Figure 1.
Numbers of descendant cells derived from precursor blastomeres of various tissues.
(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) [2]. (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.
Figure 2.
Inhibition of FGF signaling by MEK inhibitor.
(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 Table 1. (B) Muscle and mesenchyme blastomeres were isolated from 64-cell embryos that were injected with H2B:mCherry mRNA as a control (upper row), and with Tbx6 mRNA (bottom). For detailed data see Fig. 3. (C) Synthetic mRNA encoding the mCherry:Tbx6 fusion protein with the original UTRs of Tbx6 mRNA was injected into fertilized eggs. Nuclear fluorescence was detected as early as the 16-cell stage. Scale bar, 50 µm.
Table 1.
Cell counts of partial embryos treated with MEK inhibitor.
Figure 3.
Numbers of cell divisions of mesenchyme cells in Tbx6-mis-expressing embryos.
B7.3 and B7.7 mesenchyme blastomeres were isolated from 64-cell embryos that had been injected with control H2B:mCherry mRNA (gray bars) and Tbx6 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 Fig. 2B. Proportions of major specimens are given above the columns.
Figure 4.
Timing of cell divisions in the B7.4 and B7.8 muscle lineages.
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 Movie S1.
Figure 5.
(A) (upper row) In situ hybridization with the Hr-CKI-b 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 CKI-b in embryos whose cleavages were arrested at the 110-cell stage. Asterisks indicate de novo 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 CKI-b expression and cell division progression after the 64-cell stage. Mus, muscle. Not, notochord.
Figure 6.
Overexpression of CKI-b inhibits cleavage.
CKI-b mRNA was injected into eggs at approximately one hour after fertilization (bottom row). The cell cycle was prolonged at the 4-cell stage relative to normal embryos (upper), and most of them ceased dividing at the 8- or 16-cell stage.
Figure 7.
Expression of CKI-b occurs downstream of inductive cell interaction and the key transcription factor.
(A) Schematic representation of arrested 110-cell embryos, showing the position of each tissue precursor cell. Vegetal view. (B) CKI-b 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) CKI-b 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.