|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online November 9, 2007
doi: 10.1242/10.1242/dev.009282
1 Department of Molecular, Cell and Developmental Biology, Mount Sinai School of
Medicine, Box 1020, New York, NY 10029, USA.
2 Department of Craniofacial Development, Kings College, London SE1 9RT,
UK.
Authors for correspondence (e-mails:
sergei.sokol{at}mssm.edu;
jeremy.green{at}kcl.ac.uk)
Accepted 11 September 2007
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: PAR1 (MARK), aPKC, Ciliated cell, Apical-basal polarity, Xenopus, Epidermis, Notch, Ectoderm, XDelta-1 (DII1)
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
),
Drosophila neuroblasts and sensory organ precursors
(Betschinger and Knoblich,
2004; Roegiers and Jan,
2004) and have been implicated in mammalian skin stratification
and central nervous system development
(Gotz and Huttner, 2005;
Kosodo et al., 2004;
Lechler and Fuchs, 2005;
Sanada and Tsai, 2005;
Wodarz and Huttner, 2003).
Although cell polarity has been proposed to generate cell fate diversity
through asymmetric cell division (Chalmers
et al., 2002; Gotz and
Huttner, 2005; Muller and
Hausen, 1995), the molecular components of polarized cells that
are crucial in converting polarity information into cell fate determination
are largely unknown (Betschinger and
Knoblich, 2004; Cappello et
al., 2006; Imai et al.,
2006; Kosodo et al.,
2004; Ohno, 2001;
Wodarz and Huttner, 2003).
Xenopus ectoderm consists of the superficial (apical) and the
inner (basal) cell layers, which are produced as a result of asymmetric cell
divisions at blastula and gastrula stages
(Chalmers et al., 2003)
resulting in cells with different intrinsic developmental potential. The
superficial layer of non-neural ectoderm expresses several cytokeratins and
the Notch target ESR6e (Chalmers et al.,
2006; Deblandre et al.,
1999), whereas the inner layer is known to contain ciliated cells
marked by the 
-tubulin gene
(Deblandre et al., 1999;
Drysdale and Elinson, 1992).
Since frog ectoderm shows pronounced epithelial polarity with the apical and
basolateral membrane domains marked by atypical protein kinase C (aPKC) and
lethal giant larvae (LGL) (Chalmers et
al., 2005; Chalmers et al.,
2003; Dollar et al.,
2005), we used this system to assess the role of polarity proteins
on ectodermal cell fates.
Apical complex proteins, including PAR3, PAR6 and aPKC, function in the
polarization of Drosophila oocytes and mammalian epithelial cells and
may control asymmetric divisions and developmental patterning
(Betschinger and Knoblich,
2004; Ohno, 2001;
Plusa et al., 2005;
Rolls et al., 2003;
Wodarz and Huttner, 2003;
Wodarz et al., 2000). aPKC in
particular is enriched in the zygote cortex
(Nakaya et al., 2000), which
is inherited as the apical cortex of superficial cells in the Xenopus
blastula, and aPKC overexpression enhances apical character in these cells,
suppressing basolateral polarity markers, such as occludin, ß1-integrin
and LGL (Chalmers et al.,
2005; Dollar et al.,
2005). A number of biochemical (i.e. phosphorylation) targets of
aPKC have been elucidated, but it is not known which of these, if any, is
crucial for subsequent fate determination. The serine/threonine protein kinase
PAR1 [also known as MARK (MAP/microtubule affinity-regulating kinase)] is one
such target, which has been implicated in cell polarization
(Bayraktar et al., 2006;
Benton and St Johnston, 2003;
Cohen et al., 2004;
Doerflinger et al., 2003;
Pellettieri and Seydoux, 2002;
Tomancak et al., 2000). In
mammalian epithelial cells PAR1 is localized basolaterally
(Bohm et al., 1997), unlike
aPKC, PAR3 and PAR6 (sometimes referred to as the apical PAR complex).
Segregation of aPKC and PAR1 to opposite poles of epithelial cells and the
regulation of PAR1 by aPKC (Hurov et al.,
2004; Suzuki et al.,
2004; Vaccari et al.,
2005) suggest, (1) that aPKC may influence cell fates by
establishing apicobasal polarity in the ectoderm and (2) that its mechanism of
action in this regard is via local regulation of PAR1. This study provides
experimental evidence demonstrating that aPKC indeed functions to specify cell
fates in the superficial and the deep ectoderm layers and that PAR1 as a
critical molecular target of aPKC in this differential cell fate
determination. Our gain- and loss-of function data show that aPKC promotes
superficial cell fates, presumably by phosphorylating and locally inactivating
PAR1, which may modulate cell differentiation by influencing Notch
signaling.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). The
dominant negative (DN) RBP/J construct (R218>H) was generated by subcloning
a RBP/J cDNA obtained from the Ricken Bioresource Center (RDB 3022) into the
pXT7-NotI vector. Details of plasmid construction are available upon
request. Capped synthetic RNA for microinjection was generated using mMessage
mMachine kit (Ambion) and the following DNA templates: pCS2-Myc-PAR1A,
pCS2-nucßGal (Ossipova et al.,
2005), pcDNA3.1-Myc-rPKC
and pcDNA3.1-Myc-maPKC-N
(Parkinson et al., 2004),
pCS2-Delta1-Myc (Deblandre et al.,
2001), pCS2-Delta1-HA (Itoh et
al., 2003), ß-catenin, GFP-LGL1
(Dollar et al., 2005). PAR1
MOs have been previously described
(Ossipova et al., 2005),
control MO had the following sequence
5'-CCTCTTACCTCAGTTACAATTTATA-3'.
Embryo culture and microinjections, in situ hybridization and lineage tracing
Xenopus fertilization and embryo culture were performed as
described previously (Itoh et al.,
2005). Embryos were microinjected in 1/3x MMR, 3% Ficoll-400
(Pharmacia) in the animal pole with 5 nl of a solution containing 12.5 pg-1.5
ng of RNA per blastomere at the four- to eight-cell stage, and cultured in
0.1x MMR until desired stages. In loss-of-function experiments, PAR1BX
MO and PAR1B MO [referred to as PAR1BY MO in Ossipova et al.
(Ossipova et al., 2005)], or
control MO were injected at 5-40 ng per blastomere.
In situ hybridization and X-gal staining were carried out using standard
techniques (Harland, 1991)
with the following anti-sense probes: -tubulin and ESR6e
(Deblandre et al., 1999);
epidermal type I keratin (XK70)
(Winkles et al., 1985),
MyoD (Hopwood et al.,
1989). For 10 µm sections, embryos were embedded in coldwater
fish gelatin-sucrose mixture as described previously
(Fagotto and Gumbiner, 1994)
and cryosectioned using the Leica cryostat CM3050. Images were digitally
acquired on a Zeiss Axiophot microscope. Quantification of results is
presented as penetrance (PN), the percentage of embryos with a conspicuous
phenotypic change. RNA-injected embryos usually had a range of phenotype
severity. For ciliated cell quantification, embryos were scored as positively
affected if the number of ciliated cells per injected area was altered by at
least 50%. Cell numbers were determined per section of three to five
representative embryos and given as means±s.d. Results are
representative of at least three different experiments.
Immunocytochemistry
For cryosections, RNAs encoding Myc- or GFP-tagged proteins were injected
into the animal region of four-cell albino embryos. Embryos were manually
devitellinized at stage 10.5 and fixed in Dent's fixative for 2 hours.
Indirect immunofluorescence analysis on cryosections was performed essentially
as described previously (Fagotto and
Gumbiner, 1994). The following antibodies were used: anti-aPKC
(Santa Cruz, sc-216, 1:200), anti-GFP (Santa Cruz, 1:200), anti-Myc (9E10,
1:50), anti-phospho-histone 3 (Cell Signaling, 1:200), anti-acetylated tubulin
(Sigma, 1:100). When necessary, cryosections were double-stained using a
combination of monoclonal and polyclonal primary antibodies. Secondary
antibodies were Cy3-conjugated anti-rabbit (Jackson ImmunoResearch, 1:200) and
Alexa Fluor 488 (Molecular Probes, 1:200). Imaging was performed on a Zeiss
Axiophot microscope with the Apotome attachment at 400x magnification. A
representative section of an experimental group of 10-15 embryos is shown. For
hydroxyurea treatment, embryos were placed in 0.1x MMR containing 30 mM
hydroxyurea (Sigma) 1 hour after fertilization until control embryos reached
stages 10-10.5, fixed in Dent's fixative, sectioned and subjected to
immunostaining with anti-phospho-H3 antibody. The number of
phospho-H3-positive nuclei per section was determined after the analysis of
ten midsagittal sections (Saka and Smith,
2001).
Ectodermal layer separation and RT-PCR
Isolation of superficial and deep ectodermal layers was performed
essentially as described in (Chalmers et
al., 2002). Two-cell embryos were injected with PAR1 T560A (300
pg) or aPKC-CAAX (30 pg) mRNA, allowed to develop to stage 9, and animal pole
explants were dissected, dissociated in 0.7x Normal Amphibian Medium
(NAM) without calcium and magnesium (Peng et al., 1991) and allowed to
reaggregate in 1x Danilchik's buffer with BSA
(Peng, 1991). Cell aggregates
were allowed to mature until stage 14 or 19, harvested, and RNA was prepared
using an RNA purification kit (Qiagen) according to manufacturer's protocol.
First-strand cDNA synthesis was carried out using SuperScript II reverse
transcriptase (Invitrogen) according to manufacturer's instructions using 1.5
µg of RNA in a 20 µl reaction, and assayed by semi-quantitative RT-PCR.
For PCR, 2 µl of cDNA was used in a 40 µl PCR reaction. To ensure
linearity of PCR amplification, the number of PCR cycles was optimized
separately for each genetic marker in preliminary experiments. PCR primers for
ornithine decarboxylase (odc) and ESR6e were reported
previously (Chalmers et al.,
2002). Other primers were annealed at 56°C and included: for
grhl3, 5'-CGATGGAAGCACTGGCACTC-3' and
5'-CCACATCTTTGAAGATTGG-3' amplify a 520 bp fragment, 28 cycles
(based on Xenopus laevis cDNA clone xl235j16); for inca B,
5'-CCTCTCCTCAGGCGGGTTCC-3' and
5'-TAAGAATCCAGCCCCTTCG-3' amplify a 507 bp fragment, 30 cycles;
for delta-like 1 (dll1) (also known as XDelta-1),
5'-GCCTGCCGTGGTGAGTCC-3' and
5'-CACCTCTGTTGCAATGATG-3', amplify a 416 bp fragment, 30 cycles;
for XK70, 5'-CGCAGTATCTCTCAGTCG-3' and
5'-CGTCATTGATCTGGGAGCGC-3', amplify a 396 bp fragment, 21 cycles;
for -tubulin 84b, 5'-GTGGTGGAACCCTACAACGC-3' and
5'-GAGAGCTGCTCATGATAAGC-3', amplify a 319 bp fragment, 27
cycles.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Ohno, 2001;
Wodarz et al., 2000) and is
known to localize to the apical cortex of the early embryo, we hypothesized
that it may regulate cell fate decisions resulting from asymmetric divisions
of epithelial progenitors. Therefore, we examined its role in cell
specification in epidermal ectoderm, since aPKC is specifically enriched in
the superficial cell layer (Chalmers et
al., 2003). We constructed and expressed a membrane-targeted
aPKC-CAAX fusion protein, which has been described to promote self-renewal of
Drosophila neuroblasts (Lee et
al., 2006). We also studied effects of the N-terminal regulatory
domain of aPKC, which contains a pseudosubstrate site and has been shown to
function as a dominant-negative mutant
(Chalmers et al., 2005;
Parkinson et al., 2004). Using
in situ hybridization, we assessed -tubulin, a marker of ciliated cell
differentiation in the basal (inner) ectoderm layer
(Deblandre et al., 1999) in
early neurulae (Fig. 1).
aPKC-CAAX RNA produced half the number of ciliated cell progenitors on the
injected side (penetrance or percentage of conspicuously affected embryos,
PN=60%, n=104), whereas aPKC-N had the opposite effect,
leading to densely packed ciliated cell precursors on the injected side
(PN=50%, n=134; Fig.
1A-H). Embryo sections showed that ciliated cell development was
inhibited in aPKC-CAAX-expressing tissue
(Fig. 1D,E). By contrast,
aPKC-N-injected embryos contained multiple layers of ciliated cell
progenitors, with some -tubulin-positive cells positioned in the outer
ectoderm layer (Fig. 1F).
By mid to late neurula stages, ciliated cells are known to migrate into the
outer ectodermal layer and differentiate by forming multiple cilia
(Drysdale and Elinson, 1992).
We used an antibody to acetylated tubulin to assess cilia differentiation in
epidermal cells with altered aPKC expression. In stage 18 embryos, many
respecified ciliated cells formed cilia, indicating that cell differentiation
is relatively complete (Fig.
1I-K). We noted, however, that many of these cells failed to reach
the surface of the embryo, suggesting that cell migratory properties are
inhibited. Together these findings indicate that aPKC functions as an
inhibitor of ciliated cell differentiation in epidermal ectoderm.
|
), which we detected exclusively in the superficial cell
layer of epidermal ectoderm (Fig.
1L,O). XK70 was upregulated by aPKC-CAAX
(PN=82%, n=28; Fig.
1M,P,R,S) and inhibited by aPKC-N (PN=40%, n=31;
Fig. 1N,Q-S), revealing changes
complementary to the observed changes in ciliated cell differentiation. Note
that less penetrant effects of PKC-N might be due to the incomplete inhibition
of several vertebrate aPKC homologues with potentially redundant function.
Lineage tracing with coinjected lacZ RNA indicated that the effects
are cell-autonomous. Sectioning demonstrated an expansion of
XK70-positive cells into inner ectoderm layers of aPKC-CAAX
RNA-injected embryos, as compared with the superficial expression of this gene
in control embryos or reduced expression in aPKC-N RNA-injected embryos
(Fig. 1O-Q). These observations
indicate that aPKC promotes superficial layer markers at the expense of basal
layer differentiation.
aPKC regulates PAR1 localization in Xenopus ectoderm
Having established a role for aPKC in the specification of the superficial
cell layer, we wanted to identify a potential molecular target that may be
responsible for aPKC effects on ectodermal cell fates. Given that the PAR1
kinase has been shown to localize in a domain complementary to that of aPKC in
different systems (Benton and St Johnston,
2003; Bohm et al.,
1997; Ohno, 2001),
we wanted to know how PAR1 is distributed in Xenopus ectoderm and
whether this distribution is affected by aPKC. Overexpressed tagged PAR1
exhibited mostly basolateral localization in superficial ectoderm cells
(Fig. 2A). Both PAR1A and PAR1B
proteins with different epitope tags (GFP and Myc) had this localization (data
not shown). This distribution was altered by coinjected aPKC-CAAX
(Fig. 2B), resulting in a
mostly cytoplasmic staining for PAR1, consistent with the idea that aPKC
regulates PAR1 localization by phosphorylation
(Kusakabe and Nishida,
2004).
|
|
;
Suzuki et al., 2004). The
mutated T560A construct was predominantly localized at the apical surface,
although also present at the basolateral surface when introduced into early
embryos at higher levels, presumably because of saturation of the endogenous
machinery that is responsible for PAR1 localization
(Fig. 2C,C'). This
finding indicates that endogenous aPKC not only inhibits apical localization
of PAR1, in agreement with published data
(Hurov et al., 2004;
Suzuki et al., 2004), but may
also actively promote PAR1 basolateral localization. By contrast, the cortical
localization of T560A was unaffected by aPKC
(Fig. 2D), confirming that this
construct is insensitive to aPKC regulation. The overall levels of PAR1 and
T560A were not significantly affected by aPKC-CAAX in embryo lysates (data not
shown), suggesting that the observed regulation is not due to altered protein
stability. Whereas PAR1 localization was modulated by aPKC, neither
overexpressed T560A, nor previously characterized PAR1 MOs
(Kusakabe and Nishida, 2004;
Ossipova et al., 2005)
influenced aPKC distribution (data not shown). These data confirm the view
that the apical PAR protein complex functions upstream of PAR1 to regulate its
localization and, possibly, function in embryonic ectoderm
(Etemad-Moghadam et al.,
1995).
|
|
; Bohm et al.,
1997; Ohno, 2001)
(and this study), we wanted to evaluate the hypothesis that PAR1, similar to
aPKC, regulates cell fates in epidermal ectoderm. Since the deep and the
superficial cells arise by asymmetric divisions
(Chalmers et al., 2003), we
expected that PAR1 might influence cell fates in a manner opposite to that of
aPKC.
|
-tubulin-positive cells (PN=61%, n=64),
whereas ß-gal RNA (n=33) and kinase-dead PAR1 RNA
(n=26) did not have this effect
(Fig. 3A-K). Of note, a similar
enhancing effect on ciliated cell development was observed for both T560A and
wild-type PAR1 RNA, indicating that overexpression of this kinase is
sufficient to influence cell fate determination
(Fig. 3B,J). In cross-sections
of PAR1 RNA-injected embryos, -tubulin-positive cells frequently formed
a double layer instead of a single cell layer
(Fig. 3C,D and data not shown).
These ectopic -tubulin-positive cells differentiated multiple cilia,
based on immunostaining with antibodies to acetylated tubulin
(Fig. 3E,F), indicating that
the process of cell fate respecification is relatively complete. As with
aPKC-N-expressing cells many ectopic ciliated cells, formed in response to
T560A RNA injection, remained in the inner layer, in contrast to control
embryos in which ciliated cells intercalated into the superficial layer by
stage 18.
By contrast, depletion of PAR1 in ventral ectoderm with the previously
characterized PAR1B morpholino antisense oligonucleotide (MO)
(Kusakabe and Nishida, 2004;
Ossipova et al., 2005)
downregulated the -tubulin-positive cell population (PN=41%,
n=18), indicating that PAR1 is required for ciliated cell
differentiation (Fig. 3G,H).
The same effect was observed with two different PAR1 MOs, but not a control
MO, and could be rescued by PAR1 RNA, supporting specificity of these reagents
(Ossipova et al., 2005). By
contrast, ectoderm-targeted PAR1 MOs did not inhibit the mesodermal marker
MyoD (see Fig. S1D-G in the supplementary material), confirming that
the observed effects on ectoderm specification are independent of the PAR1
function in organizer formation and mesoderm development
(Ossipova et al., 2005). Thus,
the effects of aPKC and PAR1 on cell fates in the two ectoderm layers
correlate with the corresponding apical and basolateral distribution of these
proteins in embryonic cells.
|
;
Peng et al., 2000;
Plant et al., 2003)
phosphorylated by aPKC, did not influence ciliated cell development
(Fig. 4A-D). This difference
between PAR1 and LGL1 may be crucial because it provides an apparent
distinction between a polarity protein that affects cellular architecture and
shape (LGL) (e.g. Dollar et al.,
2005), from one that links polarity with cell fate (PAR1). Whereas
PAR1 has been shown to positively regulate Wnt-ß-catenin signaling
(Ossipova et al., 2005;
Sun et al., 2001), ciliated
cell development was not significantly affected by ß-catenin RNA
(Fig. 4C,D), suggesting that
the PAR1 effect on ciliated cells in not likely to be mediated by the Wnt
pathway. Both LGL and ß-catenin RNAs retained high functional activity in
these experiments, as measured, respectively, by ectoderm pigmentation
(Dollar et al., 2005) and
secondary axis induction assays (Fig.
4E-J).
aPKC acts upstream of PAR1 to specify ectodermal cell fates
The opposite effects and non-overlapping distribution of aPKC and PAR1 in
epithelial cells raise a question of the epistatic relationship between the
two proteins in the pathway leading to ciliated cell differentiation. To
assess whether PAR1 acts downstream of aPKC in cell fate specification, we
co-expressed aPKC-CAAX and T560A, and analyzed the number of ciliated cells.
We observed that aPKC-dependent suppression of ciliated cell differentiation
was rescued by T560A and by wild-type PAR1
(Fig. 5A-C,G,H, and data not
shown). aPKC-CAAX inhibited -tubulin in 75% of injected embryos
(n=36), whereas this number was reduced to 20% (n=68) in the
presence of T560A (Fig. 5G). In
a complementary loss-of-function approach, aPKC-N increased ciliated cell
differentiation in 66% of injected embryos (n=56), whereas this
number was reduced to 32% (n=34) in the presence of PAR1B MO
(Fig. 5D-F,I,J). Together,
these results indicate that aPKC functions upstream of PAR1 in cell fate
determination.
Lack of PAR1 and aPKC effects on cell cycle
Our lineage tracing experiments suggest that aPKC and PAR1 directly
modulate cell fates in the superficial and deep ectoderm layers. The
alternative explanation is that the polarity proteins affect the number of
progenitor cell divisions, resulting in the corresponding increase (or
decrease) in the number of superficial or deep ectodermal cells, as aPKC has
been shown to affect cell proliferation in Drosophila epithelial
cells (Rolls et al., 2003). We
tested this possibility by immunostaining injected embryos with anti-phosho-H3
antibodies that mark mitotic nuclei in many species including Xenopus
(Saka and Smith, 2001). Our
analyses carried out for gastrula and neurula stages showed that the expanded
number of ciliated cells in PAR1 or aPKC-N-injected embryos was not due to
increased number of mitoses (Fig.
6 and data not shown).
Opposite effects of aPKC and PAR1 on gene expression in layer separation experiments
The results of embryo injections are consistent with a model, in which aPKC
and PAR1 function to establish the difference between superficial and inner
cell layers acting effectively as cell layer determinants. This model predicts
that aPKC and PAR1 should each be capable of switching one layer-specific cell
fate to another. Alternatively, PAR1 and aPKC-CAAX may expand the
corresponding pools of the inner or superficial cells without
trans-differentiation. To address these possibilities, we studied how
overexpressed aPKC and PAR1 alter layer-specific gene expression in separated
ectodermal layers (Fig. 7A,B).
We observed that T560A upregulated inner cell layer genes -tubulin and
inca B (Luo et al.,
2007) and decreased the expression of the superficial layer genes
ESR6e and grhl3
(Chalmers et al., 2006) in
both ectodermal layers at stage 14 (Fig.
7C). Reciprocally, aPKC-CAAX inhibited -tubulin and
inca B, while inducing ESR6e and grhl3
(Fig. 7C) in the inner layer.
At stage 19, we observed that T560A inhibited the expression of XK70
and increased levels of XDelta-1 and inca B in the superficial layer,
whereas aPKC-CAAX upregulated XK70 together with ESR6e and
grhl3 in the inner layer (Fig.
7C). These results are consistent with the idea that PAR1 and aPKC
govern layer-specific gene expression. The PAR1-dependent enhancement of
-tubulin expression in inner layer cells additionally suggests that
PAR1 can modulate or override the regulation of ciliated cell precursors by
lateral inhibition in that layer (see below).
|
). To confirm these results, we examined whether PAR1 has an
effect on ESR6e expression using in situ hybridization. PAR1 RNA
downregulated ESR6e in superficial cells
(Fig. 8A,B), suggesting that
PAR1 controls cell fates through the Notch pathway previously implicated in
epidermal and ciliated cell differentiation
(Alonso and Fuchs, 2003;
Blanpain et al., 2006;
Deblandre et al., 1999).
Consistent with this interpretation, we noted that in superficial cells T560A
upregulated the expression of XDelta-1
(Fig. 7C), a marker for
suppressed Notch signaling (Deblandre et
al., 1999).
We next evaluated whether PAR1 can influence the localization and function
of XDelta-1, a Notch ligand. In the absence of PAR1, we observed basolateral
distribution of XDelta-1 in Xenopus ectoderm
(Fig. 8C). Lack of apical
localization indicates that XDelta-1 is less abundant in superficial ectoderm
as compared with deep ectoderm cells. Considering that Delta and Notch are
known to negatively affect each other's activity, lower levels of Delta would
correspond to higher levels of Notch receptor signaling, as evidenced by
higher ESR6e expression in superficial cells. In the presence of PAR1
RNA, XDelta-1 was distributed in multiple cytoplasmic vesicles
(Fig. 8D). This observation
suggests that PAR1 might influence XDelta-1 endocytosis and recycling, which
is expected to result in altered Notch signaling
(Itoh et al., 2003).
To further investigate the relationship between PAR1 and the Notch pathway,
we studied the functional interaction of PAR1 and the Notch ligand XDelta-1.
Overexpressed XDelta-1 was previously reported to induce ciliated cell
differentiation, an effect that has been attributed to the cell-autonomous or
`cis-inhibitory' activity of Delta in Notch-expressing cells
(Deblandre et al., 1999). At
lower doses, XDelta-1 or PAR1 RNAs did not significantly affect the number of
ciliated cells. However, the coinjection of both RNAs resulted in a
synergistic increase in ciliated cell number in the majority of injected
embryos (Fig. 8E-H), indicating
that PAR1 enhanced the inhibitory effect of XDelta-1 on Notch signaling. By
contrast, PAR1 did not influence the activity of dnRBP/j
(Fig. 8I,J,N,O), a dominant
intracellular inhibitor of the Notch pathway, or the Notch intracellular
domain (Notch-ICD), a constitutively active form of the Notch receptor
(Fig. 8K,L). The simplest
interpretation of these findings is that PAR1 inhibits signaling at the level
of Delta, rather than downstream of Notch. Together, our results support the
hypothesis that PAR1 functions to specify inner cell fates by downregulating
Notch signaling in the superficial ectoderm layer.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Gotz and Huttner, 2005).
Additionally, our findings provide molecular identities for the postulated
cell fate determinants, because interference with either apical (aPKC) or
basolateral (PAR1) polarity proteins changes the balance between the two cell
layers of the epidermis and results in altered cell fates. We expect that the
same pathway may control asymmetric divisions of stem/progenitor cells in
other epithelial tissues and organs.
Despite growing experimental evidence related to the establishment of the
apical-basal polarity, how cell polarity proteins influence developmental
fates of polarized progenitor cells is still poorly understood. Based on the
phenotype of the zebrafish `heart- and-soul' mutation, aPKC has been
implicated in the formation of multiple organs, including the heart, the eye
and the gut (Horne-Badovinac et al.,
2001), however, the molecular mechanism underlying its function
has remained unknown. We demonstrate that aPKC specifies the apical domain and
superficial ectodermal cell fates and suppresses inner (basal) cell fates.
Furthermore, our experiments reveal that a crucial molecular substrate for
aPKC is the PAR1 kinase, which has a complementary localization in epithelial
cells. Additionally, we show a bona fide role for PAR1 in the establishment of
the basolateral cortical domain and the corresponding cell fates. PAR1 appears
to be distinct from other basolateral determinants such as LGL, which regulate
epithelial architecture but have no effect on ciliated cell specification.
Thus, our study places PAR1 mechanistically downstream of or parallel to other
proteins, operating to specify cell polarity and cell fate following
asymmetric cell division.
At the next step, PAR1 targeted to the basolateral cortical domain by
aPKC-dependent phosphorylation may influence cell fates by modulating one or
more signaling pathways that are known to operate in early embryos. The Wnt
pathway is unlikely to play a significant role in ectodermal layer fate
determination, since ß-catenin RNA does not influence ciliated cell
differentiation. Moreover, different PAR1 MOs, which have distinct effects on
Wnt signaling (Ossipova et al.,
2005) produce similar changes in ciliated cell fate, further
supporting the idea that this process is independent of Wnt signaling. By
contrast, the Notch pathway does appear to be involved, since PAR1 modulates
XDelta-1 localization and activity, upregulates XDelta-1 expression and
inhibits the Notch target ESR6e, consistent with a recent study in
Drosophila embryos (Bayraktar et
al., 2006).
Notably, Notch signaling is known to repress ciliated cell differentiation
in Xenopus ectoderm (Chalmers et
al., 2002; Chitnis et al.,
1995; Deblandre et al.,
1999). The observation that PAR1 stimulates -tubulin gene
expression in the superficial as well as the inner ectodermal layers
demonstrates that it can override or modulate this Notch-Delta-dependent
mechanism. Although XDelta-1 is a Notch ligand and can stimulate Notch
signaling in some contexts, in Xenopus epidermis, XDelta-1
overexpression has the opposite effect from that of Notch, and stimulates
ciliated cell differentiation in embryos
(Deblandre et al., 1999). This
activity was attributed to the cis-inhibitory function of XDelta-1 in
Notch-expressing cells, also observed in other systems
(Itoh et al., 2003). PAR1
enhances the effect of XDelta-1, yet fails to modulate signaling stimulated by
the dominant negative RBP/J or Notch intracellular domain. These findings
suggest that PAR1 acts at the level of the XDelta-1 ligand, upstream of the
Notch receptor. Thus, the observed functional interaction of aPKC and PAR1 may
create unequal Delta activity in the superficial and the deep ectodermal
cells, leading to diversification of cell fates. However, since ESR6e
and other Notch target genes may have additional regulators besides Notch,
whether XDelta-1 is the primary target of PAR1 in fate determination or
whether other relevant PAR1 targets are also crucial for this process remains
to be established.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/23/4297/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Alonso, L. and Fuchs, E. (2003). Stem cells in
the skin: waste not, Wnt not. Genes Dev.
17,1189
-1200.
Bayraktar, J., Zygmunt, D. and Carthew, R. W.
(2006). Par-1 kinase establishes cell polarity and functions in
Notch signaling in the Drosophila embryo. J. Cell
Sci. 119,711
-721.
Benton, R. and St Johnston, D. (2003).
Drosophila PAR-1 and 14-3-3 inhibit Bazooka/PAR-3 to establish complementary
cortical domains in polarized cells. Cell
115,691
-704.[CrossRef][Medline]
Betschinger, J. and Knoblich, J. A. (2004).
Dare to be different: asymmetric cell division in Drosophila, C. elegans and
vertebrates. Curr. Biol.
14,R674
-R685.[CrossRef][Medline]
Blanpain, C., Lowry, W. E., Pasolli, H. A. and Fuchs, E.
(2006). Canonical notch signaling functions as a commitment
switch in the epidermal lineage. Genes Dev.
20,3022
-3035.
Bohm, H., Brinkmann, V., Drab, M., Henske, A. and Kurzchalia, T.
V. (1997). Mammalian homologues of C. elegans PAR-1 are
asymmetrically localized in epithelial cells and may influence their polarity.
Curr. Biol. 7,603
-606.[CrossRef][Medline]
Cappello, S., Attardo, A., Wu, X., Iwasato, T., Itohara, S.,
Wilsch-Brauninger, M., Eilken, H. M., Rieger, M. A., Schroeder, T. T.,
Huttner, W. B. et al. (2006). The Rho-GTPase cdc42 regulates
neural progenitor fate at the apical surface. Nat.
Neurosci. 9,1099
-1107.[CrossRef][Medline]
Chalmers, A. D., Welchman, D. and Papalopulu, N.
(2002). Intrinsic differences between the superficial and deep
layers of the Xenopus ectoderm control primary neuronal differentiation.
Dev. Cell 2,171
-182.[CrossRef][Medline]
Chalmers, A. D., Strauss, B. and Papalopulu, N.
(2003). Oriented cell divisions asymmetrically segregate aPKC and
generate cell fate diversity in the early Xenopus embryo.
Development 130,2657
-2668.
Chalmers, A. D., Pambos, M., Mason, J., Lang, S., Wylie, C. and
Papalopulu, N. (2005). aPKC, Crumbs3 and Lgl2 control
apicobasal polarity in early vertebrate development.
Development 132,977
-986.
Chalmers, A. D., Lachani, K., Shin, Y., Sherwood, V., Cho, K. W.
and Papalopulu, N. (2006). Grainyhead-like 3, a transcription
factor identified in a microarray screen, promotes the specification of the
superficial layer of the embryonic epidermis. Mech.
Dev. 123,702
-718.[CrossRef][Medline]
Chitnis, A., Henrique, D., Lewis, J., Ish-Horowicz, D. and
Kintner, C. (1995). Primary neurogenesis in Xenopus embryos
regulated by a homologue of the Drosophila neurogenic gene Delta.
Nature 375,761
-766.[CrossRef][Medline]
Cohen, D., Brennwald, P. J., Rodriguez-Boulan, E. and Musch,
A. (2004). Mammalian PAR-1 determines epithelial lumen
polarity by organizing the microtubule cytoskeleton. J. Cell
Biol. 164,717
-727.
Deblandre, G. A., Wettstein, D. A., Koyano-Nakagawa, N. and
Kintner, C. (1999). A two-step mechanism generates the
spacing pattern of the ciliated cells in the skin of Xenopus embryos.
Development 126,4715
-4728.[Abstract]
Deblandre, G. A., Lai, E. C. and Kintner, C.
(2001). Xenopus neuralized is a ubiquitin ligase that interacts
with XDelta1 and regulates Notch signaling. Dev. Cell
6, 795-806.
Doerflinger, H., Benton, R., Shulman, J. M. and St Johnston,
D. (2003). The role of PAR-1 in regulating the polarised
microtubule cytoskeleton in the Drosophila follicular epithelium.
Development 130,3965
-3975.
Dollar, G. L., Weber, U., Mlodzik, M. and Sokol, S. Y.
(2005). Regulation of Lethal giant larvae by Dishevelled.
Nature 437,1376
-1380.[CrossRef][Medline]
Drysdale, T. A. and Elinson, R. P. (1992). Cell
migration and induction in the development of the surface ectodermal pattern
of the Xenopus laevis tadpole. Dev. Growth Differ.
34, 51-59.[CrossRef]
Etemad-Moghadam, B., Guo, S. and Kemphues, K. J.
(1995). Asymmetrically distributed PAR-3 protein contributes to
cell polarity and spindle alignment in early C. elegans embryos.
Cell 83,743
-752.[CrossRef][Medline]
Fagotto, F. and Gumbiner, B. M. (1994).
Beta-catenin localization during Xenopus embryogenesis: accumulation
at tissue and somite boundaries. Development
120,3667
-3679.[Abstract]
Gotz, M. and Huttner, W. B. (2005). The cell
biology of neurogenesis. Nat. Rev. Mol. Cell Biol.
6, 777-788.[CrossRef][Medline]
Guo, S. and Kemphues, K. J. (1996). Molecular
genetics of asymmetric cleavage in the early Caenorhabditis elegans embryo.
Curr. Opin. Genet. Dev.
6, 408-415.[CrossRef][Medline]
Harland, R. M. (1991). In situ
hybridization: an improved whole-mount method for Xenopus embryos. In
Methods Cell Biol. Vol. 36 (ed.
B. K. Kay and H. B. Peng), pp. 685-695. San Diego:
Academic Press.[Medline]
Hopwood, N. D., Pluck, A. and Gurdon, J. B.
(1989). MyoD expression in the forming somites is an early
response to mesoderm induction in Xenopus embryos. EMBO
J. 8,3409
-3417.[Medline]
Horne-Badovinac, S., Lin, D., Waldron, S., Schwarz, M., Mbamalu,
G., Pawson, T., Jan, Y., Stainier, D. Y. and Abdelilah-Seyfried, S.
(2001). Positional cloning of heart and soul reveals multiple
roles for PKC lambda in zebrafish organogenesis. Curr.
Biol. 11,1492
-1502.[CrossRef][Medline]
Hurov, J. B., Watkins, J. L. and Piwnica-Worms, H.
(2004). Atypical PKC phosphorylates PAR-1 kinases to regulate
localization and activity. Curr. Biol.
14,736
-741.[CrossRef][Medline]
Imai, F., Hirai, S., Akimoto, K., Koyama, H., Miyata, T., Ogawa,
M., Noguchi, S., Sasaoka, T., Noda, T. and Ohno, S. (2006).
Inactivation of aPKClambda results in the loss of adherens junctions in
neuroepithelial cells without affecting neurogenesis in mouse neocortex.
Development 133,1735
-1744.
Itoh, K., Brott, B. K., Bae, G. U., Ratcliffe, M. J. and Sokol,
S. Y. (2005). Nuclear localization is required for
Dishevelled function in Wnt/beta-catenin signaling. J.
Biol. 4,3
.[CrossRef][Medline]
Itoh, M., Kim, C. H., Palardy, G., Oda, T., Jiang, Y. J., Maust,
D., Yeo, S. Y., Lorick, K., Wright, G. J., Ariza-McNaughton, L. et al.
(2003). Mind bomb is a ubiquitin ligase that is essential for
efficient activation of Notch signaling by Delta. Dev.
Cell 4,67
-82.[CrossRef][Medline]
Kosodo, Y., Roper, K., Haubensak, W., Marzesco, A. M., Corbeil,
D. and Huttner, W. B. (2004). Asymmetric distribution of the
apical plasma membrane during neurogenic divisions of mammalian
neuroepithelial cells. EMBO J.
23,2314
-2324.[CrossRef][Medline]
Kusakabe, M. and Nishida, E. (2004). The
polarity-inducing kinase Par-1 controls Xenopus gastrulation in cooperation
with 14-3-3 and aPKC. EMBO J.
23,4190
-4201.[CrossRef][Medline]
Lechler, T. and Fuchs, E. (2005). Asymmetric
cell divisions promote stratification and differentiation of mammalian skin.
Nature 437,275
-280.[CrossRef][Medline]
Lee, C. Y., Robinson, K. J. and Doe, C. Q.
(2006). Lgl, Pins and aPKC regulate neuroblast self-renewal
versus differentiation. Nature
439,594
-598.[CrossRef][Medline]
Luo, T., Xu, Y., Hoffman, T. L., Zhang, T., Schilling, T.,
Sargent, T. D. (2007). Inca: a novel p21-activated
kinase-associated protein required for cranial neural crest development.
Development 134,1279
-1289.
Muller, H. A. and Hausen, P. (1995). Epithelial
cell polarity in early Xenopus development. Dev. Dyn.
202,405
-420.[Medline]
Nakaya, M., Fukui, A., Izumi, Y., Akimoto, K., Asashima, M. and
Ohno, S. (2000). Meiotic maturation induces animal-vegetal
asymmetric distribution of aPKC and ASIP/PAR-3 in Xenopus oocytes.
Development 127,5021
-5031.[Abstract]
Ohno, S. (2001). Intercellular junctions and
cellular polarity: the PAR-aPKC complex, a conserved core cassette playing
fundamental roles in cell polarity. Curr. Opin. Cell
Biol. 13,641
-648.[CrossRef][Medline]
Ohshiro, T., Yagami, T., Zhang, C. and Matsuzaki, F.
(2000). Role of cortical tumour-suppressor proteins in asymmetric
division of Drosophila neuroblast. Nature
408,593
-596.[CrossRef][Medline]
Ossipova, O., Dhawan, S., Sokol, S. and Green, J. B.
(2005). Distinct PAR-1 proteins function in different branches of
Wnt signaling during vertebrate development. Dev. Cell
8, 829-841.[CrossRef][Medline]
Parkinson, S. J., Le Good, J. A., Whelan, R. D., Whitehead, P.
and Parker, P. J. (2004). Identification of PKCzetaII: an
endogenous inhibitor of cell polarity. EMBO J.
23, 77-88.[CrossRef][Medline]
Pellettieri, J. and Seydoux, G. (2002).
Anterior-posterior polarity in C. elegans and Drosophila - PARallels and
differences. Science
298,1946
-1950.
Peng, C. Y., Manning, L., Albertson, R. and Doe, C. Q.
(2000). The tumour-suppressor genes lgl and dlg regulate basal
protein targeting in Drosophila neuroblasts. Nature
408,596
-600.[CrossRef][Medline]
Peng, H. B. (1991). Xenopus laevis: practical
uses in cell and molecular biology. Solutions and protocols.
Methods Cell Biol. 36,657
-662.[Medline]
Plant, P. J., Fawcett, J. P., Lin, D. C., Holdorf, A. D., Binns,
K., Kulkarni, S. and Pawson, T. (2003). A polarity complex of
mPar-6 and atypical PKC binds, phosphorylates and regulates mammalian Lgl.
Nat. Cell Biol. 5,301
-308.[CrossRef][Medline]
Plusa, B., Frankenberg, S., Chalmers, A., Hadjantonakis, A. K.,
Moore, C. A., Papalopulu, N., Papaioannou, V. E., Glover, D. M. and
Zernicka-Goetz, M. (2005). Downregulation of Par3 and aPKC
function directs cells towards the ICM in the preimplantation mouse embryo.
J. Cell Sci. 118,505
-515.
Roegiers, F. and Jan, Y. N. (2004). Asymmetric
cell division. Curr. Opin. Cell Biol.
16,195
-205.[CrossRef][Medline]
Rolls, M. M., Albertson, R., Shih, H. P., Lee, C. Y. and Doe, C.
Q. (2003). Drosophila aPKC regulates cell polarity and cell
proliferation in neuroblasts and epithelia. J. Cell
Biol. 163,1089
-1098.
Saka, Y. and Smith, J. C. (2001). Spatial and
temporal patterns of cell division during early Xenopus embryogenesis.
Dev. Biol. 229,307
-318.[CrossRef][Medline]
Sanada, K. and Tsai, L. H. (2005). G protein
betagamma subunits and AGS3 control spindle orientation and asymmetric cell
fate of cerebral cortical progenitors. Cell
122,119
-131.[CrossRef][Medline]
Sun, T. Q., Lu, B., Feng, J. J., Reinhard, C., Jan, Y. N.,
Fantl, W. J. and Williams, L. T. (2001). PAR-1 is a
Dishevelled-associated kinase and a positive regulator of Wnt signalling.
Nat. Cell Biol. 3,628
-636.[CrossRef][Medline]
Suzuki, A., Hirata, M., Kamimura, K., Maniwa, R., Yamanaka, T.,
Mizuno, K., Kishikawa, M., Hirose, H., Amano, Y., Izumi, N. et al.
(2004). aPKC acts upstream of PAR-1b in both the establishment
and maintenance of mammalian epithelial polarity. Curr.
Biol. 14,1425
-1435.[CrossRef][Medline]
Tomancak, P., Piano, F., Riechmann, V., Gunsalus, K. C.,
Kemphues, K. J. and Ephrussi, A. (2000). A Drosophila
melanogaster homologue of Caenorhabditis elegans par-1 acts at an early step
in embryonic-axis formation. Nat. Cell Biol.
2, 458-460.[CrossRef][Medline]
Vaccari, T., Rabouille, C. and Ephrussi, A.
(2005). The Drosophila PAR-1 spacer domain is required for
lateral membrane association and for polarization of follicular epithelial
cells. Curr. Biol. 15,255
-261.[CrossRef][Medline]
Winkles, J. A., Sargent, T. D., Parry, D. A., Jonas, E. and
Dawid, I. B. (1985). Developmentally regulated cytokeratin
gene in Xenopus laevis. Mol. Cell. Biol.
5,2575
-2581.
Wodarz, A. and Huttner, W. B. (2003).
Asymmetric cell division during neurogenesis in Drosophila and vertebrates.
Mech. Dev. 120,1297
-1309.[CrossRef][Medline]
Wodarz, A., Ramrath, A., Grimm, A. and Knust, E.
(2000). Drosophila atypical protein kinase C associates with
Bazooka and controls polarity of epithelia and neuroblasts. J. Cell
Biol. 150,1361
-1374.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  K. Itoh, A. Jenny, M. Mlodzik, and S. Y. Sokol Centrosomal localization of Diversin and its relevance to Wnt signaling J. Cell Sci., October 15, 2009; 122(20): 3791 - 3798. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Sabherwal, A. Tsutsui, S. Hodge, J. Wei, A. D. Chalmers, and N. Papalopulu The apicobasal polarity kinase aPKC functions as a nuclear determinant and regulates cell proliferation and fate during Xenopus primary neurogenesis Development, August 15, 2009; 136(16): 2767 - 2777. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
