1 Wellcome Trust/Cancer Research UK Gurdon Institute, Tennis Court Road,
Cambridge CB2 1QR, UK
2 Department of Anatomy, University of Cambridge, Downing Site, Cambridge CB2
3DY, UK
3 Division of Developmental Biology, Children's Hospital Medical Center, 3333
Burnet Avenue, Cincinnati, OH 45229-3039, USA
* Author for correspondence (e-mail: np209{at}mole.bio.cam.ac.uk)
Accepted 15 December 2004
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SUMMARY |
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Key words: aPKC, Crumbs, Lgl, Epithelial polarity, Xenopus
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Introduction |
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Work in Drosophila embryonic epithelia and neuroblasts, has shown
that apical and basolateral membrane domains are defined by the antagonistic
action of protein complexes, which are localised to the apical and basal
lateral membrane domains (reviewed by
Müller and Bossinger,
2003; Tepass et al.,
2001
). The same network of interactions is conserved in the
polarity of the C. elegans zygote (reviewed by
Pellettieri and Seydoux,
2002
). Apical complexes include the Par3/Par6/aPKC/Cdc42 complex
and the Crumbs/stardust/discs-lost complex. Basolateral proteins include
Lethal Giant Larvae (LGL) and Par1. Homologues of these proteins have been
identified in vertebrates and shown to form evolutionarily conserved complexes
(Hurd et al., 2003
;
Izumi et al., 1998
;
Joberty et al., 2000
;
Lin et al., 2000
;
Plant et al., 2003
;
Roh et al., 2003
;
Yamanaka et al., 2003
). In
mammalian cultured epithelial cells, members of these complexes play a role in
polarity, primarily by regulating the formation of tight junctions (TJs)
rather than defining apical or basolateral membrane identity
(Hirose et al., 2002
;
Roh et al., 2003
;
Suzuki et al., 2002
;
Yamanaka et al., 2003
). For
example, inhibiting aPKC or Par6 blocks TJ formation during the
re-polarisation of MDCK cells but does not have an effect on already polarised
cells (Suzuki et al., 2002
;
Yamanaka et al., 2001
).
Similarly, mammalian Lgl overexpression does not affect the polarity of
polarised cells but does block the formation of TJs
(Yamanaka et al., 2003
).
Therefore, how apical and basolateral membrane identify is regulated in
vertebrates, is unknown.
Here, we investigate the roles of three molecules - aPKC, Crumbs3 and Lgl2
(each representing one of the three major protein complexes involved in cell
polarity in invertebrates) - in the polarisation of frog blastomeres. We have
shown previously that aPKC is apically localised in frog blastomeres
(Chalmers et al., 2003). We
show here that Lgl2 localised specifically to the basolateral membrane, while
Crumbs3 localised to the apical and basolateral membrane domains.
Overexpression of aPKC expands the apical membrane, correspondingly
reduces the basal side and repositions the TJs in the new apicobasal border.
Crumbs3 also expands the apical side but is less effective than
aPKC. Loss of aPKC function with a dominant-negative construct,
causes loss of apical identity and expansion of basolateral identity into the
apical side. Cells lose their polarity and tight junctions, and become similar
to inner apolar cells. Overexpressing Lgl2 phenocopies the aPKC loss
of function. Finally, aPKC and Lgl2 can inhibit the localisation of each other
and Lgl2 can rescue the over-apicalisation caused by overexpression
of aPKC. These findings suggest that aPKC/Crumbs3 and Lgl2 are
involved in polarisation of vertebrate embryonic epithelial cells by defining
apical and basolateral membrane identity. Furthermore, aPKC and Lgl2 show an
antagonistic interaction, which appears to have been evolutionarily conserved
in embryogenesis between vertebrates and invertebrates.
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Materials and methods |
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RNA overexpression
The constructs described above were used to make RNA for overexpression
using the message machine kit (Ambion) and the RNA injected into embryos at
the two-cell stage. The embryos were then cultured until the required stage
and fixed in PBSF (phosphate-buffered saline+4% formaldehyde), photographed
and if required embedded in gelatin albumen and sectioned on a vibratome
(Chalmers et al., 2002).
Alternatively the embryos were fixed in Dent's (80% methanol + 20% DMSO) at
-20°C for antibody staining. Embryos injected with GFP fusion constructs
(or GFP fusion constructs and RLDX) were fixed in 4% paraformaldeyde in PBS
for 1 hour and stored in Dent's.
Antibody staining and GFP localisation studies
Embryos injected with GFP fusion proteins were cryosectioned using the fish
gelatin protocol (Fagotto and Gumbiner,
1994), mounted in Vectashield (Vector Laboratories) and imaged
directly on the confocal (BioRad Radiance confocal). GFP and the cytoplasmic
lineage label RLDX (Rhodamine-labelled lysinated dextran, Molecular Probes)
were injected as a control.
Antibody staining was carried out on fish gelatin cryosections
(Fagotto and Gumbiner, 1994) as
described (Chalmers et al.,
2003
). The following antibodies were used. Anti-pan cytokeratin
clone C-11 (Sigma, C2931), anti-occludin
(Cordenonsi et al., 1997
), anti
ß1 integrin 8C8 (Gawantka et al.,
1992
), Developmental Hybridoma Bank), anti-cingulin
(Cardellini et al., 1996
),
anti-aPKC (Santa Cruz, nPKC
C-20 SC-216; unfortunately there seems to be
a big variation in quality between batches of this antibody), anti-GFP
(Molecular Probes, A11122). The following secondary antibodies were used anti
rabbit Alexa 568 (Molecular Probes, A11011), anti-rabbit Alexa 488 (Molecular
Probes, A11008) and anti-mouse Alexa 568 (Molecular Probes, A11004). When
Cytox Green (Molecular Probes) was used as a nuclear stain it was added with
the secondary antibody at final concentration of 1/5000.
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Results |
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The early cleavages of injected embryos were normal but at the blastula stage, the epithelium of the surface of the embryo had lost its normal appearance (Fig. 2A,B; see also whole mount in Fig. 7A), which can be seen with GFP or lacZ-injected embryos (Fig. 2C,D). The epithelial cells in aPKC-injected embryos were rounded and protruded from the surface of the embryo (Fig. 2A,B). In the animal hemisphere of wild-type Xenopus embryos only the apical surface is pigmented. In aPKC-overexpressing embryos, the pigmented surfaces of the protruding cells were expanded and the non-pigmented reduced. These protruding superficial cells were not extruded from the embryo, as they did not fall off after the removal of the vitelline membrane; manual `teasing' confirmed that they were firmly attached to their neighbours. Both mouse and Xenopus constructs showed a dose dependence in the percentage of affected embryos (Fig. 2, right panels) although, for reasons that are not clear, the mouse gene was more effective than the Xenopus one, at all concentrations tested.
|
|
This is the first time that aPKC has been shown to have the ability to
expand the apical membrane. To our knowledge, the only other molecule which
has been shown to have a similar effect in Drosophila and mammalian
epithelia is Crumbs (Roh and Margolis,
2003; Wodarz et al.,
1995
). Therefore, we tested if Crumbs3 would also cause
apicalisation in Xenopus embryonic epithelia. Over expression of
Crumbs3 did cause apicalisation similar to that observed with
aPKC, although the phenotype was weaker
(Fig. 2F).
aPKC overexpression skews the ratio of apical to basolateral membrane domain and repositions tight junctions
To characterise the effect further, we looked at alterations of apicobasal
polarity with antibodies for apical and basolateral markers in albino embryos.
As an apical marker, we used keratin, which is localised all around the cortex
of these early epithelial cells but is particularly enriched on the apical
side (Jamrich et al., 1987;
Klymkowsky et al., 1987
). As
basolateral markers we used occludin, a component of TJs, initially targeted
to the basolateral surface (Fesenko et
al., 2000
) and ß1-integrin, a basolateral transmembrane
protein (Gawantka et al.,
1992
). In aPKC-injected embryos, the keratin-enriched membrane was
expanded (Fig. 3B compare with
3A), consistent with the
pigmented surface expansion observed in pigmented embryos
(Fig. 2). By contrast, the cell
membrane that was positive for occludin and ß1-integrin was greatly
reduced (Fig. 3D compare with
3C,
Fig. 3F compare with
3E) suggesting that the apical
domain is expanded at the expense of the basolateral one. We also looked at
TJs, as aPKC has been implicated in TJ formation in mammalian epithelial
cells. As a marker, we used cingulin, a protein found in the cytoplasmic
plaque of TJs (Cordenonsi et al.,
1999
) (reviewed by D'Atri and
Citi, 2002
). Interestingly, cingulin staining was maintained but
the position was shifted to the basal side, marking the new interface of the
extended apical and reduced basolateral membrane domains
(Fig. 3H compare with
3G). Immunostaining of
aPKC-injected embryos with an aPKC antibody confirmed that the affected area
was positive for overexpressed aPKC (Fig.
3J). This analysis showed that overexpression of aPKC causes the
formation of super-apical epithelial cells, while tight junctions are
maintained but shifted in their position
(Fig. 3K). Apicobasal polarity
is maintained as the cells still have a distinct apical and basolateral
membrane. However, polarity is distorted, as the allocation of cell membrane
to the apical and basolateral sides becomes heavily biased in favour of the
apical side.
|
Third, we injected RNA at the two-cell stage for the truncated form of
aPKC, aPKC NT, which lacks the kinase domain but retains the Par6-interacting
domain (Fig. 4A). Therefore,
this fragment of aPKC can bind its normal partner Par6 but has no kinase
activity and so acts as a dominant-negative for endogenous aPKC function
(Gao et al., 2002). The
advantage of this approach is that aPKC is inhibited before the apoptotic
pathway becomes activated at MBT, so any pre-MBT effects cannot be due to
apoptosis. As expected, injection of this RNA did not cause apicalisation (see
above, Fig. 2). Instead, cells
in the injected region lost their pigmentation, indicating loss of apical
identity (Fig. 4C,D). The
effect was small in terms of percentage of embryos
(Fig. 4B) and the number of
cells effected, but reproducible. Co-expressing the wild-type aPKC with the
dominant-negative fragment reduced the number of affected embryos to almost
background levels (Fig. 4B).
This rescue confirms that the effect of the dominant-negative fragment is
caused by inhibiting aPKC and not a non-specific effect.
|
Thus, although overexpression of aPKC drives the expansion of the apical domain of the cell membrane, loss of aPKC function has the opposite effect of expanding the basolateral domain (Fig. 4K). Localisation of basolateral markers all around the cell membrane is a distinctive feature of inner apolar cells. Therefore, we conclude that the epithelial polarity of the cells is lost and that they now resemble, in their membrane characteristics, the non-polarised deep cells.
Lgl2 promotes basal lateral identity and inhibits apical identity
These experiments showed that aPKC is necessary and sufficient to define
apical domain identity. To find out whether basolateral proteins have similar
instructive roles for the basolateral side, we tested the activity of Lgl2.
Overexpression of Lgl2 caused loss of pigmentation in the outer cells
(Fig. 5). At high doses, we
also observed a defect in cytokinesis, such that it started normally but was
abandoned before completion, resulting in large non-pigmented cells.
(Fig. 5B). At lower doses,
cytokinesis proceeded normally, but pigmentation was again lost from the
apical side of the cells (Fig.
5C). Immunostaining showed that Lgl2 inhibited keratin stain
(Fig. 5I, arrow) and expanded
ß1-integrin and occludin ectopically on the apical side
(Fig. 5J,K, arrow). As in the
aPKC knockout, the effect was stronger for ß1-integrin than occludin, TJ
were lost (cingulin; Fig. 5J)
but cell adhesion was maintained.
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Discussion |
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These findings are important for two reasons. First, they have uncovered a function for these vertebrate proteins in polarisation, beyond a role in tight junction formation, that had not been appreciated from work in mammalian epithelial cells. Second, they have shown that these proteins have a role in the polarisation of cells in early vertebrate development, a prerequisite in generating cell fate diversity in the early embryo.
A role for aPKC, Crumbs3 and Lgl2 in the establishment and maintenance of polarity
In the Xenopus proto-epithelium, the apical membrane is inherited
from the egg (`old' membrane), while the basolateral membrane is newly
synthesised (`new' membrane) (Müller
and Hausen, 1995; Roberts et
al., 1992
). Therefore, effects on the apical and basolateral
membrane are indicative of effects in the maintenance and establishment of
membrane identity, respectively. The apical defects associated with aPKC
downregulation and Lgl2 overexpression suggest that the presence of aPKC
and/or the absence of Lgl2 is necessary for the maintenance of apical membrane
identity. However, the reduction of the basolateral side by aPKC and Crumbs3,
and the expansion by Lgl2, suggest that misregulation of these proteins also
affects the establishment of polarity.
In mammalian epithelial cell lines, inhibition of aPKC and overexpression
of Lgl do not have an effect on the polarity of cells that are already
polarised, but do have an effect on newly polarising cells, suggesting that
these proteins act on the establishment, rather than the maintenance of
polarity (Suzuki et al., 2002;
Yamanaka et al., 2003
). Our
findings show for the first time that, in early vertebrate embryogenesis,
these proteins are involved in both aspects of polarisation. This difference
between mature and developing epithelia is also reflected in the localisation
of aPKC protein. In confluent epithelial cells, aPKC is restricted to the
tight junctions but absent from the apical membrane. By contrast, in
Xenopus embryonic epithelia, aPKC is localised to the apical membrane
(Chalmers et al., 2003
),
consistent with the suggested role in maintaining the identity of the apical
membrane, a role additional to tight junction formation in these cells.
Interestingly, in early mouse and zebrafish, aPKC is also localised to the
apical membrane and not just the tight junctions
(Horne-Badovinac et al., 2001
;
Pauken and Capco, 2000
),
suggesting that the mechanism described here may be evolutionarily
conserved.
A model for the mechanism of action for aPKC, Crumbs3 and Lgl2 in embryonic epithelial polarity - the potential role of tight junctions and vesicle transport
Previous work in mammalian epithelial cell lines has mainly focused on the
role of polarity proteins in the formation of TJs
(Hirose et al., 2002;
Hurd et al., 2003
;
Suzuki et al., 2002
;
Suzuki et al., 2001
;
Yamanaka et al., 2001
). TJs,
apart from acting as permeability barriers, are thought to form physical
fences that prevent intermixing of apical and basolateral membrane components.
Could an effect on tight junctions explain the phenotypes that we report? When
aPKC is overexpressed, TJs are re-positioned, but not abolished. These
misplaced TJs are positioned as they normally are, at the interface of the
apical and basolateral membrane domains. As in the wild-type situation, in
experimental embryos these two domains appear cleanly segregated, but the
apical domain is expanded and the basolateral diminished. Therefore, it seems
likely that the primary effect of aPKC and Crumbs3
overexpression is on partitioning of the membrane into apical and basolateral
domains, rather than on the TJs themselves. How aPKC and Crumbs3
overexpression cause the expansion of the apical domain is not clear at
present. Perhaps some of the newly synthesised membrane acquires apical
character instead of basal, or perhaps the apical domain stretches
mechanically, or both. The apical domain is capable of constriction, brought
about, for example, by the overexpression of the actin-binding protein Shroom
(Haigo et al., 2003
), so it is
conceivable that it would also be capable of stretching.
In the aPKC knockout and Lgl2 overexpression, TJs are lost and
basolateral membrane markers spread to the apical side of the cells. In this
case, it is possible that the basolateral expansion is a consequence of the
loss of a physical barrier. Alternatively, basolateral markers could appear on
the apical side by an active mode of transport, independent on the presence or
absence the TJs. Although we cannot formally distinguish between these two
possibilities, we favour the second one. The basolateral membrane is newly
synthesised during division and it is known that ß1-integrin is inserted
into this membrane by fusion of stored vesicles
(Gawantka et al., 1992). It
seems that misdirection of such vesicles to the apical side during division,
would be a straightforward and rapid way for the insertion of ß1-integrin
to the entire apical membrane. This scenario is consistent with the
observation that mammalian Lgl biochemically interacts with syntaxin 4, a
component of basolateral exocytic machinery
(Musch et al., 2002
). It is
also important to note that Xenopus blastula cells develop and
maintain their epithelial polarity autonomously, in the complete absence of
cell-cell contacts (Chalmers et al.,
2003
; Fesenko et al.,
2000
; Müller and Hausen,
1995
) (reviewed by Müller
and Bossinger, 2003
). Therefore, in this system, functional TJs
seem to play a secondary role in the establishment and/or maintenance of
polarity and are unlikely to be the primary targets of the polarity complex
proteins. We favour a model whereby aPKC/Lgl2 maintain distinct membrane
domains not only by playing a role in TJ formation but by more direct
mechanisms, such as directing vesicle trafficking.
aPKC-, Crumbs3- and Lgl2-driven epithelial polarity underlies a conserved event of cell fate diversification in vertebrates
A conserved aspect of vertebrate embryogenesis is the polarisation of the
blastomeres and the generation of two phenotypically different cell
populations via their division (reviewed by
Müller and Bossinger,
2003). Cell polarisation is a prerequisite in generating two
phenotypically distinct populations of cells in the early embryo.
Based on the localisation of membrane markers and pigment, we have shown that in aPKC knockout and Lgl2-overexpressing embryos, the outer epithelial cells lose their polarity and become phenotypically similar to inner cells. There are several possible ways in which cells could lose their polarity, such as regionalised membrane proteins failing to localise to the membrane altogether, or mixing of apical and basal markers. Instead, what we have observed is loss of polarity by specific transformation of the apical membrane to basolateral. The transformation of outer cells to inner-like reduces cell diversity in the affected area of the embryo. Furthermore, because these outer cells have lost their polarity, they would no longer be able to generate two phenotypically distinct cell types by division, at least as far as their membrane protein localisation is concerned.
In conclusion, aPKC and Crumbs3 act to promote apical membrane and inhibit
basolateral, while Lgl2 acts to promote basolateral and inhibit apical
membrane identity. The balance between these two antagonistic activities acts
to establish and maintain apical and basal lateral membrane domains during
early vertebrate development. Similar interactions have been reported in the
establishment of embryonic epithelial polarity in Drosophila
(Hutterer et al., 2004). These
findings highlight an evolutionary conservation in the mechanisms that
generate polarity and hence phenotypic cell diversity in the early vertebrate
embryo.
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Supplementary material |
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ACKNOWLEDGMENTS |
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