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First published online 30 August 2006
doi: 10.1242/dev.02560
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Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD 21250, USA.
* Author for correspondence (e-mail: brewster{at}umbc.edu)
Accepted 2 August 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Neural tube, Convergence, Radial intercalation, Protrusive activity, Cell polarity, Adherens junction
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The cellular basis for primary neurulation has been studied extensively in
amphibia and amniotes (Colas and
Schoenwolf, 2001). In the chick, which has been used as a paradigm
for vertebrate neurulation, a broad neural plate bends, forming a groove. The
margins of the plate are raised to form neural folds that are brought into
apposition at the midline. This process is initiated by regionalized apical
constriction of the neural epithelium, resulting in the formation of a medial
hinge point and two lateral hinge points. The neural folds are `passively'
displaced medially by forces coming from the non-neural ectoderm, which drive
rotation of the neuroepithelium around the lateral hinge points. Once the
neural folds have come into close apposition, the epidermis seals over the
newly formed neural tube and neural crest cells begin to migrate.
Neurulation in the zebrafish appears different from neurulation in
amphibians and amniotes, in part because a neural groove and neural folds are
not formed. Rather, the apical surfaces of both flanks of the neural plate
become juxtaposed at the midline of the neural keel. A lumen is formed
secondarily by cavitation (Schmitz et al.,
1993). At a cellular level, neurulation in the zebrafish has been
best characterized in the trunk region. The neural tube is thought to
originate from a single cell-layered columnar neuroepithelium
(Papan and Campos-Ortega,
1994). Neural plate cells at this stage of development are not
connected by junctional complexes, suggesting that they may not be fully
epithelial (Geldmacher-Voss et al.,
2003). However, despite the lack of a clear epithelial
cytoarchitecture, several observations suggest that the neural keel is formed
by infolding of the neural plate as an organized cell layer, as in amniotes.
Neural plate cells have an elongated, epithelial-like morphology that is
maintained throughout neurulation. Moreover, cells retain their relative
positions, such that there is a close correlation between their original
mediolateral position in the neural plate and their final ventrodorsal
position in the neural tube. Finally, cells gradually transition from a
vertical position in the neural plate to a horizontal position in the neural
rod (Papan and Campos-Ortega,
1994). Because the neural tube forms from a pre-existing
epithelial substrate that undergoes a folding process, neurulation in the
zebrafish is thought to incorporate elements of `primary neurulation' observed
in other vertebrates (Papan and
Campos-Ortega, 1994; Lowery
and Sive, 2004).
Cadherins constitute a family of calcium-dependent homophilic cell-adhesion
molecules. N-cadherin (N-cad, cad 2) belongs to the subfamily of classical
cadherins, characterized by five extracellular cadherin-binding domains and an
intracellular region that binds ß-catenin (ß-cat)
(Tepass et al., 2000).
Classical cadherins are often, but not always, associated with adherens
junctions (AJs). These junctional complexes are thought to maintain both
tissue integrity and cell polarity
(D'Souza-Schorey, 2005). The
specific expression of N-cad in the neural plate following neural
induction (Hatta and Takeichi,
1986; Detrick et al.,
1990; Radice et el.,
1997) led to the hypothesis that this adhesion molecule may be
specifically required for neural tube morphogenesis. Several studies have
provided some degree of support to this hypothesis. Misexpression of
N-cad in the non-neural ectoderm in Xenopus affects neural
tube size and organization, suggesting that N-cad defines the tissue
undergoing neurulation (Detrieck et al., 1990;
Fujimori et al., 1990).
Furthermore, experiments in the chick, using function-blocking antibodies
against N-cad or expression of a dominant-negative N-cad, highlight a
role for N-cad in the maintenance of epithelial integrity, neurulation and
neural crest cell migration (Bronner-Fraser
et al., 1992; Nakagawa and
Takeichi, 1997;
Ganzler-Odenthal and Redies,
1998). However, knocking out N-cad in the mouse results
in a surprisingly subtle neurulation defect, in which a neural tube still
forms but has an abnormal, undulated appearance
(Radice et al., 1997). This
relatively weak phenotype might indicate functional redundancy with other
cadherins. Taken together, these data are consistent with a role for
N-cad in the maintenance of the neuroepithelium and in neurulation,
although the exact role of N-cad in neurulation remains unclear.
In contrast to the mouse knock-out phenotype, loss of zebrafish
N-cad function results in blockage of neural tube formation in the
midbrain-hindbrain region and several other neural defects
(Lele et al., 2002).
N-cad mutants have a characteristic `T-shaped' neural tube, which is
thought to result from the failure of lateral, but not medial, cells to
converge towards the midline. In order to more directly demonstrate the medial
convergence defect, Lele et al. (Lele et
al., 2002) transplanted N-cad mutant cells to the medial
region of the neural plate (future ventral neural tube) of wild-type host
embryos. At later stages, these cells relocated to the dorsal region of the
host neural tube, which is consistent with impaired convergence. However,
given that wild-type and mutant cells are unable to mix
(Lele et al., 2002), the above
assay may in fact reveal the ability of N-cad-positive and
N-cad-negative cells to sort away from one another
(Friedlander et al.,
1989).
We re-investigate here mechanisms of neural tube morphogenesis in the zebrafish, focusing on anterior regions, as these are most severely affected in N-cad mutants, and address the role of N-cad in this process. We demonstrate that the cellular basis for neural tube formation in the zebrafish closely resembles mechanisms used during amphibian neurulation. The zebrafish neural plate is a multi-layered structure, composed of deep and superficial cells that converge medially while undergoing radial intercalation, to form a single cell-layered neural tube. In vivo imaging of individual cell behaviors reveals that cells are polarized along the mediolateral axis and exhibit protrusive activity. In N-cad mutants, both convergence and intercalation are blocked. Moreover, although mutant cells are not defective in their ability to form protrusions, they are unable to maintain them stably. These findings thus uncover key cellular mechanisms underlying neural tube morphogenesis in teleosts and directly implicate N-cad in the polarized cell movements that may drive this process. The expression of N-cad in the neural primordium of all vertebrates following neural induction, and the similarity between mechanisms of neural tube morphogenesis in amphibians and teleosts, suggests a conservation of both cellular and molecular mechanisms.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)] was
obtained from M. Granato (University of Pennsylvania).
DNA and morpholino injections
Plasmids encoding membrane-targeted enhanced Green Fluorescent Protein
(mGFP) (R. Harland, University of California, Berkeley) and Xenopus
ß-catenin-GFP constructs (R. Moon, University of Washington) were
prepared using a Qiagen Maxi Prep Kit. Plasmid DNA (200 pg) was injected into
one- to four-cell stage embryos. N-cad translation-blocking morpholino
(Lele et al., 2002) was
obtained from Gene Tools and injected into one- to two-cell stage embryos (10
ng per embryo). Microinjection was performed using a PCI 100 Microinjector
(Harvard Apparatus).
Embryo staging
Embryos were collected at stages ranging from tailbud to 24 hpf
(Kimmel et al., 1995).
Developmental stages in the head region are as follows: neural plate/early
neural keel, tailbud-1 somite (som); neural keel, 2-3 som; early neural rod
(prior to the formation of AJs), 4-5 som; neural rod, 9-10 som; late neural
rod (following the formation of AJs), 12-13 som; and cavitation, 20 som.
However, DNA injection transiently retarded neural development, such that
injected embryos that exhibited the same number of somites as uninjected
controls were often less advanced in terms of neural tube morphogenesis. To
avoid confusion, the developmental stage, based on the progression of
neurulation, is given together with the somitic stage for all injected
specimens for which there is a discrepancy.
Embryo sectioning
Embryos were fixed in 4% paraformaldehyde at 4°C overnight, mounted in
4% low-melting point agarose (Shelton Scientific, IBI) and sectioned at 50
µm intervals using a vibratome (1500 Sectioning System, Vibratome).
Sections through the midbrain-hindbrain region were identified based on
several criteria: the stage of neurulation, the presence and size of the
notochord (beginning at 4-5 som) and otic vesicles (9-10 som onwards), and the
thickness of the underlying mesoderm.
Labeling and imaging of fixed preparations
Immunohistochemistry on floating sections was carried out according to
Westerfield (Westerfield,
2000). The following antibodies were used: mouseanti-ß-cat
(BD Biosciences) at 1:200; rabbit-
-aPKC
(C-20; Santa Cruz
Biotechnology) at 1:200; rabbit--phospho Histone3 (Upstate
Biotechnology) at 1:200; mouse -ZO-1 (Zymed laboratories) at 1:200;
rabbit--Sox3C (a gift from M. Klymkowsky, University of Colorado) at
1:2000. Secondary antibodies conjugated to Alexa-488 or Cy3 (Molecular Probes)
were used at 1:200. Alexa-488-phalloidin (Molecular Probes) was used at 1:75.
DAPI (Molecular Probes) was used according to the manufacturer's instructions.
The sections were imaged with a Zeiss 510 META confocal microscope. Single
cell analysis was performed using the LSM software (Zeiss).
Riboprobes, n-cad and dlx3 (plasmids obtained from I.
Dawid, NIH), were synthesized and whole-mount in situ hybridization was
carried out as described (Thisse et al.,
1993). Embryos were mounted in glycerol and imaged using a Zeiss
Axioscope2 microscope.
Time-lapse confocal microscopy
For time-lapse microscopy, embryos were dechorionated at 2 som. 1.2%
agarose (Shelton Scientific, IBI) was laid on a glass-bottom culture dish
(MatTek) and <1 mm holes were made in the agarose. Embryos were mounted in
these holes, oriented with the dorsal head region in contact with the glass.
They were imaged using the Zeiss Axiovert 200 with a PerkinElmer UltraView LCI
(Live Cell Imaging) System. The images were analyzed and processed using the
UltraView software, Image J (NIH) and Adobe PhotoShop.
Transmission electron microscopy
Transmission electron imaging was performed as described by Brosamle and
Halpern (Brosamle and Halpern,
2002).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), single cell labeling revealed that it is
in fact composed of two cell layers at the onset of neurulation. The
superficial layer consists of cuboidal cells, situated directly below the
enveloping layer (EVL). Cells in the deep layer are columnar, with one pole
directly apposed to the basement membrane and the other in contact with the
cuboidal cells in the upper layer (Fig.
1A). In order to determine whether the bilayered configuration of
the neural plate is specific to anterior regions, we examined the trunk and
observed a similar cellular organization (data not shown).
The multi-layered nature of the neural plate was further confirmed using
transmission electron microscopy (TEM) imaging
(Fig. 2A). However, there
remained the possibility that cells in the superficial layer represent a
transient population of dividing cells, as mitosis occurs apically in the
pseudostratified monolayered epithelium of the chick and mouse neural plate
(Sauer, 1935). To address this
possibility, we labeled embryos at the neural plate stage with -phospho
Histone3 (-PH3), a marker for dividing cells, and -Sox3C, a pan
neural marker. We observed that dividing cells are located in both superficial
and deep layers of the neural plate, and that nuclei in both layers express
Sox3C (Fig. 2B,C), further
supporting the multi-layered nature of this tissue.
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Neuroepithelial cells change orientation and elongate during neurulation
Papan and Campos-Ortega (Papan and
Campos-Ortega, 1994) previously reported that neural cells in the
trunk region transition from a vertical position in the neural plate to a
horizontal position in the neural rod, suggesting that the neural plate
infolds as an organized epithelium. A similar process was observed in the head
region of embryos injected with mGFP DNA; however, deep and superficial cells
initially exhibited distinct behaviors. The angular orientation of cells was
measured in degrees ranging from 0° to 90° (see Table S2 in the
supplementary material). Values between 0° and 20° correspond to
`horizontal' cells (cells having achieved a close to 90° rotation relative
to their initial orientation in the neural plate), values between 60° and
90° to `vertical' cells (cells that did not significantly change their
initial orientation), and values between 20° and 60° to oblique cells.
At the neural plate stage, in lateral regions, deep cells were oriented
vertically (average angle of 78±6.5°), in contrast to superficial
cells that were cuboidal (Fig.
1A). At the same stage, both superficial and deep cells in
ventral/medial regions of the neural plate were already inclined towards the
midline (average angle of 48±15° and 57±5.8°,
respectively; Fig. 1B). At the
advanced neural keel stage (4-5 som), cells in lateral regions also adopted an
oblique position (average angle of 25±3.1°;
Fig. 1C,D), whereas ventral
cells became horizontal (average angle of 11±5.4°;
Fig. 1C). By the neural rod
stage, and continuing into later stages, cells at all dorsoventral levels were
fully horizontal (Fig.
1F,G).
Concomitant with the transition to a horizontal position, cells appear to elongate significantly along their future apicobasal axis. At the neural plate stage, the average length-to-width ratio (LWR) of deep lateral cells is 3.5±0.6, whereas, at the advanced neural keel stage (4-5 som), the average LWR of lateral cells is 7.6±0.6 (see Table S2 in the supplementary material). Thus, neurulation in anterior regions involves cell elongation and a gradual change in angular orientation, which proceeds in a medial to lateral direction, consistent with an infolding mechanism.
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). Because this
mode of neurulation does not involve a folding process per se, but rather
active en masse cell movement, the term convergence rather than infolding
seems more appropriate.
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Loss of N-cad causes late but not early neural tube morphogenesis defects
Loss of N-cad was previously shown to block neurulation at the
neural rod stage (Lele et al.,
2002), which is surprisingly late given that N-cad is
broadly expressed throughout the neuroepithelium following neural induction
(Bitzur et al., 1994;
Lele et al., 2002). The late
onset of neural tube morphogenesis defects was confirmed by labeling
N-cad mutants at progressive stages of development with
dlx3, a marker for the edge of the neural plate. Embryos from a cross
between two N-cadp79emcf heterozygote parents, one quarter
of which are expected to be mutant, did not show any difference in the
expression of dlx3 at the neural plate
(Fig. 5A) and neural keel
(Fig. 5B) stages, suggesting
that the convergent extension (CE) movements that shape the neural plate
(Keller et al., 1992;
Woo and Fraser, 1995) and the
early phase of neurulation occur normally in mutants. However, by the neural
rod stage, a striking difference in the width of the neural anlage became
apparent (wild-type siblings, width of 207±17 nm;
N-cadp79emcf mutants, width of 309±2 nm;
Fig. 5C,D). These observations
confirm previous findings and suggest either that N-cad is not
required for the CE movements that shape the neural plate or that maternal
N-cad or other cadherins can compensate for loss of
N-cad.
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), in
wild type, N-cadp79emcf mutants and embryos injected with
N-cad translation-blocking morpholinos (MOs), in which both the
maternal and zygotic components should be blocked. At neural plate and early
neural rod stages, ß-cat is broadly distributed throughout the plasma
membrane of wild-type neuroepithelial cells
(Fig. 4A,C). ß-cat is also
observed at normal levels on the cell surface of MO-injected cells at the
neural plate stage (Fig. 4B),
suggesting that blockage of maternal and zygotic N-cad RNA
translation is insufficient to disrupt ß-cat plasma membrane
localization. It is therefore likely that other cadherins function to tether
ß-cat to the plasma membrane at this stage of development. However,
ß-cat expression is observed throughout the cytoplasm of
N-cadp79emcf mutants and is greatly reduced in the plasma
membrane of N-cad MO-depleted cells at the neural keel and neural rod
stages (Fig. 4D; data not
shown). The disruption of ß-cat localization thus appears to coincide
with the onset of neurulation defects observed in mutant and MO-injected
embryos, and suggests that N-cad is only required during later stages
of neural tube morphogenesis. In addition, the defects in ß-cat
localization suggest that cadherin-based cell-cell adhesion is impaired during
neurulation.
Loss of N-cad function prevents infolding of lateral neural plate cells
The characteristic `T-shaped' neural tube of N-cad mutants is
suggestive of a convergence defect in lateral regions of the neural plate. To
investigate the cellular basis of this defect, we analyzed the behavior of
single neuroepithelial cells using mGFP. We observed that the cytoarchitecture
of the neural plate appears, overall, normal in N-cad MO-injected
embryos. Cells organize into deep and superficial layers, and deep cells are
elongated and in contact with the basement membrane
(Fig. 6A). Angular measurements
(see Table S2 in the supplementary material) reveal that at early stages of
development (tb-1 som), ventral MO-injected cells, surprisingly, are more
oblique than their wild-type counterparts (19±5.2° for morphant
versus 48±15° for wild type), although LWRs do not increase
relative to wild type. At the rod stage, when neurulation in ventral regions
is complete in both wild-type and N-cad-depleted embryos, the LWR of
mutant ventral cells (4.3±0.6) is smaller than that of wild-type
ventral cells (6.6±1.4). These findings suggest that, in ventral
regions, neurulation is not impaired and may even proceed faster in
N-cad-depleted embryos, and that cell length has little impact on the
initiation or progression of this process.
Additional cellular defects became apparent at the advanced neural keel stage (4-5 som), when a striking difference in the angular orientation of cells was observed in lateral/dorsal regions relative to ventral regions (see Table S2 in the supplementary material). Cells in lateral/dorsal regions of mutant and N-cad-depleted embryos, at the neural keel (4-5 som) stage, had a near vertical orientation, with average angles of 65±8.5° and 63±8.8°, respectively (Fig. 6B,C), whereas cells in ventral regions were mostly horizontal, with average angles of 8.4±2.4° and 5.2±1.8°, respectively. By contrast, angles measured for wild-type dorsal neural keel cells were only marginally larger than angles measured for wild-type ventral cells (Table S2 in the supplementary material), indicating that cells along the entire dorsoventral (DV) axis were either oblique or horizontal. Labeling of single cells at later stages revealed that the orientation of lateral/dorsal mutant cells remained close to vertical up until 24 hpf (Fig. 6H). In addition to the cell orientation defects, cell LWRs in lateral/dorsal regions were also abnormal. Although mutant and MO-injected cells retained an epithelial-like morphology, the LWR at the advanced neural keel (4-5 som) and neural rod (6-7 som) stages revealed that N-cad-deficient cells failed to elongate as much as wild-type cells in comparable regions. For example, cells in lateral/dorsal regions of wild-type embryos at 4-5 som had an average LWR of 7.6±0.6, relative to 4.4±0.6 and 4.9±0.7 for N-cadp79emcf and N-cad MO-injected embryos, respectively. Taken together, these observations indicate that, despite the initial accelerated rate of neurulation, loss of N-cad function disrupts the medial convergence and elongation of the neuroepithelium in lateral/dorsal regions.
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As was observed in wild-type embryos, relatively few mutant and N-cad-depleted cells were in contact with both the apical and basal surfaces of the neuroepithelium at the neural plate stage (see Table S1 in the supplementary material). However, numbers were higher for medial N-cad-depleted cells (39%) relative to wild-type cells (15%), consistent with the angle measurements, suggesting that neurulation may initially proceed at a faster pace in absence of N-cad function.
Numbers of ventral N-cad-depleted cells in contact with both the apical and basal surfaces were comparable to wild type at the late neural rod stage: 74% of labeled cells for N-cad-depleted embryos relative to 72% of labeled cells for wild type. By contrast, the number of N-cad-depleted cells that achieved radial intercalation in dorsal regions (Fig. 6F) did not increase at later stages, relative to wild type. By the late neural rod stage, only 35% of labeled N-cad-depleted cells in dorsal regions established apicobasal contact, relative to 97% of labeled wild-type cells in comparable regions. Taken together, these observations suggest that N-cad is required for radial intercalation in lateral/dorsal regions. In addition, these results support the idea that radial intercalation and medial convergence are connected, as both occur properly in ventral regions of N-cad-deficient embryos and fail to take place in lateral/dorsal regions.
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) and may
explain the low number of cells in contact with apical and basal surfaces in
the neural rod. To investigate whether increased cell division can contribute
to the epithelialization defect at earlier stages, wild-type and
N-cadp79emcf embryos were labeled with the mitotic marker
-PH3. Increased mitosis was not observed at the keel and early neural
rod stages (data not shown), when radial intercalation appears defective, and
is therefore unlikely to explain the lack of cells with apicobasal
contact.
Epithelialization is partially retained in N-cad mutants
While impairment of radial intercalation may account for the failure of
most lateral/dorsal cells to establish apicobasal contact in the neural rod,
this phenotype may be enhanced by the inability of these cells to form
junctional complexes at the late neural rod/neural tube stage or to
reintegrate into the neuroepithelium upon cell division. Indeed, disruption of
N-cad function is known to cause a loss of epithelial integrity in
both the zebrafish and the chick neural tube, as cells detach from the
ventricular walls of the neural tube and populate the ventricles forming
cellular aggregates called rosettes (Fig.
4P) (Lele et al.,
2002; Ganzler-Odenthal and
Redies, 1998).
In order to further address whether the large number of cells that fail to
establish apicobasal contact can be partially explained by the inability of
cells to form junctional complexes and epithelialize, we analyzed the
expression of ß-cat, aPKC and ZO-1 (Tjp1 - Zebrafish Information
Network). aPKC is a component of the Par3/Par6/aPKC complex that is implicated
in the establishment of cell polarity
(Izumi et al., 1998;
Ohno, 2001) and ZO-1 is a
junctional protein associated with both tight junctions and AJs
(Itoh et al., 1993). In
wild-type embryos at the late neural rod stage, ß-cat is distributed
throughout the plasma membrane and is increased at the midline of the neural
rod, which corresponds to the apical surface of the neuroepithelium where AJs
form. Apical localization of ß-cat begins in ventral regions
(Fig. 4E) and extends dorsally
at later stages (Fig. 4G), as
has been previously reported
(Geldmacher-Voss et al.,
2003). During cavitation, wild-type neural progenitor cells appear
to be fully polarized, with a clear enrichment of ß-cat labeling at the
apical surface (Fig. 4I). In
N-cadp79emcf mutants, ß-cat is mislocalized
throughout the cytoplasm at the neural rod stage, in both ventral and dorsal
regions (Fig. 4D). However,
during cavitation, ß-cat localization appears enhanced at the apical
surface in ventral cells, as observed in wild-type embryos
(Fig. 4I,J), suggesting that
epithelialization occurs properly, at least in ventral regions. This
observation was confirmed by analysis of ZO-1 and aPKC distribution. In
wild-type embryos, these junctional proteins become enriched at the midline,
prior to ß-cat enhancement (Fig.
4E,K; data not shown). Interestingly, in
N-cadp79emcf mutants, ZO-1
(Fig. 4K,L) and aPKC
(Fig. 4M,N) localize to the
midline in ventral regions and to the apical surface of the neuroepithelium in
lateral/dorsal regions of advanced neural rod stage embryos. Although the
apical labeling in lateral/dorsal regions may reflect immunoreactivity of the
EVL, neuroepithelial cells located directly below the EVL also appear to be
aPKC-positive (Fig. 4N). The
ability of at least some lateral/dorsal cells to epithelialize is further
evidenced by the apical localization of phalloidin, a marker for polymerized
actin, that is closely associated with AJs
(Tsukita et al., 1992) in
lateral/dorsal regions at 24 hpf (Fig.
4O,P).
These observations suggest that ventral and some lateral/dorsal cells in
N-cadp79emcf mutants are able to undergo
epithelialization. In addition, AJs appear to form downstream of other
polarization cues in the neuroepithelium, as aPKC and ZO-1 apical localization
precedes that of ß-cat, and these markers localize properly in absence of
N-cad. This hierarchical relationship is also observed in
Drosophila epithelial cells
(Harris and Peifer, 2004).
Loss of N-cad causes abnormal protrusive activity
What can account for the inability of N-cad loss-of-function cells
to converge and undergo radial intercalation? Because protrusive activity
appears to be involved in both processes in wild-type embryos, we
investigated, using time-lapse recording of GFP-labeled cells, whether this
activity is impaired by loss of N-cad function. Consistent with this
hypothesis, deep and superficial cells exhibited protrusive activity oriented
along the mediolateral axis; however, they often retracted their cellular
extensions (Figs 7,
8; see Movie 2 in the
supplementary material). Possibly as a consequence of this abnormal protrusive
activity, deep cells did not extend sufficiently to establish contact with the
midline (Fig. 7A), and
superficial cells failed to migrate medially, elongate and make contact with
either the midline or the lateral surfaces of the neuroepithelium
(Fig. 7B; see Movie 2 in the
supplementary material). The inability of cells to elongate is consistent with
the overall decrease in cell length observed in fixed preparations (see Table
S2 in the supplementary material). In summary, although cells retain their
mediolateral polarity in the absence of N-cad, they are unable to
form the stable protrusions characteristic of polarized neuroepithelial
cells.
|
). To address whether cadherin-based adhesion
localizes to protrusive ends, we examined the localization of a ß-cat-GFP
fusion protein in wild-type embryos using time-lapse confocal imaging,
following injection of DNA encoding this construct. Interestingly, we observed
that ß-cat does not appear enhanced at the poles of cells, but rather is
evenly distributed throughout the plasma membrane of superficial cells
undergoing medial migration (see Movie 3 in the supplementary material). These
findings suggest that, if the cadherin-based cell traction model is correct,
it is the adhesive activity rather than the adhesive complex itself that
becomes localized to sites of protrusive activity.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Kimmel et al.,
1990; Kimmel et al.,
1994; Schmitz et al.,
1993; Papan and Campos-Ortega,
1994; Papan and Campos-Ortega,
1997; Papan and Campos-Ortega,
1999; Concha and Adams,
1998; Geldmacher-Voss et al.,
2003; Ciruna et al.,
2006). During neurulation, cells elongate and converge medially,
while simultaneously undergoing radial intercalation. However, deep and
superficial cells achieve this by using different mechanisms. Deep cells
gradually transition to a horizontal position and insert between superficial
cells using medially oriented protrusions. Lateral superficial cells migrate
medially, appear to establish contact with the midline first and then the
lateral edge of the neural tube, using bipolar mediolaterally oriented
protrusive activity. As cells approach the midline they interdigitate
transiently with cells in the opposing epithelium, possibly stabilizing the
forming neural keel/rod prior to the formation of junctional complexes. Once
the neural rod is formed, the neuroepithelium undergoes epithelialization in a
ventral to dorsal direction (Gelmacher-Voss et al., 2003;
Ciruna et al., 2006), resulting
in the loss of midline interdigitations and the establishment of a clear
midline. Cavitation of the neural rod follows epithelialization closely and
proceeds in the same direction (Schmitz et
al., 1993). Elements of this model contributed specifically by
this study include: (1) radial intercalation, (2) use of polarized protrusive
activity, and (3) transient interdigitation of cells at the midline.
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Role of N-cad in mediating cellular rearrangements
By a direct analysis of cellular behaviors, we demonstrate here that, in
ventral regions of N-cad-depleted embryos, neuroepithelial cells
undergo normal, if not accelerated, convergence and intercalation. The
accelerated pace of neurulation in ventral regions may be due to a
compensatory effect of other genes controlling this process or, possibly, to a
non-specific effect of the MO injection. By contrast, cellular rearrangements
are greatly impaired in lateral regions
(Fig. 9B). These defects are
likely to result from the inability of N-cad-deficient cells to
establish stable protrusive activity, despite proper mediolateral orientation.
Because the planar cell polarity (PCP) pathway is also implicated in polarized
cell behaviors (Wallingford et al.,
2000; Wallingford et al.,
2002; Myers et al.,
2002; Keller,
2002), it is tempting to speculate that it may regulate or be
regulated by the activity of N-cad during neurulation. In support of this
idea, the subcellular localization of E-cad, which is required for
gastrulation in the zebrafish (Kane et
al., 2005; Montero et al.,
2005), was recently shown to be regulated by Wnt11, a member of
the PCP pathway (Ulrich et al.,
2005).
Conservation of mechanisms of neurulation
N-cad is expressed in the neuroepithelium of all vertebrates, at
the onset of neural induction. Does this reflect a conservation of cellular
and molecular mechanisms of neurulation? At a morphological level, neurulation
in teleosts and amphibians appears quite different, given that a neural groove
and neural folds form in Xenopus but not in the zebrafish. However,
there are multiple similarities at the cellular level. The amphibian neural
plate is also composed of two layers
(Chalmers et al., 2002) that
undergo radial intercalation to form a single cell-layered neural tube
(Davidson and Keller, 1999).
Rather than passively bending towards the midline, prospective dorsal neural
cells and neural crest cells migrate using bipolar and monopolar protrusive
activity, respectively. Upon completion of neural tube formation, relumination
occurs concomitantly with re-epithelialization, in a ventral to dorsal
direction (Davidson and Keller,
1999). Thus in both amphibians and teleosts, the neuroepithelium
appears more mesenchymal than epithelial, as cellular rearrangement occurs.
Despite these similarities, why are neural folds and a neural groove not
present in the zebrafish? The formation of both of these structures is
dependent upon the ability of cells to undergo regionalized cell shape
changes, such as apical or basal constriction
(Colas and Schoenwolf, 2001).
Apical constriction involves an actomyosin network associated with junctional
complexes (Lee and Nagele,
1985; Hildebrand and Soriano,
1999; Haigo et al.,
2003; Hildebrand,
2005). However junctional complexes are absent from the zebrafish
neuroepithelium prior to neural tube closure. Thus, although amphibian and
teleost neuroepithelial cells exhibit many common behaviors, amphibian cells
may be `more epithelial' than teleost cells, particularly in the superficial
layer, which undergoes apical constriction
(Davidson and Keller, 1999).
In amniotes, neuroepithelial cells are even more distinctly epithelial, as AJs
are present during neurulation
(Aaku-Saraste et al., 1996) and
cellular rearrangements do not occur during the bending process. It is
therefore likely that the architecture of the epithelium imposes physical
constraints that dictate the mode of neurulation.
In the zebrafish, N-cad is implicated in both the cellular
rearrangements that shape the neural tube and the maintenance of
neuroepithelial integrity, upon completion of neural tube closure and
formation of AJs (Lele et al.,
2002) (this study). Thus, N-cad appears to play a dual,
early and late, role during neural tube development in teleosts. In
amphibians, this dual role is likely to be conserved, as cellular mechanisms
of neurulation are similar to those in teleosts. However, the epithelial
character of the chick neuroepithelium
(Aaku-Saraste et al., 1996)
suggests that the function of N-cad in amniotes may be shifted
towards a structural role exclusively, analogous to the late function of
N-cad in the zebrafish.
In conclusion, our studies reveal a conservation of mechanisms of neurulation in teleosts and amphibians, and highlight an unappreciated role for cadherins in the polarized cell behaviors that shape the vertebrate embryo.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/19/3895/DC1
|
ACKNOWLEDGMENTS |
|---|
-Sox3C. In addition, we would like to thank M. Halpern, M. Van Doren,
K. Chalasani and M. Harrington for comments on the manuscript. This work was
supported by NSF grant 0448432 to R.B. and an NSF ADVANCE, award number
SBE-0244880-01. |
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