Research Article
4293
PAR3 acts as a molecular organizer to define the
apical domain of chick neuroepithelial cells
Cristina Afonso1,2 and Domingos Henrique1,2,*
1
Instituto Gulbenkian de Ciência, Ap. 14, Oeiras, Portugal
Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal
2
*Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 17 July 2006
Journal of Cell Science 119, 4293-4304 Published by The Company of Biologists 2006
doi:10.1242/jcs.03170
Summary
Neural progenitors in the vertebrate nervous system are
fully polarized epithelial cells, with intercellular junctions
at the apical region. These progenitor cells remain within
the neuroepithelium throughout neurogenesis, and will
ultimately give rise to all the neurons in the mature nervous
system. We have addressed the role of the PAR polarity
complex in vertebrate neuroepithelial polarity and show
that PAR3 functions as the initial scaffold to assemble
and organize the PAR complex at the apical region of
neuroepithelial cells, coordinating also the recruitment of
additional polarity complexes and junction-associated
proteins to the same region, while restricting other polarity
Introduction
The establishment of cell polarity involves the asymmetric
localization of proteins and other molecules at distinct
membrane domains and cellular structures, organizing the cell
along a defined polarity axis. The PAR proteins are involved
in organizing this polarity and were first identified in C.
elegans, playing a fundamental role in polarizing the one-cell
stage embryo (Kemphues, 2000). One of these proteins, PAR3,
is conserved in nematodes, insects and vertebrates and has been
shown to associate with two other proteins, PAR6 and atypical
protein kinase C (aPKC), forming the core of a multiprotein
complex designated the PAR polarity complex (Ohno, 2001;
Henrique and Schweisguth, 2003).
In mammalian and Drosophila epithelial cells, the PAR
complex localizes to cellular junctions at the apical-most tip
of the lateral membrane, where it regulates a network of
molecular complexes that act together to define the apicobasal polarity of epithelial cells (Macara, 2004; Suzuki and
Ohno, 2006). In addition, the PAR complex controls the
asymmetric divisions of early C. elegans embryos and
Drosophila neuroblasts (Wodarz, 2005), also playing an
important role during specification of axonal processes in
mammalian neurons (Arimura and Kaibuchi, 2005). During
these processes, the correct localization and activity of the
complex seem to depend on PAR3, which has been suggested
to function as a scaffold to promote the assembly of the whole
complex. A subsequent interaction between PAR6 and
CDC42-GTP can activate aPKC by relieving it from PAR6mediated inhibition (Yamanaka et al., 2001). PAR3 is itself a
target of aPKC and dissociates from the complex when
phosphorylated (Nagai-Tamai et al., 2002), allowing aPKC to
proteins to the basolateral membrane. We propose that
PAR3 acts as a molecular organizer to connect the
acquisition of apico-basal polarity with the positioning and
formation of junctional structures in neuroepithelial cells,
a function of upmost importance for the morphogenesis of
embryonic neural tissue and the process of neurogenesis.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/119/20/4293/DC1
Key words: PAR polarity complex, Neuroepithelium, Apical
junctional complexes, Cell adhesion
act on other substrates, in a cascade of signalling events that
results in the segregation of different molecules to distinct
membrane domains and in the correct organization of
junctional structures within epithelial cells. Interference with
the normal function of the PAR complex, as happens in
Drosophila bazooka (encoding the fly PAR3 protein) and par6
mutants (Wodarz et al., 2000; Hutterer et al., 2004; Harris and
Peifer, 2005) or in MDCK cells expressing a dominantnegative mutant of aPKC (Suzuki et al., 2001), leads to
disruption of cellular junctions and profound alterations in
epithelial tissue architecture.
The molecular mechanisms underlying these activities of the
PAR complex are not completely understood but other protein
complexes are known to interact functionally with the PAR
complex during the establishment of epithelial polarity. One of
these is composed by the proteins Crumbs (CRB), Stardust
(PALS1) and PALS-associated tight junction protein (PATJ),
and is also localized to epithelial junctions, with PALS1
directly associating with PAR6 in a CDC42-dependent manner
(Macara, 2004). A different group of proteins is localized
basolaterally in epithelial cells, including Scribble (SCRIB),
Discs Large (DLG) and Lethal Giant Larvae (LGL), which also
contribute to the establishment of epithelial cell polarity and
adhesion (Bilder, 2004). The LGL protein can interact directly
with PAR6 and this might result in its inactivation by aPKCmediated phosphorylation (Betschinger et al., 2003; Yamanaka
et al., 2003). Furthermore, LGL controls the asymmetric
localization of Numb during divisions of neural precursors in
Drosophila (Ohshiro et al., 2000; Peng et al., 2000; Langevin
et al., 2005), in a process where PAR3 seems to also play a
crucial role (Bellaiche et al., 2001; Roegiers et al., 2001).
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Journal of Cell Science 119 (20)
A dynamic crosstalk between these polarity complexes is
involved in the establishment of epithelial polarity in both
Drosophila and mammalian cells (Macara, 2004; Suzuki and
Ohno, 2006). Although the biochemical details are still
incomplete, genetic data indicate that the SCRIB group of
proteins antagonizes the apical polarizing activity initiated by
the PAR complex, whereas the CRB complex counteracts the
activity of the SCRIB group. To maintain the apical membrane
character (Bilder et al., 2003; Tanentzapf and Tepass, 2003).
These interactions lead to the segregation of the various
polarity proteins to distinct membrane domains, with LGL,
DLG and SCRIB being excluded from the more apical domain
where the PAR and CRB complexes reside.
During neurogenesis in the Drosophila embryo, some
epithelial cells are selected to become neuroblasts, which
subsequently delaminate from the ectoderm and divide
asymmetrically to generate various neural cell types
(Betschinger and Knoblich, 2004). Although neuroblasts are
no longer epithelial cells, they maintain apico-basal polarity,
with the PAR complex localized apically. This complex serves
as polarity cue to orient neuroblast divisions, leading to the
unequal segregation of determinants between the two daughter
cells and the consequent acquisition of distinct fates. Thus, the
PAR complex in Drosophila ensures that the distinctive
polarity of epithelial cells is translated into molecular
asymmetries within delaminating neuroblasts, providing a
functional link between epithelial polarity and neurogenesis.
In vertebrates, neurogenesis occurs in a pseudostratified
epithelium and, in contrast to Drosophila neuroblasts, neural
progenitors are fully polarized epithelial cells, with apically
localized intercellular junctions and basal endfeet contacting
the basal lamina (Chenn and McConnell, 1995; Aaku-Saraste
et al., 1996). Apical Junctional Complexes (AJCs) in vertebrate
neuroepithelial (NEP) cells seem to be composed mainly of
cadherin-based adherens junctions (AJs) (Aaku-Saraste et al.,
1996), which play a structural role in maintaining epithelial
integrity and contribute to organize the characteristic apicobasal polarity of these cells (Chenn et al., 1998).
The presence of PAR3, PAR6 and aPKC proteins at the apical
region of mouse and zebrafish NEP cells (Manabe et al., 2002;
Geldmacher-Voss et al., 2003; Wei et al., 2004) suggests that the
PAR polarity complex participates also in the establishment
of apico-basal polarity in the vertebrate neuroepithelium.
Furthermore, loss-of-function mutations in aPKC or
morpholino-based inactivation of pard3 in zebrafish embryos
(Wei et al., 2004; Koike et al., 2005; Imai et al., 2006) have been
shown to disturb apico-basal polarity and cause disassembly of
cellular junctions in NEP cells, leading to alterations in neural
tissue architecture. Although these experiments show that the
PAR complex is necessary for the establishment of NEP polarity
and formation of junctional structures, the severe disruption of
the neuroepithelium has precluded a detailed analysis of the
molecular mechanisms by which the PAR complex regulates
polarity of NEP cells and how it contributes to the normal
architecture of embryonic neural tissue.
Here, we address the role of the PAR complex in the chick
embryo, using a gain-of-function approach to follow the
dynamic activity of various members of the complex, when
ectopically expressed in NEP cells. Our results show that
cPAR3 plays a central role in directing the recruitment of
various polarity and junction-associated proteins to the cortical
plasma membrane of NEP cells, excluding other polarity
proteins from the same membrane region. We propose that
cPAR3 functions as a molecular organizer that connects the
acquisition of apico-basal polarity with the formation of
junctional structures in NEP cells, thus contributing for the
proper morphogenesis of neural tissue.
Results
The PAR polarity complex is localized at the apical
region of the chick embryonic neuroepithelium
We have cloned and characterized two chick par3 genes,
named as cpar3 and cpar3L (Fig. 1A). A cpar3 full-length
cDNA clone encodes a 1,352 amino acid protein highly
homologous to the described mammalian PAR3 proteins
(Izumi et al., 1998; Joberty et al., 2000; Lin et al., 2000). Only
a partial cDNA could be obtained for cpar3L, encoding the
N-terminal 860 amino acids of cPAR3L, with a high
homology to human PAR3L (Gao et al., 2002; Kohjima et al.,
2002). The two cpar3 genes have partial overlapping
expression patterns in the embryo, with cpar3 showing higher
expression in the developing NE (see supplementary material
Fig. S1). This, and the fact that we could not obtain a fulllength cDNA for cpar3L, led us to focus our studies on cpar3.
A full-length cpar6 cDNA was also characterized, which
encodes a protein with high homology to mouse PAR6B
(Joberty et al., 2000).
To determine the subcellular localization of the PAR
complex in embryonic NEP cells, a combination of specific
antibodies and low-level expression of fluorescent protein
fusions was used. To validate this approach, we first established
that in ovo electroporation of the chick neural tube (NT) with
a vector encoding a cPAR3::GFP protein at low DNA
concentration (0.1-0.2 ␮g ␮l–1), results in a subcellular
distribution of the fusion protein identical to that revealed by
immunofluorescence (IF) with anti-PAR3 antibodies (Fig.
1Ba,b), without causing any obvious cellular defects.
Using this approach, we found that cPAR3, cPAR6 and
aPKC are localized in the apical-most region (close to the
luminal surface) of NEP cells (Fig. 1Ba-e), similar to what has
been described in mouse embryos (Manabe et al., 2002).
However, confocal microscopy analysis reveals that there is no
overlap between the localization of cPAR3 and its binding
partners cPAR6 or aPKC: while colocalization between cPAR6
and aPKC is always found (Fig. 1Be), cPAR3 is present in a
more basal domain relative to the duo cPAR6/aPKC (Fig. 1Bad). These results are different from those reported for the
mouse embryonic telencephalon, where mPAR3 was described
as colocalizing with mPAR6 and aPKC at the apical region of
NEP cells (Manabe et al., 2002). This disparity might be due
to a lower microscopic resolution of the mouse data or,
alternatively, might indicate that the localization of the PAR
complex proteins is different between mouse and chick
embryos.
We have also analyzed the distribution of other polarity
complexes in chick NEP cells, using the same approach. Our
results show that cPALS1 and cCRB3 are located at the apical
region of the neuroepithelium (Fig. 1Da,b), colocalizing with
cPAR6 (Fig. 1Dc) above the site of cPAR3 accumulation. By
contrast, LGL1 is located to the lateral membrane of NEP cells
and is excluded from the apical domain where cPAR3
accumulates (Fig. 1Ea).
PAR3 and neuroepithelial polarity
4295
Journal of Cell Science
Fig. 1. The PAR polarity complex
is localized at the apical
membrane of NEP cells.
(A) Deduced protein sequence of
cPAR3, depicting the aPKC,
KIF3A and TIAM1 binding
domains (aPKC-BD; KIF3A-BD;
TIAM1-BD). (B) Endogenous
cPAR3 localizes apically in NEP
cells (a). cPAR3 localizes basally
to cPAR6 (b,c) and aPKC (d).
cPAR6 colocalizes with aPKC
(e). (C) cPAR3 colocalizes with
N-Cadherin, Afadin and ␤Catenin at the apical tip of NEP
cells (a-c). cPAR6 and aPKC are
located apically relative to NCadherin (d,e). (D) cPAR3 is
localized basally relative to both
cPALS1 and cCRB3 (a,b).
cPAR6 colocalizes apically with
cCRB3 (c). (E) LGL1 localizes
basolaterally and is absent from
the apical cPAR3 accumulation
domain (a). (F) Schematic
representation of the localization
of polarity proteins in NEP cells.
L, lumen of the NT. Bars in
(Ba,b,d,e;C;Db,c;E), 2 ␮m; bar in
(Bc), 3 ␮m; bar in (Da), 1 ␮m.
Together, these results are consistent with a direct interaction
between cPALS1, cCRB3 and cPAR6 in NEP cells, as
previously described for other cell types (Hurd et al., 2003;
Lemmers et al., 2004). Additionally, the mutually exclusive
distribution of cPAR3 and LGL1 is consistent with published
biochemical evidence, which shows that PAR3 and LGL
compete for binding to PAR6/aPKC (Yamanaka et al., 2003).
The localization of all these proteins in chick NEP cells is
summarized in Fig. 1F.
cPAR3 is present at adherens junctions in
neuroepithelial cells
To determine how the two distinct apical domains of PAR
protein accumulation are related to junctional structures in
NEP cells, the distribution of various AJ components was
analyzed and compared with that of cPAR3, cPAR6 and aPKC.
Our results show that the AJ proteins N-Cadherin, ␤-Catenin
and Afadin are concentrated at the apical tip of NEP cells, in
the same domain as cPAR3 (Fig. 1Ca-c), but with no detectable
colocalization with cPAR6/aPKC (Fig. 1Cd,e). This suggests
that cPAR3 is a component of AJs in NEP cells, while the duo
cPAR6/aPKC occupies a more apical membrane domain.
Furthermore, co-IP assays reveal that N-Cadherin and cPAR3
are present in the same macromolecular complex (see
supplementary material Fig. S1D), confirming that cPAR3 is
an AJ-associated protein.
cPAR3 colocalizes with cNumb at the apical region of
neuroepithelial cells
In Drosophila neuroblasts and SOPs, the LGL protein controls
the asymmetric accumulation of Numb, in a process that
depends on the activity of the PAR polarity complex
(Betschinger et al., 2003; Langevin et al., 2005). In the mouse
brain, Numb was reported to be present at the apical surface of
NEP cells (Zhong et al., 1996), a localization that seems to
depend on the activity of the mouse lgl homolog, mlgl1
(Klezovitch et al., 2004). However, in the chick neural tube,
Numb has been described to accumulate in a basal crescent in
mitotic NEP cells, with no detectable apical accumulation
(Wakamatsu et al., 1999).
Our experiments, using different fixation conditions and
several distinct antibodies, could not confirm the basal
accumulation of cNumb in mitotic cells, revealing instead a
clear apical localization in NEP cells, as well as accumulation
in cytoplasmic vesicles (Fig. 2A,B). Confocal microscopy
analysis reveals colocalization of cNumb with cPAR3 at the
apical membrane of NEP cells (Fig. 2C-C⬙), raising the
hypothesis that the two proteins are present in the same
complex. To test this, IP assays were performed with antiNumb and anti-PAR3 antibodies, which revealed that the two
proteins are part of the same protein complex (Fig. 2D,E).
Furthermore, immunostaining with antibodies against NCadherin and ␤-Catenin show that apical cNumb colocalizes
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Fig. 2. cPAR3 colocalizes with
cNumb at intercellular junctions.
cNumb accumulates at the apical
membrane (arrowhead in A) and
at cytoplasmic vesicles
(arrowheads in B).
(C-C⬙) cPAR3 colocalizes with
cNumb at the apical membrane
of NEP cells. (D) cPAR3 is coimmunoprecipitated with cNumb
in NEP protein lysates. CRMP2
is used as control. Conversely,
cNumb co-immunoprecipitates
with cPAR3 (E). (F) cNumb
accumulates at the apical
membrane together with ␤Catenin and N-Cadherin (G). L,
lumen of the NT. Magnification
in (A), 20⫻. Bar in (B), 6 ␮m;
bars in (C-C⬙), 3 ␮m; bars in
(F,G), 2 ␮m.
Journal of Cell Science
with both proteins (Fig. 2F,G), suggesting that cNumb is also
an AJ-associated protein.
Ectopic cPAR3 accumulates in punctate clusters at the
cortical plasma membrane of neuroepithelial cells
To study the function of the PAR complex in NEP cells, a
gain-of-function approach was used to generate ectopic
localization of the cPAR3 and cPAR6 proteins, and
evaluate the effects on the cellular architecture of the
neuroepithelium. Electroporation of chick embryos with
high concentrations of the cPAR3::GFP or GFP::cPAR6
vectors allowed us to follow the spatio-temporal
accumulation of the ectopic proteins in NEP cells and the
effects they have on the dynamics of endogenous polarity
proteins. While ectopic cPAR6 accumulates diffusely in the
cell and has no detectable effect on the localization of
endogenous polarity proteins (data not shown), ectopic
cPAR3 accumulates in discrete clusters along the lateral
plasma membrane of NEP cells (Fig. 3A,B) and causes a
Fig. 3. Ectopic cPAR3 accumulates in punctate
clusters at the lateral membrane of NEP cells. (A) 12
hours post-electroporation, cPAR3::GFP accumulates
in clusters along the plasma membrane of NEP cells
(arrows) and also apically (arrowhead). (B) A single
electroporated cell with multiple clusters of
cPAR::GFP accumulation along the plasma
membrane. (C) At 24 hours, cells with high levels of
cPAR3 accumulate at the basal region of the NT
(arrow) and ectopic mitotic cells (red, pHistH3positive) are also present in this region (arrowheads in
D). (E) At 48 hours, cPAR3 accumulates in rosettelike structures at the basal part of the NE (square),
with punctate clusters in other cells (arrow).
(F,F⬘) Detail of a rosette-like group of cells [outlined
by the dotted line; enlarged from square in (D)], with
cPAR3 accumulating at the center (arrow). (G) The
cPAR3-⌬N::GFP mutant still localizes apically
(arrowhead), but does not form punctate clusters. L,
lumen of the NT. Magnification in (A,D), 40⫻; in
(C,E,G), 63⫻. Bars in (B) and (F,F⬘), 10 ␮m.
redistribution of other polarity proteins (see below). Cells
with ectopic cPAR3::GFP clusters concentrate in the
basal region of the neuroepithelium 24 hours postelectroporation (Fig. 3C) and some of these cells undergo
mitosis (Fig. 3D). Later, at 48 hours, cells with cPAR3::GFP
punctates are still accumulated basally (Fig. 3E) but, in
addition, we could detect the formation of rosette-like groups
of cells in this region of the neuroepithelium. cPAR3::GFP
concentrates at the center of these rosettes (Fig. 3F,F⬘) and is
almost absent from the remaining plasma membrane.
Overexpression of untagged cPAR3, followed by detection
with anti-PAR3 antibodies, gave essentially the same results
(data not shown).
To address whether formation of cPAR3 punctate clusters
results from protein oligomerization (Benton and St Johnston,
2003; Mizuno et al., 2003), a form of cPAR3 without the CR1
conserved region (cPAR3-N::GFP) was overexpressed in the
chick embryonic neural tube. The results show that this
truncated protein is still partially localized at the apical tip of
PAR3 and neuroepithelial polarity
Journal of Cell Science
NEP cells (Fig. 3G), but is unable to form punctate
accumulations at the membrane, being distributed
homogeneously in the cytoplasm (compare Fig. 3G with
3C,D). Thus, the CR1 region is essential to promote cPAR3
oligomerization, although it is dispensable for the apical
localization.
Cells overexpressing cPAR3 are misplaced within the
neuroepithelium
During neurogenesis, NEP cells undergo a characteristic
interkinetic nuclear movement (INM) (Fujita, 1960),
associated with the various phases of cell cycle. At S phase,
the nucleus is located in a more basal position and, as cells
enter G2, it moves apically and reaches the luminal surface,
where cells divide. After division, the nucleus of the two
daughter cells moves basally, as these cells enter S phase.
Surprisingly, cPAR3-overexpressing cells in mitosis can be
detected at the basal region of the neuroepithelium, contrasting
with the normal, non-electroporated half of the neural tube,
where mitosis always occur close to the lumen. The number of
mitotic cells is very similar in the two sides of the neural tube
(data not shown), discarding the hypothesis that cPAR3
overexpression might cause cell cycle alterations. Another
hypothesis is that cPAR3 overexpression might arrest the
normal INM of NEP cells, resulting in cells entering mitosis
at ectopic positions in the basal region of the neuroepithelium.
To test this, we followed cPAR3-overexpressing cells by live-
Fig. 4. cPAR3 overexpression
affects positioning of dividing
cells within the NE.
(A) Normal NEP cells,
expressing a membranetethered GFP, move their
nucleus apically and always
divide near the luminal
surface (red and white
arrowheads). (B) Illustrative
example (n=12 cells) of a
GFP-gpi NEP cell
overexpressing cPAR3
dividing away from the apical
surface (red arrow). Bar in
(A), 10 ␮m; (B), 20 ␮m.
4297
imaging confocal analysis. A neural tube slice culture system
was developed that allows visualization of NEP cells along the
various phases of their INM, through incorporation of a GPIanchored GFP (GFP-gpi) at the plasma membrane. To assess
the effects of cPAR3 overexpression, a group of embryos
(n=12) was electroporated with a mixture of GFP-gpi vector
and a vector encoding a non-tagged cPAR3 protein, while
control embryos (n=12) were electroporated with the GFP-gpi
vector only.
Our results show that, in control embryos, NEP cells
undergo normal INM and always enter mitosis at the luminal
surface (Fig. 4A, see supplementary material Movie 1).
However, in embryos electroporated with cPAR3,
overexpressing cells show increased adhesiveness and seem
‘trapped’ within the NE, unable to accomplish INM. As a
consequence, when these cells enter mitosis, the nucleus is still
basally located, far from the luminal surface (Fig. 4B, see
supplementary material Movie 2).
cPAR3 drives local accumulation of junction-associated
proteins at the neuroepithelial cell membrane
This behavior of cPAR3-overexpressing cells might involve the
formation of new intercellular adhesion sites, primed by the
ectopic accumulation of cPAR3 and subsequent recruitment
of other proteins that control the assembly of junctional
complexes. In support of this hypothesis, ectopic N-Cadherin
is readily detectable in cPAR3-overexpressing cells 12 hours
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Fig. 5. Ectopic cPAR3 is sufficient to mislocalize polarity and junction-associated proteins in NEP cells. At the electroporated side of the NT,
N-Cadherin accumulates ectopically (white bracket in A,A⬘), colocalizing with cPAR3::GFP punctates (arrowheads in B,B⬘). The same applies
to ␤-Catenin (arrowheads in C-D⬘) and aPKC (arrowheads in E-F⬘). Triple-labelling of cPAR3, cPAR6 and aPKC shows colocalization at
ectopic clusters (G,G⬘). Members of the CRB complex also mislocalize to cPAR3 punctates (arrowheads in H-K⬘). (L) LGL1 is excluded from
the ectopic sites of cPAR3 accumulation at the lateral membrane (arrowheads). b, basal side of the NE. Magnification in
(A,A⬘,C,C⬘,E,E⬘,H,H⬘,I,I⬘), 40⫻. Bars in (B,B⬘,G,G⬘), 2 ␮m; bars in (D,D⬘,F,F⬘,J,J⬘,K,K⬘), 10 ␮m; bar in (L), 1 ␮m.
after electroporation, being distributed along the cell
membrane (Fig. 5A-B⬘), instead of normally concentrated at
the apical tip of NEP cells. Confocal analysis shows a clear
correspondence between cPAR3::GFP punctates and NCadherin accumulation (Fig. 5B,B⬘), suggesting that cPAR3 is
able to recruit N-Cadherin. Consistent with this, coIP studies
show that N-Cadherin and cPAR3 are normally present in
the same macromolecular complex in NEP cells (see
supplementary material Movie 1). In addition, ␤-Catenin is
also seen to accumulate ectopically in cPAR3-overexpressing
cells, showing a good correlation with ectopic cPAR3::GFP
localization (Fig. 5C-D⬘). However, not all cPAR3 puncta show
ectopic accumulation of ␤-Catenin (and of other recruited
proteins, see below), which might be due to limited amounts
of available endogenous proteins.
Together, these results indicate that cPAR3 overexpression
interferes with the distribution of junction-associated proteins
and drives the formation of ectopic accumulations of both NCadherin and ␤-Catenin, thereby triggering the formation of
ectopic adhesion structures in NEP cells.
cPAR3 coordinates the recruitment of other polarity
proteins to the membrane of neuroepithelial cells
The recruitment of junctional proteins to ectopic positions and
the subsequent establishment of new adhesion sites is likely to
involve the concerted activity of other polarity proteins which
function together with PAR3 to control apico-basal polarity in
epithelial cells. We therefore asked whether PAR6, aPKC, and
members of other polarity complexes are also present at ectopic
sites of cPAR3 accumulation. Our results reveal that both
aPKC and PAR6 are ectopically distributed in cPAR3overexpressing cells, overlapping with some of the membrane
associated cPAR3::GFP punctates (Fig. 5E-G⬘). Similarly,
cCRB3 and cPALS1 are also mislocalized in cPAR3overexpressing cells (Fig. 5H-I⬘), with a subcellular
distribution that coincides with ectopic cPAR3 clusters (Fig.
5J-K⬘).
We thus conclude that cPAR3 is able to trigger the assembly
of apical polarity complexes in NEP cells, implying that it
normally functions as a scaffold to coordinate the ordered
recruitment of various polarity proteins to the cortical
membrane. This function is unique to cPAR3 and depend on
its capacity to oligomerize, as overexpression of cPAR6B or
cPAR3-N::GFP in NEP cells does not cause any of the
molecular or cellular defects due to ectopic cPAR3 (data not
shown).
Other polarity proteins are known to be involved in
defining the basolateral membrane domain of epithelial cells,
like LGL (Wodarz, 2005). As described above, LGL1 is
normally distributed along the lateral membrane of chick
Journal of Cell Science
PAR3 and neuroepithelial polarity
4299
Fig. 6. PAR complex formation and cNumb subcellular distribution are affected by the presence of excess cPAR3. (A) Western blot shows that
overexpression (OE) produces an 80⫻ increase in of cPAR3 levels. (B) Endogenous cPAR6 and aPKC levels are unaffected when cPAR3 is
overexpressed. (C,D) Overexpression of cPAR3 recruits more aPKC molecules to the PAR complex. (E) cPAR6/aPKC complex formation is
increased in the presence of excess cPAR3. (F) cLGL1 and cNumb protein levels are unaffected when cPAR3 is overexpressed. (G-G⬙) At 24
hours post-electroporation, cNumb accumulates at the apical region (arrowhead) and in ectopic punctates (arrow). (H,H⬘) Higher magnification
of square in (G⬙) shows that cNumb colocalizes with ectopic cPAR3 punctates (arrowheads), with cNumb vesicles accumulating at the cell
periphery (upper half of the image). In the bottom half of the image, showing non-electroporated cells, cNumb-positive vesicles are uniformly
distributed in the cytoplasm. Magnification in (G-G⬙), 20⫻. Bars in (H,H⬘), 6 ␮m.
NEP cells and is excluded from the apical cPAR3 domain. In
cPAR3-overexpressing cells, we observed that LGL1 is also
excluded from the ectopic cPAR3 punctates at the cortical
plasma membrane (Fig. 5L). This effect is likely mediated by
the activity of aPKC at the ectopic cPAR3 clusters, with
LGL1 being phosphorylated and excluded from the
membrane, as it happens in Drosophila epithelial cells
(Hutterer et al., 2004).
Taken together, these results suggest that ectopic activity of
cPAR3 leads to the establishment of new membrane domains
with apical character, at the sites where ectopic apical polarity
complexes assemble and from where LGL is excluded.
cPAR3 promotes the formation of ectopic
cPAR3/cPAR6/aPKC complexes
To address whether the effects on the localization of apical and
basolateral proteins are due to an increase in their synthesis or
to a redistribution of the pre-existing pool, the various proteins
were quantified by western blot. Our results show that there is
no significant increase in the amount of endogenous polarity
proteins, despite an 80-fold increase in cPAR3 levels (Fig.
6A,B). Nevertheless, the presence of more cPAR3 molecules
in NEP cells seems to promote formation of additional PAR
complexes, as shown by IP with anti-PAR6 antibody, followed
by western blot with the anti-aPKC antibody (Fig. 6E). This is
further supported by IP experiments with anti-PAR3 and antiaPKC antibodies, which reveal the presence of more aPKC
molecules in a complex with cPAR3 (Fig. 6C,D). Taken
together, these results indicate that excess cPAR3 drives
formation of additional PAR complexes in NEP cells, and show
that ectopic localization of cPAR6 and aPKC is a consequence
of protein mislocalization, involving recruitment by cPAR3.
Similarly, western blot data confirm that LGL1 mislocalization
(and cNumb, see below) is due to protein redistribution in NEP
cells (Fig. 6F).
Ectopic cPAR3 causes a redistribution of cNumb in
neuroepithelial cells
LGL phosphorylation by aPKC is important to position Numb
correctly at the basal pole of Drosophila neuroblasts
(Betschinger et al., 2003). Furthermore, the asymmetric
localization of Numb during SOP division is controlled by
BAZ (Bellaiche et al., 2001; Roegiers et al., 2001), with the
LGL protein being also involved in this process (Langevin et
al., 2005). Given the defects in LGL1 distribution caused by
cPAR3 overexpression, we asked whether cPAR3 also controls
cNumb localization in NEP cells. Analysis of embryonic
neural tubes where cPAR3::GFP was overexpressed revealed
that there is a clear alteration in cNumb distribution, which
becomes highly concentrated at the apical membrane (Fig. 6G-
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Journal of Cell Science 119 (20)
G⬙), and appears also as ectopic punctates in cells at the basal
region of NE (Fig. 6G-G⬙). Furthermore, confocal analysis
reveals a striking accumulation of cNumb at the membrane of
cPAR3-overexpressing cells, with a clear overlap with ectopic
cPAR3 clusters (Fig. 6H,H⬘). Concurrently, a reduction of
cytoplasmic cNumb is observed (compare upper and bottom
halves of Fig. 6H,H⬘) which, since the overall intracellular
concentration of cNumb is not affected (Fig. 6F), suggests that
ectopic cPAR3 causes a redistribution of cNumb from
cytoplasmic vesicles to the cell membrane. Taken together,
these results show that cPAR3 regulates the subcellular
localization of cNumb in NEP cells. This might involve an
interaction between the two proteins, a hypothesis supported
by our finding that cPAR3 and cNumb can be detected in the
same molecular complex in NEP cells (Fig. 2D,E).
Journal of Cell Science
cPAR3 overexpression causes the formation of
neuroepithelial rosettes
Forty eight hours after electroporation, cPAR3-overexpressing
embryos show a dramatic disruption of the neuroepithelium.
Ectopic mitosis is often detected and BrdU incorporation
indicates the presence of several proliferating cells in the basal
region, which normally should contain only post-mitotic
neurons (Fig. 7A,A⬘). The most striking phenotype is the
appearance of rosettes in the basal region of the NE (Fig. 3E),
formed by groups of cells organized concentrically around a
lumen (Fig. 3F,F⬘). In rosette cells, cPAR3 concentrates in the
region close to the ‘luminal’ surface, together with aPKC and
cPAR6 (Fig. 7B-B⬙). This suggests that a defined apical domain
is present in rosette cells, which is supported by the presence
of centrosomes close the same membrane domain (Fig. 7C,C⬘).
N-Cadherin and ␤-Catenin also occupy the region closest to
the luminal space, colocalizing with cPAR3 (Fig. 7D-E⬘),
implying that junctional complexes are present at the apical
region of rosette cells and might function to organize the cells
around the central lumen. In this aspect, rosette cells behave
like normal NEP cells, forming polarized structures similar to
neural tubes.
To test whether rosette cells are also engaged in
neurogenesis, the expression of various members of the Notch
Fig. 7. Characterization of rosette-like structures in the NE of cPAR3-overexpressing embryos. (A,A⬘) Cycling cells (BrdU+) are present
outside the VZ (bars). (B-B⬙) cPAR3 is localized in a central ring inside the rosettes, together with cPAR6/aPKC. (C,C⬘) Rosette cells (dotted
line) contain centrosomes close to the central lumen (dotted circle). (D,D⬘) ␤-Catenin (arrowhead) and N-Cadherin (arrowhead in E,E⬘) localize
with cPAR3::GFP at the center of rosettes. (F,G) cPAR3 overexpression (left side of the NT) causes an expansion of the cNotch1 and ches5-1
expression domains (white brackets). (H) Ectopic cDelta1 expressing cells can be detected in the basal region of the cPAR3-electroporated NT
(white brackets). (I,I⬘) Rosette cells express ches5-1. (J,J⬘) TUJ1- and HU-positive neurons (K,K⬘) accumulate outside rosettes. Magnification
in (A,A⬘), 40⫻; magnification in (F-H), 20⫻. Bars in (B-B⬙), 4 ␮m; bars in (C-D⬘,J,J⬘), 10 ␮m; bars in (I,I⬘), 5 ␮m; bars in (K,K⬘), 20 ␮m.
PAR3 and neuroepithelial polarity
Journal of Cell Science
pathway was examined in cPAR3-overexpressing embryos,
using in situ hybridization. Expression of cNotch1 and ches51, two genes active in neural progenitors (Fior and Henrique,
2005), could be detected in numerous cells in ectopic positions
outside the ventricular zone (VZ) (Fig. 7F,G), including rosette
cells (Fig. 7I,I⬘). In addition, the cDelta1 gene, which is
expressed in nascent neurons, is also transcribed in some
rosette cells (Fig. 7H). Together, this indicates that the Notch
pathway is active in rosette cells, some of which stop cycling
and start to differentiate. This is confirmed by the presence of
neuronal markers (HU and TUJ1) in cells at the periphery of
rosettes (Fig. 7J-K⬘), some of them still maintaining an apical
process (Fig. 7J). In conclusion, these results show that rosette
cells are engaged in neurogenesis, with newborn neurons
accumulating outside the rosette environment, in the same way
that differentiating neurons in the neural tube migrate out of
the VZ and accumulate in the mantle layer.
Discussion
In this work, we address the central role of PAR3 in defining
the apico-basal polarity of NEP cells in chick embryos. Our
data support a model in which PAR3 functions as the initial
scaffold to assemble the PAR polarity complex at the apical
region of NEP cells, coordinating also the recruitment of
additional polarity complexes and junction-associated proteins
to the same region, while restricting other polarity proteins to
the basolateral membrane. We propose that PAR3 acts as a
molecular organizer to connect the acquisition of apico-basal
polarity with the positioning and formation of junctional
structures in NEP cells, a function of upmost importance for
the correct morphogenesis of embryonic neural tissue and the
subsequent process of neurogenesis.
cPAR3 as a molecular organizer of apico-basal polarity
in neuroepithelial cells
The role of the PAR complex in controlling the establishment
and maintenance of cell polarity in different cell types, from
various organisms, is well established (Henrique and
Schweisguth, 2003; Macara, 2004). The main biochemical
output of the PAR complex is provided by aPKC activity,
whereas PAR6 regulates aPKC in a CDC42-dependent manner
and PAR3 is usually seen as a structural scaffold. However,
analysis of protein localization during polarization of the C.
elegans one-cell embryo suggests that PAR3 might play a more
active role in localizing the PAR complex (Cuenca et al., 2003),
and recent results in Drosophila reveal that PAR3 acts
upstream of aPKC and PAR6 as an essential polarity cue during
epithelial cellularization (Harris and Peifer, 2004; Harris and
Peifer, 2005). Furthermore, in MDCK cells PAR3 has been
shown to act independently of aPKC/PAR6 during epithelial
polarization, regulating the activity of the GTP exchange factor
TIAM1 to control actin dynamics (Chen and Macara, 2005).
Thus, the contribution of PAR3 to the mechanisms that
generate cellular polarity is still not totally known.
Although loss-of-function experiments in zebrafish embryos
indicate that PAR3 is necessary for the correct morphogenesis
of neural tissue, analysis of these mutants could not reveal the
specific role of PAR3 in the polarity mechanisms, due to the
severe disorganization of the neuroepithelium and widespread
NEP cell death (Geldmacher-Voss et al., 2003; Wei et al.,
2004).
4301
The data presented in this paper, using ectopic expression of
cPAR3 in chick NEP cells, unveil a new perspective on the
function of PAR3 during establishment of NEP polarity. We
show that PAR3 is able to oligomerize and form punctate
clusters at the cortical plasma membrane of NEP cells, where
it can nucleate new PAR complexes by recruiting endogenous
cPAR6 and aPKC. Biochemical analysis confirms that cPAR3
ectopic expression results in higher number of PAR complexes
within the cell and that this is due to a redistribution of
endogenous cPAR6 and aPKC. Recruitment of aPKC to
ectopic cPAR3 sites is most likely mediated by cPAR6, since
overexpression of a mutant cPAR3 without the putative aPKCbinding domain still results in the accumulation of aPKC (and
cPAR6) at cPAR3 punctates (data not shown), also indicating
that the ability of cPAR3 to nucleate the PAR complex is
independent of its phosphorylation state.
The PAR complex seems to be highly dynamic,
disassembling quickly after its initial formation so that the
constituent proteins can subsequently interact with other
polarity complexes. This is suggested by our finding that
cPAR3 and the duo cPAR6/aPKC are localized to distinct
apical membrane domains in NEP cells, with the assembled
PAR complex being detected only by the more sensitive coIP
assays (and at very low levels). Similar observations have been
recently reported in the forming epithelia of the Drosophila
embryo (Harris and Peifer, 2005) and imply that the interaction
between PAR3 and the duo PAR6/aPKC is indeed very
transient, with the proteins localizing to different membrane
domains after dissociation of the complex.
Phosphorylation of cPAR3 by aPKC might be responsible
for such dynamic behavior of the PAR complex, as
phosphorylated PAR3 is known to have reduced affinity for
aPKC and can dissociate from the complex (Nagai-Tamai et
al., 2002). After dissociation, either cPAR3 or the duo
cPAR6/aPKC (or both) could then quickly relocate and occupy
a distinct subcellular localization. Experiments using cPAR3
proteins without the aPKC phosphorylation site reveal that
unphosphorylated cPAR3 occupies the same position as
endogenous cPAR3 in relation to the duo cPAR6/aPKC (data
not shown). This implies that the final localization of cPAR3
is independent of the interaction with aPKC and subsequent
phosphorylation, thus excluding the hypothesis that cPAR3
moves basally following aPKC-mediated phosphorylation. Our
results are more compatible with the alternative model in which
the duo cPAR6/aPKC achieve its final localization after
interaction with cPAR3 at the apical membrane, followed by
phosphorylation of this protein and release of PAR6/aPKC to
a more apical position. There, the duo PAR6/aPKC might then
serve to localize the CRB complex, which we have shown to
be present in the same apical domain.
Our finding that endogenous cCRB3 and cPALS1 are also
recruited to sites of cPAR3 ectopic accumulation is consistent
with this model. In cPAR3-overexpressing cells, after the initial
recruitment of cPAR6/aPKC to ectopic cPAR3 punctates and
subsequent cPAR3 phosphorylation, the duo cPAR6/aPKC
might relocate and become available to bind members of the
CRB complex, thereby mimicking the cascade of protein
assembly that normally occurs at the apical membrane.
These results also raise the hypothesis that cPAR3 is able to
confer an apical character to the membrane regions where it
accumulates, which is further supported by our finding that the
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Journal of Cell Science 119 (20)
Journal of Cell Science
basolateral LGL protein is excluded from the sites of ectopic
cPAR3 accumulation. Strikingly, overexpression of cPAR6
failed to cause similar reorganization of polarity complexes in
NEP cells (data not shown), emphasizing the unique and
central role of cPAR3 in this process.
In conclusion, our data highlight the central role of cPAR3
in coordinating the spatio-temporal organization of the various
polarity complexes in NEP cells, triggering a series of dynamic
interactions that lead to the segregation of different molecular
domains in the plasma membrane.
cPAR3 and assembly of intercellular junctions
Acquisition of apico-basal polarity and formation of cellular
junctions are connected processes in epithelial cells, but it is
generally accepted that the establishment of cell-cell contacts
is the initiating cue for polarity generation (Nelson, 2003;
Perez-Moreno et al., 2003). The process starts with the
assembly of the AJC at the apical end of the lateral membrane,
which mediates cell adhesion and triggers a series of
biochemical events that will lead to the delimitation of distinct
apical and basolateral membrane domains in epithelial cells.
However, recent work in Drosophila demonstrated that this is
not a universal mechanism and that the activity of BAZ
precedes AJC formation, accumulating at the apical domain of
epithelial cells even in the absence of junctional complexes
(Harris and Peifer, 2004).
Our data in the chick neuroepithelium point to a similar
hierarchy in polarity mechanisms, with cPAR3 acting upstream
of AJCs during establishment of apico-basal polarity in NEP
cells. Indeed, we show that cPAR3 is able to direct the
formation of new AJCs at the cell membrane, as revealed by
the accumulation of N-Cadherin and ␤-Catenin at the sites
of ectopic cPAR3 localization. The consequent higher
adhesiveness of cPAR3-overexpressing cells might be
responsible for the appearance of mitotic cells at abnormal
positions within the NE. As our time-lapse analysis reveals, the
INM of cPAR3-overexpressing cells is arrested, probably due
to additional adhesion sites with their neighbors, and this
results in cells entering division at ectopic positions.
The ability of cPAR3 to promote the assembly of AJCs at
the membrane of NEP cells might involve various mechanisms.
cPAR3 might contribute to the positioning of AJCs through its
organizing activity of the apical polarity complexes, which
results in the definition of a boundary between membrane
regions with apical and basolateral characteristics, where AJCs
will form and develop into mature junctions. In addition,
cPAR3 might have an active role in recruiting AJC components
to the sites where it first accumulates, as suggested by our
overexpression experiments and by the fact that cPAR3 and
N-Cadherin can be detected in the same macromolecular
complex. This is also consistent with recent findings in the
Drosophila embryonic epithelium (Harris and Peifer, 2005),
which demonstrated that BAZ can form complexes with DEcadherin and ␤-Catenin, directing their apical recruitment
during polarity establishment.
An additional mechanism might involve the function of
cNumb, which we show to localize at AJs in NEP cells and
to be recruited to the sites of ectopic cPAR3 accumulation.
Numb has been linked to the ARF6 membrane recycling
pathway (Smith et al., 2004), which is a key modulator of
Cadherin endocytosis from AJs (Palacios et al., 2001), so it is
tempting to speculate that apical cNumb acts downstream of
cPAR3 as part of the mechanism that regulates AJ dynamics
in NEP cells.
The primary role of PAR3 as polarity cue, upstream of AJC,
in the mechanisms that generate apico-basal polarity of
epithelial cells, raises the question of how is the protein
targeted to the plasma cell membrane in the first place. During
cellularization of the Drosophila embryo, dynein-mediated
transport along microtubules and subsequent capture by the
apical actin scaffold have been shown to be involved in the
initial targeting of BAZ (Harris and Peifer, 2005). The question
has not yet been addressed in vertebrate epithelial cells, but a
good candidate to mediate the transport of PAR3 to the apical
membrane is the kinesin motor KIF3, which can interact with
PAR3 (Nishimura et al., 2004) and is involved in the
localization of the PAR complex to the cilia of MDCK cells
(Fan et al., 2004) and to the axonal tip of polarizing
hippocampal neurons (Shi et al., 2004). The KIF3 motor is also
involved in N-Cadherin transport and localization to the apical
region of mouse NEP cells, and a mutation of Kap3, which
encodes an essential component of the KIF3 motor, causes a
severe disruption of the NE’s polarized architecture (Teng et
al., 2005). Although these defects were attributed to the lack
of N-Cadherin function at AJCs, it is also possible that the
apical targeting of PAR3 is affected in Kap3 mutants, and this
might have an important contribution to the defects observed
in NEP polarity.
cPAR3, neuroepithelial polarity and cell fate
determination
Another cellular phenotype due to cPAR3 overexpression in
chick neuroepithelium was the late formation of aggregates of
NEP cells organized around a small lumen (rosettes), which
were often found in the basal part of the NT. Within these
rosettes, ectopic cPAR3 concentrates at the region of the
membrane close to the lumen, where other apical polarity
proteins also accumulate, indicating that this region constitutes
the apical domain of rosette cells. This is further supported by
the finding that AJ proteins, like N-Cadherin and ␤-Catenin,
are also concentrated at the same region, and that centrosomes
are present in the cytoplasm just below this apical domain, like
in normal NEP cells. Taken together, these results indicate that
cPAR3-overexpressing cells are able to reorganize their
polarity and define a single apical domain, close to the luminal
surface, with associated junctional structures allowing cells to
adhere to their neighbors and form rosettes. In addition, rosette
cells are progressing through the cell cycle and engaged in
neurogenesis, indicating that these structures behave like small
ectopic neural tubes. The function of PAR3 in organizing the
apical domain and correctly positioning intercellular junctions
in NEP cells is therefore of upmost importance for
neurogenesis, as it allows cells to establish intercellular
communication and participate in the normal process of cell
fate determination. This is supported by the finding that a
partial loss-of-function of the zebrafish par3D gene causes a
profound disorganization of the retinal neuroepithelium, a
block of neurogenesis and a consequent reduction in neuronal
differentiation (Wei et al., 2004).
In Drosophila, the PAR3 homolog BAZ also plays a central
role in defining neuroblast polarity, underlying the asymmetric
divisions that give rise to the various neuronal and glial cells
PAR3 and neuroepithelial polarity
that make up the fly nervous system (Wodarz, 2005). Thus,
although vertebrate and Drosophila neurogenesis are
apparently very different processes, with neural progenitors
possessing distinct polarity characteristics (epithelial in
vertebrates and mesenchymal in Drosophila), the function of
PAR3 in coordinating polarity establishment is essential in
both types of progenitors for the normal process of neural fate
determination.
Materials and Methods
Plasmids
Plasmids for expression of GFP-gpi (Keller et al., 2001), mRFP::cPAR6, cPAR3,
cPAR3::GFP and cPAR3-N::GFP (corresponding to a deletion of the first 228 amino
acids of cPAR3) were constructed using the pCAGGS vector (Niwa et al., 1991).
Accession numbers for cPAR3, cPAR3L and cPAR6B are, respectively, DQ683186,
DQ683187 and DQ683188.
The mLGL1 plasmid (mLGL1pFLAGCMV2) was a kind gift from Dr Tony
Pawson, and was used at a final concentration of 1 ␮g ␮l–1. The mRFP cDNA
(Campbell et al., 2002) was kindly provided by Dr Roger Tsien.
Journal of Cell Science
In ovo electroporation of chick embryos
Embryos at HH11-12 were microinjected in the NT with plasmid DNA and
electroporated using a BTX ECM830 electroporator. Embryos were incubated at
38°C and then harvested and processed for criostat sectioning. BrdU labeling was
done as previously described (Fior and Henrique, 2005). Plasmids were
electroporated at 0.1-0.4 ␮g ␮l–1 for localization analysis and at 2 ␮g ␮l–1 for gainof-function studies.
Immunofluorescence on cryostat sections
Embryos were fixed in either 1% or 4% paraformaldehyde, for 15 minutes or 2
hours at room temperature, respectively. After embedding in 7.5% gelatin, 8 ␮m
criostat sections were prepared for IF as described previously (Manabe et al., 2002).
Results were analyzed in a Leica DMR microscope, with both 20⫻ (PlanApo NA
0.7) and 40⫻ (PL Fluotar NA 0,75) objectives, and photographed with a Leica
DC350F digital camera. Confocal analysis was done using a Zeiss LSM510
microscope with 63⫻ (Plan-Apochromat; NA 1.4) objectives and LSM 510 AIM
software. Adobe Photoshop 6.0 was used to adjust input levels, brightness and
contrast.
Antibodies
The antibodies used in this work were: rabbit anti-mPAR3 and anti-phosphohistone
H3 (Upstate); rabbit anti-aPKC␨ and goat anti-actin (SCB); rabbit anti-PALS1 and
rabbit anti-CRB3 (Makarova et al., 2003); rabbit anti-mLGL1 (Musch et al., 2002);
mouse anti-N-cadherin, mouse anti-␥-tubulin, mouse anti-BrdU and rabbit anti-␤Catenin (Sigma); mouse anti-Numb (CMNB1, DSHB) mouse anti-HU (Molecular
Probes); mouse anti-TUJ-1 (BabCo); rabbit anti-PAR6␤ (Yamanaka et al., 2003);
rabbit anti-CRMP2 (Nishimura et al., 2003). Secondary antibodies were HRPconjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG (Promega); HRPconjugated anti-goat IgG (SCB): Alexa594-goat anti-rabbit, Alexa594-donkey antimouse and Alexa488-goat anti-rabbit (Molecular Probes).
Live imaging of NT slices
Embryonic NT was electroporated in ovo with the GFP-gpi vector (0.4 ␮g ␮l–1),
alone or in combination with the cPAR3-vector (2 ␮g ␮l–1), and embryos were
harvested 4 hours later. Slices (100 ␮m) were prepared using a McILTwain Tissue
Chopper, and embedded in 1% low-melting agarose in embryo media (Ham’s/F12
with 4 mM glutamine, 1 mM sodium pyruvate, 5% fungizone, 50 U ml–1 penicillin
and 8 mg ml–1 streptomycin). Slices were mounted in a glassbottom Petri dish,
covered with embryo media and overlayed with mineral oil. Dishes were then
incubated at 38°C in an inverted microscope and Z-series images were collected at
1.5, 2, 2.3 or 3.3 ␮m-steps using a laser confocal microscope Zeiss LSM510, at
various time intervals, with a 20⫻ objective (Plan-NeoFluor; NA 0.5). Images were
processed using the Zeiss LSM AIM software. ImageJ and Jasc Animation Shop2
software were used to convert image sequences to QuickTime movies. A total of
18 slices (six with GFP-gpi alone and 12 with a mix of GFP-gpi and cPAR vectors)
was followed for periods between 8 and 24 hours. We analyzed a total of 12 cPAR3overexpressing cells in detail, all with a similar behavior (Fig. 4 and see
supplementary material Movie 2).
In situ hybridization
Chicken embryos were collected and fixed in 4% paraformaldehyde/PBS at 4°C.
Whole-mount in situ hybridization was done as described (Fior and Henrique,
2005). For hybridization on cryostat sections, fixed embryos were cryoprotected in
15% sucrose in PBS, embedded in 7.5% gelatin/15% sucrose/PBS and
4303
cryosectioned (12 ␮m). Hybridization on cryostat sections was done as previously
described (Fior and Henrique, 2005), with Fast-Red (Roche) as Alkaline
Phosphatase substrate, producing a red fluorescent precipitate. Detailed protocols
are available upon request.
Immunoprecipitation
NTs from electroporated embryos were dissected and sectioned longitudinally along
the midline. Protein lysates from each half, or from entire NTs of HH18 and HH23
embryos, were prepared by homogenizing the tissue in cold Lysis Buffer containing
150 mM NaCl, 1% Triton, 50 mM HEPES pH 7.5, 2 mM EDTA, 10% glycerol, 50
mM NaF, 1 mM Na3VO4 and protease inhibitors (Boehringer). Protein extracts
were incubated with antibodies overnight at 4°C. Protein G Sepharose (Amersham)
or ProteinA/G PLUS-Agarose IP Reagent (SCB) beads were added to the extract
and incubated for 3 hours at 4°C. After washing in Lysis Buffer, extracts were
subjected to western blot analysis with different antibodies. Signal quantification
was performed by densitometry using ImageQuant® software (Molecular
Dynamics).
We thank S. Ohno, B. Margolis, A. LeBivic, T. Pawson, R. Tsien,
K. Simons, A. Musch, K. Kaibuchi and the Developmental Studies
Hybridoma Bank for providing antibodies and DNA constructs. We
thank Alina Costa for excellent technical help. We thank P. Ingham,
D. Ish-Horowicz, F. Schweisguth and O. Pourquié for critical
reading of the manuscript. This work was supported by FCT
(POCTI/SAU/48783). C.A. was recipient of a FCT fellowship.
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