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First published online 27 July 2004
doi: 10.1242/dev.01261

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Real-time lineage analysis reveals oriented cell divisions associated with morphogenesis at the shoot apex of Arabidopsis thaliana

¹ California Institute of Technology, Division of Biology, MC 156-29, 1200 E. California Boulevard, Pasadena, CA 91125, USA
² Department of Plant Biology, Carnegie Institution of Washington, Stanford, CA 94305, USA

View larger version (178K): [in a new window]   Fig. 1. Cell division markers. (A-F) 35S::YFP29-1 expression in different clonal layers of SAMs. (G-H) 35S:H2B:mYFP expression in reconstructed L1 layer. (A,C,E,G) The first time points (0 hour) and (B,D,F,H) represent the same SAMs after the elapsed time indicated on top right-hand corner of each panel. (A,B) The reconstructed Z-series revealing all the cells in the L1 layer. Primordia at different stages of development are marked as P1, P2, P3. Arrows indicate the same cells prior to and after division. (C,D) Cells in the L2 layer (8 µm deep); the arrow points to cell division events. (E,F) Cells in the corpus (12 µm deep); the arrows point to cell division events. (G,H) The reconstructed images of the L1 layer expressing 35S::H2B:mYFP. Arrows in G indicate cells in division, and arrows in H are the same cells after an hour, showing decondensed chromatin after completion of mitosis. (I-L) Reconstructed views of different growth stages of the same SAM after 24-hour intervals. The earliest visible bulge on the meristem flank is considered as stage P1 (J,K). The extension of the bulge in the x/y axis and the appearance of a first sign of a groove between the SAM and the primordium is considered stage P2. Scale bar: 20 µm.

View larger version (102K): [in a new window]   Fig. 3. Differential mitotic cyclin expression in SAMs. (A-C) The same optical section of an L2 layer from a time-lapse series. (A) First time point; (B,C) subsequent time points. The total time elapsed is indicated at the top right-hand corner. In all panels, red represents FM4-64 staining of plasma membranes, and green represents expression of cyclinB1;1:GFP. Arrows in (A-C) point to the same cells over time. (D-F) The reconstructed L1 layer of SAMs from different plants. Arrows point to cyclin-positive cells in SAMs and the arrowheads point to cyclin-positive cells in flower buds.

View larger version (53K): [in a new window]   Fig. 2. Temporal variation in mitotic activity in SAMs. (A) Raw data on the number of dividing cells in the L1 layer of four different SAMs (SAM1-SAM4) imaged at 6-hour intervals. In case of SAM2, the data were analyzed only until interval 14 and in case of SAM3 and SAM4 only until interval 11. (B) Data from a plant imaged every 12 hours. (C) Comparison of cell division activity in the L1 layer with that in the L2 layer and corpus of the same plant, imaged every 1 hour and 15 minutes. The number of cell divisions within each time interval is presented. The time at the beginning of the experiment and the cell division marker used are indicated in each case. In each case, note the differences in y-axis dimension, which represents number of cell divisions.

View larger version (135K): [in a new window]   Fig. 4. Spatial distribution of mitotic activity over time. (A-C) A reconstructed L1 layer of the same plant separated by 12-hour intervals. Cells that have divided in each of the intervals are differentially color-coded. Red dots represent cells that divided in the first 12-hour window, yellow dots the following 12 hours, and blue dots the final 12 hours. (D-G) Individual optical sections from the same plant, depicting cells located in the L2 and the corpus from the same time point as in (C), and the color code remains the same. The overlapping dots indicate a second round of cell division (arrows). (H-J) Reconstructed views of the L1 layer of the same SAM followed over 72 hours. The total elapsed time is marked in individual panels. Different colored sectors represent regions of primordium development marked as stages in transition from P-2 to P-1, from P-1 to P0, from P0 to P1, from P1 to P2 and from P2 to P3. The numbers expressed as percentages in (J) represent averaged cumulative mitotic index calculated for every 24-hour interval over 72 hours, in sectors representing primordia at the same stages. The numbers in parentheses indicate the number of cell divisions over the total number of cells.

View larger version (119K): [in a new window]   Fig. 6. Location of primordium progenitor cells. (A-E) Reconstructed view of the L1 layer of a shoot apex expressing 35S::YFP29-1, with (A) being the first time point and elapsed time in (B-E) marked on individual panels. (A) Initials that give rise to individual primordia (P-1, P-2, P-3 and P-4) are shown in enclosed areas marked in white. The progenitors of a given primordium are color-coded. (B-E) Primordia that have developed from the progenitors marked in A. Individual lineages within a primordium are color-coded. The same color has been used for each progenitor and its descendants.

View larger version (99K): [in a new window]   Fig. 5. Frequency and spatial distribution of cell cycle length. (A-C) Reconstructed views of the L1 layer of the same plant, expressing 35S::YFP29-1, over a period of time. (D) Frequency distribution of cell cycle length among cells in the L1 and in the L2 and corpus together. Each of the color-coded bars represents a window of cell cycle length and each one of them is separated by 6 hours. Such individual events are mapped onto the L1 layer of the SAM in A-C, by marking both of the siblings. In cases where a single dot can be seen, the cells divided in the subsequent time interval. The color code employed in the spatial representations is the same as that used in representing the frequency distribution. The stages in A represent the ones at the beginning of the experiment. The stages marked in B and C are the ones in transition from P2 to P3 and P3 to P4, respectively. Elapsed time is indicated on each panel. White arrows point to cells in primordial regions P0, P-1 and P-2. White arrowheads point to cells in the intervening region located between P-1 and P-2. Yellow arrows point to cells in the intervening region, located between P0 and P-1. The cells marked in regions P-1 and P-2 (A, white arrows) are also represented in the next panel (B, white arrows) to maintain continuity.

View larger version (156K): [in a new window]   Fig. 7. Cell division patterns in primordium progenitors and their descendents. (A-F) Reconstructed views of the L1 layer of the same SAM expressing 35S::YFP29-1, over a period of time. The elapsed time is indicated in individual panels. Cells of individual developing lineages are marked in different colors. (G-L) represents a similar analysis of the P-2 region of the same shoot apex. The lateral (L in red) and the medial (M in yellow) axis of the developing flower primordium are marked in (D). The cell division axis in primordial progenitors is referenced as parallel to the lateral axis of the flower primordium.

View larger version (86K): [in a new window]   Fig. 9. Cell division patterns in cells located between developing primordia. (A,B) Reconstructed view of the shoot apex of the same plant at the same point in a time series. Elapsed time from the initial observation is marked. The lineages that result in a primordium in transition from P1 to P2 are marked in (A), while the lineages in the intervening region (IR) between two successive primordial regions P1P2 and P0P1 are marked in (B). Individual lineages are differentially color-coded.

View larger version (173K): [in a new window]   Fig. 8. Cell division patterns in the L2 layer. The cell division patterns that occurred in the L2 layer of a primordial region is presented. Such cell division patterns are projected onto the final time point. (A-D) Sections at different depths in the SAM. The structure of the lineages leading to each primordium (white boxed region) is represented. Three different lineages are color-coded and the arrows point to sibling cells located at different depths.

View larger version (197K): [in a new window]   Fig. 10. Cell expansion patterns and cell behavior at the boundary region. (A-C) Reconstructed views of a SAM expressing 35S::YFP29-1, from a time series. Total elapsed time is indicated in individual panels. Arrows point to the same cells in the PZ over a period of time, and they divide along their long axis. Arrowhead points to a cell in the CZ dividing along its short axis. The white-boxed area in (B) represents the boundary region. Magnified view of a time-lapse series of the boundary region is shown in (D-G). The total elapsed time is marked on individual panels. Arrows indicate the same cells in the boundary region across time. Arrowheads indicate adjacent cells in the developing flower primordium. The lateral (L) and the medial (M) axis of the developing flower primordium are marked in B. Scale bar: 20 µm in A-C; 10 µm in D-G.

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