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First published online 9 April 2008
doi: 10.1242/dev.013847
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1 Department of Biological Sciences, Stanford University, Stanford, CA 94305,
USA.
2 Department of Physiology, University of California, San Francisco, San
Francisco, CA 94143, USA.
3 Lilly Research Laboratories, Indianapolis, IN 46285, USA.
* Author for correspondence (e-mail: suemcc{at}stanford.edu)
Accepted 13 March 2008
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: MALS, LIN7, Apicobasal polarity, Neurogenesis, Proliferation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Astrom and Webster, 1991;
Chenn et al., 1998;
Zhong et al., 1996). This
intrinsic polarity is exploited when NPCs undergo asymmetric mitotic divisions
to produce apical and basal daughters with different cell fates
(Chenn and McConnell, 1995;
Chenn et al., 1998;
Gotz and Huttner, 2005;
Huttner and Kosodo, 2005).
Imaging studies reveal that the apical cell surface is distributed unequally
during asymmetric division of NPCs, suggesting that apically localized
molecules might play a crucial role in determining the fates and mitotic
behaviors of newly generated daughter cells
(Huttner and Kosodo, 2005;
Kosodo et al., 2004).
The fate of daughter cells that retain apical components remains
controversial. Some studies suggest that these cells continue to proliferate
and remain as NPCs (Miyata et al.,
2001), whereas other studies suggest that they differentiate into
neurons (Huttner and Kosodo,
2005; Kosodo et al.,
2004; Wodarz and Huttner,
2003). Despite this disagreement, it is clear that only one
daughter of an asymmetric division inherits apical components, and that this
daughter has a fate distinct from that of the other
(Miyata et al., 2001;
Noctor et al., 2001). These
observations suggest that determinants at the apical membrane [such as PAR6
(PARD6A - Mouse Genome Informatics) and PAR3 (PARD3 - Mouse Genome
Informatics)] or associated with adherens junctions might influence the fates
of daughter cells (Cappello et al.,
2006; Imai et al.,
2006).
Numerous proteins, including Numb, PAR3, CDC42, prominin 1 (CD133), ASPM
and afadin (MLLT4 - Mouse Genome Informatics), are localized apically in NPCs
(Gotz and Huttner, 2005), and
many of these play crucial roles in corticogenesis by regulating apicobasal
polarity (Fish et al., 2006;
Junghans et al., 2005;
Petersen et al., 2002). For
example, conditional disruption of CDC42 causes a loss of apical
markers and adherens junctions in NPCs, which become mislocalized to the basal
VZ and show altered fates and mitotic behavior
(Cappello et al., 2006). The
brains of transgenic mice that overexpress the junctional protein
β-catenin are much larger than those of wild-type mice, because of an
increase in the number of cycling progenitors
(Chenn and Walsh, 2003),
whereas loss of the basally localized protein LGL results in severe brain
dysplasia and proliferation defects during corticogenesis
(Klezovitch et al., 2004).
These studies suggest that the establishment and maintenance of polarity in
NPCs is crucial to normal corticogenesis.
Studies in MDCK cells have identified three complexes (CRB3/PALS1/PATJ,
PAR3/PAR6/aPKC and MALS/PALS1) that are crucial for maintaining polarity
(Margolis and Borg, 2005).
MALS (also known as LIN-7 or Veli) is a PDZ domain-containing protein that
specifies cell fates in C. elegans by controlling the basal
localization of the EGF receptor LET-23 in vulval precursor cells
(Kaech et al., 1998). LET-23
is mislocalized to the apical membrane in lin-7 mutants, leading to a
mis-specification of vulval cell fates
(Kaech et al., 1998). In
vertebrates, there are three MALS genes, MALS-1, -2 and
-3. Silencing MALS-3 (LIN7C - Mouse Genome
Informatics) in MDCK cells results in defective tight junctions and the loss
of several MALS-3 binding partners, such as PALS1
(Margolis and Borg, 2005;
Olsen et al., 2005a). PALS1
(MPP5 - Mouse Genome Informatics) forms a complex with CRB3 and PATJ (INADL -
Mouse Genome Informatics), and can also interact with PAR6, thus linking the
PAR6 signaling complex to other apically localized proteins such as CRB3 and
MALS-3 (Roh et al., 2002).
Together, these proteins are positioned apically to link transmembrane
signaling proteins with cytoskeletal structures and/or cell nuclei
(Roh and Margolis, 2003).
Despite progress in understanding the role of MALS proteins in MDCK cells
in vitro, less is known about their roles in vivo. MALS-1 and MALS-2 are
expressed at synapses (Misawa et al.,
2001), but MALS-1/MALS-2 double knockout mice upregulate
MALS-3 and show no obvious synaptic defects
(Misawa et al., 2001). The
analysis of mice lacking all three MALS genes revealed that MALS proteins play
a crucial role in presynaptic vesicle recycling; the triple knockout (TKO)
resulted in reduced synaptic transmission and neonatal lethality
(Olsen et al., 2005a;
Olsen et al., 2006). Here, we
explore these proteins in NPCs and find that MALS-3 is localized to the apical
surface of progenitors during neurogenesis, where it interacts primarily with
PALS1 and CASK (LIN-2). Analyses of MALSTKO embryos
revealed that MALS-3 is required for the normal regulation of NPC
proliferation and differentiation at the onset of neurogenesis, and for the
continued apical localization of PATJ and PALS1 proteins.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Polyclonal
antibodies were generated by immunizing rabbits (BAbCo Laboratories, Berkeley,
CA) with full-length MALS-3 or Mint protein in BL21 pLysS. MALS-3 antibodies
were affinity-purified using MALS-3-GST fusion proteins coupled to Affi-Gel 10
beads (Bio-Rad Laboratories, CA). Immunohistochemistry (IHC) was performed as
described previously (Cappello et al.,
2006). Other antibodies used were as follows (dilutions used for
IHC are given first, those for western blots (WB), second): MALS (LIN-7; Ben
Margolis, University of Michigan, Ann Arbor, MI; 1:2000; 1:5000), PALS1 (Ben
Margolis; 1:500; 1:2000), CRB3 (Ben Margolis; 1:500; 1:2000), ZO1 (Zymed,
Invitrogen, Carlsbad, CA; 1:100; 1:500), ZO2 (Zymed; 1:100; 1:500),
β-catenin (BD Transduction Laboratories, San Jose, CA; 1:200; 1:8000),
DLG (BD Transduction Laboratories; 1:250; 1:500), 
-catenin (BD
Transduction Laboratories; 1:250; 1:250), pan-cadherin (SIGMA, St. Louis, MO;
1:250; 1:250), PATJ (Andre Le Bivic, Université de la
Méditerranée, Marseille, France; 1:100; 1:500), CASK (Upstate
Biochemicals, Waltham, MA; 2 µg/ml; 1 µg/ml), PAR6 (James Nelson,
Stanford, CA; WB 1:50) TGN38 (ABCAM, Cambridge, MA; WB 1:500), Actin
(Chemicon, Billerica, MA; WB 1:1000), 3-subunit of
Na+/K+ ATPase (James Nelson; WB 1:500).
Cell fractionation and western analysis
Dorsal telencephalon was dissected from E14 rat embryos and homogenized in
CSK homogenization buffer (50 mM NaCl, 150 mM sucrose, 10 mM Pipes, pH 6.8, 3
mM MgCl2 with 1x Sigma protease inhibitor) by sonication on
ice for 2 minutes, at 20% duty cycle, power setting 3. Cellular debris was
removed by centrifugation at 1000x g for 10 minutes at
4°C.
High-speed centrifugation was used to pellet membranes and
membrane-associated protein from the postnuclear supernatant (PNS), and to
separate Triton X-100-soluble material from Triton X-100-insoluble material in
the membrane pellet. Dorsal telencephalon from 10 E14 embryos was homogenized
in 2 ml CSK or 2 ml CSK sans Mg+2 supplemented with EDTA. PNS (1
ml) was centrifuged at 100,000x g for 40 minutes to
pellet membranes. The supernatant (S100) was removed and the pellet
resuspended in an equal volume of CSK with 0.5% Triton X-100 at 4°C and
centrifuged again at 100,000x g to pellet Triton
X-100-insoluble material. The supernatant (TX100) was removed and the pellet
from this spin resuspended in 2 ml SDS-PAGE sample buffer; the previous
supernatants were brought to 2 ml with 2xSDS-PAGE sample buffer
(Vogelmann and Nelson,
2007).
Continuous density-gradient centrifugation of homogenates from the
embryonic rat telencephalon, followed by a quantitative analysis of proteins
present in each fraction following western blot analysis was performed as
described by Vogelmann and Nelson
(Vogelmann and Nelson, 2007).
Particles migrated in the gradient to a position that matched their density,
with cytosolic proteins remaining in the densest bottom third of the gradient
(fractions 16-21) and buoyant particles in the lighter upper two-thirds of the
gradient (fractions 1-15; see Fig. S4 in the supplementary material). The
protein concentration was determined for each fraction and then plotted in
arbitrary units normalized to the maximum protein concentration (in fraction
17; see Fig. S4 in the supplementary material). In addition, markers for
distinct sub-cellular compartments were used to distinguish fractions
containing cytosol (actin), membranes derived from the Golgi apparatus
(TGN38), and the plasma membrane (Cadherin, Na+/K+
ATPase; Fig. S4 in the supplementary material). Proteins from samples were
separated by SDS-PAGE (Laemmli,
1970) and processed for western blotting
(http://www.licor.com).
The signal intensity of each band in western blots from each fraction was
normalized to the maximal value obtained for each protein; these values were
then plotted to represent the relative amount of protein present in each
fraction from the gradient (see Fig. S4 in the supplementary material). Three
independent gradients were analyzed for each study. Graphs of protein
distribution throughout density gradients were generated using Microsoft
Excel. Integrated intensity data and protein concentration for each set of
fractions were converted linearly to arbitrary units from zero to maximum of
100.
In utero electroporation
In utero electroporation was performed as described
(Kawauchi et al., 2003).
Embryos were collected 2 or 5 days post-surgery for analysis; five embryos
were analyzed for each time point.
Cell cycle analyses
For labeling index (LI) studies, pregnant mice were injected with BrdU at
E11.5 or E13.5, and embryos were collected 2 hours later. The LI (fraction of
VZ cells in S phase) was calculated by determining the fraction of VZ cells
that were BrdU+. A modified LI (a relative measure of cell-cycle
length) was calculated by determining the fraction of Ki67+ cycling
cells that were BrdU+. For quit fraction (QF) analyses, pregnant
mice were injected with BrdU at E12.5 and embryos were collected 24 hours
later (n=3 for each analysis). The QF was determined by dividing the
number of BrdU+/Ki67- cells by the total number of
BrdU+ cells.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When we compared the localization of MALS with that of F-actin
(concentrated near adherens junctions at the boundary of the basolateral and
apical regions) (Chenn et al.,
1998), we found that MALS-3 occupies a domain largely supra-apical
to that of F-actin (Fig. 1E).
En face views of the ventricular surface revealed that F-actin rings the
apical endfeet of NPCs (Fig.
1C). MALS-3 overlaps partially with these F-actin rings, but is
also seen in regions that are both more apical and continuous
(Fig. 1C), suggesting that
MALS-3 is distributed in a cup-like pattern at the apical ends of NPCs.
Comparison of the MALS-3 localization with that of other apically localized
proteins [ZO1 (TJP1 - Mouse Genome Informatics), ZO2 (TJP2 - Mouse Genome
Informatics), pan-cadherin, β-catenin, PALS1 and CRB3]
(Calegari et al., 2005;
Cappello et al., 2006;
Imai et al., 2006) revealed
partially or entirely overlapping apical expression domains
(Fig. 1A,B;
Fig. 2). Of particular interest
was the overlapping expression with PALS1
(Fig. 2G-I), a protein that
binds directly to MALS-3 via an L27 interaction domain. Thus, MALS-3 and
several proteins with which it can interact biochemically are expressed in,
and localized to, the apical domains of NPCs during neocortical
development.
MALS-3 is associated with cell membranes
Although MALS-3 does not contain a transmembrane domain, in other systems,
it associates with membranes via interactions with other proteins
(Roh and Margolis, 2003;
Straight et al., 2006). To
ascertain whether the MALS-3 in VZ cells is associated with the plasma
membrane, we used high-speed centrifugation and density-gradient separation to
separate membranes and membrane-associated proteins from the cytosol.
High-speed centrifugation revealed that MALS-3 exists in two pools in the
telencephalon: approximately two-thirds of the total MALS-3 was present in the
cytosolic fraction (S100), whereas one-third was distributed between
Triton-soluble (TX100) and Triton-insoluble (SDS) membranes
(Fig. 3A). This suggests that a
substantial fraction of the MALS-3 in NPCs is associated with membranes. To
compare the distribution of MALS-3 with that of other proteins with which it
might potentially interact, we probed the same fractions with antibodies
against CASK, Mint (LIN-10; APBA1 - Mouse Genome Informatics), PALS1, and
several apically localized proteins. These studies revealed that CASK is
associated primarily with Triton-insoluble membranes, whereas Mint was found
mostly in the cytosol (Fig.
3A). PALS1, DLG, β-catenin, cadherin and
Na+/K+ ATPase were found primarily in membrane
fractions, whereas p38
/SAPK3 was almost exclusively in the cytosol
(Fig. 3A).
|
|
).
Markers for distinct sub-cellular compartments were used to distinguish
fractions containing cytosol, Golgi membranes and plasma membranes (see Fig.
S4 in the supplementary material). The signal intensities of bands in western
blots were normalized to the maximal value obtained for each protein then
plotted to represent the relative amount of protein in each fraction from the
gradient (Fig. S4 in the supplementary material). These experiments revealed
that, as predicted from the high-speed centrifugation, MALS was present in
both cytosolic fractions (peak, fraction 17) and fractions containing plasma
membrane (fractions 5 and 14; Fig.
3B,C). MALS and CASK had nearly identical distributions, with an
overall maximum at fraction 17, and local maxima at fractions 5 and 14
(Fig. 3B,C). These
distributions suggest that CASK and MALS are each found in the cytosolic and
plasma membrane pools, consistent with the possibility that these proteins
interact biochemically. Mint was distributed in a single peak centered at
fraction 17, suggesting that Mint is primarily cytosolic. Thus, in contrast to
the close association between LIN-7, LIN-2, and LIN-10 in C. elegans,
only MALS and CASK localize to plasma membranes in NPCs (although all three
proteins might still interact in the cytosol).
|
;
Roh et al., 2002;
Straight et al., 2004),
exhibited a major peak at fraction 14 and a minor peak at fraction 5, and a
broad distribution across cytosolic fractions. It is likely that MALS and
PALS1 interact biochemically, as PALS1 can be co-immunoprecipitated with
antibodies to MALS-3 (see Fig. S3C in the supplementary material).
Interestingly, -catenin, β-catenin and cadherin were similarly
distributed in density gradients (Fig.
3D,E), suggesting that each is associated with plasma
membranes.
The junctional proteins ZO1 and ZO2 had a major plasma membrane peak at
fraction 14, but only ZO2 had a second peak in the cytosol (fraction 17;
Fig. 3D,E). DLG, which is
commonly considered to be basolaterally localized but is found at tight
junctions in MDCK cells (Albertson and Doe,
2003; Peng et al.,
2000), exhibited peaks in plasma membrane fractions 5 and 14, and
cytosolic fraction 18. DLG co-immunoprecipitated with MALS-3 from these
gradients (see Fig. S3C in the supplementary material), which is consistent
with recent reports that DLG1 interacts with all three MALS proteins via
associations with the PALS1 family member MPP7
(Bohl et al., 2007). The
polarity proteins PAR6A and PAR6B were found primarily in cytosolic fractions,
although PAR6B also displayed a small plasma membrane peak (fraction 5).
Although previous studies suggest that PAR6B localizes to the cytosol in MDCK
cells, and that PAR6A associates with the tight junction protein ZO1
(Gao and Macara, 2004), these
relationships were not apparent from our study. Finally, PATJ was found almost
exclusively in cytosolic fraction 18 (Fig.
3D). This is surprising because PATJ associates with tight
junctions in MDCK cells (Shin et al.,
2005), where it interacts with PALS1
(Roh et al., 2002;
Straight et al., 2006) and
Crumbs (Makarova et al.,
2003).
In sum, of the potential MALS interactors studied here, the distribution of
CASK in density gradients was nearly identical to that of MALS, and the
distribution of PALS1 was similar in lighter plasma membrane fractions. These
observations are consistent with evidence that CASK and PALS1 interact with
MALS in MDCK cells (Straight et al.,
2006). In addition, analyses of homogenates from postnatal brains,
in which MALS-1 and MALS-2 are found at synapses, have revealed that MALS
associates with CASK, Mint and liprins in postmitotic neurons
(Olsen et al., 2005a).
|
). Mice
that lack MALS-1 and MALS-2 appear normal, owing to the
upregulation of MALS-3 in neurons, and were therefore used as
controls in our study. Mice in which only MALS-3 is mutated also lack
obvious defects, presumably because of compensation by the two other MALS
isoforms (Misawa et al.,
2001). MALS triple knockout (MALSTKO) mice
were bred and genotyped as described earlier
(Olsen et al., 2005a).
MALSTKO pups die soon after birth
(Olsen et al., 2005a), and
examination of their brains at P0 revealed no obvious morphological
abnormalities, such as smaller brain size or defects in cortical lamination
(as evidenced by immunostaining with layer-specific antibodies; see Fig. S5 in
the supplementary material).
Although it comprises less than 2% of the entire membrane surface
(Huttner and Kosodo, 2005;
Kosodo et al., 2004), the
apical domain of NPCs represents a region that is actively targeted by several
proteins, including signaling molecules such as β-catenin and aPKC
(Chenn and Walsh, 2002;
Chenn et al., 1998;
Imai et al., 2006), and EGFR
((Sun et al., 2005). Because
the MALS homolog LIN-7 plays an essential role in the basolateral targeting of
EGF receptors in C. elegans vulval precursors
(Kaech et al., 1998), we
examined EGFR localization in MALSTKO brains but observed
no differences between controls and mutants (data not shown). Because MALS-3
regulates apicobasal polarity in MDCK cells, we hypothesized that MALS-3 might
play a similar role in NPCs (Straight et
al., 2006). At E13.5, the VZ appeared intact in
MALSTKO brains (Fig.
4A,B). However, MALSTKO mutants showed a
complete loss of PATJ staining (Fig.
4H), which is normally concentrated apically
(Fig. 4G). The localization of
other apical proteins, including PALS1, was unaltered
(Fig. 4; data not shown)
(Kamberov et al., 2000). The
loss of PATJ staining by E13.5 was surprising as MALS-3 associates with PATJ
through its interaction with PALS1, a part of the CRB3/PALS1/PATJ complex.
Moreover, our density centrifugation experiments showed distinct patterns for
MALS-3 and PATJ, suggesting that they occupy distinct intracellular domains
(Fig. 3). Nevertheless, PATJ
was completely absent from the apical region of MALSTKO
NPCs.
Similar analyses of E18.5 MALSTKO mutants revealed
that, in addition to the loss of PATJ apically
(Fig. 4O,P), NPCs showed a loss
of PALS1 immunoreactivity from their apical domains
(Fig. 4K,L). This suggests that
MALS-3 is not required for the initial apical targeting of PALS1 in NPCs but
is important for maintaining its apical localization. In many mutants, the
distribution of CRB appeared normal (Fig.
4M,N), but we occasionally observed a reduction in the intensity
of apical CRB staining (data not shown). Because the antibody used to detect
CRB3 also detects CRB2 (data not shown), it is possible that changes in the
localization of one isoform might be masked if that of the other were
unaltered. No changes were observed in the distributions of PAR3, PAR6 or
aPKC
(Fig. 4I,J; data not
shown), suggesting that MALS does not affect the PAR6 signaling pathway in
NPCs. Despite the loss of at least three proteins (MALS-3, PATJ, PALS1, and
occasionally CRB) from the apical surface, the VZ still appeared intact at
E18.5, suggesting that none of these proteins is crucial for maintaining the
structural integrity of the VZ. Western blots revealed no obvious changes in
the total protein levels of PALS1 or PATJ
(Fig. 4Q), suggesting that MALS
is required for the apical localization, but not the overall stability, of
PALS1 and PATJ in NPCs. This result contrasts with studies in which silencing
of MALS-3/LIN-7 in MDCK cells resulted in a loss of PATJ from tight
junctions because of altered protein stability
(Straight et al., 2006).
|
). Although
NPCs in vivo do not form tight junctions
(Aaku-Saraste et al., 1996),
they do form adherens junctions where ZO1 and ZO2 colocalize. The intact
appearance of the VZ in MALSTKO embryos suggested that
adherens junctions were maintained. This was confirmed by the continued
expression of ZO1 and ZO2 in mutant embryos
(Fig. 5A,B; data not shown). We
also assessed the localization of cadherins, an integral component of adherens
junctions, and the signaling protein β-catenin, as changes in the
localization of the proteins would likely have profound consequences on NPCs
(Chenn and Walsh, 2003).
Again, no differences in protein localization were observed in the
MALSTKO cortex (Fig.
5C-F). Collectively, these results suggest that MALS is not
required for the establishment or maintenance of adherens junctions in the
developing brain.
Analysis of NPC proliferation and differentiation in MALSTKO mice
To ascertain whether the loss of MALS-3 and, subsequently, PATJ and PALS1
altered neurogenesis, we immunostained brains with antibodies that distinguish
NPCs (nestin), neurons (TuJ1, NeuN and TBR1) and glial cells (GFAP; see
Fig. 5; data not shown). At
E13.5, staining for nestin and GFAP appeared similar in mutant and control
brains (Fig. 5G,H). However, we
observed a broadened TuJ1 expression domain in MALSTKO
mutants compared with controls (Fig.
5I,J), suggesting that the production of neurons might be expanded
or initiated prematurely in MALS mutants.
To ascertain whether the loss of MALS genes altered NPC proliferation, we
first performed BrdU injections to assess the labeling index (LI, representing
cells in S phase). At E11.5, the LI in MALSTKO mice
(0.36±0.05) was significantly lower than in controls (0.44±0.03,
P<0.03; Fig. 6A). A
reduction in LI suggests that either fewer VZ cells were cycling in the
mutant, or the same number of cells were cycling more slowly. The limited
availability of MALSTKO mice precluded us from performing
cumulative labeling studies to ascertain the total length of the cell cycle.
Instead, we assessed BrdU incorporation in actively cycling cells only by
immunostaining embryos for BrdU and Ki67 (expressed by all cycling cells). If
the altered LI were due to decreased numbers of cycling cells, we should see
no difference in fraction of Ki67+ cells that were also
BrdU+ in mutants. However, if the decrease in LI arose from a
lengthened cell cycle, fewer BrdU+/Ki67+ cells would be
expected (Chenn and Walsh,
2003; Siegenthaler and Miller,
2005). Indeed, we observed a significant decrease in the fraction
of Ki67+ cells that were BrdU+ (control,
0.80±0.04; MALSTKO, 0.63±0.06;
P<0.01), suggesting that MALS-deficient progenitors cycle more
slowly than controls.
During early corticogenesis, wild-type NPCs divide rapidly and most
divisions are symmetric, producing more progenitors; later, the cell cycle
slows and the fraction of daughter cells that differentiate into neurons
increases (Calegari et al.,
2005). The lower LI in E11.5 MALSTKO brains
and the broadened domain of TuJ1 immunoreactivity suggested that early NPCs
might prematurely enter the neurogenic phase of development. To assess the
fraction of cells that differentiated following early divisions in
MALSTKO mutants, we calculated the Quit Fraction (QF) of
cells that exited the cell cycle. The QF in MALSTKO
embryos (0.53±0.03) was significantly higher than in littermate
controls (0.45±0.03, P<0.03;
Fig. 6B), demonstrating that
MALSTKO progenitors differentiate prematurely during early
neurogenesis. As the orientation of the mitotic spindle can affect the outcome
of NPC divisions (Chenn and McConnell,
1995), we investigated whether the increase in neurogenic
divisions was correlated with changes in spindle orientation. However, no
significant differences in the orientations of E11.5 VZ cells in anaphase of
the cell cycle were observed between control and MALSTKO
embryos (data not shown), suggesting that MALS-3 does not regulate
neurogenesis by controlling mitotic spindle orientation.
Interestingly, MALSTKO progenitors appeared to synchronize with their control counterparts by E13.5. BrdU injections at this age revealed no differences in the LI of MALSTKO mice compared with controls (Fig. 6C). Thus, although the loss of MALS-3 alters NPC proliferation during early stages of neurogenesis, by mid-neurogenesis NPCs resumed normal cell cycle dynamics. We presume that this recovery explains the relatively normal size and appearance of the cortex in newborn MALSTKO mice.
|
By contrast, embryos electroporated with CA-myr_MALS-3 showed
striking morphological abnormalities (Fig.
7P-R; see also Fig. S6C in the supplementary material). The
integrity of the VZ was compromised, and the lateral ventricles were
infiltrated with TuJ1+ cells
(Fig. 7R). Within 2 days of
electroporation, at E15.5, breaks were observed in the apical staining
patterns of ZO1 (Fig. 7E,F),
PATJ (Fig. 7H,I), PALS1
(Fig. 7K,L) and CRB
(Fig. 7N,O) near
EGFP+ cells, suggesting that adherens junctions were disrupted at
these sites. Consistent with this interpretation, we also observed breaks in
β-catenin staining (see Fig. S6C in the supplementary material). The
ventricle contained misplaced cells that were positive for nestin at E15.5
(although in smaller numbers than at E18.5), suggesting they were delaminated
NPCs (data not shown). These phenotypes are reminiscent of the conditional
loss of β-catenin in the developing forebrain, in which adherens junction
integrity is compromised, leading to a delamination of NPCs and the appearance
of ectopic cells in the lateral ventricles
(Junghans et al., 2005).
Interestingly, we did not observe any changes in β-catenin localization
in MALSTKO brains (Fig.
4, Fig. 5C,D),
suggesting that MALS-3 is not normally required for the apical localization of
β-catenin.
Finally, we calculated the LI and QF for electroporated VZ cells at E14.5 (one day after electroporation) and E15.5, respectively. We observed no significant change in the QF upon expression of CA-myr_MALS-3 (data not shown); however, the LI was significantly reduced in cells expressing CA-myr_MALS-3 (0.31±0.06) compared with controls (0.55±0.04; P<0.006). Calculation of the fraction of Ki67+ cells that incorporated BrdU revealed that cells expressing CA-myr_MALS-3 also showed a significant reduction of the LI (0.39±0.06) compared with GFP+ controls (0.67±0.01; P<0.001), indicating that the overexpression of CA-myr-MALS-3 caused cells to cycle more slowly (as was observed in MALS-3TKO mice). In conjunction with the changes in apical protein localization described above, these alterations in cell-cycle length suggest that CA-myr-MALS-3 dominantly interferes with apicobasal polarity in NPCs.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and β-catenin, suggesting that adherens junctions remained
intact throughout corticogenesis. The loss of MALS genes also slowed the cell
cycle and resulted in premature differentiation during early (but not later)
stages of neurogenesis. These differences did not visibly alter the overall
construction of the cortex, which appeared normal at P0. Finally, we found
that overexpression of a myristoylated form of MALS severely disrupted the
apicobasal polarity of NPCs. Collectively, these data suggest that MALS is
required for the maintenance of apicobasal polarity and the normal control of
proliferation in the developing forebrain.
|
). By
contrast, in mammalian NPCs, MALS is localized apically, as is EGFR
(Sun et al., 2005). However,
MALS does not appear to regulate the apical targeting of EGFR in NPCs, as the
distribution of EGFR was not obviously altered in mutants. This is perhaps not
surprising in light of previous observations showing that EGFR is expressed
during, and plays important functions only at later stages of, cortical
development (Sun et al.,
2005), compared with the earlier role for MALS demonstrated
here.
It appears that the subcellular localization of MALS proteins is tissue
dependent in vertebrates. For example, renal epithelial cells express all
three MALS proteins, but their subcellular distributions vary among cell
types, from primarily cytosolic in intercalated cells to predominantly basal
or apical in the collecting ducts (Olsen
et al., 2005b). By contrast, MDCK cells localize MALS-3/LIN7C,
PALS1 and CRB3 to tight junctions (Roh and
Margolis, 2003). It is not yet clear why MALS-3 and its partners
show distinct localization patterns in different cell types; one possibility
is that MALS-3 becomes localized to regions of cell-cell contact, including
tight junctions in epithelial cells and adherens junctions in NPCs. However,
in NPCs, MALS-3 clearly occupies a domain supra-apical to that of F-actin and
adherens junctions. PALS1 is unlikely to mediate this localization because
MALS-3 expression is not altered in MDCK cells silenced for PALS1
(Straight et al., 2004). One
possibility is that β-catenin mediates the junctional localization of
MALS-3, as β-catenin appears to be involved in actively localizing MALS-3
from the cytosol to calcium-mediated cadherin junctions in MDCK cells, and to
early synapses in hippocampal neurons
(Perego et al., 2000).
Many MALS-3 biochemical partners appear to be highly conserved in NPCs. In
MDCK cells, MALS-3 stabilizes the PALS1 complex at tight junctions; cells
silenced for MALS-3 lose the apical localization of PALS1 and ZO1, which
delays the formation of tight junctions
(Straight et al., 2006). This
phenotype was fully rescued by introducing exogenous PALS1, providing strong
evidence that MALS-3 function is mediated by PALS1. This interaction is not
reciprocal, as MALS-3 expression is maintained in the absence of PALS1
(Straight et al., 2004).
Consistent with these previous studies, MALSTKO VZ cells
also showed a loss of PALS1, but only at later stages of neurogenesis. This
suggests that MALS-3 is required not for the initial localization but for the
maintenance of PALS1 apically in NPCs. We observed an earlier loss of apical
PATJ from the apical surfaces of VZ cells, which was surprising in the absence
of direct biochemical interactions between these two proteins. The loss of
MALS did not appear to affect the expression levels of PALS1 or PATJ,
suggesting that MALS-3 is primarily involved in maintaining the apical
localization of the PALS1 complex. By contrast, MDCK cells silenced for LIN7C
(MALS-3) displayed a loss of both PALS1 and PATJ owing to rapid protein
turnover. The authors speculated that MALS-3/LIN7C is required to stabilize
PALS1 apically. Because PATJ interacts with PALS1, the loss of PALS1 might
result in the loss of PATJ from these cells
(Straight et al., 2006).
However, our data from MALSTKO mutant brains suggest that
the role for MALS in apical protein localization can be separated from its
role in protein stabilization.
In contrast to the role of MALS in maintaining polarity, PALS1 appears to
play a crucial role in establishing apicobasal polarity. In MDCK cells, PALS1
is required for the formation of both tight and adherens junctions
(Wang et al., 2007). These
observations, together with data from MDCK cells silenced for LIN7C/MALS-3
(Straight et al., 2006),
provide strong evidence that PALS1 functions upstream of MALS-3, and that
PALS1 can compensate to a large extent for the loss of MALS-3 during cell
polarization. As we did not observe a loss of PALS1 from the apical surfaces
of NPCs until later stages of corticogenesis, we suspect that PALS1
compensated functionally for the loss of MALS-3. On the basis of these
observations, we would predict that the loss of PALS1 in NPCs should
generate a more profound disruption of corticogenesis than does the loss of
all three MALS genes.
NPC proliferation and differentiation depend on apicobasal polarity but not the integrity of adherens junctions during cortical development
The relationship between apicobasal polarity and cell-cell junctions in
controlling NPC proliferation has been a subject of recent interest,
particularly in light of the role of junctions in mediating intercellular
signaling. In Drosophila, intact junctions are essential for normal
NPC proliferation (Lu et al.,
2001); however, this requirement appears to be less strict in
vertebrate neurogenesis. For example, loss of the Rho family GTPase
CDC42 results in a loss of adherens junctions and the mislocalization
of mitotic progenitors from the apical to the basal region of the cortical VZ;
however, misplaced cells continued to cycle despite their aberrant positions
(Cappello et al., 2006).
Similarly, conditional mutations of aPKC
(a key component of
the apically localized PAR complex) produced a complete loss of adherens
junctions yet failed to disrupt neurogenesis
(Imai et al., 2006). These
observations suggest that maintenance of adherens junctions is not absolutely
required for normal NPC proliferation.
In the MALSTKO mutants, we observed a complementary situation. Adherens junctions were intact in mutants, demonstrating that MALS-3 is not required for assembling or maintaining these junctions. However, we observed significant abnormalities in both the proliferation and differentiation of NPCs. First, MALSTKO mutants exhibited a lower LI and a lengthening of the cell cycle at the onset of corticogenesis, although proliferation recovered a few days later. Second, MALSTKO mutants displayed premature neuronal differentiation at early times during neurogenesis. Interestingly, these alterations were also temporary: the cortex as a whole appeared relatively normal at P0, and MALSTKO mutants did not display gross morphological changes, such as smaller brain size or loss of select neuronal populations, at birth.
Although these transient changes in proliferation present a puzzle, we note
that, in normal animals, the transition from diffuse basolateral to strictly
apical localization of MALS-3 occurs at 
E11.5, the same time at which we
observed abnormal proliferation in mutant NPCs. We hypothesize that early NPCs
are particularly vulnerable to alterations in the composition of their apical
complexes, and that, at later stages, other apical polarity proteins restore
normal patterns of proliferation. We further hypothesize that the higher QF
observed at E13 is a result of the earlier shift in LI. Previous studies
suggest that lengthening the cell cycle in NPCs can cause a shift from
proliferative to neuron-generating divisions
(Calegari et al., 2005). Thus,
the more slowly cycling progenitors in E11 MALSTKO mice
might differentiate into neurons earlier than control counterparts once they
complete their cell cycle, thus generating a higher QF by E13.
In some mutant lines, higher QFs can be accompanied by a loss of NPCs,
depleting the progenitor pool and reducing brain sizes owing to a loss of
later-born neurons and/or glial cells. For example, loss of the chromatin
remodeling protein BRG1 leads to precocious differentiation, a premature
depletion of NPCs prior to gliogenesis, and a severe loss of glial cells
(Lu et al., 2001).
Gliogenesis, however, appeared normal in MALSTKO mice,
further emphasizing that the consequences of altered QFs at E13.5 were subtle,
most likely because of the rapid recovery in NPC proliferation.
It is not clear how the loss of MALS causes a shift towards neuronal
differentiation at early stages of cortical development. One possibility is
that MALS-3 is important for the asymmetric distribution or localization of a
cell-fate determinant that regulates NPC proliferation and/or differentiation.
For example, in epithelial cells, the tight junction protein ZO1 influences
the G1/S-phase transition by sequestering a complex formed by CDK4 and the
transcription factor ZONAB (Csda - Mouse Genome Informatics) away from the
nucleus (Balda et al., 2003;
Sourisseau et al., 2006). It
is plausible that the disruption of MALS proteins in NPCs disrupts the
localization of similar cell-cycle regulators, and thus leads to premature
cell-cycle exit, although the molecular mechanisms underlying this phenomenon
are as yet unknown.
Deliberate mistargeting of MALS-3 results in a breakdown of VZ integrity
Although a complete loss of MALS-3 did not affect the structural integrity
of the VZ or intercellular junctions, the overexpression of MALS-3 tagged with
an N-terminal myristoylation sequence profoundly altered the localization of
PALS1 and PATJ, and triggered a loss of adherens junction proteins. The
mislocalization of MALS-3 also resulted in a slowing of the cell cycle,
suggesting that the myristoylated MALS-3 dominantly interfered with
proliferation. Many electroporated cells delaminated from the VZ and spilled
into the lateral ventricles, suggesting that the integrity of the adherens
junctions was severely compromised by the mislocalization of MALS-3. We
suspect that this mislocalization directly affects the localization of other
proteins, particularly the MALS-3 direct-binding partner PALS1, but also the
aPKC and PAR proteins, which require PALS1 for their normal localization
(Margolis and Borg, 2005).
Loss of these proteins from the apical surface might trigger the dissolution
of adherens junctions (Imai et al.,
2006), which could lead to the delamination of NPCs. However,
MALSTKO mutant brains showed no alteration in aPKC
staining, suggesting that MALS-3 is not required for the normal localization
of the PAR3/PAR6/aPKC complex. Collectively, these results suggest that,
although a loss of MALS-3 is tolerated by NPCs, its mislocalization is not,
with mislocalization causing far more detrimental effects on apical
polarity.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/10/1781/DC1
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ACKNOWLEDGMENTS |
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