and numbers of descendants cells derived from each are shown. Blue bars connect sister blastomeres on the left half of the embryo. Images showing the descendant cells are reproduced from Nishida (1987) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090188#pone.0090188-Nishida1" target="_blank">[2]</a>. (B) Mechanism of asymmetric cell divisions that is induced by FGF signaling in the anterior and posterior parts of embryos, respectively. Mch, mesenchyme. Mus, muscle. Not, notochord. NC, nerve cord.</p

    Inhibition of FGF signaling by MEK inhibitor.

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    <p>(A) Muscle and mesenchyme blastomeres were isolated from 64-cell embryos treated with DMSO as a control (upper row), and with MEK inhibitor (bottom). Resulting partial embryos were gently squashed on glass slides by compressing them with cover slips until the constituent cells spread into a monolayer. Nuclei (asterisks) were stained with DAPI, and numbers of constituent cells were counted. The number of cell divisions was then calculated. Approximate numbers of cell division rounds in controls and their changes resulting from treatment are indicated below the photos. Numbers of partial embryos observed are shown in parentheses. Specimens that showed altered numbers of cell divisions are indicated by red squares. For detailed data see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090188#pone-0090188-t001" target="_blank">Table 1</a>. (B) Muscle and mesenchyme blastomeres were isolated from 64-cell embryos that were injected with <i>H2B:mCherry</i> mRNA as a control (upper row), and with <i>Tbx6</i> mRNA (bottom). For detailed data see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090188#pone-0090188-g003" target="_blank">Fig. 3</a>. (C) Synthetic mRNA encoding the mCherry:Tbx6 fusion protein with the original UTRs of <i>Tbx6</i> mRNA was injected into fertilized eggs. Nuclear fluorescence was detected as early as the 16-cell stage. Scale bar, 50 µm.</p

    Timing of cell divisions in the B7.4 and B7.8 muscle lineages.

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    <p>Progression of cell division was monitored by time-lapse video. The B7.4 and B7.8 muscle precursors were isolated from 64-cell embryos and recorded. Snapshots from the resulting time-lapse video are shown at the bottom. The duration of each cell cycle up to the 64-cell stage was observed in whole embryos. The duration of each cell cycle after the 64-cell stage was averaged from 5 independent time-lapse recordings. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090188#pone.0090188.s002" target="_blank">Movie S1</a>.</p
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