Download Lecture 6: Cell Fates

Lecture 6: Cell Fates
Summary: Asymmetric cell division and intercellular signalling play a role in the patterning
of different cell types in the epidermis.
Recommended reading:
How do cells know what they want to be when they grow up? Lessons from epidermal
patterning in Arabidopsis. Larkin JC, Brown ML, Schiefelbein, J. Annual Reviews of Plant
Biology 54:403-30 (2003).
Cell-fate specification in the epidermis: a common patterning mechanism in the root and
shoot. John Schiefelbein, Current Opinion in Plant Biology, 6:74-78, (2003).
Patterning of the Arabidopsis root epidermal cells.
The Arabidopsis root epidermis is composed of two cell types. Root hair or trichoblast cells
develop over underlying anticlinal walls of the cortex cells, while the remaining epidermal
cells develop as atrichoblast or non-hair cells. Current models for root hair differentiation
suggest 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 (H) cell; if it does express this pathway, then it develops as
a non-hair (N)cell. The expression of this pathway appears to be partly controlled by the
relative abundance of two MYB-type transcription factors, WEREWOLF (WER) and
CAPRICE (CPC). Each of these is proposed to interact with the same bHLH (basic helixloop-helix) transcription factor and the TRANPARENT TESTA GLABRA (TTG) protein,
with the WER protein able to generate an active transcription complex and the CPC protein
generating an inactive complex.
TRANSPARENT TESTA GLABRA (TTG). The homozygous recessive ttg mutants
possess root hairs on essentially every root epidermal cell (see figure to the right). This
implies that the normal role of the TTG product is either to promote specification of the nonhair 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.
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.
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.
Together, these findings suggest that (1) an R-like bHLH protein exists in Arabidopsis to
promote non-hair cell specification and (2) this R-like bHLH protein acts downstream from
the TTG in the specification pathway.
WEREWOLF (WER). Homozygous recessive wer mutations cause nearly all root epidermal
cells to be specified as root-hair cells. The WER gene encodes a MYB protein of the typical
R2R3 class. In situ RNA hybridization as well as GUS- and GFP-reporter gene fusions to
show that WER is preferentially expressed in N cells, which are the cells whose fate is
disrupted by wer mutations. 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. 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 WERbHLH 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.
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 nonhair cell specification pathway. The GL2 gene encodes a homeodomain transcription factor
protein (Rerie et al. (1994) Genes Dev. 8:1388-1399). A GL2::GUS reporter and in situ RNA
hybridization have been used to show that GL2 is preferentially expressed in the
differentiating non-hair epidermal cells within the meristematic and elongation regions of the
root.
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. Furthermore, the phenotypic effect of the
35S::R transgene is GL2-dependent. A 500 bp fragment of the GL2 promoter that is critical
for directing position-dependent expression contains putative MYB-binding site elements.
TTG activates an R-like bHLH transcription factor which in turn positively regulates the
expression of GL2 in a cell position-dependent manner to specify the non-hair cell type.
CAPRICE (CPC), TRIPTYCHON (TRY). The cpc and try mutants produce fewer roothair cells than the wild type, implying that CPC and TRY are positive regulators of root-hair
cell specification. The gl2 mutation is epistatic to cpc, which suggests that CPC promotes
root-hair cell specification by acting as a negative regulator of GL2. An explanation for
CPC/TRY's negative action is provided by the nature of its gene product. CPC and TRY
encode a small protein with a MYB-like DNA binding domain but without a typical
transcriptional activation domain. CPC and WER appear to compete with one another for the
ability to activate 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. Further,
wer cpc double mutants exhibit an epidermal phenotype and GL2 expression pattern that is
intermediate between the wer and cpc single mutants, indicating that the MYB encoded by
WER and the truncated MYB encoded by CPC exert opposing effects on root epidermis
patterning.
Epidermal Cell Fate Specification in Other Arabidopsis Organs Employs the Same
Components. 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. 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. This hypocotyl cell 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. 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.
In addition to affecting root and hypocotyl cell fate, 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.
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 Together, this suggests that cell specification in the root and shoot
epidermis employs a similar set of components, with the related WER and GL1 proteins
fulfilling the same role in the two different tissues.
Ectopic cell fates in the epidermis: the tornado mutant.
The cell layers of the Arabidopsis primary root are arranged in a simple radial pattern. The
outermost layer is the lateral root cap and lies outside the epidermis that surrounds the ground
tissue. The files of epidermal and lateral root cap cells converge on a ring of initials (lateral
root cap/epidermis initial) from which the epidermal and lateral root cap tissues of the
seedling are derived, once root growth is initiated after germination. Each initial gives rise to
a clone of epidermal cells and a clone of lateral root cap cells. These initial divisions in the
epidermal/lateral root cap initial are defective in tornado1 (trn1) and trn2 plants indicating a
requirement for TRN1 and TRN2 for initial cell function. Furthermore, lateral root cap cells
develop in the epidermal position in trn1 and trn2 roots indicating that TRN1 and TRN2 are
required for the maintenance of the radial pattern of cell specification in the root. The death of
these ectopic lateral root cap cells in the elongation zone (where lateral root cap cells
normally die) results in the development of gaps in the epidermis. These observations indicate
that TRN1 and TRN2 are required to maintain the distinction between the lateral root cap and
epidermis and suggest that lateral root cap fate is the default state. It also suggests that TRN1
and TRN2 repress lateral root cap fate in cells in the epidermal location. Furthermore, the
position-dependent pattern of root hair and non-root hair cell differentiation in the epidermis
is defective in trn1 and trn2 mutants. Together these results indicate that TRN1 and TRN2 are
required for the maintenance of both the radial pattern of tissue differentiation in the root and
for the subsequent circumferential pattern within the epidermis.
Stomatal patterning
Stomatal development in wild-type Arabidopsis involves three successive types of stomatal
precursor cells: meristemoid mother cells, meristemoids, and guard mother cells (GMCs). The
first two types divide asymmetrically, whereas GMCs divide symmetrically. Analysis of cell
wall patterns indicates that meristemoids can divide asymmetrically a variable number of
times. Before meristemoid division, the nucleus and a preprophase band of microtubules
become located on one side of the cell, and the vacuole on the other. Meristemoids are often
triangular in shape and have evenly thickened walls. GMCs can be detected by their roughly
oval shape, increased starch accumulation, and wall thickenings on opposite ends of the cells.
Because these features are also found in developing stomata, stomatal differentiation begins
in GMCs. The wall thickenings mark the division site in the GMC since they overlie a
preprophase band of microtubules and occur where the cell plate fuses with the parent cell
wall. Stomatal differentiation in Arabidopsis resembles that of other genera with kidneyshaped guard cells. This identification of stages in stomatal development in wild-type
Arabidopsis provides a foundation for the analysis of relevant genes and of mutants defective
in stomatal patterning, cell specification, and differentiation.
Patterning mutants.
Stomata are consistently patterned so that they are not in contact. This patterning is violated
in the too many mouths (tmm) and four lips (flp) mutations of Arabidopsis. which have
stomatal clusters in the first-formed leaves. To clarify the function of both genes in stomatal
initiation and patterning, the phenotypes of many different organs were quantified. The flp
mutation affects dorsiventral and cylindrical organs differentially with respect to the
frequency of clustering. The tmm mutation has a more complex region-specific phenotype in
that some regions lack stomata entirely, other regions have excess stomata, and the flower
stalk exhibits an apex-to-base gradient from excess to no stomata. This suggests that TMM
represents an unusual type of gene regulating plant cell development in that it can either
influence stomatal initiation in a positive or negative fashion depending on region. Since the
frequencies of initiation and clustering can be uncoupled in tmm, these two functions are
under separate region-specific control. Analysis of double mutants shows that tmm and flp in
some cases show region-specific interactions in both cluster formation and initiation, and that
there may be subpopulations of stomata under different genetic control. Recent isolation of
the TMM gene suggests that it is a membrane receptor component. In addition, a mutant of
the STOMATAL DENSITY & DISTRIBUTION (SDD) gene has a similat phenotype to flp, and
has been shown to encode a serine protease, which may be required for proteolytic processing
of a signalling intermediate.
Trichome patterning.
The plant epidermis is a multifunctional tissue playing important roles in water relations,
defence and pollinator attraction. This range of function is performed by a number of
different types of specialized cells, which differentiate from the early undifferentiated
epidermis in adaptively significant patterns and frequencies. These various cells show
different degrees of morphological specialization, but there is evidence to suggest that even
the less specialized cell types may require certain signals to ensure their correct
differentiation and patterning. Epidermal cells may potentially adopt certain fates through a
cell lineage based mechanism or a cell interaction mechanism. Work on stomatal
development has focused on the cell lineage mechanism and work on trichome differentiation
has focused on the cell interaction model. Recent work on the Arabidopsis trichome suggests
that interactions between neighbouring cells reinforce initial differences, possibly in levels of
gene expression or cell cycle stage, to commit cells to different developmental programmes.
Arabidopsis trichomes are large unicellular structures that develop on the surface of most
shoot-derived organs. In leaves, the number, spacing and shape of trichomes is tightly
regulated, and this process has been used as an experimental system to study the control of
cell fate and pattern formation. The general approach to understanding trichome development
generally involves:
(i) Description of the cellular events that take place during trichome initiation and
differentiation. (What’s going on?)
(ii) Screening for Arabidopsis mutants that affect this process. (Which genes control this
process?)
(iii) Isolation of the gene sequences. (What type of proteins are they?)
(iv) Promoter fusion and in situ hybridisation experiments, to determine which cells the genes
are transcribed in. (Where are the proteins acting?)
(v) Gene misexpression and protein interaction studies (What’s the control network?)
Screening for genes that affect trichome development has produced:
Model for Trichome Patterning in Arabidopsis.
(A) Two epidermal cells in a balanced situation. The R and GL1 genes act together to
promote trichome initiation, with TTG regulating the R gene. The double heterozygous
combinations of gl1 and ttg alleles suggest an interaction between GL1 and TTG, probably
with GL1 functioning as a positive regulator of TTG. TTG and TRY act together in the
inhibition of neighboring cells, with TRY acting as a repressor of trichome initiation.
(B) Two epidermal cells in a biased situation. The black cell has gained more competence,
and as a result, this cell expresses more GL1 and R and produces more TRY activity to inhibit
trichome initiation in surrounding cells. The dashed line represents reduced interactions.
In (A) and (B), arrows indicating activation of TTG by GL1 are dotted to indicate that this
interaction is postulated based on theoretical considerations and that evidence for this
interaction is only circumstantial. Solid-line arrows indicate positive interactions.