Institut für Entwicklungsbiologie, Universität zu Köln, 50923 Köln, Germany
* Author for correspondence (e-mail: jose.campos{at}uni-koeln.de)
Accepted 14 May 2003
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SUMMARY |
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Key words: Zebrafish, Neurulation, Mitoses, Orientation, Spindle rotation, Neuroepithelial polarity, ASIP, Histone2A.F/Z:GFP, tau:GFP
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INTRODUCTION |
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During neurulation, mitotic divisions of neuroepithelial cells are
polarised. On the basis of single cell labelling experiments, Kimmel et al.
(Kimmel et al., 1994) reported
that mitotic orientation of dividing neural progenitor cells changes from
preferentially anteroposterior at embryonic division 15 to mediolateral at
division 16, this latter mitosis leading to bilateral segregation of progeny
cells. Papan and Campos-Ortega (Papan and
Campos-Ortega, 1994
) followed fluorescein-labelled neural plate
cells that gave rise to progeny disposed on either side of the midline of the
neural rod. Concha and Adams (Concha and
Adams, 1998
) used time-lapse microscopy and Nomarski optics to
follow individual cells in the epiblast during gastrulation and early
neurulation stages, and showed that mitoses in the neural plate are planar,
oriented parallel to the apical surface of the epithelium, and occur initially
in a preferred anteroposterior direction. However, at the onset of neural
plate infolding, mitoses were oriented preferentially in the mediolateral
direction. In addition, Concha and Adams
(Concha and Adams, 1998
)
concluded that `The orientation of cell division for those cells that will
contribute to the CNS will tend towards several mutually orthogonal alignments
during the course of gastrulation and neurulation.'.
To follow the behaviour of zebrafish neuroepithelial cells in vivo, we
established strains carrying stable transgenic insertions of the zebrafish
histone2A.F/Z gene fused to a DNA fragment encoding the green
fluorescent protein (GFP) (Pauls et al.,
2001). Expression of the Histone2A.F/Z:GFP fusion provides an
ubiquitous chromatin marker from the 7th-8th cleavage onwards. To visualise
mitotic spindles directly, we injected mRNA encoding tau:GFP. We find that
mitotic spindles show a stereotypic orientation throughout neurulation, lying
parallel to the plane of the neuroepithelium. However, we find that in
divisions during the keel/rod stage the mitotic spindle itself rotates by
90° so that it lies perpendicular to the apical surface; the ensuing
divisions are therefore oriented orthogonally.
A rotation of the mitotic spindle has also been observed at two different
instances in neural development in Drosophila. Neuroectodermal cells
undergo planar mitoses, but in delaminated neuroblasts the spindle rotates so
that it is perpendicular to the neuroectoderm
(Kaltschmidt et al., 2000).
Similarly, in the lineage of sensory bristles, the progenitor cell pIa divides
within the plane of the epithelium, whereas the spindle in pIIb changes its
orientation and the cell divides along the apicobasal axis
(Gho and Schweissguth, 1998
;
Roegiers et al., 2001
).
Recently, Lu et al. (Lu et al.,
2001
) and Le Borgne et al. (Le
Borgne et al., 2002
) presented evidence for a role of junctional
proteins in controlling the shift in spindle orientation in the divisions of
neuroblasts and pIIb cells, respectively.
In Caenorhabditis elegans, a protein complex consisting of two PDZ
proteins, PAR-3 and PAR-6, and an atypical protein kinase C (aPKC) controls
the polarity of the first cleavages
(Kemphues et al., 1988;
Cheng et al., 1995
;
Guo and Kemphues, 1996
). This
complex is phylogenetically conserved, and is localised at the adherens
junctions of neuroepithelial cells of vertebrates and in the subapical region
of Drosophila epithelial cells
(Horne-Badovinac et al., 2001
;
Manabe et al., 2002
) (for
reviews, see Ohno, 2001
;
Wodarz, 2002
). ASIP (aPKC
specifically interacting protein), a vertebrate homologue of PAR-3,
contributes to the control of epithelial polarity
(Izumi et al., 1998
;
Suzuki et al., 2001
). A
similar role has been postulated for the Drosophila PAR-3 homologue
Bazooka (Kuchinke et al.,
1998
; Wodarz et al.,
1999
; Wodarz et al.,
2000
) (see also Müller
and Wieschaus, 1996
). We have analysed the expression of a
zebrafish ASIP homologue, as well as that of aPKC
/
and several
other apical markers in the neuroepithelium. We find that these markers become
apically localised for the first time late in the neural keel stage. The
orientation of the cleavage plane is weakly but significantly impaired
following injection of morpholinos directed against ASIP. Defects in mitotic
orientation of similar degree are found in heart and soul mutants
(hasm129) (Schier et
al., 1996
; Stanier et al., 1996;
Malicki et al., 1996
), which
express an inactive aPKC
(Horne-Badovinac et al., 2001
;
Peterson et al., 2001
).
Therefore, stereotypic mitotic orientation depends, at least in part, on the
integrity of cell junctions. However, the rotation that the mitotic spindles
undergo in dividing neural keel/rod cells appears not to require firmly
established cell junctions.
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MATERIALS AND METHODS |
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Multilevel confocal time-lapse imaging and statistical analysis
An LSM 410 confocal microscope (Zeiss) attached to an inverted Zeiss
Diavert microscope with 40x and 63x immersion objectives was used
to collect up to 12 different vertical stacks of images (z-series) at
intervals of 45 or 180 seconds, each stack separated by between 0.75 and 7
µm from the next, depending on the experiment. z-series were
transferred to a Macintosh Power PC for image processing using NIH-Image and
Adobe Photoshop 6.0. Anaphase orientation was determined as described by
Concha and Adams (Concha and Adams,
1998) for cytokinesis. The midline of the neural plate was first
labelled on each frame and its mediolateral extent was subdivided into three
equal parts. The orientation of each anaphase was determined by drawing a line
parallel to the plane of cleavage and projecting it to the midline. The angles
subtended at the midline of the neural plate were measured and mitoses within
each region of the plate were grouped in intervals of 10°. Circular
statistics for multimodal samples
(Batschelet, 1981
) were
employed. Angles were measured on both sides of the neural plate and data were
doubled to obtain a circular distribution of the angles. The average length of
the vector was calculated and a Raleigh significance test
(Batschelet, 1981
) was
applied.
Orthogonal sections (z-scans) of the neural plate of experimental embryos were obtained from sequences of 250 frames each (60 second intervals). A line was determined across the neural plate, and all sections from each sequence were referred to that line and transformed by the software of the confocal microscope.
Cloning of a zebrafish ASIP homologue and design of morpholinos
Based on the published 319 bp sequence of the zebrafish EST clone
fe48f11.y1 (dbEST Id: 3215681), we designed the PCR primers
5'-TATCGTAGTCTTCCTCGTGATG-3' and
5'-CACTGAAGGGACAACATGGAT-3' to amplify a 276 bp fragment encoding
amino acids 203 to 294 of the predicted ASIP homologue. A cDNA library was
screened using this PCR fragment as a probe and a 3.8 kb cDNA clone was
obtained. Alignment of this sequence with the corresponding sequences from
human and rat revealed that the cDNA clone contained the complete coding
region of a zebrafish ASIP gene, specifying a protein of 1127 amino acids
(Accession Number AF465629).
A morpholino-modified antisense ASIP oligonucleotide
(Nasevicius and Ekker, 2000),
designed according to the manufacturer's recommendations, was obtained from
Gene Tools, LLC. The `standard control oligonucleotide' provided by the
manufacturer was used as a negative control. Their sequences were as follows:
ASIP-Morpholino, 5'-ACACCGTCACTTTCATAGTTCCAAC-3'; standard control
oligo, 5'-CCTCTTACCTCAGTTACAATTTATA-3'. Stock solutions of the
ASIP morpholino (5 mM; 41.7 ng/nl) and the standard control oligo (1 mM) were
prepared by resuspension in water. Stock solutions were diluted to working
concentrations ranging from 2.6 ng/nl to 20.9 ng/nl in 1x Danieau medium
containing 0.2% Phenol Red, and 5 nl aliquots of the different working
solutions were injected into the yolk of Tg(H2AF/Z)kca37
and Tg(H2AF/Z)kca66 zygotes. To test their efficacy in
blocking translation, the morpholino was also injected (10.4 ng/nl) into
wild-type zygotes together with mRNA encoding ASIP:GFP. Western blots were
incubated with rabbit anti-GFP antibody (Torrey Pine Biolabs, Houston) and
goat anti-rabbit HRP. Antibodies were detected by chemiluminescence ECL
(Amersham Life Science, Arlington Heights, IL).
GFP fusion proteins and mRNA injections
For mRNA injections, four different constructs were synthesised, three of
which encoded variants of ASIP fused to the enhanced variant of GFP (EGFP)
(Cormack et al., 1996), while
the fourth coded for tau:EGFP. An expression vector was made by cloning the
Eco47III-XhoI fragment encoding the enhanced GFP variant
from the pEGFPC1 vector (Clontech Laboratories, Accession Number U55763) into
the StuI+XhoI-digested pCS2+ vector
(Turner and Weintraub, 1994
).
The coding region of zebrafish ASIP was amplified by PCR from the full-length
cDNA clone using the primer pair ASIP-5Eco
5'-TCGGGAATTCGTGTTGGAACTATG-3', ASIP-3Sal
5'-AAGCGTCGACGTACCTGTCTGAAG-3'.
The PCR fragment was digested with EcoRI/SalI and cloned
into the EcoRI+SalI-digested pEGFP-N3 vector (Clontech
Laboratories, Accession Number U57609). The ASIP fragment was excised with
EcoRI/XmaI and cloned into the
EcoRI+AgeI-digested pCS2+/EGFP vector, resulting in a
construct encoding amino acids 1-1127 of ASIP fused to the N terminus of EGFP.
Starting from this full-length asip:egfp clone, we made the following
pair of deletion constructs. The codons for amino acids 688-1127 were deleted,
resulting in a construct
(asip-3':egfp) encoding a fusion of
residues 1-687 to the N terminus of EGFP; and a fragment encoding the
C-terminal part of ASIP (asip-
5':egfp)
was cloned into the pCS2+/EGFP vector, resulting in a construct encoding a
fusion of amino acids 658-1127 to the N terminus of EGFP. For the
tau:egfp construct, a EcoRI-XhoI fragment encoding
a bovine tau fused to EGFP from pCaspeR (UAS:tau-mEGFP), kindly
provided by Andrea Brand and Nick Brown (Cambridge, UK) (see
Kaltschmidt et al., 2000
), was
cloned into the pCS2+ vector.
Capped RNA was synthesised in vitro by transcription with SP6 polymerase from the constructs described above. mRNAs were injected in 5 nl aliquots (2.5 ng) into the yolk of zygotes of either wild type or Tg(H2AF/Z)kca37 and/or Tg(H2AF/Z)kca66.
Immunohistochemistry
For immunohistochemistry, we used the following antisera: rat polyclonal
anti-PKC (C20) at 1:500 (Santa Cruz Biotechnology); rabbit
anti-ß-catenin at 1:500 (a gift from P. Hausen) (see
Schneider et al., 1996
); mouse
anti-ZO-1 at 1:100 (a gift of S. H. Tsukita) (see
Itoh et al., 1993
); and a
mouse anti-phosphotyrosine antibody (PY20) at 1:100 (Transduction
Laboratories). Secondary antibodies for detection were conjugated to Cy2 and
Cy3 (Amersham Pharmacia). For whole-mount antibody staining, embryos were
fixed for 2 hours at room temperature in 4% paraformaldehyde in PBS. All
incubations for antibody staining were performed in solutions containing 5%
DMSO and 1-1.5% normal goat serum. For DNA staining, Sytox Green (Molecular
Probes) was used according to the manufacturer's protocol. To visualise
F-actin, embryos were incubated in rhodamine-conjugated phalloidin (Molecular
Probes).
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RESULTS |
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Our analysis of the neural plate was restricted to the one-to three-somite
stage, immediately preceding midline infolding. Owing to convergence, the
mediolateral extent of the neural plate encompassed in our LSM photographs
changed from 150 µm at the one-somite to 90 µm at the three-somite
stage; the depth of the epithelium increased from 10-15 µm to 25-36 µm.
As mitoses take place apically, a single level of focus was chosen to study
mitoses during the plate stage. A total of 327 mitoses were analysed in this
stage in four different embryos, and all were found to be planar, i.e.
essentially parallel to the plane of the neuroepithelium
(Fig. 4, see Movies 1 and 2 at
http://dev.biologists.org/supplemental/).
Most of the metaphase plates observed underwent striking planar rotations of
10° to 90°, which ceased when the cells entered anaphase
(Fig. 4A,B,F,G). These
movements are similar to, although not as pronounced as, the rotations of the
metaphase plate observed by Adams (Adams,
1996) in the ventricular zone of the developing cerebral cortex in
the rat, which extended up to 360°. Nevertheless, as in the rat cortex,
chromosomal complements separate at anaphase without major additional changes
in spindle orientation. For statistical analysis, the neural plate was
subdivided into three equal parts and the orientation of anaphase was measured
with respect to the midline. The anaphase can be oriented in any direction
with respect to the mediolateral or anteroposterior axes of the
neuroepithelium. However, mitoses within the medial region of the neural plate
are preferentially oriented in a mediolateral direction at
86.6°±35.9° (P=0.05). Mitoses within the other two
regions of the neural plate did not show any preferential orientation
(68.2°±39° 0.27<P<0.7 for intermediary and
12.38°±37.94° 0.24<P<0.49 for lateral
regions).
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Irregularities in the neural plate during infolding to form the neural keel
precluded observations of the transition from plate to keel stage. However,
the change from the orthogonal orientation of divisions in the neural keel/rod
to the planar orientation in the neural tube could be readily observed (see
Movie 6 at
http://dev.biologists.org/supplemental/).
This change occurred progressively, rather than suddenly. Mitoses with a
pronounced oblique orientation first appeared in the ventral-most levels of
the spinal cord, gradually progressing to dorsal levels; from the 15-somite
stage onwards, planar mitoses appeared gradually, again beginning at ventral
levels. Daughter cells resulting from mitoses with oblique orientation
remained on the same side of the neurocoel and were not distributed
bilaterally. As neurocoel formation follows the same ventral to dorsal course
(Schmitz et al., 1993), these
observations suggest that the transition from orthogonal to planar divisions
occurs when the two halves of the neural anlage separate from each other.
Postmitotic cells migrate into subventricular regions of the neural
tube
In the developing cerebral cortex of the ferret, the behaviour of
neuroepithelial cells after mitosis correlates with the orientation of the
mitosis (Chenn and McConnell,
1995). Roughly 15% of all mitoses detected were oriented
perpendicular to the ventricular surface and produced progeny that behaved
differently, in that one daughter cell remained at the ventricular surface
whereas the other migrated into subventricular regions. As no perpendicular
mitoses were seen in neural tube stage in the developing zebrafish spinal
cord, we wondered about the behaviour of the daughter cells resulting from
mitoses during this stage. In 52 of the divisions whose progeny could be
followed for a relatively long time, we observed that one of the daughter
cells migrated into subventricular regions, whereas the other remained at the
neurocoel (Fig. 6, see Movie 7
at
http://dev.biologists.org/supplemental/).
It is very likely that the migrating cells differentiated as neurons or glia
cells, as the subventricular region into which they moved contained
differentiating cells and was clearly separated from the region with
proliferating cells by a layer of neuropil
(Fig. 6). Unfortunately, we
could not ascertain whether any of the cells that remained at the neurocoel in
these 52 mitoses divided again. The observations therefore indicate that
mitotic spindle orientation does not correlate with the behaviour of daughter
cells in the developing spinal cord of the zebrafish.
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ASIP, a mammalian homologue of PAR-3, associates with the atypical protein
kinase C (aPKC) and plays an important role in controlling epithelial polarity
(Izumi et al., 1998;
Suzuki et al., 2001
;
Ohno, 2001
). A zebrafish
asip homologue, whose product shows 65% identity to ASIP from the
rat, was cloned and used to make three different reporter constructs:
asip:egfp includes the entire coding sequence,
asip-
5':egfp is an amino-terminal
deletion, and asip-
3':egfp is a
C-terminal deletion. These constructs were used to synthesise mRNA, which was
injected into the yolk of zygotes of either wild-type or
Tg(H2AF/Z)kca37; Tg(H2AF/Z)kca66
heterozygotes, and the injected embryos were observed by confocal microscopy
to follow ASIP:EGFP expression in vivo. As an additional control, mRNA
encoding EGFP only was injected into other embryos. After injections of
egfp mRNA, strong cytoplasmic labelling was observed in embryonic
cells from mid-gastrula stages on. A similar cytoplasmic distribution can be
observed following the injection of
asip-
5':egfp mRNA. By contrast,
injection of asip-
3':egfp or
asip:egfp mRNA resulted in membrane labelling. Whereas the C-terminal
deletion occasionally also labelled other structures, such as the mitotic
spindles, the full-length protein resulted in specific labelling of the apical
cell membrane (data not shown; Fig.
7J-L).
|
The localisation of aPKC can be followed with an antibody that recognises
two different isoforms, aPKC and aPKC
(Horne-Badovinac et al.,
2001
). In the neural plate (not shown), as well as dorsally in the
neural keel, which corresponds to the region of infolding of the neural plate
(Fig. 7M), we failed to detect
any specific staining. In the neural keel, weak staining was observed apically
in the neuroepithelial cells restricted to ventral levels
(Fig. 7N) (i.e. the region
where one wall of the neural keel has already made contact with the opposite
wall). At rod and tube stages, the epitope detected by this antibody was found
at the apical pole of the neuroepithelial cells, and the intensity of the
staining increased with the age of the embryos.
Other markers of adherens and tight junctions (revealed using phalloidin to
visualise F-actin and antibodies against ß-catenin, phosphorylated
tyrosine, and ZO-1) were found to behave in the same manner as ASIP:EGFP and,
in part, aPKC/
. The markers were diffusely distributed at the
cell membrane in neural plate stage, and became concentrated at the apical
pole of the neuroepithelial cells when the two halves of the neural primordium
were in apposition at the neural keel stage
(Fig. 8, and not shown). The
markers continued to concentrate at the apical pole in the neural tube stage.
All these observations indicate that important components of adherens and
tight junctions are either expressed in an unpolarised fashion or even absent
altogether at the neural plate stage, and that cell junctions mature over the
period from the keel to the tube stage.
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DISCUSSION |
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On the orientation of mitoses during neurulation
Technical differences make it difficult to compare our data to those
described by Kimmel et al. (Kimmel et al.,
1994). The latter authors injected rhodamine-dextran into
individual blastomeres and followed the development of the resulting clones
within the prospective hindbrain and spinal cord. They found that neural
progenitors undergoing the 15th embryonic division are oriented preferentially
along the anteroposterior axis, whereas cells at the 16th division adopt a
mediolateral orientation. The authors explicitly state that division 14 takes
place in early gastrula, whereas division 16 splits the clones bilaterally.
However, there is no direct indication as to when division 15 occurs during
neurulation. Division 16 should take place either in keel or in rod stage, as
only divisions in keel/rod stage lead to bilateral progeny
(Papan and Campos-Ortega,
1994
; Papan and Campos-Ortega,
1997
; Concha and Adams,
1998
) (this study, see below). Consequently, in the 11 developing
cell clones studied by Kimmel et al.
(Kimmel et al., 1994
) the 15th
embryonic division might have taken place in neural plate stage. As this
division was oriented anteroposteriorly, it might have occurred early in plate
stage, because, according to Concha and Adams
(Concha and Adams, 1998
),
mitoses in the early neural plate are oriented anteroposteriorly. Therefore,
according to this interpretation, the material analysed by Kimmel et al.
(Kimmel et al., 1994
) did not
reveal any mediolaterally oriented mitoses in neural plate stage.
Concha and Adams (Concha and Adams,
1998) analysed the direction of cytokinesis, whereas we have
observed anaphases; however, we can readily compare our data with theirs, as
we use the same points of reference. The authors found changes in orientation
during the neural plate stage, from anteroposterior (early) to mediolateral
(late). The material we used for the analysis of mitoses in plate stage, which
was restricted to the late neural plate (one to three somite), was processed
in the same manner as that studied by Concha and Adams, and we can confirm
their results: at the one- to three-somite stage, anaphase figures tend to be
oriented mediolaterally in cells located medially in the neural plate, whereas
in the remaining neural plate anaphase figures exhibit random orientation. We
would like to emphasise that divisions medially in the neural plate, although
oriented mediolaterally, are unrelated to those that lead to bilateral progeny
[the 16th division according to the terminology of Kimmel et al.
(Kimmel et al., 1994
)]; the
latter divisions are also oriented mediolaterally, but take place in the
keel/rod stage and are orthogonal following a 90° shift of the spindle
(see below), whereas the former are planar.
With respect to the neuroepithelium, mitoses are planar (parallel) in plate
and tube stage (Concha and Adams,
1998) (this study), and orthogonal (perpendicular) in keel/rod
stage (Kimmel et al., 1994
;
Papan and Campos-Ortega, 1994
;
Concha and Adams, 1998
). Our
present observations are compatible with the assumption that all mitoses in
plate stage are planar, for we observed no exceptions to this. However, the
late neural plate is engaged in energetic convergence movements and it is
frequently difficult to establish the orientation of a cell with respect to
the epithelium. Therefore, exceptions are conceivable. Our material strongly
suggests that all divisions in keel/rod stage are orthogonal, all of them
leading to bilateral progeny, and that all mitoses in tube stage are again
planar [as is implicit in figure
10 by Concha and Adams (Concha
and Adams, 1998
)]. As we observed the neural primordium from
above, it was difficult to establish any preferential orientation of mitoses
in tube stage along the dorsoventral and anteroposterior axes.
What determines mitotic orientation during neurulation in the zebrafish?
Concha and Adams (Concha and Adams,
1998) have discussed how mechanical tension and other forces may
contribute to steer the direction in which cells move in the epiblast and to
align mitotic divisions during gastrulation and neurulation. Indeed, the
planar orientation of mitoses in neural plate stage might well be mechanically
determined by factors acting on the neuroepithelium, for example from the
overlying enveloping layer. Concha and Adams
(Concha and Adams, 1998
)
emphasise stretching of the epiblast as a major element in orienting mitoses,
and propose that cells cleave in the direction of greatest elongation.
Although stretching may well be operative in this respect earlier in the
epiblast, the strong convergence movements that precede neural keel formation
seem to preclude stretching in plate stage. However, convergence itself might
contribute to the preferential mediolateral orientation of planar mitoses
within the medial one-third of the neural plate. Similarly, in keel/rod stage,
mechanical forces may be exerted by one half of the neural primordium on the
other half, with which it is in direct contact, and thus contribute to spindle
formation parallel to the zone of apposition of the two halves. Other factors
may also operate in the neural tube to determine the planar orientation of the
spindle in this stage.
Epithelial polarity and mitotic orientation
An additional factor might be represented by the polarity of the
epithelium. The well-established function of the Par3/Par6/aPKC protein
complex in controlling cell polarity in several animal species prompted us to
ask whether this complex might be involved in controlling mitotic orientation
in zebrafish neurulation. Indeed, Horne-Badovinac et al.
(Horne-Badovinac et al., 2001)
have described abnormalities in the orientation of dividing, has
(aPKC defective) retina cells. For convenience, we concentrated on the neural
tube stage, in which mitoses are always planar. Injection of ASIP morpholinos
caused the appearance, in tube stage, of a few perpendicular mitoses as well
as a relatively high proportion of obliquely oriented mitoses. Perpendicular
mitoses were never seen in the neural tube of uninjected embryos, we found
only one after injection of a control morpholino, and oblique mitoses were
less abundant and less pronounced in uninjected and control embryos. The
observations on the developing spinal cord of has mutants uncovered a
similar behaviour. The two extant alleles of the has gene,
m129 and m567, which encode C-terminal truncations of the
aPKC
that lack 73 and 69 amino acids, respectively, and have lost
kinase activity, are assumed to be null alleles
(Horne-Badovinac et al., 2001
;
Peterson et al., 2001
).
Homozygosity for the hasm129 allele leads to mild
disruption of spindle orientation in neural tube stage in the time span
encompassed by our observations: up to 10% perpendicular and another 9%
oblique mitoses were found. One possible explanation for the weak effects of
the ASIP morpholinos and those found in the spinal cord of has
mutants is functional redundancy, as two isoforms of the aPKC are known, which
are likely to be encoded by different genes. It is possible that additional
ASIP genes may also exist. Therefore, we conclude that ASIP and aPKC
are involved in controlling stereotypic spindle orientation in the
neuroepithelium, but that they represent only part of the controlling
machinery.
Orientation of mitotic divisions and behaviour of the daughter
cells
During neurogenesis in other animal species [for example in the cerebral
cortex of the mouse (Smart,
1973) and ferret (Chenn and
McConnell, 1995
) and in the retina of the rat
(Cayouette et al., 2001
) or the
zebrafish (Horne-Badovinac et al.,
2001
)], mitoses show a variety of orientations with respect to the
neuroepithelium. Thus, in the cerebral cortex of the ferret, planar mitoses
accounted for 85% of the cases, producing progeny that remained at the
ventricle and continued to divide. These mitoses were termed symmetrical. An
orientation orthogonal to the neuroepithelium was observed in 15% of all
mitoses (Chenn and McConnell,
1995
). Moreover, perpendicular mitoses were referred to as
asymmetrical because the progeny behaved differently: one of the daughter
cells continued to divide whereas the other migrated into subventricular
regions to differentiate. In his study of the cerebral cortex of the rat,
Adams (Adams, 1996
) failed to
find unambiguous evidence for perpendicular mitoses. Here, the mitotic spindle
was found to be oriented invariably parallel to the plane of the
neuroepithelium. Our present results indicate that perpendicular mitoses, if
they occur at all, are extremely rare in the developing spinal cord of the
zebrafish. Nevertheless, although all mitoses in tube stage were planar, one
of the daughter cells was seen to migrate away from the ventricular region in
52 cases. This figure appears very low in light of the fact that we analysed
several thousand mitoses in the neural tube. However, it is a minimal
estimate, as it corresponds only to those cases in which both progeny cells
remained within the same plane of focus and could be followed. In many other
cases, the two daughter cells were seen to separate from each other for a
given distance, but one cell then left the plane of focus and could not be
followed any further. We thus conclude that during zebrafish neurogenesis
spindle orientation cannot be correlated with equivalent or dissimilar
behaviour of the progeny cells.
Mitotic spindles rotate by 90° in the neural keel/rod stage
The orthogonal orientation of mitoses in keel/rod stage is brought about by
a 90° rotation of the mitotic spindle. In most divisions (89%) during this
stage, the spindle formed parallel to the plane of the neuroepithelium.
However, the spindle apparatus rotated by 90° and the ensuing division was
orthogonal. The striking correlation between apposition and separation of the
two halves of the neural anlage and orthogonal or planar orientation of
mitoses suggests a causal link between these phenomena. One can imagine that
in keel/rod stage a signal passes from one wall of the neural anlage to the
other and elicits the rotation of the spindle. This hypothetical signal would
be interrupted as the neurocoel forms and the mitotic spindle would not rotate
but remain planar in tube stage.
The 90° rotation of the mitotic spindle in neural keel/rod stage raises two questions. One is related to the mechanism and the molecular control of the rotation; the other is whether the change from a planar to an orthogonal orientation of the mitosis actually has any functional significance.
Two cases of mitotic spindle rotation occur during neural development in
Drosophila, which are strikingly similar to the rotation described
here. Although Drosophila neuroectodermal cells divide in the plane
of the epithelium, after delamination, spindle orientation in the neuroblasts
turns by 90° immediately prior to mitosis, and division takes place
orthogonal to the neuroectoderm
(Kaltschmidt et al., 2000)
(for a review, see Knoblich,
2001
). Two different mechanisms are involved in the control of
mitoses in the neuroectoderm and the neuroblasts. The first mechanism is
provided by adherens junctions and by the subapical protein complex, which
exert control over the planar divisions in the neuroectoderm
(Lu et al., 2001
). When the
neuroblasts delaminate in wild-type embryos, the junctions disappear and the
rotation of the mitotic spindle is brought about by the second mechanism, i.e.
the apical localisation of Inscuteable
(Kraut and Campos-Ortega,
1996
; Kraut et al.,
1996
), in association with Bazooka
(Schober et al., 1999
;
Wodarz et al., 1999
), Partner
of Inscuteable (Pins) (Yu et al.,
2000
; Schaefer et al.,
2000
), Drosophila Par-6 (Petronzcki and Knoblich, 2001)
and Drosophila aPKC (Wodarz et
al., 2000
). Lu et al. (Lu et
al., 2001
) have reported that in crumbs mutants or after
misexpression of crumbs, the junctions disappear and, consequently,
the plane of division of the neuroectodermal cells is randomised. A similarly
decisive role has been proposed for junctional proteins in directing spindle
orientation in dividing pIIa and pIIb cells in the lineage of
Drosophila sensory bristles (Le
Borgne et al., 2002
). The pI progenitor divides asymmetrically in
the plane of the epithelium, giving rise to pIIb, which delaminates partially,
and pIIa, which remains in the epithelium. A strong accumulation of
Drosophila E-Cadherin,
-Catenin and ß-Catenin is observed
in pIIb at its point of contact with pIIa, while the localisation of these
markers in pIIa is the same as in the progenitor pI. The division of pIIa is
also planar, suggesting that the junctional protein complex directs the
orientation of the division. By contrast, in the partially delaminated pIIb
cell, there is a strong accumulation of junctional proteins in an apical stalk
that remains in contact with pIIa. In the stalk, Bazooka recruits Inscuteable
and Partner of Inscuteable apically in the neighbourhood of the junctional
complex, and thus ensures that the mitosis takes place orthogonally to the
epithelium (Le Borgne et al.,
2002
).
We are reluctant to draw any firm conclusions as to whether junctional
proteins play any role in controlling the rotation of the mitotic spindle in
the zebrafish neural keel/rod. The present observations indicate that adherens
and tight junctions mature in the period between neural keel and neural tube,
i.e. during the phase in which mitotic spindles undergo the 90° rotation.
Therefore, spindle rotation in the neural keel/rod does not seem to require
perfectly polarised cell junctions, and the mechanism controlling spindle
rotation in the zebrafish neuroepithelium might well be different from that in
the two examples from Drosophila. However, the change in expression
of ASIP:EGFP and aPKC/
during mitoses in keel/rod and tube
stages is very striking, although its significance is not yet clear. Whereas
apical localisation of these proteins in cells in keel/rod stage is lost upon
entry into prophase, the apical localisation is retained when the cells of the
neural tube divide. This may be simply a reflection of the fact that cell
junctions are not firmly established and well polarised until the late rod or
early tube stage. But one cannot exclude the possibility that the mitotic
spindle is reoriented as soon as the junctional proteins lose their apical
localisation in dividing cells in keel/rod stage, thus determining the 90°
rotation. Therefore, the question of causal relationships between junctional
proteins and spindle rotation in the neuroepithelium of the zebrafish must
remain open.
Functional significance of the shift of mitotic spindle
orientation
The orthogonal divisions in neural keel/rod stage are obviously related to
the bilateral distribution of progeny. As mentioned, cell clones with a
bilateral distribution have been described at several levels on the zebrafish
neuraxis after single cell labelling
(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
). Our current results indicate that most, if not all, mitoses
that take place in neural keel/rod stage lead to bilateral progeny. As sister
cells within cell clones differentiate into distinct cell types on either side
of the spinal cord (Papan and
Campos-Ortega, 1997
; Papan and
Campos-Ortega, 1999
), perpendicular mitoses leading to bilateral
progeny might well be accompanied by a differential distribution of
cytoplasmic determinants. Experimental support for this hypothesis is
pending.
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ACKNOWLEDGMENTS |
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Footnotes |
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