PMC

Tet2 knockout hematopoietic precursors show distinct division pattern. a The frequency of symmetric self-renewal, symmetric differentiation, and asymmetric division of GFP⁺ precursors from wild-type (WT) mouse is shown. The data are a summary from three independent experiments. Error bars represent standard error of the mean (SEM). b The frequency of symmetric self-renewal, symmetric differentiation, and asymmetric division of wild-type GFP⁺ co-cultured with OP9 stromal cells is shown. OP9 stromal cells were plated in the culture dish 24 h before the time-lapse experiment. Then GFP⁺ hematopoietic precursors were sorted and plated on OP9 stromal cells for tracking of the division of HSCs. The data are a summary from three independent experiments. Error bars represent SEM. c The frequency of symmetric self-renewal, symmetric differentiation, and asymmetric division of Tet2 knockout (KO) GFP⁺ cells is shown. The data are a summary from three independent experiments. Error bars represent SEM. d The frequency of symmetric self-renewal, symmetric differentiation, and asymmetric division of Tet2 knockout GFP⁺ cells co-cultured with OP9 stromal cells is shown. OP9 stromal cells were plated in the culture dish 24 h before the time-lapse experiment. Then GFP⁺ hematopoietic precursors were sorted and plated on OP9 stromal cells for tracking of the division of HSCs. The data are a summary from three independent experiments. Error bars represent SEM. e The frequency of symmetric self-renewal, symmetric differentiation, and asymmetric division of Tet2 knockout GFP⁺ cells treated with 5-aza-2′-deoxycytidine (DAC) 500 nM is shown. The data are a summary from three independent experiments. Error bars represent SEM

(a) Cellular events that occurred prior to multipolar cell division were analyzed by tracing the cell lineage backward. Pattern 1a (cell fusion [CF] ➛ multipolar cell division [MD]): cell fusion occurred prior to multipolar cell division. Pattern 1b (➛ MD): no cell fusion occurred. The numbers of each pattern occurring in 50 cell lineages are shown. Percentages of Patterns 1a and 1b in Control and p53 RNAi cells are also shown. (b) Cells that underwent multipolar cell division were identified. If the cells were generated by cell fusion, the origin of the fused cells was searched by tracing the cell lineage backward. Pattern 2a (BD ➛ sibling CF ➛ MD): bipolar cell division occurred, cells produced by division were fused, followed by multipolar cell division. Pattern 2b (BD ➛ non-sibling CF ➛ MD): one sibling produced by bipolar cell division was fused with another non-sibling cell, followed by multipolar cell division. The numbers of each pattern in 50 cell lineages are shown. Percentages of Patterns 2a and 2b in Control and p53 RNAi cells are also shown. (c) Progeny produced by multipolar cell division was tracked and the number of reproductive progeny that underwent bipolar cell division was determined. The numbers of reproductive progeny found in 100 cell lineages and the percentages of such progeny among the total number of progeny produced by multipolar cell divisions are shown. (d) The number of multipolar cell division events was plotted against the number of bipolar cell division events. Numbers in (d) indicate doses of MNNG. (a–d) Because the number of multipolar cell divisions in Control cells was low, we searched three videos to find multipolar cell divisions and then traced the cells back to their progenitors to determine the events preceding multipolar cell division. To normalize the values, we assumed that multipolar cell division occurred at a similar frequency to that recorded in the cell-lineage database. BD: bipolar cell division; MD: multipolar cell division, CD: cell death; and CF: cell fusion.
Figure 6—source data 1.Events leading to the induction of multipolar cell division.Data used in .

(A–C) Schematic illustration of division plane orientation. Division plane of the first and the second generation are colored as dark blue and pink, respectively. (D–F) Frequency of cell polygonal types during cell proliferation by three choices of division plane orientation. Random orientation reaches a hexagonal frequency of about . Orthogonal orientation produces a higher percentage of hexagons of about . Largest side orientation generates a distribution with a hexagonal frequency of about . (G–I) Visualization of cell shapes by three division plane orientations. Random orientation generates some cells with irregular shape (marked by red ellipse). Orthogonal orientation produces regular cell shapes with a high percentage of hexagons. Largest side orientation generates regular cell shapes. Pentagons, hexagons, and heptagons are colored yellow, magenta, and blue, respectively. Gray indicates other polygonal types.

Presence of BASL post-ACD ensures cell fate asymmetry and coordinates sister cell divisions. (A) Schematic and examples of how excessive spacing divisions form small cell clusters. Top row gives examples of normal lineages in which meristemoid amplifying divisions are associated with limited SLGC spacing divisions. Bottom row shows examples of both amplifying and spacing divisions occurring in neighboring sisters. Tracked stomatal lineage cells are false-colored in green. (B) Division frequencies are coordinated between sister cells when overall divisions are elevated through increased SPCH dosage (SPCH+). Frequencies are calculated by tracing the subsequent division behaviors of the daughters of an asymmetric division (n=3 cotyledons/genotype; n>300 pairs/cotyledons tracked from 3-5 dpg). (C) Quantification of observed spacing division frequency (Obs[SPA]), observed amplifying division frequency (Obs[AMP]), observed frequency of both amplifying division and spacing division (Obs[SPA+AMP]), and the expected frequency of amplifying division and spacing division (Exp[SPA+AMP]) under the assumption that these two divisions were independent (calculated by multiplying Obs[AMP] by Obs[SPA]; n>500 pairs/genotype). (D) Suppression of entry and spacing divisions by overexpression of EPF2 (p35S::EPF2) in BASL^+/− plants. (E) Suppression of entry divisions, but not spacing divisions, by p35S::EPF2 in basl^−/−. (F,G) Quantification of entry divisions (F; estimated by the number of actively dividing pairs of sister cells at 3 dpg) and sister cell division coordination (G; estimated by the spacing division frequency when amplifying division occurs in sister meristemoid) from the genotypes shown in D,E. Both results in F,G are quantified from whole-leaf lineage-tracing experiments (n=3 cotyledons/genotype tracked from 3-5 dpg; n>120 pairs/cotyledons in p35S::EPF2 lines; n>300 pairs/cotyledons in lines not expressing p35S::EPF2). In D,E, cell outlines are visualized by the plasma membrane reporter pATML1::RCI2A-mCherry and small cell clusters are labeled with red braces. For all the box plots, the box represents the first and third quartiles with the middle line indicating median; the upper or lower whisker extends from the box to the largest or smallest value no further than 1.5 times of the interquartile range. All P-values are calculated by Student's t-test due to small sample size. Scale bars: 10 μm.