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
* Author for correspondence (e-mail: meyerow{at}its.caltech.edu)
Accepted 11 May 2004
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SUMMARY |
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Key words: Live imaging, Lineage analysis, Shoot apical meristem
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Introduction |
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The shoot apical meristem (SAM) of Arabidopsis thaliana presents
an elegant system for the study of cell behavior in a morphogenetic context.
The essential function of the SAM is to produce the cells that comprise the
aboveground plant parts. The SAM is a multilayered structure consisting of
three clonally distinct layers of cells. The outermost L1 and the subepidermal
L2 are single layers in which anticlinal divisions occur, while the underlying
corpus forms a multilayered structure with cell division in periclinal as well
as anticlinal planes (Steeves and Sussex,
1989). Within this framework, the SAM can be divided into
cytological zones, where the central zone (CZ) is at the very apex, the
peripheral zone (PZ) is on the sides, and the rib meristem (RM) is in the
central part of the meristem. The CZ has been thought to harbor a set of
initials, which divide and displace their daughters into the PZ, where they
are incorporated into the primordia of leaves and flowers at defined
locations. Starting just after germination, the first four leaves are formed
as opposite pairs, and then subsequent leaves and flowers are formed in a
spiral pattern with an angle close to 137.5° between consecutive primordia
(Callos et al., 1994
). The SAM
retains a nearly constant size from germination to senescence, despite a
constant flux of cells from the meristem to newly established lateral organs
and underlying stem. Thus the SAM has to coordinate two independent but
related functions, first to ensure a constant cell number in different regions
of the SAM, and at the same time to allow cells in defined locations to
differentiate and become part of primordia. A tight coordination between cell
division and displacement of the progeny, both within and across clonally
distinct layers of the SAM, has been proposed to be a major factor in
regulating the size of the SAM and in generating the radial pattern of the
shoot apex (Meyerowitz, 1997
).
In plants, such a coordination must be achieved predominantly through
controlled patterns of cell division and expansion, as common animal
mechanisms such as programmed cell death and cell migration do not operate
during SAM morphogenesis.
Genetic studies have revealed signaling mechanisms involved in meristem
maintenance. Mutations in CLAVATA (CLV) genes (CLV1, CLV2
and CLV3) result in larger meristems, while mutations in WUSCHEL
(WUS) result in a failure to maintain a functional meristem
(Clark et al., 1993;
Clark et al., 1995
;
Laux et al., 1996
;
Kayes and Clark, 1998
).
Several studies have contributed to a model involving positive and negative
feedback loops to maintain meristem size
(Clark et al., 1997
;
Mayer et al., 1998
;
Fletcher et al., 1999
;
Brand et al., 2000
;
Schoof et al., 2000
). The
function of WUS, a homeodomain transcription factor, is required to maintain a
constant stem-cell pool in the CZ and at the same time CLV1, a receptor
kinase, and CLV3, a small secreted protein, function to repress WUS activity.
The function of SHOOT MERISTEMLESS (STM) adds another layer of regulation in
SAM establishment and/or maintenance (Long
et al., 1996
). stm mutants fail to develop a functional
SAM, and STM has been proposed to function in maintenance of cell
proliferation by repressing genes such as ASYMMETRIC LEAVES1 (AS1),
which are specific to developing leaves at the periphery
(Byrne et al., 2000
). Auxin
distribution has been shown to mediate the placement of primordia within the
PZ (Reinhardt et al., 2003b
).
These studies demonstrate a role for short-range signaling between adjacent
groups of cells, both within and across clonally distinct layers of cells.
Understanding how these signaling mechanisms interface with cell division
patterns is central to understanding meristem maintenance and
morphogenesis.
Our knowledge of cell division patterns in SAMs is limited, though it has
been a subject of intense analytical investigation since early in the last
century (Steeves and Sussex,
1989; Meyerowitz,
1997
). Early studies based on cytological appearance and also on
counts of mitotic figures in different regions of the SAM have reached a broad
consensus that cells in the CZ divide more slowly than cells in the PZ.
Efforts have also been made to record cell behavior in living shoot apices
(Ball, 1960
;
Soma and Ball, 1964
). These
studies did reveal cell displacement patterns but visualized only epidermal
cells. The studies on the surface expansion of the SAMs have yielded
quantitative description of cell expansion behavior
(Hernandez et al., 1991
;
Dumais and Kwiatkowska, 2002; Kwiatkowska
and Dumais, 2003
; Kwiatkowska,
2004
). A comprehensive morphometric analysis in Arabidopsis
thaliana has revealed spatial patterns of mitotic activity in different
regions of the SAMs (Laufs et al.,
1998
). A recent in-vivo study in Arabidopsis thaliana,
based on optical imaging, has revealed the effects of anti-mitotic drugs and
DNA synthesis inhibitors on differentiation and morphogenesis at the shoot
apex (Grandjean et al., 2003
).
Sector boundary analysis has predicted the number of initial cells for both
the leaf and flower formation (Irish and
Sussex, 1992
; Bossinger and
Smyth, 1996
). Although all these studies have yielded insights at
several levels, a comprehensive dynamic view of cell behavior is lacking.
In this study we have analyzed cell division patterns in living and actively growing wild-type SAMs of Arabidopsis thaliana. One of the major challenges in observing cells in living SAMs has been the accessibility of these cells, as they are shrouded by developing primordia. We have designed a live-imaging technique based on confocal microscopy and have employed a variety of cell division and cell structure markers to observe cell division and cell expansion in real time. We have used the time-lapse imaging data to reconstruct events in time by utilizing image registration algorithms to visualize morphogenesis in relation to cell behavior. Finally, we have incorporated time-lapse data to serially reconstruct lineages in real time. Our analysis reveals that distinct cell behavior is associated with different stages of primordium morphogenesis. We show that the amount of cell division is comparable across successive primordial regions. Oriented cell divisions, in primordial progenitors and in cells located proximal to them, is associated with initial primordial outgrowth, followed by a rapid and coordinated burst of cell expansion and cell division to transform this extension into a three-dimensional flower bud. This study provides a dynamic spatio-temporal analysis of cell division in SAMs that can form a basis for future quantitative studies of meristematic cell behavior.
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Materials and methods |
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Live imaging
Plants were germinated on MS-agar plates and allowed to grow for 10 days
before they were transferred into clear plastic boxes containing MS-agar. The
plants were maintained in aseptic conditions until bolting. Upon bolting, when
the shoot apex emerged out of the rosette, the plants were prepared for
time-lapse imaging. The MS-agar surface was overlaid with 1% agarose to
minimize contamination. The older floral buds were carefully removed or spaced
out in order to expose the SAM. The rosette was stabilized by applying 1.5%
molten agarose onto the stem. FM4-64 (50 µg/ml), when used, was applied
directly onto the SAM 30 minutes prior to imaging.
Microscopy and image processing
Plants were imaged by using a Zeiss 310 or Zeiss 510 upright confocal
microscope using a 63x water dipping achroplan lens, which has a working
distance of 2 mm. Plastic boxes were filled with water to submerge the plant
prior to each imaging session, which lasted for 30 seconds to 1 minute. The
water was then discarded and the plants were returned to normal growth
conditions. This process was repeated for imaging intervals as described for
individual experiments. YFP was stimulated with an argon laser at 514 nm at
25-50% of its output, and by using neutral density filters at 4-7% to
attenuate the laser line. The emission was filtered by using a 530-590 nm
band-pass filter. The confocal Z-stacks across time points were aligned by
using a multimodality image registration program, MIRIT, which utilizes
information theory to maximize mutual information across image stacks to
register at sub-pixel resolution (Maes et
al., 1997). The registered stacks were reconstructed in three
dimensions, rendered and animated to play continuous movies by using either
the Zeiss LSM3.2 or VOLOCITY software (Improvision). The cells in the L1
layer, located at various depths on the curved surface, were projected onto a
single reconstructed view by using maximum intensity projection in the
VOLOCITY software (Improvision).
Validation of the technique
The older flower buds were removed prior to imaging and the plants were
imaged repeatedly at regular intervals. The following criteria were used to
assess the performance of plants in our imaging conditions. Dissecting
early-stage floral buds can result in desiccation, and such plants were easily
recognized and removed from experiments. The vertical growth of the plant was
measured at the end of each imaging session by recording the growth in the
Z-axis. The plants that stopped growing were not imaged thereafter. The plants
that continued to grow but exhibited a gradual and continuous decrease in both
the SAM size and the total number of cells were excluded from the analysis.
That the conditions used allowed normal meristematic activity was indicated by
the following: in all the plants analyzed, no deviation from clonal
restriction of cell division patterns in the L1 layer was noticed and the
cells continued to divide anticlinally. As in earlier studies, cells in the PZ
divide at a faster rate than cells in the CZ. Two studies involving
noninvasive sector boundary analysis have shown that the lateral sepals are
rarely sectored (Bossinger and Smyth,
1996; Furner and Pumfry,
1993
). The sector configurations of flower buds observed in this
study correspond well with the configurations obtained in noninvasive methods.
The cell expansion behavior observed at the boundary regions mirrors the cell
expansion behavior described during the partitioning of leaf primordium from
the SAM in Anagallis arvensis
(Kwiatkowska and Dumais,
2003
). The total duration of imaging varied with the imaging
intervals, so that when shorter intervals such as 1-1.5 hours and 3 hours were
used, the plants were imaged no more than 40-66 hours (n=11 plants).
With longer observation intervals such as 6 hours and 12 hours, imaging was
performed for 72 (n=5) to 144 hours (n=2).
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Results |
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In order to test whether the apparent variations are in part due to the
markers used, a cyclinB1;1:GFP construct was used to visualize mitotic cells
at various time points. CyclinB1;1:GFP expression dynamics in SAMs were
determined by counter-labeling cyclinB1;1:GFP shoot apices with FM4-64, a
water-soluble lipophilic dye, to visualize cell divisions. In an individual
cell cyclinB1;1::GFP is seen as a bright nuclear label prior to division
(Fig. 3A). It then accumulates
as a thin line, resembling chromatin at the metaphase plate
(Fig. 3B). An hour later
cyclin:GFP expression disappears, with concomitant appearance of a new
cross-wall (Fig. 3C). Several
plants were tested for cyclinB1;1:GFP expression, and a wide range in the
number of cyclin-positive cells across different SAMs was observed
(Fig. 3D-F). Plants with very
few cyclin-expressing cells (Fig.
3D) to intermediate (Fig.
3E) to high levels (Fig.
3F) were observed. A similar variation in mitotic activity across
plants has been reported in earlier studies
(Laufs et al., 1998;
Grandjean et al., 2003
).
Therefore it can be concluded that the observed temporal variation in mitotic
activity is not due to the markers used, but represents real variation.
Spatial distribution of cell division
Primordia arise in a temporal sequence from definite locations within the
PZ. Therefore we represented the temporal sequence of cell divisions in space.
To do this, time-lapse data were utilized to integrate every cell division
event in successive 12- hour windows. These data were projected onto the final
time point to generate a spatial map of the mitotic progression in the L1
layer (Fig. 4A-C). The striking
feature in such a representation is that cell division is uniformly
distributed across the meristem, without any preference for the regions of
primordial specification marked P0 (the youngest primordium), P-1 (the
position where the primordium after P0 will form) and P-2. A few cells undergo
a second round of division within a 36-hour window
(Fig. 4C-G, arrows). Time-lapse
data (144 hours) from four different plants were analyzed and showed a similar
distribution. No spatial preference of cell division activity was observed
during both the low (Fig. 4A)
and the peak (Fig. 4C) phases.
Within this global uniform distribution, however, adjacent cells located in a
discrete spatial domain can divide simultaneously or within a short time
frame, suggesting a role for local signals in communicating cell division
information within a layer (Fig.
4A-C; Movies 1 and 2 at
http://dev.biologists.org/supplemental).
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The distribution of cell cycle length in the CZ revealed a pattern in striking contrast to that in the PZ. The range of cell cycle duration in the CZ is much wider than that in the PZ, starting from 36-72 hours. The most slowly dividing cells in the CZ are not arranged in a well-defined concentric circle.
Adjacent cells in the CZ can show very different cell cycle lengths. For example, a cell with a previous cell cycle duration of 66-72 hours can share its walls with cells whose previous cycle duration ranges between 36-66 hours. One of the reasons for such a wide range of cell cycle duration could be that the location of the CZ is not constant. Future experiments involving an operational marker for the CZ such as CLV3 might yield insights into the cell division behavior of these cells. In summary, the cell cycle duration in the PZ is relatively uniform compared with that in the CZ. Within the PZ, cell cycle duration is marginally shorter in early primordial regions compared with the cells in the intervening region, with a dramatic decrease in P3 and later stages. This analysis is consistent with the earlier observation that the amount of cell division is similar in regions where successive primordia arise.
Lineage analysis on time-lapse observations
One of the poorly understood aspects of meristem morphogenesis is the cell
behavior associated with the origin and subsequent development of primordia
from specialized regions in the PZ. The plant hormone auxin and its
distribution have been implicated in the selection of sites of primordium
specification (Benkova et al.,
2003; Reinhardt et al.,
2003b
). Lineage analysis has indicated the number of primordium
initials (Bossinger and Smyth,
1996
). Among the questions that remain are: Where are the
primordium initials located? How do they divide? What makes these cells
different from the intervening cells located between successive primordia?
What is the cell behavior associated with the separation of primordia from the
SAM? These questions were addressed by lineage reconstruction from time-lapse
observations.
The 35S::YFP29-1 transgene is uniformly expressed throughout the shoot apex, its use as a transgenic fluorescent marker facilitated the tracking of lineages originating from the regions close to the CZ and ending in differentiated primordia (Fig. 6). Time-lapse data from plasma membrane-localized YFP taken at 6-hour intervals over a period of 5 days were used to serially reconstruct lineages in the SAM. Data taken at 3-hour intervals for a period of close to 3 days were used to create continuous four-dimensional movies to reveal essential features of morphogenesis in relation to cell behavior (see Movies 1 and 2 at http://dev.biologists.org/supplemental).
Primordial progenitor cells map close to the slowly dividing cells in the CZ
The time-lapse series allowed the tracking of individual lineages that
result in primordial development from very early stages, even before any
visible appearance of primordial outgrowth. The time series of an entire
three-dimensional Z-stack was registered sequentially onto subsequent time
points by using a multimodality image registration algorithm in order to
achieve cell-by-cell alignment at sub-pixel resolution. This allowed
superimposition of the corresponding cells in the SAM across time points. Such
aligned stacks were used to trace lineages in the meristem that lead to floral
primordia. The L1 lineages incorporated into the successive primordia within
one SAM are depicted in Fig. 6.
Each one of the color-coded dots represents a progenitor cell in the 0 hour
image (Fig. 6A), and the
lineages that result from the divisions of these cells and their descendents
are marked with the same color code (Fig.
6B-E). We compared the location of the 0-hour progenitor cells to
that of the slowly dividing cells in the CZ. In most cases they abut these
cells. The number of cells that give rise to P-1 and P-2 was higher and with a
progressive decrease with P-3 and P-4 (Fig.
6A), as expected from the continued division of cells from P-4 to
P-1. The progenitor cells are arranged in either two rows in a radial arc (P-1
and P-2) or at earlier stages as a single row (P-4). Although we were unable
to determine the differentiation status of the primordium progenitor cells,
the mapping of progenitor cells close to the CZ allowed us to follow the
entire sequence of PZ cell behavior from early divisions to primordium
development.
Oriented cell divisions associated with axis of primordial outgrowth
A full-time series of projected sections depicting successive cell
divisions in L1 cells that gave rise to two primordia is represented
(Fig. 7A-F). Successive
divisions in the earliest cells and their descendents were oriented away from
the CZ and divided parallel to the lateral axis of primordial outgrowth as
shown for P-1 (Fig. 7D). The
first set of oriented cell divisions was observed between 12-18 hours within
the P-1 sector. Such oriented cell divisions in the following 24-30 hours
resulted in a column-shaped lineage and in extension growth of the primordium
from P-1 to P1. A similar pattern was visualized for the subsequent primordia:
for example, P-2 in the same shoot apex
(Fig. 7G-L). While most of the
progenitors consistently divide parallel to the axis of primordial outgrowth,
the first divisions in the most laterally located cells (white and pink in the
figure) are random with later divisions oriented parallel to the axis of
primordial outgrowth (Fig.
7G-L). Similar observations were made for the P-3 region (data not
shown). The division orientation of the cells in the PZ located between
primordial progenitors was also measured; in this case the region is marked as
P-4 and it is located between P-1 and P-2
(Fig. 6A). The cell division
analysis in these cells revealed a contrasting pattern in comparison with the
cells that form the next set of primordia. The cell division patterns of P-4
cells that occurred over a period of 66 hours are projected onto the final
time point in Fig. 9A,B. Cells
in the intervening regions divided in random orientation and the resultant
lineages appeared as square blocks rather than as columns of cells. For
simplicity in comparison, the lineages in the rest of the meristem are not
represented, although similar differential patterns of cell division did occur
there. However, not all of the lineages that follow oriented cell divisions
ultimately became part of primordia (for example, the lineage marked in blue
in Fig. 7L, which was retained
in the SAM proximal to the primordium, and only a part of the lineage marked
in yellow became part of the primordium). This observation suggests that the
axis of growth does not entirely constrain the fate of these lineages. Seven
plastochrons from two different plants were examined and all of them revealed
similar cellular behavior. Similar analysis was carried out on cells in the L2
layer, and the resultant lineages were projected onto the final time point.
Since it was not possible to represent all the cells located at different
depths in the L2 layer in one section, the cells were represented in
individual optical sections at different depths
(Fig. 8B-D). This analysis
revealed that cells preferentially divided parallel to the axis of primordium
growth. Even though individual cell divisions in the corpus could be followed,
it was not possible to serially reconstruct complete lineages due to a lack of
resolution in the distal-most regions, owing to the curvature of the SAM.
However, cells located in the corpus region of primordia in P3 stages could be
mapped; this corresponds to the time the flower primordium begins to acquire
height. During these stages, periclinal divisions could be observed in the
corpus. In summary, this analysis links cell division orientation changes to
the initial stages of primordium outgrowth.
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Cell expansion patterns
Cell expansion is another critical aspect of growth, and it has been
proposed to play a key role in SAM morphogenesis. Cell expansion patterns in
different stages of primordium development were examined. The cells in the
primordial regions expanded along the medial axis of primordial outgrowth and
divided perpendicular to the axis of cell expansion
(Fig. 7A-D). We have not yet
developed software to quantitate cell size. However, rapid cell expansion
could be observed during P2 and later stages, in continuous movies, as the
distance between adjacent nuclei increased rapidly (see Movie 1 at
http://dev.biologists.org/supplemental).
At the P3 stage the cells located in the primordium continued to expand
rapidly compared with cells located in the groove between the primordium and
the SAM, which is referred to as the boundary region
(Fig. 10B, boxed area, and C;
see Movies 1 and 2 at
http://dev.biologists.org/supplemental).
This behavior can be readily seen in higher magnification images
(Fig. 10D-G). The cells
located in the boundary region failed to expand along the medial axis of the
flower bud following division, while they appeared to expand along the lateral
axis (arrows in Fig. 10D-G).
The differential cell expansion resulted in a column of cells that appeared
elongated along the lateral axis and contracted along the medial axis of the
flower bud. Meanwhile, the cells that lined the medial edge of the primordium
expanded rapidly in both directions (arrowheads in
Fig. 10E-G point to the same
cells over a period of time). This rapid cell expansion was followed by a
burst of coordinated cell division in the cells of the primordium (Movie 2 at
http://dev.biologists.org/supplemental;
the red open arrow points to the boundary region, while the closed red arrow
points to cells in the primordium). It was during this time that the flower
primordium 2 (P2), which was only an extension of the meristem in the X-Y
dimension, began to acquire height, eventually leveling off at the height of
the SAM. The sustained growth of the flower primordium P3, thereafter bordered
by a non-growing boundary region, ultimately resulted in complete separation
of the primordium from the meristem, with the boundary region forming a
continuous layer with the pedicel (see Movie 2 at
http://dev.biologists.org/supplemental,
P3). Similar cell behavior could be seen in cells that formed the boundary
regions in layers below the L1 (data not shown).
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Discussion |
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Cell behavior and primordium development
We have given a dynamic description of several aspects of growth: the
amount of cell division, cell division orientation and cell expansion patterns
associated with SAM morphogenesis. Analysis reveals that it requires a minimum
of 4 days of continuous observation to reveal the essential aspects of cell
behavior associated with primordium growth. We have mapped the location of
primordium progenitor cells to a point early enough that they are close to the
slowly dividing cells in the CZ. Sector boundary analysis in inflorescence
meristems (IM) of Arabidopsis thaliana has led to the prediction that
a flower bud arises from a set of four progenitor cells
(Bossinger and Smyth, 1996).
Our observations show that number of primordium progenitor cells varies across
successive plastochrons, as must be true if the cells continue to divide. The
progenitor cells that represent the primordium at stages P-1 and P-2 are
higher in number than those comprising primordia at stages P-3 and P-4.
Therefore, number of primordium progenitors in our time-lapse experiments is
determined by the stage at which the progenitor cells are observed.
Ultimately, a lineage originating from a single cell might result in a
primordium, as proposed by Bossinger and Smyth
(Bossinger and Smyth, 1996
).
The limited duration of our time-lapse observations prevented us from
observing such a cell. Furthermore, our results do not reveal when primordial
progenitor cells become determined to a primordial fate. However, we can ask
the question: what is the stage at which the progenitor cells begin to divide
predominantly in an oriented manner, and become exclusively primordial in
fate? The answer is no later than a stage between P-1 and P0
(Fig. 6A,B), when the
progenitor cells exhibit oriented cell divisions, and all the lineages
originating from them become part of a flower bud
(Fig. 7A-F). Oriented cell
divisions are not necessarily a useful criterion for the first signs of
primordial differentiation or determination, however, because not all the
lineages that exhibit such a division pattern ultimately become part of the
primordium. Answers to questions of time of the earliest differentiation of
primordial cells will require a combination of a range of carefully defined
cell-type-specific markers with cell division analysis. Such an effort has
been made in a recent study by observing LEAFY(LFY)-expressing cells
through a live-imaging approach (Grandjean
et al., 2003
). It was shown that the LFY expression
domain is established gradually through cell recruitment, and LFY was
found to be expressed in a broader domain than the primordial progenitors.
Therefore, it was not possible to determine the number or exact location of
primordial initials. Recent studies have demonstrated that auxin distribution
in the SAM, mediated by auxin efflux carrier PINFORMED1 (PIN-1)
predicts the sites of primordium initiation
(Benkova et al., 2003
;
Reinhardt et al., 2003b
). It
has also been shown that PIN-1 expression is established at least a
plastochron earlier than LFY expression
(Reinhardt et al., 2003b
).
Therefore, early auxin response genes might act as more useful markers.
Since we were able to identify the progenitor cells early in primordial
development by tracing back to early stages, we could follow the cell behavior
associated with primordium development in relation to the rest of the cells in
the SAM. Our analysis of both the temporal and spatial patterns of cell
division activity, and of cell cycle length revealed that the amount of cell
division is comparable in regions of primordial development. This observation
is in contrast with that described earlier for Arabidopsis SAMs
(Laufs et al., 1998). The
analysis in that paper indicated that cell division is not uniformly
distributed in a SAM, and that twice the amount of cell division occurs in the
predicted P0 sector compared with the P-1. The apparent contradiction could be
due to the differences in technique, as the earlier analysis was not dynamic
and the temporal variations in mitotic rate within a given SAM might introduce
errors upon averaging single time point observations taken from different
plants. Other possibilities, such as differences in staging of the primordia
and our averaging cell divisions over 24-hour intervals, which may not
precisely correlate to each plastochron, might contribute to the different
observations made in this study. However, such possibilities should not have
influenced the conclusions drawn in this study because the primordial regions
were followed simultaneously, in continuous observations. The striking
similarities in the rates of cell division at each one of the time windows,
irrespective of averaging, can be seen by following individual cell divisions
in primordial regions P-1 and P-2 starting from 0 hours to 66 hours (compare
Fig. 7A-D with
7G-J), and such a similarity is
also reflected in similar cell cycle lengths
(Fig. 5A,B, white arrows). The
definition of primordial boundaries is based on lineage restriction to the
flower bud. It was possible to determine the primordial boundaries at a single
cell resolution in cases such as P-1 (Fig.
6B), but it was not the case in the P-2 region, as only a subset
of the lineages marked in yellow and blue became part of the flower bud
(Fig. 6C). Therefore, although
the determination of future primordial boundaries cannot be done at
single-cell resolution earlier than stage P-1, this may not affect the
conclusions drawn in this study. An additional question is: what is the rate
at which the PZ cells divide in the intervening region, located between
successive primordial regions? The differences in cell cycle lengths in the
intervening region located between the P0 and P-1 sectors were not strikingly
different when compared to either one of the primordial regions
(Fig. 5A,B, compare regions of
P0 and P-1, marked in white arrows, with the intervening region marked in
yellow arrows). However, a subset of cells in primordial regions P-1 and P-2
(Fig. 5A,B, white arrows)
exhibited a marginally shorter cell cycle length compared with the cells in
the intervening region located between them
(Fig. 5B, arrowheads). The cell
division rates in all the primordial progenitor cells and their descendents
are not uniform. For example, during early primordial growth, a cell with a
previous cell cycle length of 18-24 hours may shift to divide every 30-36
hours or even 42-48 hours. A subset of primordium progenitor cells divide
relatively infrequently compared with the rest of the progenitor cells,
resulting in lineages of different sizes
(Fig. 7D,E, compare the
lineages marked in white and blue with the rest). Similar differences in cell
division rates among primordial progenitors and their descendents can be
inferred by comparing the sizes of individual lineages in the flower buds
(Fig. 6B-E). Such a
heterogeneity in cell cycle duration within a floral primordium has been
described (Grandjean et al.,
2003
). Asynchronous cell division among primordial progenitors has
been predicted, as an explanation of sector configurations observed in clonal
analysis (Bossinger and Smyth,
1996
). The comparison of sizes of individual lineages in P2 with
that of the intervening region (IR) (Fig.
9A,B) revealed that the lineages in P2 are only marginally larger
than those in the IR, which corresponds with the slow formation of the
primordial bulge on the meristematic flank (see Movie 1 at
http://dev.biologists.org/supplemental;
watch for the growth in the P-1 region). However, it can also be argued that
the transient local increase in cell division rates in primordial regions
could result in the primordial bulge, which in itself is an inconspicuous
growth. Our data on cell division rates support such an argument, although
such transient differences could not be resolved in the mitotic index
analysis.
There is a striking pattern of oriented cell divisions parallel to the axis
of primordial outgrowth. Such a pattern of cell division is accompanied by an
initial extension of the primordium. Not all the cells that showed such a
behavior were ultimately incorporated into a primordium. This suggests that
not only the growth axis of primordial progenitor cells, but also the growth
axis in cells located proximal to the progenitor cells, might facilitate
outward growth. In this context, our results are not in complete agreement
with Bossinger and Smyth (Bossinger and
Smyth, 1996), with respect to the proposed spatial arrangement of
progenitor cells on the meristem flank, and cell division patterns. They
propose that the four progenitor cells are arranged as a block and the four
cells then divide to generate a concentric group of cells. Such cell division
patterns would not explain the outward growth noticed in initial stages of
primordium development. Several studies have indicated that structured cell
division patterns play a critical role in differentiation and morphogenesis.
Recent growth models of Antirrhinum petal lobes reveal that the
generation of asymmetric shape depends on the direction of growth rather than
on regional differences in growth rates
(Rolland-Lagan et al., 2003
).
It has been shown that the induction of non-anticlinal divisions in the tunica
layers of tobacco shoot apices leads to alterations in key regulators of SAM
maintenance but fails to induce morphogenesis
(Wyrzykowska and Fleming,
2003
). However, it can be argued that the maintenance of
structured anticlinal divisions in the tunica layer, such as the ones
described in this study, might be essential for morphogenesis to occur.
Surgical ablation of the L1 layer has been shown to affect cell division
patterns in layers below and also to affect primordium development
(Reinhardt et al., 2003a
). Our
results, indicating regular patterns of cell division from early primordial
growth, support the idea that planes as well as numbers of cell divisions, are
under tight control in the SAM.
We have observed cells dividing perpendicular to the expanding axis in
regions of primordial development (Fig.
7A-D). It was not possible, however, to quantify cell expansion
patterns. Therefore, we cannot say whether the initial set of oriented cell
divisions are a cause or an effect of growth in the primordial regions. The
role of cell expansion in SAM morphogenesis has been explored
(Pien et al., 2001;
Reinhardt et al., 1998
).
Expansin proteins are upregulated at the sites of incipient primordium
formation in tomato meristems, and the local expression of expansins has been
shown to induce leaf development in tobacco meristems
(Pien et al., 2001
). Based on
these observations, it has been argued that cell-division-independent
mechanisms play a role in morphogenesis. The oriented cell divisions
associated with primordium outgrowth in our study could be due to regulated
regional expansion resulting in oriented cell divisions. Although regulated
cell expansion might initiate such patterning, the gradients of local cell
division patterns have to be maintained to sustain further growth. It should
be possible to seek a causal link between regulated patterns of cell expansion
and orientation of cell division by studying them in real time, with the
methods introduced here, after development of image processing methods for
measurement of cell size. The studies on the surface expansion of the SAMs, in
Arabidopsis thaliana and in other species, have yielded quantitative
description of cell expansion behavior (Dumais and Kwiatkowska, 2002;
Kwiatkowska and Dumais, 2003
;
Kwiatkowska, 2004
). Studies of
the vegetative shoot apex of Anagallis arvensis and the inflorescence
apex of Arabidopsis thaliana have shown that the surface expansion in
the CZ is slow and nearly isotropic in comparison with that of the PZ, which
is found to be greater and anisotropic
(Kwiatkowska and Dumais,
2003
).
The surface geometry and expansion patterns during early stages of leaf
primordium development have been described. Our analysis of cell expansion
patterns is not yet quantitative. However, some qualitative observations can
be made. We have noticed that cells in the CZ exhibit a relatively
proportional expansion in all directions compared with the cells in the PZ
(see Movie 2 at
http://dev.biologists.org/supplemental).
It has been shown that cell expansion is slow in regions that give rise to the
leaf axil, with cells expanding along the axil while contracting across it
(Kwiatkowska and Dumais,
2003). It has been proposed that such cell behavior on the adaxial
leaf margins of developing primordium would partition the leaf primordium from
the surface of the SAM. We have described a similar cell behavior in the
boundary regions between flower primordium and the SAM, with cells failing to
expand along the medial axis of the flower bud. Therefore, such cell expansion
behavior at boundary regions could be a common theme utilized in developmental
contexts that require partitioning of actively growing regions, where
mechanisms such as programmed cell death (PCD) are not utilized.
Toward a `digital shoot apex'
One of the major limitations in understanding growth in both plants and
animals has been the inability to monitor cell behavior in real time. Several
studies have tried to address this issue, starting from inference of cell
behavior from clonal analysis, to generative modeling of growth through
computer simulations (Resino et al.,
2002; Rolland-Lagan et al.,
2003
). Our analysis of growth in real time circumvents the
requirements for inference in studies of clonal growth, or for theoretical
growth simulations. Once cell positions can be extracted by cell-finding
algorithms, it should be possible to integrate cell coordinates in time-lapse
observations. Such efforts are currently in progress
(www.computableplant.org).
The challenge for the future is to superimpose models of gene regulatory
networks on such models of growth, and to integrate with these models the
cell-cell interactions involved in meristem maintenance and morphogenesis
(Shapiro et al., 2003
).
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ACKNOWLEDGMENTS |
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Footnotes |
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