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Research in the Schiefelbein Lab
- What We Have Learned |
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This page summarizes some of the recent findings
from our lab. Other aspects of our research are described in the
Introduction and the
Where We Are Going sections.
Our research is focused on defining the molecular mechanisms that control cell
fate specification in the Arabidopsis root epidermis. To put it another way,
we have been trying to determine what genes and molecules cause epidermal cells
in the H position to always become hair cells and epidermal cells in the N
position to become non-hair cells. Putting together the results from our work
so far, we have come up with a simple model to explain how the process might
work. This model is shown below:
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| Model for the
molecular control of root epidermal cell fate in Arabidopsis. Arrows
indicate positive control and blunted lines indicate negative action.
Click on any word to go to a detailed description of that
component.
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To summarize the model, our research suggests that the fate of an epidermal cell
is determined by whether or not it expresses a genetic pathway that inhibits hair
cell differentiation. If a cell does not express this inhibitory pathway, then
it develops as a hair cell; if it does express this pathway, then it develops as
a non-hair cell. The expression of this pathway appears to be controlled by the
relative abundance of two MYB-type transcription factors, WER and CPC. Each of
these is proposed to interact with the same bHLH transcription factor and
the TTG protein, with the WER protein able to generate an active transcription
complex and the CPC protein generating an inactive complex. The epidermal cell
pattern is proposed to result from the WER activity being relatively concentrated
in the N position cells (which leads to expression of the inhibitory pathway) and
the CPC activity being concentrated in cells in the H position (which inactivates
the inhibitory pathway and, thus, causes the H cells to differentiate as hair
cells). The SCRAMBLED (SCM) receptor-like kinase protein is involved in
the differential accumulation of the WER and CPC activities in a
position-dependent manner.
Additional information about each of the components of this model is presented
below:
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Photograph of a
cross-section of a ttg mutant root showing root hair cells in both
the H and the N positions.
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TRANSPARENT TESTA GLABRA (TTG). The homozygous recessive ttg mutants possess
root hairs on essentially every root epidermal cell (see figure at the right).
This implies that the normal role of the TTG product is either to promote
specification of the non-hair cell type or to repress specification of the
root-hair cell type in cells located in the N position. Because ttg mutations
alter all aspects of non-hair cell differentiation (including early developmental
characteristics like cytoplasmic density, vacuolation, and cell division rate),
TTG is likely to be an early-acting component in the cell specification process.
The TTG gene has recently been cloned and found to encode a protein with four
WD repeats (Walker et al. (1999) Plant Cell 11:1337-1349). Although the
protein sequence does not provide an immediate mechanistic understanding of
TTG's action, other WD-repeat proteins mediate protein-protein interactions,
which implies that TTG may interact with proteins (e.g. transcription factors)
that specify epidermal cell fate. At present, we do not know the pattern of TTG gene expression in the root.
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Photograph of a ttg
mutant seedling (left) compared to a ttg seedling containing the
35S::R transgene.
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Basic Helix-Loop-Helix Protein (bHLH). bHLH transcription factors are
defined by a basic/helix-loop-helix motif originally identified in the product of
the proto-oncogene c-myc. In collaboration with Alan Lloyd's group, we found
that the ttg mutant can be functionally complemented by expressing the cDNA of
the maize R
bHLH gene under the control of the strong cauliflower mosaic virus 35S promoter
in Arabidopsis. In addition to restoring the normal specification of the
epidermal cells in the N position, this 35S::R transgene causes cells in the H
position to develop as non-hair cells (see figure at the right). Together,
these findings suggest that an R-like bHLH protein exists in Arabidopsis to
promote non-hair cell specification. Indeed, we have found that
three
Arabidopsis bHLH genes have a role in the root epidermis (GL3,
EGL3, and MYC1). In particular, the GL3 and EGL3 play largely
redundant roles in specifying the non-hair cell fate.
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Photograph of a root
bearing a WER::GFP transgene. GFP expression (green flourescence)
occurs in the developing N cells of the epidermis as well
as the lateral root cap cells.
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WEREWOLF (WER). Homozygous recessive wer mutations cause nearly all root
epidermal cells to be specified as root-hair cells. Using a map-based approach,
we cloned the WER gene and discovered that it encodes a MYB protein of the
typical R2R3 class. Furthermore, we employed in situ RNA hybridization as well
as GUS- and GFP-reporter gene fusions to show that WER is preferentially
expressed in N cells (see figure at the right), which are the cells whose
fate is disrupted by wer mutations. Thus, WER is likely to be an early-acting
transcriptional regulator that acts within the N-position cells to ensure they
adopt the non-hair cell fate.
We further discovered that WER action is closely associated with a bHLH protein.
A functional WER gene is required for the maize R bHLH protein (35S::R) to
influence root epidermis cell specification, and the WER protein physically
interacts with the R bHLH protein in the yeast two-hybrid assay. Thus, a
WER-bHLH interaction is likely to be required for the transcriptional regulation
of genes necessary to define the non-hair fate in the N cell position of the
Arabidopsis root epidermis.
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Root tip (left and
center) and root cross-section (right) showing
GUS activity (blue staining) in an Arabidopsis root bearing the
GL2::GUS transgene. GUS expression is restricted to specific files
of epidermal cells in the N position.
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GLABRA2 (GL2). Homozygous recessive gl2 mutants cause nearly all root
epidermal
cells to produce root hairs. However, in contrast to the ttg or wer mutations,
gl2 mutations do not affect any of the other non-hair cell phenotypes; that is,
the usual position-dependent differences in cytoplasmic density, vacuolation,
and cell division rate are observed in the developing gl2 root epidermis. This
implies that GL2 only regulates one branch of the non-hair cell specification
pathway. The GL2 gene encodes a homeodomain transcription factor protein
(Rerie et al. (1994) Genes Dev. 8:1388-1399). In collaboration with David
Marks' laboratory, we used a GL2::GUS reporter and in situ RNA hybridization to
show that GL2 is preferentially expressed in the differentiating non-hair
epidermal cells within the meristematic and elongation regions of the root
(see the photo at the right).
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Photograph of wild-type
(left), ttg mutant (center), and wer mutant (right) roots bearing the
GL2::GUS transgene. Note that ttg and wer dramatically
reduce GL2 gene expression.
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We also found that GL2 gene expression is influenced by TTG, WER, and by the
maize R bHLH protein, because ttg and wer mutations cause a reduction in GL2
promoter activity and 35S::R expression causes ectopic GL2 promoter activity (see
figure at the right). Furthermore, we showed that the phenotypic effect of the
35S::R transgene is GL2-dependent. Finally, in collaboration with David Marks'
laboratory, we defined the region of the GL2 promoter critical for directing
position-dependent expression to a 500 bp fragment that includes putative
MYB-binding site elements. Together, our findings suggest that TTG activates an
R-like bHLH transcription factor which in turn positively regulates the
expression of GL2 (and probably other as yet unidentified genes) in a cell
position-dependent manner to specify the non-hair cell type.
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CAPRICE (CPC). The cpc mutant and CPC gene were identified by the Okada
laboratory (Wada et al. (1997) Science 277:1113-1116). The cpc mutant produces
fewer root-hair cells than the wild type, implying that CPC is a positive
regulator of root-hair cell specification. Interestingly, the gl2 mutation is
epistatic to cpc, which suggests that CPC promotes root-hair cell specification
by acting as a negative regulator of GL2. Futhermore, the WER-bHLH-TTG factors are positive transcriptional regulators of the CPC gene.
An explanation for CPC's negative action is provided by the
nature of its gene product. CPC encodes a small protein with a MYB-like DNA
binding domain but without a typical transcriptional activation domain. Thus,
CPC may be able to bind to the GL2 promoter and block its activation. Taking
this one step forward, this explanation suggests that CPC and WER compete with
one another
for the ability to bind to the GL2 promoter, with CPC "winning" in the H cells
and WER "winning" in the N cells. Consistent with this explanation, expression
of the CPC gene under control of the constitutive 35S promoter (35S::CPC)
induces root-hair cells in the N position. Furthermore, we found that the
wer cpc double mutant exhibits an epidermal phenotype and GL2 expression pattern
that is intermediate between the wer and cpc single mutants, which indicates
that the MYB encoded by WER and the truncated MYB encoded by CPC exert opposing
effects on root epidermis patterning.
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SCRAMBLED (SCM). The SCM gene encodes a leucine-rich
repeat receptor-like kinase that is required for the proper
position-dependent cell-type pattern to form. The scm mutants
produce an altered pattern of hair/non-hair cell types, and gene
expression patterns (see figure below), which implies that SCM helps
developing epidermal cells to interpret their position. The
SCM gene is expressed throughout the developing root, including
epidermal cells in both the N and H positions, which suggests that
its activity is regulated differently in the N vs. H positions (e.g.
via binding of its putative ligand) and this leads to differential
accumulation of the WER and CPC transcription factors. In addition, we have recently discovered a transcriptional feedback loop, whereby SCM gene expression is negatively affected by the accumulation of the WER-bHLH-TTG transcriptional group. This loop causes the SCM receptor to persist in developing H cells for a longer period, relative to developing N cells, which likely enhances its ability to establish the cell type pattern.
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Epidermal Cell Fate Specification in Other Arabidopsis Organs Employs the Same
Components. During the course of our work on the root epidermis, we
discovered
that similar cell specification mechanisms operate during epidermis development
in the above-ground parts of the Arabidopsis plant. For instance, we found that
the same position-dependent mechanism is used to generate a pattern of epidermal
cells in the hypocotyl (the seedling stem). Although hypocotyl epidermal cells
do not produce root hairs, there are two types of epidermal cells in the
hypocotyl;
cells that generate stomatal complexes and cells that do not. Amazingly, these
two cell types arise in the same position-dependent manner as the hair and
non-hair cells in the root epidermis. Stomatal cells preferentially arise
outside an anticlinal cortical cell wall (analogous to the H position in the
root), whereas cells located outside a periclinal cortical cell wall (analogous
to the N position) preferentially develop as non-stomatal cells. Furthermore,
this hypocotyl cell
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Side view (left and center) and transverse section (right) of
hypocotyls from a three-day-old Arabidopsis seedlings stained for GUS activity and
containing the GL2::GUS transgene. The center image shows a stomatal complex in a
cell file that does not express the GL2 gene.
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pattern is controlled by the same molecular components as
the root pattern. The ttg, wer, and gl2 mutations significantly alter the
patterning of the hypocotyl cell types, causing a greater proportion of ectopic
stomata. Also, the GL2 and WER genes are preferentially expressed in epidermal
cells located outside the periclinal cortical cell wall of the hypocotyl (see
figure at left). This parallel pattern of cell types and gene activities
indicates that a common position-dependent cell specification mechanism is
employed during development of both the root and hypocotyl epidermis of the
Arabidopsis seedling.
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Trichomes on the surface of an Arabidopsis leaf containing the
GL2::GUS transgene and stained for GUS activity.
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In addition to affecting root and hypocotyl cells, the TTG and GL2 genes are
required for proper trichome (leaf hair) development on the Arabidopsis shoot.
Furthermore, a MYB gene, GLABRA1 (GL1), that is related to WER is involved in
specifying trichome cell fate. Unexpectedly, we discovered that although the
WER and GL1 genes control cell fate in distinct tissues, they encode functionally
equivalent proteins. Also, both GL1 and WER appear to regulate GL2 expression
in a similar fashion (see figure at right). Together, this suggests that
cell specification in the root and shoot epidermis employs a similar set of
components, with the related WER and GL1 protiens fulfilling the
same role in the two different tissues.
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Now that you've read about our past research, you can find out what we have
planned for the future in the Where We Are Going section
or, if you want more detailed information about this research, check out our
recent publications.
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