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First published online 4 March 2009
doi: 10.1242/dev.032508
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Max-Planck Institut für Entwicklungsbiologie, Department of Genetics, Spemannstraße 35, Tuebingen, D-72076, Germany.
* Author for correspondence (e-mail: mahendra.sonawane{at}tuebingen.mpg.de)
Accepted 9 February 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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seems
dispensable for the localisation of Itga6 during hemidesmosome formation,
knockdown of E-cadherin function leads to an Lgl2-dependent increase in the
localisation of Itga6. Thus, Lgl2 and E-cadherin act antagonistically to
control the localisation of Itga6 during the formation of hemidesmosomes in
the developing epidermis.
Key words: Lgl2 (Llgl2), E-cadherin (Cadherin 1), Hemidesmosome formation, Epidermis, Zebrafish
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Knust and Bossinger,
2002; Humbert et al.,
2003; Shin et al.,
2006; Suzuki and Ohno,
2006).
The balanced activities of the Crumbs, Par3 (Bazooka, Pard3)-atypical
Protein kinase C (aPKC) and Lgl-Scrib-Dlg pathways are essential for the
formation of adherens junctions or tight junctions in epithelia. Whereas the
Par3-aPKC pathway is required for the maintenance of the apical domain, the
Lgl pathway provides the basolateral cues. In the absence of basolateral cues,
the apical domain expands at the expense of the basolateral domain, and vice
versa, thus affecting the formation of adherens junctions or tight junctions
(Bilder et al., 2003;
Tanentzapf and Tepass, 2003;
Yamanaka et al., 2003;
Chalmers et al., 2005). The
localisation of Lgl to the basolateral membrane domain depends on its
phosphorylation status, which is regulated by the apical Par3-aPKC pathway
(Betschinger et al., 2003;
Plant et al., 2003;
Yamanaka et al., 2003;
Hutterer et al., 2004;
Betschinger et al., 2005).
Drosophila Lgl and its vertebrate orthologues, Lgl1 and Lgl2, possess
conserved serine residues that can be phosphorylated by aPKC
(Betschinger et al., 2003;
Plant et al., 2003;
Yamanaka et al., 2003;
Sonawane et al., 2005). It has
been proposed that aPKC phosphorylates Lgl at the apical domain, leading to
its release from the cortex. Because the Par3-aPKC complex is absent from the
basolateral domain, Lgl cannot be phosphorylated in this domain and thus
remains localised to the cortex
(Betschinger et al., 2003).
In Drosophila neuroblasts, basolaterally localised Lgl [L(2)gl -
FlyBase] is essential for the targeting of proteins such as Miranda, a
determinant of ganglion mother cell fate, to the basolateral cortex
(Ohshiro et al., 2000;
Peng et al., 2000;
Betschinger et al., 2003). It
has been proposed that this targeting is actomyosin-dependent
(Ohshiro et al., 2000;
Peng et al., 2000). In
mammalian epithelia, Lgl1 interacts with syntaxin 4, a t-SNARE involved in the
fusion of post-Golgi vesicles to the target membranes
(Musch et al., 2002). In
yeast, Lgl homologues interact with the exocyst complex and are involved in
polarised exocytosis (Zhang et al.,
2005). Thus far, it is not clear whether Lgl is essential for
targeting any component to the basolateral domain in epithelia, be it by
polarised exocytosis or by an actomyosin-dependent mechanism.
The interaction between the Lgl pathway and the formation of adherens
junctions is reciprocal. The function of Drosophila Lgl is essential
for localising adherens junctions (Bilder
et al., 2003; Tanentzapf and
Tepass, 2003), and these junctions are also necessary for the
segregation of Dlg to the basolateral domain to establish epithelial cell
polarity (Harris and Peifer,
2004). In addition, the formation of adherens junctions is
essential for the maintenance of apical-basal cell polarity and for the
formation of other cellular junctions. E-cadherin has been shown to be
essential for the formation of tight junctions in MDCK cells and in the mouse
epidermis (Gumbiner et al.,
1988; Tunggal et al.,
2005; Capaldo and Macara,
2007). Another adherens junction component, 
-catenin, has a
function in the maintenance of cell polarity in the mouse epidermis
(Vasioukhin et al., 2001). The
formation of focal adhesions is linked to the loss of adherens junctions and
the endocytosis of E-cadherin (Balzac et
al., 2005). Moreover, β1 integrin, a transmembrane component
of focal adhesions, influences cadherin-dependent intercellular junction
formation (Gimond et al.,
1999). These latter observations indicate an interaction between
cadherins and integrins (reviewed by Chen
and Gumbiner, 2006). However, it is not clear whether the
formation of hemidesmosomes is regulated by E-cadherin in any way.
We are investigating the role of polarity genes in the formation of
cellular junctions in the larval epidermis of the zebrafish. In zebrafish
larvae, the epidermis is bi-layered, consisting of the outer periderm and the
underlying basal epidermis (see Fig.
1A). The basal epidermal cells exhibit three distinct plasma
membrane domains. The basal domain connects the cells to the extracellular
basal lamina, the lateral domains are in contact with the neighbouring basal
epidermal cells, and the apical domain attaches to the outer peridermal cells
(Fig. 1A). The basal domains of
epidermal cells that cover the larval head and flanks are equipped with
hemidesmosomes, whereas the lateral and apical domains of all epidermal cells
contain adherens junctions and desmosomes
(Fig. 1A,B). A number of
mutants exhibiting defects in the larval epidermis have been isolated
(van Eeden et al., 1996). In
the penner (pen) mutant, the larval epidermis detaches from
the basal lamina. We have shown that pen encodes Lgl2. Strikingly, in
pen mutant larvae, hemidesmosomes are absent from the basal epidermal
cells (Sonawane et al., 2005).
However, the precise function of Lgl2 in hemidesmosome formation remained to
be elucidated.
We have analysed the functions of Lgl2, aPKC and E-cadherin (Llgl2, Prkci and Cadherin 1, respectively - ZFIN) in the formation of cellular junctions in basal epidermal cells in zebrafish larvae. We show that Lgl2 and E-cadherin localise to the lateral domain in basal epidermal cells. At the lateral domain, Lgl2 promotes the formation of hemidesmosomes by mediating the targeting of a hemidesmosomal component, Integrin alpha 6 (Itga6), to the plasma membrane. By contrast, E-cadherin negatively regulates hemidesmosome formation, presumably by regulating the Lgl2-mediated Itga6 targeting. Thus, Lgl2 and E-cadherin localised at the lateral domain act antagonistically in controlling the formation of hemidesmosomes at the basal membrane domain.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) were used as donors and embryos from
albino fish as recipients.
Generating antibodies against Lgl2 and Itga6
The cDNA corresponding to amino acids 994-1094 of Lgl2 was cloned into the
pQE-30 UA vector in M15(pREP4) cells. The expressed His-tagged protein was
isolated and affinity purified under native conditions (The Qiaexpressionist,
Qiagen) and used for immunisation. A peptide polyclonal antibody against
zebrafish Itga6 (accessions: XP_683444, XP_707029, XP_707030), corresponding
to the sequence KRKHKILSCSGDAR, was generated in guinea pigs (Genosphere
Biotechnologies). The sera were used in immunohistology without any further
purification.
To test its specificity, the anti-Itga6 serum was diluted (1:500), preincubated with different concentrations of the peptide epitope in PBS for 2 hours at room temperature, centrifuged at 16,100 g for 10 minutes and used for immunostaining. The specific signal was completely eliminated at a peptide concentration of 1000 µg/ml.
Morpholino injections and cell transplantations
Antisense morpholino oligos (Gene Tools, Corvallis) against lgl2
(Sonawane et al., 2005),
e-cadherin (Knaut et al.,
2005) and aPKC
(Horne-Badovinac et al.,
2001), along with their corresponding five-base mismatch control
morpholinos, were used at the following concentrations: lgl2,
5'-GCCCATGACGCCTGAACCTCTTCAT-3' (200 µM); lgl2
control, 5'-GCACATAACGCCTCAACCTGTTAAT-3' (200 µM);
e-cadherin, 5'-ATCCCACAGTTGTTACACAAGCCAT-3' (100 µM);
e-cadherin control, 5'-ATCCGACACTTCTTACAGAACCCAT-3' (100
µM); aPKC, 5'-TGTCCCGCAGCGTGGGCATTATGGA-3' (750 µM
yielded consistent knockdown in clones at 5 dpf).
For transplantations, morpholino oligos were injected into β-actin::GFP donor embryos (1- to 2-cell stage). For transplantation from has (prkci) mutants, embryos were injected with Alexa 488-dextran (Molecular Probes/Invitrogen). At the blastula stage, cells were transplanted to recipient albino embryos that were at the same stage to obtain epidermal clones. For electron microscopy analysis of clones, GFP was detected using rabbit anti-GFP antibody and biotinylated anti-rabbit antibody with the Elite ABC System (Vectastain) and DAB.
Brefeldin A (BFA) treatment
Three-day-old albino larvae were incubated in 1.8, 2.7 and 3.6 mM
BFA in the fish medium until 5.5 dpf and analysed for morphological phenotype.
The larvae raised in 3.6 mM BFA showed a distinct blistering phenotype over
the head and were fixed in 4% paraformaldehyde (PFA).
Immunohistochemistry and lectin GS-II staining
Larvae were fixed overnight in 4% PFA in PBS for anti-Lgl2, monoclonal
anti-E-cadherin, anti-GFP (Torrey Pines, Roche), anti-aPKC (Santa Cruz
Biotechnology), anti-Itga6 and anti-Alexa 488 antibodies. For anti-Cytokeratin
II (Ks-pan1-8, Progen Biotechnik) staining, larvae were fixed in Dent's
fixative overnight at -20°C. Larvae were incubated overnight at 4-6°C
in the following antibody dilutions: anti-Lgl2 (1:400), anti-E-cadherin
(1:100), anti-aPKC (1:1000), anti-Cytokeratin (1:10), anti-Itga6 (1:500),
anti-GFP (1:200), anti-Alexa 488 (1:250). Afterwards, larvae were washed in
PBT and incubated with appropriate secondary antibodies conjugated with Cy3,
Cy5, Alexa 488 or Alexa 546. Larvae were washed, developed using the ABC Elite
Kit and DAB as required, post-fixed in 4% PFA and either mounted in glycerol
or embedded for sectioning.
For immunoelectron microscopy, larvae were prestained with anti-Itga6 antibody and anti-guinea-pig antibody conjugated with HRP (1:500). For detection, DAB (0.2 mg/ml in PBS containing 0.1% Tween 20) was supplemented with nickel ammonium sulphate (10 mg/ml) and Imidazole (1 mM). After staining, larvae were post-fixed and processed for electron microscopy.
For Golgi staining, anti-Itga6-stained larvae were embedded in 2% agarose, sectioned (100 µm) on a Vibratome and incubated for 4 hours in lectin GS-II (Molecular Probes/Invitrogen) diluted (1:250) in PBS containing 1 mM CaCl2. After the incubation, sections were washed with PBS with CaCl2 and mounted in 70% glycerol for confocal microscopy.
Histology: fluorescence and electron microscopy
Immunostained larvae were embedded in Technovit 7100 for sectioning, and in
Epon for electron microscopy as previously described
(Sonawane et al., 2005).
Image acquisition
Almost all fluorescence microscopy images were acquired on a Zeiss LSM 510
Meta confocal microscope at 40x/1.3 with a 2x digital zoom. Images
of DAPI-counterstained sections were acquired on a Zeiss Axioplan 2 microscope
at 40x/0.75 using a digital Axiocam camera without digital zoom.
Quantification of fluorescent signal intensities
The z-stacks were obtained by scanning the epidermis every 0.4
µm by confocal microscope with the pinhole adjusted to 83 µm. The
fluorescent intensity scans were performed on a single section from a
z-stack using LSM 510 software (Zeiss). To quantify Itga6 staining
intensity, the cell area was demarcated and intensity histograms obtained. The
software displays the data in tabular form as the mean pixel intensity and
standard deviation for the selected cell area. The mean intensities for three
cells from the mutant clone and three host cells were obtained and represented
in a histogram. We further compared the mean of each of these clonal cells
with each host cell in a 3x3 matrix using Student's t-test.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
However, its cellular localisation and precise role in hemidesmosome formation
have yet to be resolved. To analyse the localisation of Lgl2 during epidermal
development, we generated an antibody against the C-terminal epitope of Lgl2.
This antibody stains basal epidermis and periderm in developing wild-type
larvae (Fig. 1C,H). The
lgl2 (pen) mutant larvae do not exhibit staining in the
basal epidermis or periderm (Fig.
1D), indicating that the antibody specifically recognises Lgl2. In
histological sections, we never observed Lgl2 localisation at the basal cortex
of the basal epidermal cells, where hemidesmosomes form
(Fig. 1H). Instead, Lgl2 was
predominantly localised to the lateral domain in the basal epidermal cells
(Fig. 1H). The apparent apical
staining in the basal epidermal cells might in fact represent the basolateral
localisation of Lgl2 in juxtaposed peridermal cells.
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;
Plant et al., 2003;
Yamanaka et al., 2003;
Hutterer et al., 2004;
Betschinger et al., 2005). It
was not clear whether aPKC is expressed in basal epidermal cells in zebrafish
and whether it regulates the localisation of Lgl2 in a similar manner. We used
an anti-aPKC antibody (Horne-Badovinac et
al., 2001) to analyse aPKC localisation in the epidermis. In the
basal epidermis, aPKC expression begins at 3 days post-fertilisation (dpf),
peaks at 
4-5 dpf and then declines at 6 dpf
(Fig. 1E and data not shown).
Interestingly, histological sections revealed that aPKC localises to the basal
domain (Fig. 1I). To test
whether the antibody is specifically recognising the aPKC isoform, we
injected a morpholino against aPKC
(Horne-Badovinac et al.,
2001). These morpholino-injected larvae did not develop after 2
dpf. Therefore, we transplanted cells (marked with GFP) from
morpholino-injected embryos into wild-type recipient embryos at the blastula
stage to obtain clones in the basal epidermis
(Fig. 1F). The aPKC expression
was strongly attenuated in these clones, indicating that the aPKC
isoform is expressed in the epidermis (Fig.
1G). We then asked whether aPKC localisation coincides with the hemidesmosomal components at the basal domain. We found that Integrin alpha 6 (Itga6) localises to the basal domain and that aPKC localisation overlaps with that of Itga6 (Fig. 1J,K). Surprisingly, a minor fraction of Itga6 also localised to the lateral domain along with Lgl2 (Fig. 1L-N) and E-cadherin (Fig. 1O-Q) in 4-day-old larvae. We further analysed the localisation of Lgl2, E-cadherin and Itga6 in histological sections to check whether any of these localise to a sub-domain of the lateral membrane domain. However, all three proteins localised to the entire lateral domain and also exhibited perfect overlap with each other (Fig. 1R-T'). In histological sections, Itga6 localisation was apparent in the apical side of the basal cell, just like Lgl2 and E-cadherin. It is not clear whether this apical staining represents the basal localisation in the peridermal cells. The Itga6 staining at the basal, as well as lateral, membrane domain proved specific in a competition assay using the peptide antigen (data not shown).
We analysed the temporal changes in Itga6 localisation in the basal
epidermis during larval development. Whereas electron-dense hemidesmosomes
become apparent after 4 dpf in the developing larval epidermis
(Sonawane et al., 2005), Itga6
localised to the basal domain as early as 2.5 dpf (data not shown). The signal
intensity for Itga6 at the basal domain increased considerably between 3 and 4
dpf and reached a plateau thereafter (Fig.
2A-C). By contrast, the intensity of Itga6 staining at the lateral
domain diminished after 4 dpf and was difficult to detect in larval epidermis
at 5 dpf (Fig. 2A-C). We
performed immunoelectron microscopy on larvae prestained with anti-Itga6
antibody. At 3.5 dpf, although hemidesmosomes were not apparent
(Fig. 2D), DAB precipitate was
clearly seen clustered at the intermediate filaments at the basal cortex
(Fig. 2F). By 4 dpf,
electron-dense hemidesmosomes became apparent in the epidermis
(Fig. 2E) and the DAB
precipitate was associated with the hemidesmosomes that were being assembled
(Fig. 2G). Based on these
observations, we propose that the increase in the amount of Itga6 at the basal
domain between 3 and 5 dpf indicates additional targeting of Itga6 to the
basal domain, and that this increase is essential for driving Itga6, which is
clustered at intermediate filaments at 3.5 dpf, into hemidesmosomal plaques by
4-5 dpf. To test whether Itga6 is indeed targeted to the membrane between 3
and 5 dpf, we treated 3-day-old larvae with Brefeldin A (BFA), which inhibits
Golgi function (reviewed by Nebenführ
et al., 2002). In the BFA-treated larvae, Itga6 exhibited a broad
accumulation around the nucleus at 5 dpf, and Itga6 localisation at the basal
domain was lost (Fig. 2H-J).
These observations (Fig.
2A-C,H-J) clearly indicate that substantial amounts of Itga6 are
newly synthesised and targeted to the basal domain between 3 and 5 dpf, and
that this later targeting is essential for stabilising the early Itga6
fraction, which is targeted before 3 dpf, at the basal domain.
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function is dispensable for the localisation of Itga6 to the basal membrane domain, which might be serving as the primary
regulator of hemidesmosome formation because it co-localises with Itga6 during
hemidesmosome formation. To test this hypothesis, we asked whether
aPKC localisation is altered in the absence of lgl2 function
in the basal epidermal cells. Indeed, in the lgl2 mutant larvae, the
basal localisation of aPKC was disrupted
(Fig. 3A,B), raising the
possibility that the loss of hemidesmosomes is a consequence of the loss of
aPKC localisation. To test this, we analysed hemidesmosome formation in
aPKCMO clones (Fig.
3C). Interestingly, Itga6 exhibited normal basal localisation in
aPKCMO clones, indicating that hemidesmosome formation does not
require aPKC (Fig. 3D).
Similarly, we did not observe any significant difference in the localisation
of Itga6 in aPKC (heart and soul) mutant clones
(Fig. 3E,F). These results
indicate that aPKC function is dispensable for Itga6 localisation
during hemidesmosome formation, and that Lgl2 does not act in hemidesmosome
formation via aPKC.
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At 3.75 dpf, several mutant larvae exhibited selective loss of the
lateral membrane staining of Itga6, whereas the basal localisation appeared
largely unperturbed (see Fig. S1G-L in the supplementary material).
Interestingly, however, at 4-5 dpf, lgl2 mutant larvae exhibited
faint cytoplasmic Itga6 staining with some enrichment around the nucleus
(Fig. 4A,B,D; compare with
Fig. 2A-C). Furthermore,
analysis of lgl2MO clones at 6 dpf indicated that, as in lgl2 mutant
larvae, Itga6 accumulates in the cytoplasm (data not shown). GSII lectin
staining revealed that Itga6 does not accumulate in the Golgi apparatus
(Fig. 4E). Rather, it appeared
to accumulate in post-Golgi compartments such as recycling endosomes or
exocytotic vesicles, which play important roles in protein targeting. At these
time points, we never observed Itga6 staining at the lateral or basal domain
in lgl2 mutant larvae (Fig.
4C), indicating that Lgl2 is involved in mediating the targeting
of Itga6 to the plasma membrane as well as in its maintenance at the plasma
membrane.
Next, we analysed the distribution of Itga6 vesicles in 4-day-old wild-type and lgl2 mutant larvae by immunoelectron microscopy. The Itga6 vesicles were difficult to locate in the cytoplasm of basal epidermal cells of wild-type larvae, possibly owing to the rapid targeting of exocytotic vesicles to the membrane (Fig. 4F). Nevertheless, we occasionally observed Itga6 vesicles in the vicinity of the lateral membrane domain (Fig. 4F'). By contrast, Itga6 vesicles were abundant in the basal epidermal cells of lgl2 mutant larvae (Fig. 4G-I'). Moreover, in lgl2 mutant larvae, these Itga6 vesicles were distributed in the lateral and apical cortical region of the basal epidermal cells (Fig. 4G-I'). In the lgl2 mutant larvae, which exhibit a slightly stronger phenotype, Itga6 vesicles were seen to accumulate in the cytoplasm (Fig. 4I,I').
To conclude, in lgl2 mutant larvae, the basal and lateral Itga6 localisation is lost after 4 dpf and Itga6 vesicles accumulate in the lateral cortical region as well as in the cytoplasm in general, further indicating that Lgl2 function is essential for mediating the targeting of Itga6 to the plasma membrane beyond 3.5-4 dpf. Since the basal and lateral localisation of Itga6 is not altered prior to 3.5 dpf in the absence of lgl2 function, we conclude that Lgl2 is dispensable for the initial Itga6 targeting.
E-cadherin negatively regulates the formation of hemidesmosomes during epidermal development
In addition to its role in adherens junction formation, E-cadherin is
involved in the maintenance of epithelial cell polarity and in the formation
of other cellular junctions such as desmosomes and tight junctions
(Gumbiner et al., 1988;
Tunggal et al., 2005;
Capaldo and Macara, 2007). We
tested whether E-cadherin plays a role in hemidesmosome formation and in the
localisation of Lgl2 in the developing basal epidermis. E-cadherin has
essential functions in many early developmental processes, and mutation or
morpholino knockdown of e-cadherin (half-baked, cadherin 1)
leads to an early phenotype that is lethal
(Kane et al., 2005).
Therefore, we analysed late e-cadherin knockdown phenotypes in clones
of epidermal cells (ecadMO clones). ecadMO clones from 5- to 6-day-old larvae
did not exhibit any appreciable E-cadherin expression
(Fig. 5A,B). Of 120 clones
(n=10), 76 exhibited complete knockdown, whereas 22 exhibited partial
knockdown of E-cadherin expression by 6 dpf. Surprisingly, further analysis of
ecadMO clones revealed augmented levels of Itga6 localisation to the basal
domain (Fig. 5C,D). We analysed
35 clones from 5- to 6-day-old larvae (n=27) of which 28 exhibited
this augmented localisation phenotype. Quantification revealed that the mean
intensity for Itga6 in ecadhMO clones was consistently higher than that in the
surrounding host cells (t-test, P
0.001; see Materials
and methods for details), a phenomenon never exhibited by clones carrying
control morpholinos (Fig.
5E,F,I,J). We then investigated whether this increase in Itga6
represents an increase in the number of hemidesmosomes. Electron microscopy
analysis revealed that whereas in wild-type cells hemidesmosomes appear as
discrete electron-dense punctae, in ecadMO clones the number of hemidesmosomes
was so high that these punctae coalesced to form an electron-dense mat at the
basal domain (Fig. 5K,L). We
observed intermediate filaments projecting out of these electron-dense mats,
indicating that these mats are formed from functional hemidesmosomes
(Fig. 5K). We further argued
that if increased Itga6 levels represent increased numbers of hemidesmosomes,
this should lead to an increase in the hemidesmosome-associated keratin
cytoskeleton. By contrast, if Itga6 is associated with its other partner,
Integrin beta 1, in ecadMO clones then keratin levels should not change. In
75% of ecadMO clones (n=16), more keratin localisation was observed
as compared with the surrounding basal epidermal cells
(Fig. 5G,H). We conclude that
Itga6 localisation to the basal domain increases in ecadMO clones and that
this increased fraction is assembled in hemidesmosomes, leading to the
increase in the number of hemidesmosomes.
Because aPKC colocalises with Itga6 at the basal domain and Lgl2 is
involved in targeting Itga6 during hemidesmosome formation, we asked whether
aPKC or Lgl2 localisation is altered in ecadMO clones. Whereas Lgl2
localisation was unaltered in ecadMO clones
(Fig. 5M,N), we observed a
clear increase in the basal localisation of aPKC in ecadMO clones
(Fig. 5O,P).
Our data show that E-cadherin negatively regulates Itga6 localisation during the formation of hemidesmosomes. Although the number of hemidesmosomes is increased in the absence of E-cadherin, we did not find elevated levels of Lgl2 at the lateral domain. Thus, we hypothesised that existing Lgl2 levels are sufficient for the increased basal localisation of Itga6 when negative regulation by E-cadherin is lost. To test this hypothesis, we reduced the levels of Lgl2 in ecadMO clones by co-injecting lgl2 morpholino at a concentration (100 µM) that reduces, but does not completely eliminate, Lgl2 levels (Fig. 6C,D). Analysis of these lgl2-ecadMO clones revealed that the reduced levels of Lgl2 normalised the basal localisation of Itga6 in 82% of clones (29 out of 35) analysed from 6-day-old larvae (n=28) (Fig. 6B,F). Furthermore, punctate hemidesmosomal morphology was restored in lgl2-ecadMO clones (Fig. 6E). This experiment demonstrated a quantitative requirement of Lgl2 in Itga6 localisation during hemidesmosome formation, and proved that the increased Itga6 localisation observed in the absence of E-cadherin is mostly Lgl2-dependent.
Finally, we asked whether E-cadherin function is essential for Itga6 localisation prior to 3 dpf. We did not observe a difference in Itga6 localisation in ecadMO clones (10 out of 12) in 3- to 3.5-day-old larvae (n=6), indicating that the early Itga6 localisation is independent of E-cadherin function (Fig. 6G,H).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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in the formation of
hemidesmosomes in the developing basal epidermis. Our data indicate that Lgl2
regulates the formation of hemidesmosomes by mediating the targeting of a
hemidesmosomal component, Itga6, to the membrane beyond 3.5 dpf. Lgl has been
shown to mediate the basolateral targeting of cell fate determinants in
Drosophila neuroblasts (Ohshiro
et al., 2000; Peng et al.,
2000; Betschinger et al.,
2003; Betschinger et al.,
2006). However, it was not clear whether Lgl2 functions in
targeting any protein in epithelia. Here, we have identified Itga6 as the
first membrane protein whose targeting is mediated by Lgl2.
Similar to Lgl2 and Itga6, E-cadherin is localised to the lateral domain in
basal epidermal cells. Given the role of E-cadherin in the formation of
cellular junctions, such as desmosomes and tight junctions
(Gumbiner et al., 1988;
Tunggal et al., 2005;
Capaldo and Macara, 2007), we
analysed the role of E-cadherin in hemidesmosome formation. Interestingly, the
loss of E-cadherin in the basal epidermis leads to increased Itga6
localisation at 5-6 dpf, in turn leading to an increase in the number of
hemidesmosomes. The reduction of Lgl2 levels in E-cadherin knockdown clones
rescues this hemidesmosomal phenotype. Our analysis has led to the surprising
discovery that the lateral domain of the basal epidermal cells harbours two
signals that act antagonistically to regulate the formation of hemidesmosomes
in the basal domain. This is the first evidence indicating that in developing
epidermal cells, an interaction between proteins localised at the lateral
domain is essential to regulate the formation of junctions in the basal
domain.
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).
Since, in lgl2 mutant larvae, the Itga6 fraction targeted beyond 3.5
dpf fails to reach the lateral membrane domain and thus also the basal domain,
the existing levels of Itga6 at the basal domain (localised before 3.5 dpf)
remain insufficient to form functional hemidesmosomes.
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In mouse, E-cadherin deficiency has thus far not been correlated with
increased hemidesmosome formation, although beta 4 integrin localisation
studies have been performed in E-cadherin knockout mice
(Tinkle et al., 2004;
Tinkle et al., 2008). In the
light of our data, these knockout mice models should be re-evaluated by
constructing mutant-wild type chimeras to examine quantitative differences in
hemidesmosome formation and to check whether the mechanism we have described
in zebrafish is conserved in mammals. Our analyses demonstrate a clear,
quantifiable effect of the loss of e-cadherin function on
hemidesmosomes in a basal vertebrate. Such analyses will be important in the
quest to understand how mechanisms that establish polarity and hemidesmosome
formation have evolved in vertebrates.
Although hemidesmosomes are not formed, Itga6 is localised to the basal domain as early as 2.5 dpf. At 3.5 dpf, Itga6 exhibits a clustered association with intermediate filaments, and this appears to be an intermediate step prior to hemidesmosome formation. This early basal localisation of Itga6, as well as the lateral localisation, were mostly unperturbed in lgl2 mutant larvae at 3.5 dpf and in lgl2 and e-cadherin morphant clones. Thus, the initial targeting of Itga6, prior to 3.5 dpf, is Lgl2- and E-cadherin-independent. This early Itga6 fraction that is localised to the basal domain prior to 3.5 dpf is lost in lgl2 mutant larvae at 4-5 dpf. This indicates that in addition to its primary function in targeting, Lgl2 function is also essential for the maintenance of Itga6 at the basal domain beyond 3.5 dpf. It is not clear what happens to the early Itga6 fraction, which is localised to the basal domain, in lgl2 mutant larvae. One plausible explanation is that the turnover of Itga6 that is not yet assembled in hemidesmosomes is high, leading to its endocytosis and degradation at subsequent stages.
|
; Tanentzapf and Tepass,
2003; Yamanaka et al.,
2003; Hutterer et al.,
2004; Chalmers et al.,
2005). In Xenopus blastula epithelium, beta 1 integrin, a
basolateral marker, has been shown to mislocalise to the apical domain when
the function of aPKC is inhibited or Lgl2 is overexpressed
(Chalmers et al., 2005).
Interestingly, however, Itga6 localisation is not altered in the
aPKC mutant or when aPKC is knocked down.
Thus, Itga6 does not respond to polarity cues like beta 1 integrin and
adherens as well as tight junction components do. However, there is a strong
correlation between Itga6 localisation, the formation of hemidesmosomes and
the localisation of aPKC during development. Moreover, the loss of
hemidesmosomes in lgl2 mutants and the increase in the number of
hemidesmosomes when E-cadherin function is knocked down also correlate with
the absence and increase, respectively, in aPKC localisation.
Therefore, we propose that aPKC localisation is dependent on hemidesmosome
formation.
To summarise, our analysis has revealed hitherto unidentified steps in the
formation of hemidesmosomes and in the establishment of polarity in the
developing basal epidermis of vertebrates
(Fig. 7). Prior to 3.5 dpf,
neither Lgl2 nor E-cadherin function is essential for the targeting of Itga6
to the basal domain. During 3.5-5 dpf, the assembly of hemidesmosomes requires
progressive targeting of Itga6 to the membrane. This latter Itga6 targeting
depends on Lgl2, which localises to the lateral domain. We propose that after
its synthesis, Itga6 is targeted to the lateral domain first and from there it
is translocated to the basal domain, where it participates in hemidesmosome
formation. Whereas Lgl2 positively regulates hemidesmosome formation by
mediating Itga6 targeting and maintaining its localisation, E-cadherin
negatively regulates the Lgl2-mediated Itga6 targeting. These antagonistic
signals control the precise levels of Itga6 at the basal domain during
hemidesmosome formation in the developing zebrafish epidermis. The
localisation of aPKC in the basal epidermis is tightly correlated
with, and might be dependent on, Itga6 localisation and hemidesmosome
formation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/8/1231/DC1
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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