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First published online May 16, 2007
doi: 10.1242/10.1242/dev.000885
1 Developmental Biology Program, Sloan-Kettering Institute, 1275 York Avenue,
New York, NY 10021, USA.
2 The Hospital for Sick Children, Arthur and Sonia Labatt Brain Tumor Research
Center and Department of Medical Biophysics, University of Toronto, Toronto,
Ontario M5G 1X8, Canada.
3 Department of Cell and Systems Biology, University of Toronto, Toronto,
Ontario M5S 3G5, Canada.
* Author for correspondence (e-mail: k-anderson{at}ski.mskcc.org)
Accepted 20 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: FERM, Epithelial morphogenesis, EMT, Cytoskeleton, Gastrulation, Actin, Crumbs, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Mangeat et al., 1999). The
mouse genome encodes at least 50 FERM proteins, but the functions of only a
few, including the actin-binding proteins ezrin (also known as Vil2 - Mouse
Genome Informatics), radixin, moesin, Nf2 (also known as merlin) and the
erythrocyte protein band 4.1, have been characterized
(Bretscher et al., 2002;
Mangeat et al., 1999). Of
these proteins, only Nf2, the product of the Nf2 tumor suppressor
gene, has been shown to be required for early embryonic development; embryos
that lack Nf2 die shortly after the onset of gastrulation due to defects in
the epithelial organization of the extra-embryonic ectoderm
(McClatchey et al., 1997).
Here we identify Lulu (Epb4.1l5) as another FERM protein that is essential for
mouse development, and show that it has central roles in early embryonic
morphogenesis.
During early postimplantation development, the single-layered columnar
epithelium of the mouse epiblast is transformed over the course of 2 days into
a three-layered embryo with a well-defined body plan
(Tam and Gad, 2004). Beginning
at embryonic day (E)6.5, cells at the primitive streak undergo an
epithelial-to-mesenchymal transition (EMT) to give rise to the mesodermal and
endodermal germ layers. Once generated, each germ layer undergoes a
characteristic set of morphological changes: the mesenchymal cells of the
mesodermal layer migrate around the embryonic circumference, endodermal cells
form an epithelium that folds to generate the gut tube, and cells of the
neural epithelium bend and contract their apical surfaces to generate a closed
neural tube.
In an N-ethyl N-nitrosourea (ENU)-mutagenesis screen for mutations that
affect the morphology of the mid-gestation mouse embryo, we isolated a
mutation that we named limulus (lulu) based on the flat,
plate-like morphology of the mutant embryos
(García-García et al.,
2005). lulu embryos have defects in the morphogenesis of
all three germ layers: very little paraxial mesoderm is specified, the
endoderm fails to form the gut tube, and the neural plate has an irregular
shape and does not generate a neural tube. Based on positional cloning, we
find that lulu is a null allele of the gene encoding the mouse
FERM-domain protein erythrocyte protein band 4.1-like 5 (Epb4.1l5); the allele
is designated Epb4.1l5lulu and we refer to the allele here
as lulu (Epb4.1l5 has also been called YMO1)
(Laprise et al., 2006).
Homologs of Epb4.1l5 play roles in specific aspects of
Drosophila and zebrafish development
(Hoover and Bryant, 2002;
Hsu et al., 2006;
Jensen and Westerfield, 2004;
Laprise et al., 2006). The
Drosophila ortholog of Epb4.1l5, named yurt, is
required for epithelial polarity and dorsal closure in the embryo, and for
photoreceptor morphogenesis (Hoover and
Bryant, 2002; Laprise et al.,
2006). Yurt appears to act as a negative regulator of Crumbs, a
key determinant of the apical domain of epithelial cells
(Tepass et al., 1990;
Wodarz et al., 1995). The
zebrafish epb41l5 (also known as mosaic eyes; moe)
gene is required for the layering of the retina and inflation of the brain
ventricles, and, like Yurt, regulates the size of the apical membrane domain,
possibly also via the negative regulation of Crumbs
(Hsu et al., 2006;
Jensen et al., 2001;
Jensen and Westerfield,
2004).
Analysis of the tissues affected in lulu mutant embryos reveals that Lulu has specific roles in the EMT at gastrulation and in the organization of the pseudo-stratified epithelium of the neural plate. In both contexts, the defects in tissue organization are associated with dramatic changes in the organization of the actin cytoskeleton. In contrast to the results in zebrafish and Drosophila, Crumbs localization appears not to be affected in lulu mutants. The results indicate that Lulu is crucial for the dynamic rearrangements of the actin cytoskeleton that occur during the morphogenesis of epithelial tissues.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
García-García et al.,
2005; Kasarskis et al.,
1998). The name limulus refers to the embryonic shape at
E8.5, which resembles the horseshoe crab Limulus polyphemos. The
GT1 and GT2 alleles of Epb4.1l5 correspond to
BayGenomics lines AE0088 and XC282, respectively. Embryonic stem (ES) cells
were injected into C57BL/6J blastocysts to produce chimeras, which were
screened for transmission of the gene trap. Carriers were genotyped by PCR
amplification of the neomycin resistance gene using primer sets from the
Jackson Laboratories.
Mapping and identification of lulu
lulu was mapped by backcrossing to C3HeB/FeJ, using linkage to
flanking simple sequence length polymorphism (SSLP) markers from the MIT
database or new markers that we generated
(http://mouse.ski.mskcc.org/).
lulu was mapped to a 650 kb interval by analyzing approximately 1000
informative recombination opportunities. The ENSEMBL and Celera databases were
used for mapping and candidate gene selection; Epb4.1l5, RalB and
Ptpn4 were sequenced because of their predicted associations with the
actin cytoskeleton. Complementary (c)DNAs of candidate genes were made from
lulu mutant and C57BL/6J embryos using Superscript OneStep
(Invitrogen). Products were subcloned into the Invitrogen pCR 2.1-TOPO vector
and sequenced using T7 and SP6 primers. No sequence changes were found in the
coding regions of RalB and Ptpn4; a single C to T transition
was identified in the Epb4.1l5 coding sequence, in multiple
lulu mutant embryos. The lulu mutation abolishes a
BsaJI restriction site, which was used to confirm the mutation in
carrier mice.
Phenotypic analysis
Whole-mount in situ hybridization, X-gal staining and immunohistochemistry
were performed using standard protocols
(Belo et al., 1997;
Hogan et al., 1994;
Yamada et al., 1993). All
wild-type controls were littermates of the mutant embryos. Embryos were
dissected in phosphate-buffered saline (PBS)/0.4% bovine serum albumin (BSA)
and fixed in PBS/4% paraformaldehyde at 4°C overnight for in situ
hybridization, or for 1 hour at room temperature for immunohistochemistry,
with the exception that embryos used for N-cadherin staining were fixed for 10
minutes on ice in 100% methanol. Fixed embryos were embedded in OCT and
cryosectioned at 8 µm thickness. Mutant and wild-type littermates were
dissected and fixed together, and then embedded and sectioned in one block.
Single slides containing wild-type and mutant sections were used for staining.
Whole-mount embryos were imaged using a Zeiss Axiocam HRC digital camera on a
Leica DM1RE2 inverted confocal microscope. Sections were imaged on a Leica
MZFLIII microscope. In all cases, identical microscope and camera settings
were used when imaging wild-type and mutant samples. Confocal datasets were
analyzed using the Volocity software package (Improvision); 3D reconstructions
were created using the Amira package (Mercury Computer Systems). The mitotic
index was defined as the ratio of phospho-histone-H3-positive cells to total
cells in anterior neural plate sections. Phospho-histone-H3-positive nuclei
located 1 or more nuclear diameters away from the apical surface were scored
as ectopic. Nuclear length and width were measured from 3D
reconstructions.
Cell culture and cDNA transfections
HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum. For transfection, cells were grown
in six-well dishes and seeded onto glass coverslips at 60-70% confluency.
pEGFP empty vector and lulu-pEGFP constructs were transfected into
the cells using 1.5 µl of Lipofectamine 2000 and 0.5 µg of DNA per well
in Opti-MeM. At 5 hours after transfection, the nucleotide-Lipofectamine
mixture was removed and replaced with normal growth media. Immunocytochemistry
was performed at approximately 24 hours after transfection.
Immunohistochemistry
The antigen used to produce the 
-Lulu antibody, amino acids 669-731
of isoform B (Laprise et al.,
2006), lies C-terminal to the stop codons in the lulu,
Epb4.1l5GT1 and Epb4.1l5GT2 alleles. The
-crumbs homolog 3 (-Crb3) antibody was raised against human CRB3
and recognizes all zebrafish Crumbs proteins
(Hsu et al., 2006;
Makarova et al., 2003); it is
therefore likely to recognize all mouse Crumbs proteins. Other antibodies:
rabbit -Lulu antibody, 1:300
(Laprise et al., 2006); rabbit
anti-Crb3, 1:250 (Makarova et al.,
2003); rabbit anti-Sox2, 1:1000 (Chemicon); rabbit
anti-phosphohistone H3, 1:200 (Upstate); rat anti-E-cadherin, 1:500 (Sigma);
rabbit anti-Myosin IIB, 1:500 (Covance); rabbit anti-phospho-ERM, 1:50 (Cell
Signaling); rabbit anti-laminin, 1:50 (Sigma); mouse anti-N-cadherin, 1:500
(BD Biosciences); mouse anti-ZO-2 (anti-Tjp2), 1:250 (BD Biosciences); rabbit
anti-Pals1 (anti-Mpp5), 1:100 (Upstate); FITC- and TRITC-phalloidin, 10 U/ml
(Molecular Probes).
Cells were fixed in 4% paraformaldehyde, 30 mM sucrose in PBS for 30 minutes at room temperature, permeabilized with 0.1% Triton X-100 and blocked with 2% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were incubated with a rhodamine-conjugated phalloidin (Invitrogen, Carlsbad, CA) for 30 minutes at 37°C. Confocal images were acquired using an LSM510 microscope (Carl Zeiss MicroImaging, Thornwood, NY). Quantification was based on counting 93 cells for both the pEGFP control and the pEGFP-lulu transfections and scoring cells that showed increased phalloidin staining at the cell cortex.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). lulu mutants had shortened trunks and lacked
visible somites. The neural plate of E8.5 lulu embryos was raised in
a series of transverse folds and failed to close to form the neural tube
(Fig. 1A,B). Extra-embryonic
tissues were also affected: the yolk sac appeared ruffled and the allantois
did not fuse with the chorion (data not shown). Mesoderm and definitive endoderm were specified, but abnormal, in lulu embryos. Analysis of molecular markers showed disruptions in axial, paraxial and cardiac mesoderm in lulu embryos. Brachyury (T) expression marks axial mesoderm and this gene was expressed discontinuously in the midline of E8.5 lulu mutants (Fig. 1E,F). Meox1, which marks somitic and presomitic mesoderm, was expressed in a greatly reduced, unsegmented domain in lulu mutants (Fig. 1G,H). Nkx2.5 (also known as Nkx2-5), a marker for cardiac mesoderm, was expressed in the position of the lateral cardiac anlage; however, the lateral anlage failed to move to the midline to form a single heart tube (data not shown). In contrast to these tissues, the lateral plate mesoderm, assayed by Twist1 expression, appeared normal in lulu embryos (data not shown). Definitive endoderm, marked by the expression of Cerl (also known as Dand5) at E7.5 and later by the gut markers Hex1 (Hhex) and Shh, was also specified (data not shown). Despite normal specification, the endoderm failed to move ventrally to form the gut tube.
The broad anterior tissue of lulu mutants was identified as neural ectoderm, based on its expression of the pan-neural marker Sox2 (Fig. 1A,B). Despite its abnormal morphology, anterior-posterior patterning of the neural plate was normal. Krox20 (also known as Egr2 - Mouse Genome Informatics), which marks rhombomeres 3 and 5 of the hindbrain, was expressed in two parallel stripes in the neural plate (Fig. 1C,D). Similarly, the anterior markers Hesx1, Otx2, Wnt1 and En1 were expressed in the correct anterior-posterior order (data not shown). Thus, the phenotypes seen in lulu mutant embryos appeared to reflect defects in morphogenesis rather than in cell type specification.
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). We
identified a C to T transition mutation in lulu embryos that creates
a premature stop codon near the C-terminal end of the FERM domain in
Epb4.1l5 (Fig. 2A).
Epb4.1l5 produces two alternatively spliced isoforms of 505 and 731
amino acids, which share a 444 amino acid N-terminal domain (including the
entire FERM domain) but have divergent C-termini
(Fig. 2A). The lulu
mutation lies within the FERM domain and thus disrupts both isoforms. Mouse
lulu is orthologous to the zebrafish moe gene, which encodes
two isoforms of the same structure as lulu
(Jensen and Westerfield, 2004)
(www.ensembl.org).
Yurt is the most closely related FERM-domain protein in Drosophila;
its FERM domain is 68% identical to the Lulu FERM domain. The Yurt sequence
C-terminal to the FERM domain is unrelated to Lulu; however, Yurt and both
isoforms of Lulu terminate with putative PSD-95/Dlg/ZO-1 (PDZ)-binding
motifs.
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lulu (Epb4.1l5) mRNA was ubiquitously expressed at E7.5 and E8.5 (data not shown). Lulu protein was broadly expressed but was apically enriched in epithelial tissues (Fig. 2D,E). The apical concentration of Lulu mirrored that of the Epb4.1l5GT2-ß-galactosidase fusion protein, which contained an intact FERM domain and was also localized to the apical surface of embryonic epithelia (Fig. 2G). At gastrulation, Lulu protein localization shifted from an apical localization in the epiblast to a position around the circumference of ingressing cells (Fig. 2D, inset).
Lulu is important for the epithelial-to-mesenchymal transition at the primitive streak
One of the most striking aspects of the lulu phenotype was the
deficit in paraxial mesoderm, seen in the dramatically reduced expression
domains of Meox1 (Fig.
1G,H) and of the presomitic mesoderm marker Tbx6
(Fig. 3A,B). Mesoderm
generation depends on successful completion of the EMT at the primitive
streak. Downregulation of E-cadherin (also known as Cdh1 - Mouse Genome
Informatics) is required for this EMT and for mesoderm migration away from the
streak region (Burdsal et al.,
1993; Ciruna and Rossant,
2001). Cells in the mesodermal layer of E7.5 lulu embryos
correctly downregulated E-cadherin (Fig.
4A,B). However, transverse sections of the E8.5 streak showed that
lulu embryos did have a defect in the EMT: although Tbx6 was
expressed in the nascent mesoderm beneath the primitive streak in both
wild-type and lulu embryos (Fig.
3C,D), some mutant cells that had delaminated from the epithelial
layer of the streak were located in an abnormal bulge in the region above the
Tbx6-expressing mesoderm (Fig.
3D, arrow). The cells in the primitive streak bulge expressed
E-cadherin and Sox2, two markers of the epiblast
(Burdsal et al., 1993;
Damjanov et al., 1986),
although they did not have an epithelial morphology
(Fig. 3E,F). Thus, although
some lulu epiblast cells could undergo the EMT, downregulate
E-cadherin and generate mesoderm, other cells at the lulu primitive
streak apparently initiated the EMT but retained some epiblast character and
were trapped in the streak.
Gastrulation appeared to initiate normally in lulu embryos, but defects in the primitive streak could be detected in phalloidin-stained embryos by E7.75. By this stage, mesoderm had spread around the embryonic circumference in lulu embryos, as in wild type (Fig. 4C,D), but the anterior mesodermal wings were only a single cell thick, in contrast to three to four cells thick in wild-type littermates (Fig. 4E,F). This suggested that, by E7.75, the number of mesodermal cells was already reduced, but that mutant mesodermal cells that escaped the streak region could migrate effectively. The ability of mutant mesoderm cells to migrate was confirmed in E7.5 primitive streak explants, where lulu mutant mesoderm cells migrated efficiently away from the streak (data not shown).
The primitive streak of E7.75 lulu embryos was thicker and wider than the wild-type streak (Fig. 4C,D,G,H). Laminin marks the basement membrane that separates the epiblast and mesoderm, and there is a gap in laminin staining at the position of the wild-type primitive streak (Fig. 4I, bracket). In lulu mutants, the gap in laminin expression was wider than in the wild type (Fig. 4J, bracket). Laminin staining also highlighted the increased thickness of the E7.75 streak in lulu mutants compared with wild type (Fig. 4I,J, arrows), similar to the increased thickness that was seen at E8.5 (Fig. 3D).
|
).
Therefore, it was possible that the EMT defect in lulu might
represent a disruption of apical-basal polarity. We examined the expression of
Crumbs proteins at the primitive streak using a pan-Crumbs antibody
(Hsu et al., 2006;
Makarova et al., 2003). In
both wild-type and lulu embryos, Crumbs proteins were expressed
apically in the epiblast at E7.5, and were absent from nascent mesoderm cells
(Fig. 4K,L). Crumbs proteins
were not detected in the cells that accumulated in the abnormal bulge at the
lulu streak, which indicated that these cells had lost this
characteristic of the epiblast. These results show that Lulu is required
neither for apical localization of Crumbs in the epiblast nor for the
downregulation of Crumbs in nascent mesoderm.
Phalloidin staining of E7.5 embryos revealed defects in actin organization
in the mutant primitive streak. In the wild-type primitive streak, F-actin was
enriched around the periphery of cells that were ingressing through the streak
(Fig. 4G, arrowheads), in a
pattern remarkably similar to the distribution of Lulu protein
(Fig. 2D). By contrast, the
circumferential F-actin was not present in cells of the lulu streak;
instead, puncta of F-actin were prominent in the cells that accumulated in the
lulu streak at E7.5 (Fig.
4H, arrowheads) and E8.5 (data not shown). Because some
FERM-domain proteins bind actin (Bretscher
et al., 2002), this abnormal distribution of F-actin could
represent the primary defect that prevents the normal EMT in the lulu
primitive streak.
Abnormal organization of the lulu anterior neural plate
The other tissue with particularly striking defects in lulu
embryos was the neural plate. The anterior neural plate of lulu
embryos was broad, open and folded into irregular ridges
(Fig. 1B,D), and it appeared
thicker than the wild-type neural plate
(Fig. 5A,B). To evaluate
whether the apparently large size of the neural plate could reflect excessive
proliferation in that tissue, we counted the total number of cells in the
anterior neural plate. Sox2 is expressed in the pseudo-stratified neural
ectoderm and the columnar gut endoderm, whereas E-cadherin is expressed in the
gut endoderm and the cuboidal surface ectoderm
(Fig. 5A,B); therefore neural
cells were defined as Sox2-positive and E-cadherin-negative (Sox2+,
E-cadherin-). The total number of Sox2+,
E-cadherin- cells in the anterior lulu neural plate was
approximately the same as in wild-type littermates
(Table 1). We also assayed the
fraction of neural cells in mitosis, based on staining with
anti-phospho-histone H3 antibodies (Fig.
5C,D and Table 1).
The mitotic index in the lulu anterior neural plate was
indistinguishable from that of wild type
(Table 1). We conclude that the
abnormal shape of the neural plate in lulu embryos is not caused by
abnormal proliferation or by an increase in the number of neural cells;
instead, the abnormal shape represents a defect in the organization of the
neural tissue.
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Although the mitotic index in the lulu neural plate was normal,
phospho-histone H3 staining revealed another defect in the organization of the
neural epithelium (Fig. 5C,D).
Nuclei in columnar and pseudo-stratified epithelia, such as the neural plate,
migrate along the apical-basal axis of the cell during the cell cycle, and
mitosis takes place when nuclei are at the apical surface
(Götz and Huttner, 2005).
We observed that 20% of phospho-histone H3-positive nuclei in the
lulu neuroepithelium were located away from the apical surface
(Fig. 5D, arrows), whereas only
5% of phospho-histone H3-positive nuclei were located away from the apical
surface in the wild-type neural plate
(Table 1).
Markers of apical junctions are localized correctly in the lulu neural plate
It was possible that the abnormally positioned mitoses in the lulu
neural plate reflected a defect in the apical-basal polarity of the
epithelium, which depends on the proper organization of apical junctions
(Imai et al., 2006;
Koike et al., 2005;
Wei and Malicki, 2002). We
therefore examined the organization of tight junctions and adherens junctions
in the lulu neural plate. We found that the tight junction
transmembrane proteins occludin and claudin 1, as well as the tight
junction-associated proteins ZO-1 (Tjp1) and ZO-2 (Tjp2), were expressed and
apically localized in lulu mutant neural plates
(Fig. 6A,B and data not shown).
N-cadherin, the major cadherin present in the neuroepithelium
(Radice et al., 1997), and its
associated protein, ß-catenin, were also enriched in the apical neural
plate of both wild-type and lulu E8.5 embryos
(Fig. 6C,D and data not
shown).
Because Lulu can bind Crumbs proteins
(Hsu et al., 2006;
Jensen and Westerfield, 2004;
Laprise et al., 2006;
Omori and Malicki, 2006), we
tested whether components of the Crumbs complex were localized normally in the
lulu neural plate. Crumbs proteins were detected at the apical
surface of the neuroepithelium in wild-type, lulu and
Epb4.1l5GT1 homozygous embryos
(Fig. 6E,F and data not shown).
Pals1 (also known as Mpp5 - Mouse Genome Informatics), a MAGUK protein that
binds Crumbs and the zebrafish Lulu ortholog, Moe
(Hsu et al., 2006), was
expressed on the apical surface, as well as in part of the lateral surface
between adjacent cells, in both the wild-type and the lulu neural
plate (Fig. 6G,H). The
Par6-Par3-aPKC (aPKC is also known as Prkci - Mouse Genome Informatics)
complex interacts with the Crumbs complex
(Hurd et al., 2003;
Lemmers et al., 2004); we
found that Par3 was present apically in both the wild-type and lulu
neural plate (data not shown). Thus, these markers of apical-basal polarity
appeared to be localized correctly in the lulu neural plate.
|
|
;
Tsukita and Yonemura, 1999)
and because we observed ectopic F-actin in the lulu primitive streak,
we examined the organization of F-actin in the lulu neural
epithelium. In wild-type E8.5 neural plate, phalloidin staining showed that
F-actin formed a dense band near the apical surface of each cell
(Fig. 7A,C). By contrast,
although F-actin was enriched apically in the lulu neural plate, it
was also present ectopically at more basal positions
(Fig. 7B,D, arrowheads). Myosin
IIB is a major non-muscle myosin and participates in apical constriction of
the actin ring, an essential step during neural tube closure
(Haigo et al., 2003;
Hildebrand, 2005). Myosin IIB
was highly enriched at the apical surface of the wild-type neural plate
(Fig. 7E). By contrast, Myosin
IIB was seen both apically and in ectopic basal positions in the lulu
neural plate (Fig. 7F,
arrowheads). Spnb2 (ßII-spectrin), an F-actin-binding protein
(Liu et al., 1987), showed
similar ectopic staining within the neuroepithelium (data not shown).
Phospho-ERM (P-ERM) antibodies, which recognize the activated, F-actin-binding
forms of ezrin, radixin and moesin (Gary
and Bretscher, 1995; Matsui et
al., 1998), strongly labeled the apical surface of the wild-type
neural plate (Fig 7G),
consistent with previous reports of their apical localization in other
epithelia (Berryman et al.,
1993; Morales et al.,
2004). By contrast, P-ERM was found both apically and more basally
in the neuroepithelium of lulu mutants
(Fig. 7H), which again
indicated the presence of ectopic F-actin at basal positions in the
lulu neural epithelium. To investigate whether Lulu might regulate the actin cytoskeleton directly, we expressed EGFP-tagged Lulu protein in HeLa cells. EGFP-Lulu was concentrated at the plasma membrane and we observed increased phalloidin staining at the cell cortex in 66% of the transfected cells, whereas only 30% of EGFP-control transfected cells had phalloidin staining concentrated at the cell periphery (Fig. 8). In addition, compared with the transfected control cells, the HeLa cells that overexpressed EGFP-Lulu appeared more spread out and showed increased numbers of small actin-rich protrusions. Thus, overexpression of Lulu was sufficient to increase cortical actin and alter the morphology of these epithelial cells.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Jensen and Westerfield, 2004).
The milder moe phenotype could reflect the large contribution of
maternal gene products to zebrafish development, although translation-blocking
morpholinos recapitulate the moe mutations
(Jensen and Westerfield,
2004). Alternatively, the cellular behaviors required for
gastrulation and neural tube formation are distinct in zebrafish and mouse
(Adams and Kimmel, 2004;
Geldmacher-Voss et al., 2003)
and may depend on different molecular machines.
Lulu acts at an intermediate step in the epithelial-mesenchymal transition
The absence of Lulu leads to dramatic defects in the production and
morphogenesis of paraxial mesoderm, including the complete absence of somites.
Some mesoderm forms in the absence of Lulu, but fewer cells are present in the
mesodermal wings as soon as they surround the embryo. These defects are the
consequence of the abnormal organization of the primitive streak, where cells
begin to accumulate as early as E7.5.
During the EMT at the primitive streak, cells must lose epithelial cell
junctions, escape the epithelial layer and acquire the properties of
mesenchymal cells (Shook and Keller,
2003). These morphological transitions are accompanied by changes
in gene expression, as cells downregulate expression of epithelial genes, such
as Sox2 and E-cadherin, and upregulate mesodermal genes, such as
Tbx6. The early steps of the EMT (breakdown of the basement membrane,
loss of cell-cell junctions and downregulation of Crumbs) do not require Lulu.
However, in the absence of Lulu, many cells accumulate at the primitive
streak. Although these abnormal streak cells have lost their epithelial
organization, they continue to express the epithelial markers Sox2 and
E-cadherin, and do not express the mesodermal marker Tbx6. Thus,
these lulu mutant cells appear to be trapped at an intermediate state
in the EMT.
Mutations in Fgfr1, Snail (also known as Snai1) and
p38IP (also known as D3Ertd300e) all block the gastrulation EMT and,
in each case, the defect has been attributed to the inability to downregulate
expression of E-cadherin (Ciruna and
Rossant, 2001; Carver et al.,
2001; Zohn et al.,
2006). By contrast, although E-cadherin is expressed in the cells
trapped at the lulu primitive streak, E-cadherin is downregulated in
the mesodermal wings in lulu mutant embryos
(Fig. 4B). This suggests that
the lulu phenotype defines a previously unrecognized step of the EMT
that requires a Lulu-dependent reorganization of the actin cytoskeleton. The
change in Lulu protein localization during the EMT suggests that it plays an
active role in the EMT: Lulu is enriched at the apical surface of the epiblast
and then relocalizes to the periphery of ingressing cells. This change in
localization parallels the rearrangements seen in F-actin, which is also
apically localized in the epiblast and surrounds the ingressing cells in the
streak. In lulu mutants, this rearrangement of F-actin fails and,
instead, cells in the mutant streak show ectopic foci of F-actin. We therefore
propose that Lulu helps anchor F-actin to the surface of ingressing cells and
that the architecture of the cells at this transition state is important for
the changes in adhesion and motility that allow these cells to acquire a
mesenchymal character. Some FERM domains have been shown to bind F-actin
(Bretscher et al., 2002), and
overexpressed Lulu is sufficient to reorganize the morphology and actin
cytoskeleton in HeLa cells (Fig.
8). We therefore suggest that Lulu regulates the F-actin
cytoskeleton during the EMT, either via the direct binding of actin or via an
intermediary protein.
Lulu and the organization of the neural epithelium
The second striking defect in lulu mutant embryos is that the
anterior neural plate is broader and thicker than wild type; the irregular
folding pattern in lulu mutants is strikingly different than the
median and dorsolateral folds that lead to wild-type neural tube closure.
These defects are not due to differences in cell proliferation or cell number,
nor are they due to a loss of the apical junctions that are a hallmark of the
apical domain of epithelial cells. Instead, we observe that the abnormal
morphology of the neural plate is coupled to defects in organization of the
apical actin network: F-actin and F-actin-binding proteins are present at
basal positions in the epithelium of the anterior neural plate, in addition to
their normal apical location.
Mouse Lulu, like its Drosophila and zebrafish homologs, binds
Crumbs proteins, which are key determinants of the apical domain of epithelial
cells (Laprise et al., 2006).
Recent work in both Drosophila and zebrafish has indicated that the
function of these Lulu homologs is to regulate the size of the apical domain
of epithelial cells via regulation of either the localization or the activity
of Crumbs (Hsu et al., 2006;
Laprise et al., 2006). The
effect of the loss of Drosophila Yurt varies between tissues: in the
ventral ectoderm, the domain of Crumbs expression is expanded in yurt
mutants, while, in yurt mutant photoreceptors, the apical domain is
expanded in a Crumbs-dependent fashion without a change in the domain of
Crumbs expression (Laprise et al.,
2006). Loss of zebrafish moe function prevents tight
junctions from forming in the retina
(Jensen and Westerfield,
2004), possibly due to loss of apical Crumbs proteins in the
moe retina (Hsu et al.,
2006). In contrast to these results, we find that loss of Lulu
does not affect Crumbs localization or apical junction formation
(Fig. 6), and en-face imaging
did not reveal differences in the size of the apical domain of mutant cells in
the neural epithelium (data not shown).
The lulu neural plate does not have detectable defects in apical junctions, but does show a clear disruption in the organization of the apical F-actin network. One possible explanation for these findings is that the loss of Lulu leads to a partial disruption in apical-basal polarity, so that some cells lose their connections to the apical surface of the pseudo-stratified neural epithelium and the F-actin network associated with these cells appears as ectopic, basal F-actin. However, the disruption of cellular organization in the neural epithelium does not appear to be limited to a sub-population of neural cells. For example, P-ERM, which marks apical F-actin, is ectopically localized throughout the mutant neural epithelium. Similarly, the nuclei throughout the mutant neural epithelium are less elongated than wild type and are not correctly oriented with respect to the apical-basal axis of epithelium (Fig. 5F, Table 1), which is likely to reflect a general change in cellular architecture. Thus, we conclude that there is a global disruption of cell organization in the lulu neural plate.
We suggest that Lulu is required to link the F-actin cytoskeleton to
plasma-membrane protein complexes that are important during the dynamic cell
rearrangements that take place during both the EMT at gastrulation and the
folding of the neural plate. One model that reconciles the demonstrated
ability of Crumbs to bind Lulu with our findings would be that mouse Crumbs
acts upstream of Lulu, and Lulu helps to link Crumbs to the actin
cytoskeleton. Alternatively, Lulu may act independently of Crumbs to anchor
the F-actin cytoskeleton as it rearranges or contracts. In the future,
characterization of the functions of the mouse Crumbs complex proteins and of
genes that produce lulu-like phenotypes
(García-García et al.,
2005) should distinguish among these possibilities.
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ACKNOWLEDGMENTS |
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