Epidermal patterning genes are active during embryogenesis in Arabidopsis
Silvia Costa and
Liam Dolan*
Department of Cell and Developmental Biology, John Innes Centre, Norwich,
NR4 7UH, UK
*
Author for correspondence (e-mail:
liam.dolan{at}bbsrc.ac.uk)
Accepted 24 February 2003
 |
SUMMARY
|
---|
Epidermal cells in the root of Arabidopsis seedling differentiate
either as hair or non-hair cells, while in the hypocotyl they become either
stomatal or elongated cells. WEREWOLF (WER) and
GLABRA2 (GL2) are positive regulators of non-hair and
elongated cell development. CAPRICE (CPC) is a positive
regulator of hair cell development in the root. We show that WER, GL2
and CPC are expressed and active during the stages of embryogenesis
when the pattern of cells in the epidermis of the root-hypocotyl axis forms.
GL2 is first expressed in the future epidermis in the heart stage
embryo and its expression is progressively restricted to those cells that will
acquire a non-hair identity in the transition between torpedo and mature
stage. The expression of GL2 at the heart stage requires WER
function. WER and CPC are transiently expressed throughout
the root epidermal layer in the torpedo stage embryo when the cell-specific
pattern of GL2 expression is being established in the epidermis. We
also show that WER positively regulates CPC transcription and GL2
negatively regulates WER transcription in the mature embryo. We
propose that the restriction of GL2 to the future non-hair cells in
the root epidermis can be correlated with the activities of WER and
CPC during torpedo stage. In the embryonic hypocotyl we show that
WER controls GL2 expression. We also provide evidence
indicating that CPC may also regulate GL2 expression in the
hypocotyl.
Key words: Arabidopsis embryos, Epidermis, CAPRICE, WEREWOLF, GLABRA2
 |
INTRODUCTION
|
---|
Epidermal cells enclose the cortex and differentiate in a
position-dependent manner along the root-hypocotyl axis of
Arabidopsis. In the root, epidermal cells located over two cortical
cell files develop as hair cells while those overlying a single cortical cell
file develop as non-hair cells (Dolan et
al., 1994
; Galway et al.,
1994
). In the hypocotyl, epidermal cells lying over two files of
cortical cells develop as stomatal complexes while those epidermal cells
overlying a single cortical cell file develop as elongated cells
(Gendreau et al., 1997
;
Berger et al., 1998a
). The root
meristem, from which the seedling root is derived, forms at the basal end of
the root-hypocotyl axis and the cellular organisation of the meristem is in
place by the late heart stage of embryogenesis
(Scheres et al., 1994
). A
combination of genetic determinants and positional signals specifies the
identity of epidermal cells in the seedling root
(Galway et al., 1994
;
Lee and Schiefelbein, 1999
;
Masucci et al., 1996
;
Wada et al., 1997
;
Berger et al., 1998b
).
Enhancer-trap and promoter-reporter gene studies indicate that the root
epidermal pattern is established during embryogenesis
(Berger et al., 1998b
;
Lin and Schiefelbein,
2001
).
Characterisation of mutants with defects in the specification of epidermal
cell types has defined a pathway for epidermal patterning in the root and in
the hypocotyl of the seedling. GLABRA2 is a homeodomain protein required for
non-hair fate in the root, and elongated epidermal cell fate in the hypocotyl
(Masucci et al., 1996
;
Di Cristina et al., 1996
;
Berger et al., 1998a
;
Hung et al., 1998
). Positive
regulators of GL2 transcription in seedlings include
WEREWOLF (WER), which encodes a MYB protein
(Lee and Schiefelbein, 1999
)
and TRANSPARENT TESTA GLABRA (TTG), which encodes a WD40
protein (Galway et al., 1994
;
Walker et al., 1999
).
GL2 and WER are expressed in non-hair cells in the root and
elongated cells in the hypocotyl of the seedling
(Masucci et al., 1996
;
Di Cristina et al., 1996
;
Lee and Schiefelbein, 1999
)
and the accumulation of GL2 transcript in these cells requires
WER function (Lee and
Schiefelbein, 1999
; Lee and
Schiefelbein, 2002
). CAPRICE (CPC) is a MYB protein and is
required for the specification of root hair cell identity
(Wada et al., 1997
), yet it is
expressed in non-hair cells (Lee and
Schiefelbein, 2002
; Wada et
al., 2002
). In cpc mutants only a few hairs are formed on
the root (Wada et al., 1997
),
but no mutant phenotype has been described for the hypocotyl.
TRIPTYCHON (TRY) encodes a CPC-related MYB protein and acts
in a partially redundant manner with CPC to specify root hair cell
identity (Schellmann et al.,
2002
).
It has been shown, on the basis of double mutant phenotypes and
promoter-reporter gene studies, that a reciprocal transcriptional control
between WER and CPC contributes to the establishment of the
position-dependent expression of GL2 in the seedling root
(Lee and Schiefelbein, 2002
).
WER positively regulates the expression of both GL2 and CPC
in non-hair cells, while CPC moves into hair cells and represses the
transcription of WER and GL2 in these cells
(Lee and Schiefelbein, 2002
;
Wada et al., 2002
). Lee and
Schiefelbein (Lee and Schiefelbein,
1999
) proposed that a WER:CPC protein ratio could determine the
fate of epidermal cells in the root. A high WER:CPC ratio would result in the
specification of a non-hair cell while a low ratio would result in the
formation of a hair cell. In the seedling, positional cues may be responsible
for the expression of WER in non-hair cells and a high WER/CPC ratio,
which initiates the cascade of molecular events that leads to
position-dependent cell type specification in the root epidermis
(Lee and Schiefelbein, 2002
).
The WER, CPC and GL2 genes have been shown to be required
for the maintenance of pattern in the post-embryonic seedling
(Berger, 1998a
;
Masucci et al., 1996
;
Di Cristina et al., 1996
;
Lee and Schiefelbein, 1999
;
Lee and Schiefelbein, 2002
).
Lin and Schiefelbein (Lin and
Schiefelbein, 2001
) have shown that GL2 is expressed
during embryogenesis and is controlled by WER and TTG. Here
we show the embryonic expression of WER, CPC and GL2 and
describe how the complex transcriptional interaction between these genes
starts in the embryo and defines the specific expression of GL2,
which leads to epidermal cell differentiation in the root-hypocotyl axis of
the seedling.
 |
MATERIALS AND METHODS
|
---|
Plant material
Wild-type Landsberg erecta (Ler) ecotype and
gl2-1 (in Ler) were obtained form the Nottingham Stock
Centre, cpc (in WS) was kindly provided by K. Okada (Kyoto
University, Japan), and wer-1 (in Col-o) was kindly provided from J.
Schiefelbein (University of Michigan, USA).
RNA in situ hybridisation
Tissue fixation and wax embedding were performed as described by Long and
Barton (Long and Barton,
1998
). Tissue sectioning and in situ hybridisation of
digoxigenin-UTP-labelled RNA probes were performed as described by Coen et al.
(Coen et al., 1990). Antisense GL2 probe was generated using T7 RNA
polymerase to transcribe the pGL5 cDNA clone that spans exon 5 of the
GL2 gene after linearising with BamHI. Sense GL2
probe was transcribed with T3 RNA polymerase from the same clone linearised
with EcoRI. Antisense WER probe was generated using T7 RNA
polymerase to transcribe the pWER1 clone, containing a 450 bp WER
cDNA fragment that spans exons 2 and 3, after linearisation with
BamHI. Sense WER probe was synthesised with T3 RNA
polymerase from the same clone linearised with EcoRI. Antisense
CPC probe was generated using T7 RNA polymerase, from the full-length
CPC cDNA clone linearised with EcoRI, and the CPC
sense probe was generated by transcription with T3 RNA polymerase, of the PCR
amplified insert of the same clone. Antisense GFP probe was generated
using T7 RNA polymerase to transcribe the clone containing the GFP5
cDNA, after linearising with XhoI, and the sense GFP probe
was transcribed with T3 RNA polymerase from the same clone, but linearised
with BamHI. All the clones are in pBluscript SK+ (Stratagene).
For each gene, serial sections through embryos at different developmental
stages were probed by in situ hybridisation and the experiments were repeated
on independent samples.
Digital images were captured with a Nikon Cool Pix 995 using differential
interference contrast (DIC) optics and assembled using Photoshop 5.
Confocal analysis of embryos
Embryos at different developmental stages were dissected from the seed
coats with a hypodermic needle and aligned on the surface of a thin layer of
Murashige and Skoog medium supplemented with 0.5% (w/v) Phytagel and 1% (w/v)
sucrose to avoid embryo desiccation. Embryos were then imaged with a MRC600
Biorad confocal microscope using a 488 nm excitation line, and a 523 nm
short-pass filter to monitor the expression of GFP. Optical sections of 2.5-3
µm were collected, processed with the NIH Image program
(http://rsb.info.nih.gov/nih-image/)
and assembled using Photoshop 5. At least ten embryos for each of the heart,
torpedo and mature developmental stages of the J2301 marker line and of the
homozygous gl2/gl2 J2301/J2301, cpc/cpc
J2301/J2301, and wer/wer J2301/J2301 lines were
analysed.
 |
RESULTS
|
---|
GL2 expression is initiated in the heart stage embryo
In the seedling GL2 is expressed in non-hair cells of the root and
elongated cells of the hypocotyl (Hung et
al., 1998
; Berger et al.,
1998a
). These two cell types overlie the wall of single cortical
cells and GL2 is required for their position-dependent specification
(Masucci et al., 1996
). To
investigate the earlier temporal and spatial pattern of GL2
expression during embryogenesis, GL2 mRNA was detected by in situ
hybridisation on transverse and longitudinal sections of the root and
hypocotyl of wild-type embryos at different developmental stages, as
classified by Jürgens and Mayer
(Jürgens and Mayer,
1994
). We consistently detected GL2 expression in heart
stage embryos (Fig. 1). Series
of consecutive longitudinal sections through wild-type heart stage embryos
were hybridised with the GL2 antisense probe and alternate sections
displayed a faint GL2 expression in protodermal cells at the base of
the embryo (arrows in Fig.
1A,B). This suggests that protodermal cells, which are not in
contact with each other, first express GL2 at the basal end of the
embryo during the heart stage. During the transition between heart and torpedo
stage, periclinal divisions in the protodermal cells give rise to an outer
lateral root cap layer of cells and an inner epidermal layer
(Scheres et al., 1994
). At the
torpedo stage GL2 transcript is detectable throughout root cap,
lateral root cap and epidermal layer in transverse sections of the root
(Fig. 1C,Da). In the hypocotyl,
GL2 is detected in most of the epidermal cells that overlie a single
cortical cell when viewed in transverse section
(Fig. 1C,Db). At the mature
stage, in both root and hypocotyl, GL2 is expressed in epidermal
cells that overlie a single cortical cell and will become non-hair cells in
the root and elongated cells in the hypocotyl
(Fig. 1E,F). These data suggest
that GL2 expression is first established in isolated protodermal
cells at the base of the heart stage embryo, and by the torpedo stage the
expression spreads to every cell of the root cap and epidermis of the root.
Between the torpedo and the mature stage GL2 expression is restricted
to those cells that will become non-hair cells in the seedling. In contrast,
in the hypocotyl, GL2 expression is already established in an
alternate pattern by the torpedo stage, which is then maintained throughout
the subsequent stages. The temporal difference in the establishment of the
cell-specific expression pattern of GL2 between the root and
hypocotyl suggests that during embryogensis the two epidermal cell types are
defined earlier in the hypocotyl than in the root.

View larger version (121K):
[in this window]
[in a new window]
|
Fig. 1. In situ hybridisation of GL2 mRNA in developing wild-type embryos.
In the schematics in B, D and F, RNA localisation is indicated in orange. (A)
Serial longitudinal sections through a heart stage embryo. Weak expression of
GL2 mRNA is detected at the basal end of the embryo, in the
protodermal layer, in alternate sections (arrows). Inset: a longitudinal
section hybridised with GL2 sense probe shows no signal. (B)
Schematic representation of the GL2 expression pattern detected in
one of the sections in A. (C) Transverse sections through the torpedo stage
embryo. From the left to the right of the panel the sections displayed were
taken from: the root pole at the base of the embryo, the root close to the
root pole, the root close to the hypocotyl, the hypocotyl. At the root pole,
GL2 mRNA is present in the root cap, lateral root cap and throughout
the epidermis. In the hypocotyl, GL2 mRNA is present in epidermal
cells that overlie a single cortical cell. Inset: section hybridised with
GL2 sense probe. (D) A schematic representation of the longitudinal
organisation of the embryo at torpedo stage and the radial organisation in the
embryonic root (a) and hypocotyl (b) as indicated. (E) Transverse sections
through the mature stage embryo. From the left to the right of the panel the
sections displayed were taken from: the root pole at the base of the embryo,
the root close to the root pole, the root close to the hypocotyl, the
hypocotyl. In the root, GL2 is expressed only in those cells that
overlie the walls of single cortical cells that will develop into non-hair
epidermal cells in the seedling. In the hypocotyl GL2 is only
expressed in epidermal cells that will become elongated, non-stomatal cells
overlying the walls of single cortical cells. Inset: section hybridised with
GL2 sense probe. (F) A schematic representation of the longitudinal
organisation of the embryo at the mature stage and the radial organisation of
the root (a) and the hypocotyl (b) as indicated. Scale bars: 40 µm.
|
|
WER and CPC are transiently expressed in the epidermis of the torpedo
stage embryo
WER is required to repress the formation of hairs and stomatal
complexes in those cells that contact a single underlying cortical cell in the
root and the hypocotyl, respectively (Lee
and Schiefelbein, 1999
). To define when WER expression is
established during embryonic development, in situ hybridisation studies were
performed on wild-type embryos at different developmental stages. WER
expression was not detected in globular or heart stage embryos
(Fig. 2Aa,Ab), but was first
detected in torpedo stage embryos. At this stage WER mRNA is
consistently present in consecutive transverse sections throughout the root
cap, the lateral root cap and the epidermal layer in the root
(Fig. 2B,Ca) and throughout the
epidermal layer in the hypocotyl (Fig.
2B,Cb). Later in embryogenesis, in mature embryos, no WER
expression is detected in any cell type of either the root or the hypocotyl
(Fig. 2D,E). Therefore, high
levels of WER expression may be required for only a short period
during embryogenesis.

View larger version (96K):
[in this window]
[in a new window]
|
Fig. 2. Localisation of WER mRNA in developing wild-type embryos. (A)
Longitudinal sections through a globular (a) and a heart (b) stage embryo and
schematic cellular organisation. No expression of GL2 mRNA is
detected in either embryonic stage. Inset: a longitudinal section hybridised
with WER sense probe shows no signal. (B-E) Transverse sections
through embryos at different developmental stages hybridised with WER
antisense RNA probe, and schematic representations of the cellular
organisation of root and hypocotyl. The localisation of mRNA is indicated in
orange on these schematics. From the left to the right of B and D the sections
displayed were taken from: the root pole at the base of the embryo, the root
close to the root pole, the root close to the hypocotyl, the hypocotyl. (B)
Torpedo stage. WER mRNA is detected in lateral root cap and epidermis
at the root pole. In the hypocotyl WER expression is detected
throughout the epidermal layer. Inset: section hybridised with WER
sense probe. (C) A schematic representation of the longitudinal organisation
of the embryo at torpedo stage and the radial organisation in the embryonic
root (a) and hypocotyl (b) as indicated. (D) Mature stage. WER
expression was not detected in either the root or the hypocotyl. Inset:
section hybridised with a WER sense probe. (E) A schematic
representation of the longitudinal organisation of the embryo at the mature
stage and the radial organisation of the embryonic root (a) and hypocotyl (b)
as indicated. Scale bars: 40 µm.
|
|
CPC is required for hair cell specification in the root
(Wada et al., 1997
) and so far
its function has not been characterised in the hypocotyl. Based on the role
and expression of the other genes involved in epidermal patterning in the root
and the hypocotyl we suspected that CPC might also be expressed
during embryogenesis. CPC expression was first detected in the
torpedo stage embryo and no expression was detected in earlier embryonic
stages (Fig. 3Aa,Ab,Ac). At the
torpedo stage, CPC mRNA was detected throughout the root in sections
close to the basal end (Fig.
3B,Ca). CPC expression was also detected in epidermal and
cortical cell layers of the hypocotyl (Fig.
3B,Cb). In the mature embryo CPC mRNA was present in the
root cap and lateral root cap of the root and was also detectable at a low
level throughout the root (Fig.
3D,Ea). In the hypocotyl CPC mRNA was only present in the
vascular tissues (Fig.
3D,Eb).

View larger version (94K):
[in this window]
[in a new window]
|
Fig. 3. Pattern of CPC mRNA localisation in wild-type embryos. (A)
Longitudinal sections through a globular (a), an early-heart (b) and a
mid-heart (c) stage embryo and schematic cellular organisation. No expression
of CPC mRNA is detected in any embryonic stage. Inset: a longitudinal
section hybridised with CPC sense probe shows no signal. (B-E)
Transverse sections through embryos at different developmental stages probed
with CPC antisense RNA and schematic representation of the cellular
organisation of root and hypocotyl. The localisation of mRNA is indicated in
orange on these schematics. From the left to the right of B and D the sections
displayed were taken from: the root pole at the base of the embryo, the root
close to the root pole, the root close to the hypocotyl, the hypocotyl. (B)
Torpedo stage. CPC mRNA is present throughout the root. In the
hypocotyl CPC mRNA is present throughout the epidermal and cortical
layers. Inset: a transverse section hybridised with CPC sense probe
shows no signal. (C) A schematic representation of the longitudinal
organisation of the embryo at torpedo stage and the radial organisation in the
embryonic root (a) and hypocotyl (b) as indicated. (D) Mature stage.
CPC is preferentially expressed in root cap and lateral root cap
cells in the root. In the hypocotyl CPC expression is confined to the
vascular tissues. Inset: transverse section hybridised with CPC sense
probe. (E) A schematic representation of the longitudinal organisation of the
embryo at mature stage and the radial organisation in the embryonic root (a)
and hypocotyl (b) as indicated. Scale bars: 40 µm.
|
|
WER and GL2 regulate each others transcription
It has previously been shown that WER positively regulates
GL2 expression in seedlings (Lee
and Schiefelbein, 1999
). To test if WER controls
GL2 in the embryo, the expression of each gene was determined in
wer and gl2 mutant embryos by in situ hybridisation. No
GL2 mRNA was detected in the root or hypocotyl of mature wer
embryos (Fig. 4A,B) but we
found that in wild-type embryos GL2 is expressed in epidermal cells
that overlie single cortical cells (Fig.
1F). This indicates that WER is required for the
establishment and maintenance of GL2 expression in the embryo.
Surprisingly, WER expression was detected throughout the epidermal
cell layer of gl2 mutant mature embryos in both root and hypocotyl
(Fig. 4C,D), whereas no
expression of WER was previously detected in mature wild-type embryos
(Fig. 2D,E). This suggests that
GL2 is required to repress WER transcription in the mature
embryo. Even though WER expression was not detectable in early
embryonic stages, i.e. globular and heart stage
(Fig. 2A), we wanted to test if
WER is active and required for the onset of GL2 expression
at the heart stage. No GL2 mRNA was detected in wer heart
stage embryos (Fig. 4E,F). This
data indicate that at the heart stage the level of WER expression is
below detection but that the gene is active and promotes GL2
expression.

View larger version (95K):
[in this window]
[in a new window]
|
Fig. 4. Localisation of GL2, WER and CPC mRNA in transverse
sections through mutant embryos at different developmental stages. From the
left to the right of A,C,K,M the sections displayed were taken from: the root
pole at the base of the embryo, the root close to the root pole, the root
close to the hypocotyl, the hypocotyl. E and G are consecutive sections
throughout heart stage embryos. (A) GL2 expression in wer
mature embryo. No GL2 mRNA was detected in either the root or the
hypocotyl. Inset: transverse section hybridised with GL2 sense probe.
(B) Schematic representations of the cellular organisation in transverse
sections of the root (a) and the hypocotyl (b). (C) WER expression in
gl2 mature embryo. WER mRNA is detected in every epidermal
and lateral root cap cell in the root and throughout the epidermal layer in
the hypocotyl. Inset: transverse section hybridised with WER sense
probe. (D) Schematic representations of the cellular organisation in
transverse sections of the root (a) and the hypocotyl (b) and the localisation
of WER mRNA (orange). (E) GL2 expression in wer
heart stage embryo. No expression of GL2 mRNA is detected. Inset: a
longitudinal section hybridised with GL2 sense probe shows no signal.
(F) Schematic representation of the cellular organisation of one of the
sections in E. (G) GL2 expression in cpc heart stage embryo.
GL2 mRNA is detected in protodermal cells both in the centre and at
the basal end of the embryo (arrows). Inset: transverse section hybridised
with GL2 sense probe. (H) Schematic representation of the
GL2 expression pattern (orange) detected in one of the sections in G.
(I) GL2 expression in cpc mature embryo. From the left to
the right of the panel the sections displayed were taken from: the root pole
at the base of the embryo, the root close to the root pole, the hypocotyl.
GL2 mRNA is detected throughout the root cap, lateral root cap and
epidermal layers in the zone above the initials. In the hypocotyl GL2
mRNA is detected in epidermal cells that overlie single cortical cells, as it
is in the wild-type embryo. Inset: transverse section hybridised with
GL2 sense probe. (J) Schematic representations of the cellular
organisation in transverse sections of the root (a) and the hypocotyl (b) and
the localisation of GL2 mRNA (in orange). (K) CPC expression
in gl2 mature embryo. CPC mRNA is present in lateral root
cap of the root and in the vascular precursor cells in the hypocotyl. Inset:
transverse section hybridised with CPC sense probe. (L) Schematic
representations of the cellular organisation in transverse sections of the
root (a) and the hypocotyl (b) and the localisation of CPC mRNA (in
orange). (M) CPC expression in gl2 torpedo stage embryo.
CPC mRNA is present in lateral root cap of the root and in the
vascular precursor cells in the hypocotyl. Inset: transverse section
hybridised with CPC sense probe. (N) Schematic representations of the
cellular organisation in transverse sections of the root (a) and the hypocotyl
(b) and the localisation of CPC mRNA (in orange). (O) CPC
expression in wer embryo. No CPC mRNA is detected in
transverse sections in the root (a) and hypocotyl (b). (P) WER
expression in cpc embryo. No WER mRNA is detected in
transverse sections in the root (a) and hypocotyl (b); inset, section
hybridised with WER sense probe. Scale bars: 40 µm.
|
|
CPC controls GL2 expression
To determine if CPC regulates GL2 expression in the
developing embryo we examined the expression of GL2 in cpc
mutant embryos. In cpc heart stage embryos GL2 expression is
detected in protodermal cells at the basal end of the embryo, as observed in
wild type (Fig. 4G,H;
Fig. 1A,B) and in some
protodermal cells in the central region (future hypocotyl) of the embryo. In
mature cpc mutant embryos GL2 is expressed at a low level in
the lateral root cap and in every epidermal cell of the root
(Fig. 4I,Ja). This contrasts
with the pattern of GL2 expression in wild-type in which epidermal
GL2 expression is restricted to the future non-hair cells
(Fig. 1Fa). To determine if
GL2 can control CPC, the expression of CPC was
examined in mature gl2 mutant embryos. The pattern of CPC
mRNA localisation in mature gl2 mutant embryos is very similar to
that found in wild-type embryos, i.e. it is located in the lateral root cap
(Fig. 4K,La;
Fig. 3D,E). Therefore,
GL2 does not control the accumulation of CPC mRNA whereas
CPC is required for the establishment of the specific pattern of
GL2 expression in the root of the mature embryo but not for the
initial expression of GL2 at the heart stage.
In the hypocotyl of mature cpc embryos GL2 mRNA was
present in some epidermal cells that were located over the wall of single
cortical cells (Fig. 4I,Jb), as
in wild type (Fig. 1Fb). In
gl2 embryos, CPC expression was present in the vascular
precursor cells as in wild-type embryos
(Fig. 4K,Lb;
Fig. 1Fb). This indicates that
GL2 does not require the activity of CPC for the maintenance
of its expression in the hypocotyl of the mature embryo. Furthermore, as it is
found in the root, GL2 does not control CPC expression in
the hypocotyl.
CPC transcription requires WER activity
To determine if WER controls CPC we examined the
expression of CPC in the root and in the hypocotyl of wer
embryos. In wer torpedo stage embryos, the same CPC
expression was detected as in wild-type mature stage embryos
(Fig. 4M,N;
Fig. 3D,E). In wer
mature stage no CPC mRNA was detectable in any sections through
wer mutant embryos hybridised with the CPC antisense probe
(Fig. 4O), in contrast to
wild-type mature embryos where CPC is expressed in the lateral root
cap and in the provascular tissue in the hypocotyl
(Fig. 3E) and WER is
not expressed (Fig. 2E). These
data indicate that WER positively regulates CPC transcription. To
examine if there is a reciprocal control between WER and CPC we analysed the
expression of WER in cpc mature embryos; no WER
mRNA was detectable, either in the root or in the hypocotyl
(Fig. 4P). As WER was
also not expressed in the wild-type mature embryo
(Fig. 2E), this suggests that
CPC does not negatively regulate WER transcription at this
stage.
GL2, CPC and WER are active in the embryo
We have shown that GL2, CPC and WER are expressed and
active in the embryo. To further investigate their role during embryogenesis
and to confirm that they are functional in the wild-type embryo, we analysed
the GFP expression pattern of the root-hypocotyl epidermal marker line J2301
in gl2, cpc and wer mutants.
In seedlings the GFP enhancer trap line J2301
(http://www.plantsci.cam.ac.uk/haseloff/Home.html)
expresses GFP in non-hair cells of the root and in elongated cells of the
hypocotyl (Berger et al.,
1998a
). Berger et al. (Berger
et al., 1998b
) indicated that GFP is expressed at the basal end of
the mid-heart stage embryo in the derivatives of the hypophysis in J2301
enhancer trap embryos. Later, from the mid-torpedo stage onwards, GFP is
expressed in the root epidermis and root cap cells. We repeated this analysis
by confocal microscopy, but consistently detected no GFP expression in any
embryonic stage prior to the torpedo stage
(Fig. 5Aa). Instead we
consistently found that isolated cells expressed GFP at the torpedo stage
(Fig. 5Ab,Ad). It was difficult
to determine if these GFP-expressing cells were located in the base of the
hypocotyl or in the upper part of the root
(Fig. 5Ab,Ac). By the end of
the torpedo stage, GFP was expressed in alternate longitudinal files of two to
three cells (Fig. 5Ad). At the
root end of mature stage embryos every cell expressed GFP and above this zone
cells expressed GFP in an alternative pattern
(Fig. 5Ae).

View larger version (107K):
[in this window]
[in a new window]
|
Fig. 5. Pattern of GFP expression in J2301 enhancer trap embryos. (A) Confocal
images showing the GFP expression in J2301 embryos dissected from the seed
coats at different developmental stages. From left to right: heart stage (a),
early, mid and late torpedo stage (b,c,d) and bent cotyledons stage (e).
Arrowheads point to cells expressing GFP. (B) In situ hybridisation of
GFP mRNA in transverse sections of a late torpedo stage embryo of the
J2301 enhancer trap line. From the left to the right the sections displayed
were taken from: the root pole at the base of the embryo, the root close to
the root pole, the root close to the hypocotyl, the hypocotyl. GFP is
expressed in lateral root cap cells in the root and in epidermal cells in the
hypocotyl. (C) Schematic representations of the cellular organisation in
transverse sections of the root (a) and the hypocotyl (b) and the localisation
of mRNA (in orange). Scale bars: 40 µm.
|
|
It was not possible by confocal analysis to determine with certainty the
identity of the cells that expressed GFP along the radial axis. It was also
difficult to resolve the root from the hypocotyl. Therefore, we analysed the
expression pattern of GFP by in situ hybridisation. Consecutive
transverse sections from the root tip to the hypocotyl of late torpedo stage
embryos were hybridised using an antisense GFP mRNA probe. In the
root we detected GFP mRNA exclusively in the lateral root cap
(Fig. 5B,Ca), while in the
hypocotyl we detected GFP mRNA in those epidermal cells that overlay
single cortical cells (Fig.
5B,Cb). These results indicate that the enhancer trap J2301 is
first expressed in the embryo at the torpedo stage in isolated cells, and
later, GFP expression continues in the lateral root cap cells of the root and
in those cells of the hypocotyl that overlie the anticlinal cortical cell
walls.
After characterising the GFP expression pattern of the J2301 enhancer trap
we generated plants that carried the enhancer trap in gl2, cpc and
wer mutant backgrounds. No GFP expression was detected by confocal
microscopy in any embryonic stage of gl2 mutants
(Fig. 6A), similarly, no GFP
expression was detected in cpc and wer mutants (data not
shown). To confirm these results we analysed the GFP expression by in
situ hybridisation on longitudinal sections of mature embryos of GFP marker
line J2301 in wild type, gl2 and cpc mutants. GFP
mRNA was detected in the lateral root cap and hypocotyl of wild type
(Fig. 6B,C), but none was
detected in gl2 (Fig.
6D,E) or cpc mutants
(Fig. 6F,G). These data
indicate that GL2, CPC and WER genes are required to
establish the expression of the GFP in the J2301 enhancer trap in
both the root and the hypocotyl of the mature embryo. Therefore, these genes
not only control their reciprocal expression in both the root and the
hypocotyl, but also the expression of the epidermal marker line J2301. The
results further prove that GL2, CPC and WER are functional
during embryogenesis in the epidermis and lateral root cap of the root and in
the epidermis of the hypocotyl.

View larger version (66K):
[in this window]
[in a new window]
|
Fig. 6. Embryonic expression of the J2301 enhancer trap in wild-type, cpc,
gl2 and wer mutants. (A) Confocal images showing the absence of
GFP expression in gl2 mutant embryos carrying the J2301
enhancer trap dissected from the seed coat at different developmental stages.
Scale bar, 60 µm. The dark green colour in whole embryos does not represent
GFP expression. The image was obtained using and open iris and high
gain during the collection of the optical sections with the confocal
microscope in order to visualise the embryos. (B-G) Detection of GFP
expression at the basal pole of mature wild-type, cpc and
gl2 mutant embryos carrying the J2301 enhancer trap using confocal
microscopy (B,D,F) and by in situ hybridisation (C,E,G). (B) In wild type,
strong GFP expression was detected in cells at the basal end of the
embryo, which in median longitudinal section (C) was shown to be precisely
located in lateral root cap cells. In cpc embryos (D,E) and
gl2 embryos (F,G) GFP expression was not detected. Scale
bars: 60 µm.
|
|
 |
DISCUSSION
|
---|
We show that the WER, CPC and GL2 genes, which are
required for the patterned differentiation of cells along the root-hypocotyl
axis in the seedling (Galway et al.,
1994
; Masucci et al.,
1996
; Di Cristina et al.,
1996
; Wada et al.,
1997
; Berger et al.,
1998a
; Hung et al.,
1998
; Lee and Schiefelbein,
1999
; Walker et al.,
1999
; Wada et al.,
2002
), are expressed and active during embryogenesis. We show that
these genes are expressed along the root-hypocotyl axis of the developing
embryo and propose that a complex interaction of transcriptional regulation
exists among WER, CPC and GL2 genes during the formation of
epidermal pattern in the embryo.
Model for the establishment of GL2 position-dependent expression
during embryogenesis
We propose a model based on the regulatory interactions between WER,
CPC and GL2 that occur during embryogenesis
(Fig. 7). We have shown that
WER positively regulates GL2 expression at the heart stage.
Then, by the torpedo stage, GL2 expression has spread to all cells in
the future epidermis. WER and CPC expression is then
detectable and WER promotes GL2 expression throughout the
epidermis. CPC is in turn required for the preferential accumulation
of GL2 transcript in future non-hair cell, perhaps by negatively
regulating GL2 transcription in hair cells position. In the mature
embryo GL2 negatively regulates WER transcription and
WER positively regulates CPC expression from the torpedo to
mature stages. These events result in GL2 being expressed at high
levels in the future non-hair cells and absent from the future hair cells in
the mature embryo. The pattern of GL2 expression is then maintained
in the root of the seedling and accounts for the pattern of hair cells and
non-hair cells in the root epidermis, where GL2 negatively regulates
hair formation in cells located in the non-hair position. The demonstration
that WER activity is required for the transcription of GL2
at the heart and mature stage and for the expression of CPC at the
mature stage is at odds with the fact that we cannot detect WER mRNA
at either stage by in situ hybridisation. It is possible that WER
transcript accumulates at levels that are too low to be detected by in situ
hybridisation or that the protein is stable and persists despite the fact that
the WER mRNA has disappeared. Further experiments are required to
explain this observation.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 7. Schematic representation of the expression of patterning genes in the
developing root during embryogenesis (upper row) and the proposed model for
their transcriptional regulation (lower row). Cortex is shown in yellow,
epidermis in white and lateral root cap in grey. (Upper row) GL2 is
first expressed in the heart stage embryo in a subset of cells in the
protoderm. By the torpedo stage, GL2 and WER are expressed
in all cells of the epidermis and CPC is expressed in the cortex,
epidermis and lateral root cap (C). In mature stage embryos GL2 is
expressed in the future non-hair cells and CPC is expressed in the
root cap. (Lower row) WER positively regulates GL2
transcription in heart stage embryos. At the torpedo stage WER
positively regulates GL2 transcription while CPC negatively
regulates GL2 transcription in future hair cells, which overlie the
cleft between two cortical cells. In the mature embryo GL2 negatively
regulates WER transcription that in turn positively regulates
CPC transcription. CPC is required for preferential
accumulation of GL2 in future non-hair cells.
|
|
In the hypocotyl, GL2 is required to specify the fate of elongated
epidermal cells (Berger et al.,
1998a
; Hung et al.,
1998
; Lin and Schiefelbein,
2001
) and from the early stages of embryogenesis, GL2
expression is restricted to those epidermal cells that will become elongated
cells (this study). WER is required for the control of GL2
expression in the hypocotyl of the embryo
(Lin and Schiefelbein, 2001
)
(this study) and of the seedling (Lee and
Schiefelbein, 1999
). We show that CPC may also regulate
GL2 transcription at the heart stage
(Fig. 4G,H). Further support
for this role of CPC comes from the observation that the J2301
enhancer trap is not expressed in the cpc hypocotyls in the mature
embryo (Fig. 6F,G). This
indicates that during embryogenesis, both WER and GL2 are
required for the specification of epidermal cell identity throughout the
entire length of the root-hypocotyl axis. In contrast CPC does not
control the position-dependent expression of GL2 expression in the
embryonic hypocotyl, as it does in the root. It is therefore likely that other
MYB-related proteins are required for the restriction of GL2
expression in the hypocotyl and that CPC may have other functions not
related to epidermal cell specification in the hypocotyl since it is also
expressed in the provascular region.
Different timing of establishment of epidermal patterning along the
root-hypocotyl axis
During embryogenesis the different domains of the embryo are progressively
laid down and by the late heart stage, the cellular organisation of the future
root is in place (Scheres et al.,
1994
) and GL2 mRNA is detectable. Tissues are
concentrically organised in the root (Dolan
et al., 1993
) and it is likely that the radial organisation of the
tissue layers is laid down first, and then later, the epidermal patterning is
superimposed on the pre-existing radial elements. The epidermal pattern is
then completed when GL2 expression is restricted to the cells in the
non-hair cell position of the root at the mature stage and to the cells in the
elongated cell position of the hypocotyl at the torpedo stage. In the
seedling, the pattern established during embryogenesis is then propagated in a
position-dependent manner, under the control of information that might be
located in the cell wall of the underlying layer of cortical cells
(Berger et al., 1998b
). The
model that we propose for the establishment of the root and the hypocotyl
epidermal pattern differs from those reported by Lin and Schiefelbein
(Lin and Schiefelbein, 2001
)
and Berger et al. (Berger et al.,
1998b
). Based on GL2 promoter-reporter gene expression
studies, Lin and Schiefelbein proposed that the epidermal cell patterning
mechanism in the root initiates during the early heart stage and it occurs
before the establishment of a functional meristem. This interpretation is
clearly correct for the patterning of the hypocotyls but contentious for the
patterning of the root because it is impossible to resolve the precise
patterns of expression in the root pole from the published images.
Furthermore, Berger et al. (Berger et al.,
1998b
) proposed that domains of positional information in the root
epidermis are established by the torpedo stage. This study was based on a
confocal microscope analysis of the GFP expression pattern of the J2301 line,
an enhancer trap that is expressed in non-hair cells of the root and elongated
cells of the hypocotyl in the seedling. We have now shown that in the mature
stage embryo the enhancer trap J2301 expresses GFP in the lateral root cap and
epidermis of the hypocotyl. Hence, the gene expression pattern observed by
Berger et al. (Berger et al.,
1998b
) was probably in the epidermis of the basal end of the
hypocotyl and could not have been in the root epidermis. This is consistent
with our view that the epidermal cells of the hypocotyl are patterned by the
torpedo stage and the root pattern is established later.
We can conclude from the results of these two previous studies and this one
that in the hypocotyl the initiation of epidermal pattern occurs in the heart
stage and is completed by the torpedo stage. The development of cell
patterning in the root epidermis is also initiated in the heart stage embryo,
but it is not completed until the embryo reaches the mature stage. The
difference between root and hypocotyl in the timing of the establishment of
epidermal pattern may reflect the presence of a gradient of positional
information along the apical-basal axis of the embryo. Alternatively, it may
reflect the presence of an equivalent source of positional information along
the apical-basal axis that it is interpreted at different developmental stages
in the root and in the hypocotyl.
The dynamic development of epidermal pattern is revealed by GL2
expression
GL2 mRNA is first present in isolated epidermal cells at the root
pole in the heart stage embryo. By the torpedo stage GL2 is present
in every epidermal cell and by the end of the mature stage it is restricted to
cells that will develop as non-hair epidermal cells. This restriction of
GL2 expression to future non-hair cells is regulated by genes whose
transcripts do not accumulate in the same cell-specific manner as
GL2, i.e. in non-hair cells. For example CPC transcripts are
found in a number of different cell layers and in every cell of the future
epidermis. This suggests the spatial distribution of WER and
CPC transcript alone cannot account for the pattern of GL2
transcript accumulation and suggests that cell to cell movement of a signal
that is controlled by CPC or WER may play an important role
in the establishment of epidermal pattern. Since the cell to cell movement of
CPC protein in the epidermis of seedling roots has been demonstrated,
CPC is a strong candidate for such a mobile signal
(Wada et al., 2002
).
The restriction of GL2 transcript to non-hair cells indicates that
the development of the pattern in the epidermis is progressive there
is early widespread GL2 expression throughout the epidermis that is
later restricted to cells in the future non-hair location. This progressive
restriction of gene expression is analogous to the process that occurs during
the development of sensory bristles in Drosophila. Early in
development the proneural genes are expressed in large fields of cells only to
become restricted to individual cells later
(Simpson et al., 1999
). The
restriction of proneural expression to individual cells is mediated by
cell-cell interactions. It is therefore likely that cell-signalling events may
act in the Arabidopsis embryo between the heart and torpedo stages.
The molecular basis of these interactions remains to be elucidated.
 |
ACKNOWLEDGMENTS
|
---|
We thank Des Bradley for help, advice and critical reading of the
manuscript, Fred Berger for advice and Nigel Kilby for providing the
CPC cDNA clone. This research was supported by the Gatsby Charitable
Foundation and the BBSRC.
 |
REFERENCES
|
---|
Berger, F., Linstead, P., Dolan, L. and Haseloff, J.
(1998a). Stomata patterning on the hypocotyl of Arabidopsis
thaliana is controlled by genes involved in the control of root epidermal
patterning. Dev. Biol.
194,226
-234.[CrossRef][Medline]
Berger, F., Haseloff, J., Schiefelbein, J. and Dolan, L.
(1998b). Positional information in root epidermis is defined
during embryogenesis and acts in domains with strict boundaries.
Curr. Biol. 8,421
-430.[Medline]
Coen, E., Romero, J. M., Doyle, S., Elliott, R., Murphy, G. and
Carpenter, R. (1999). FLORICAULA: A homeotic
gene required for flower development in Antirrhinum majus.Cell 63,1311
-1322.
Di Cristina, M., Sessa, G., Dolan, L., Linstead, P., Baima, S.,
Ruberti, I. and Morelli, G. (1996). The Arabidopsis
Athb-10 (GLABRA2) is an HD-Zip protein required for regulation of root hair
development. Plant J.
10,393
-402.[CrossRef][Medline]
Dolan, L., Janmaat, K., Willemsen, V., Linstead, P., Poethig, R.
S., Roberts, K. and Scheres, B. (1993). Cellular
organisation of the Arabidopsis thaliana root.
Development 119,71
-84.[Abstract/Free Full Text]
Dolan, L., Duckett, C., Grierson, C., Linstead, P., Schneider,
K., Lawson, E., Dean, C., Poethig, S. and Roberts, K.
(1994). Clonal relation and patterning in the root epidermis of
Arabidopsis. Development
120,2465
-2474.[Abstract]
Galway, M. E., Masucci, J. D., Lloyd, A. M., Walbot, V., Davis,
R. W. and Schiefelbein, J. W. (1994). The TTG
gene is required to specify epidermal cell fate and cell patterning in the
Arabidopsis root. Dev. Biol.
166,740
-754.[CrossRef][Medline]
Gendreau, E., Traas, J., Desnos, T., Grandjean, O., Caboche, M.
and Hofte, H. (1997). Cellular basis of hypocotyl
growth in Arabidopsis thaliana. Plant Physiol.
114,295
-305.[Abstract/Free Full Text]
Hung, C. Y., Lin, Y., Zhang, M., Pollock, S., Marks, M. D.
and Schiefelbein, J. W. (1998). A common
position-dependent mechanism controls cell-type patterning and
GLABRA2 regulation in the root and hypocotyl epidermis of
Arabidopsis. Plant Physiol.
117, 73-84.[Abstract/Free Full Text]
Jürgens, G. and Mayer, U. (1994).
Arabidopsis. In Embryos. Colour Atlas of
Development (ed. J. Bard), pp 7-21.
London: Wolfe Publications.
Lee, M. M. and Schiefelbein, J. (1999).
WEREWOLF, a MYB-related protein in Arabidopsis, is a
positional-dependent regulator of epidermal cell patterning.
Cell 99,473
-483.[Medline]
Lee, M. M. and Schiefelbein, J. (2002). Cell
pattern in the Arabidopsis root epidermis determined by lateral
inhibition with feedback. Plant Cell
14,611
-618.[Abstract/Free Full Text]
Lin, Y. and Schiefelbein, J. (2001). Embryonic
control of epidermal cell patterning in the root and hypocotyl of
Arabidopsis. Development
128,3697
-3705.[Abstract/Free Full Text]
Long, J. A. and Barton, M. K. (1998). The
development of apical embryonic pattern in Arabidopsis.Development 125,3027
-3035.[Abstract/Free Full Text]
Masucci, J. D., Rerie, W. G., Foreman, D. R., Zhang, M., Galway,
M. E., Marks, M. D. and Schiefelbein, J. W. (1996).
The homeobox gene GLABRA2 is required for position-dependent cell
differentation in the root epidermis of Arabidopsis thaliana.Development 122,1253
-1260.[Abstract/Free Full Text]
Schellmann, S., Schnittger, A., Kirik, V., Wada, T., Okada, K.,
Beermann, A., Thumfahrt, J., Jurgens, G. and Hulskamp, M.
(2002). TRIPTYCHON and CAPRICE mediate lateral
inhibition during trichome and root hair patterning in Arabidopsis.EMBO J. 21,5036
-5046.[Abstract/Free Full Text]
Scheres, B., Wolkenfelt, H., Willemsen, V., Terlouw, M., Lawson,
E., Dean, C. and Weisbeek, P. (1994). Embryonic origin
of the Arabidopsis primary root and root meristem initials.
Development 120,2475
-2487.[Abstract/Free Full Text]
Simpson, P., Woehl, R. and Usui, K. (1999). The
development and evolution of bristle patterns in Diptera.
Development 126,1349
-1364.[Abstract/Free Full Text]
Wada, T., Tachibana, T., Shimura, Y. and Okada, K.
(1997). Epidermal cell differentiation in Arabidopsis
determined by a Myb homolog, CPC. Science
277,1113
-1116.
Wada, T., Kurata, T., Tominaga, R., Koshino-Kimura, Y.,
Tachibana, T., Goto, K., Marks, M. D., Shimura, Y. and Okada, K.
(2002). Role of a positive regulator of root hair development,
CAPRICE, in Arabidopsis root epidermal cell differentiation.
Development 129,5409
-5419.[Abstract/Free Full Text]
Walker, A. R., Davison, P. A., Bolognesi-Winfield, A. C., James,
C. M., Srinivasan, N., Blundell, T. L., Esch, J. J., Marks, M. D. and
Gray, J. C. (1999). The TRANSPARENT TESTA GLABRA1
locus, which regulates trichome differentiation and anthocyanin biosynthesis
in Arabidopsis, encodes a WD40 repeat protein. Plant
Cell 11,1337
-1349.[Abstract/Free Full Text]