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daughter of the stem cell, but rather took place in cells
further up the root (
Figure 20.4
B). The onset of asym-
metric divisions at 6 hours suggested that genes respon-
sible for these divisions should be activated in that
timeframe. Therefore, a time course experiment was per-
formed, taking samples every 3 hours after the addition of
dexamethasone. To focus on genes activated in the cell
layer undergoing the asymmetric cell divisions, a marker
was introduced into the line that expresses GFP specifi-
cally in the cell layer prior to division and in both cell
layers after the division (
Figure 20.4
B). This was used for
cell sorting, followed by hybridization of the RNA from
the sorted cells to microarrays.
Analysis of the time course data identified a cluster of
co-regulated genes whose expression went up markedly at
6 hours after the addition of dexamethasone
[17]
. Among
the genes in the cluster were some that had been previously
identified as direct targets of SHR, including SCR. A
surprising finding was that a cell cycle component, a D-type
cyclin, was also in the cluster. Because there are many cells
undergoing division in the root, there was no reason to
believe that a protein involved in regulating the cell cycle
would be specifically regulated during a particular asym-
metric division. Further characterization showed that both
SHR and SCR could bind to the promoter region of the D-
type cyclin, and that it was not expressed in every dividing
cell but only in cells undergoing asymmetric divisions
dependent on SHR and SCR (
Figure 20.4
C). Mutation of
the D-type cyclin resulted in a delay in the asymmetric
division, while mis-expression in the mutant layer of shr
plants could partially rescue the loss of the asymmetric
division. Taken together these results showed how
a systems-level analysis with cell type-specific resolution
over time could lead to the identification of a dedicated
circuit regulating a specific asymmetric cell division crit-
ical for patterning of the root
[17]
.
Auxin Flow and Stem Cell Specification
Auxins are a class of small molecules with indole acetic
acid as a major representative. They serve as plant growth
regulators whose distribution conveys patterning informa-
tion and have been identified as key players in many
developmental processes. In roots, high levels of auxin are
correlated with stem cell specification, and membrane
proteins of the PIN-FORMED (PIN) family that facilitate
auxin transport are key factors for the accumulation of
auxin in the stem cell niche
[18,19]
. The auxin transport
system plays an important role in the ability of stem cells to
regenerate after removal from the root
[20]
. How is this
system of stem cells maintained in such a way that it can be
rebuilt during regeneration? Answers to this question do
not follow from the molecular connections between the
main players in stem cell specification by themselves, but
require an analysis of these links at the cellular level.
Construction of a 'digital root', where cells were repre-
sented using a formalism that allows them to grow and
divide, allowed an understanding of how auxin transport
and auxin diffusion in roots translate polar localization of
PIN proteins into a stable gradient of auxin with a peak at
the stem cell position
[21]
. Simulation of growth and cell
division, both under the control of auxin, revealed that such
an auxin gradient is stable at the multicellular level while
cells grow, divide and expand. Simulation of surgical
manipulations and laser ablations, known to regenerate
stem cells, revealed that the model had the capacity to
emulate regeneration. This model represented an early step
towards understanding dynamic pattern formation in roots.
Important future additions are needed, which explicitly
explain the coordinated polarization of the PIN proteins.
This polarization is expected to be in part self-organizing,
but the molecular components that govern polarization are
only partially understood, which makes it difficult to
formalize their activity and derive models that can be tested
by experiment. Next-generation models should explicitly
include self-organizing mechanisms that explain formation
of the root stem cell system.
How does auxin program the stem cell state? Auxin-
responsive transcription factors are required in the embryo
for the expression of transcription factors of the
PLETHORA (PLT) family, which maintain root identity
and the stem cell state in the root apex
[22,23]
. Some PLT
members can ectopically induce roots when expressed in
shoot primordia, indicating that they are necessary and
sufficient for root stem cell specification and maintenance
[23]
. While auxin accumulation is necessary to activate
PLTexpression, PLT function is required for the subsequent
expression of genes encoding PIN auxin transporters
[23]
.
Therefore, transcription factors expressed under the influ-
ence of auxin signal transduction in turn regulate auxin
flow. Finally,
Computational Modeling of Root
Development
Detailed knowledge on the transcriptome of an organ
identifies new factors that contribute to development and
allows us to elaborate the essential gene regulatory
networks. However, the logic of developmental regulation
is often not immediately apparent from a description of
the dynamics of the molecules that guide it. In the Ara-
bidopsis root, two examples illustrate this point well. First,
we discuss the relationship between the major driving
signal for stem cell specification, auxin, and the tran-
scription factors that are required for stem cell specifica-
tion. Second, we describe a lateral inhibition process that
leads to a regular pattern of hair-bearing and hairless
epidermal cells.
induction of PLT expression is auxin
