Wellcome Trust/Cancer Research UK Institute, Tennis Court Road, Cambridge
CB2 1QR, UK
Department of Anatomy, Downing Street, University of Cambridge,
Cambridge, UK
Author for correspondence (e-mail:
np209{at}mole.bio.cam.ac.uk)
Accepted 6 March 2003
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SUMMARY |
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Key words: Xenopus, Superficial cells, Deep cells, Oriented cell division, Neurogenesis, Competence, aPKC, Par proteins, Epithelial polarity, Occludin
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INTRODUCTION |
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The deep versus superficial cell distinction is particularly important in
the development of the frog nervous system because postmitotic primary
neurones, which differentiate early, are derived solely from the deep cells,
while the superficial cells give rise to longer dividing precursors
(Hartenstein, 1989;
Chalmers et al., 2002
). We have
previously investigated whether the different fate between inner and outer
cells is due to an intrinsic difference or is externally imposed. We have
shown that the two cell populations are intrinsically different, already at
late blastula/early gastrula stage
(Chalmers et al., 2002
).
Specifically, deep-layer cells isolated from a late blastula stage embryo are
receptive, while the superficial layer cells are refractory, to signals that
promote neuronal differentiation. At the neurula stage, a number of genes are
differentially expressed between the two cell types and the expression of at
least one gene, the hairy/enhancer of split related ESR6e, is
restricted to superficial cells from the onset of zygotic transcription
(Chalmers et al., 2002
;
Deblandre et al., 1999
).
ESR6 inhibits primary neurogenesis when it is misexpressed in deep
cells and may therefore underlie the low differentiation potential of
superficial cells (Chalmers et al.,
2002
).
One mechanism to generate deep and superficial cells would be ingression or
delamination of superficial cells to the interior of the embryo. However,
timelapse video microscopy has shown that this is not the case in the frog
embryo (Keller, 1978). An
alternative method would be orientation of the mitotic spindle perpendicular
to the surface of the embryo, thereby generating a superficial cell and a deep
cell upon division. Oriented cell divisions, where the spindle is aligned with
an axis of cell polarity, play an important role in cell fate diversification
in many other systems. Two well-studied examples are the division of
Drosophila neuroblasts and the C. elegans zygote (reviewed
by Chia and Yang, 2002
;
Jan and Jan, 2001
;
Guo and Kemphues, 1996
;
Wodraz, 2002). In both cases, cell fate determinants are localised along an
axis of polarity in the dividing cell and are differentially inherited by the
daughter cells, which then acquire different fates.
In this work, we sought to investigate whether oriented cell divisions play a similar role in the frog embryo. We asked three main questions: do oriented divisions take place, what are their properties and do they underlie the fate diversification of deep versus superficial cells? We show that the divisions of frog blastomeres can be grouped into three classes based on the orientation of the spindle relative to the surface of the embryo and the arrangement of the resulting daughter cells: parallel, oblique and perpendicular. Perpendicular divisions generate inner cells, starting at the sixth cleavage (32-64 cell) and continue to do so until the start of gastrulation. We show that equal numbers of perpendicular divisions occur in each quadrant of the embryo but the spatial distribution of perpendicular divisions is variable between embryos. However, in each embryo, cells with a small apical surface and a long apicobasal axis, show a very high probability of dividing perpendicularly in the next division. This suggests that the choice of division plane orientation correlates with cell shape and tends to bisect the long axis of the cell.
To investigate whether the progeny of perpendicular divisions are
molecularly distinct, we examined the distribution of aPKC, an important
molecule in oriented divisions of polarised cells in other systems (reviewed
by Knoblich, 2001). We show
that aPKC is localised to the apical membrane throughout the early cleavage
stages and the membrane localised aPKC is inherited only by superficial cells.
Finally, to prove that the differences between deep and superficial cells can
be traced back to the perpendicular division that generated them, we isolated
64-cell blastomeres, separated their progeny and cultured them in vitro. We
show that only clones derived from cells that have inherited the apical
membrane express ESR6e.
Based on these findings, we propose that orientated divisions generate deep and superficial cells in the Xenopus blastula and play a key role in directing these cells towards different fates.
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MATERIALS AND METHODS |
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Histology
Embryos were fixed in MEMFA [0.1 M MOPS (pH 7.4), 2 mM EGTA, 1 mM
MgSO4, 3.7% formaldehyde] dehydrated through an ethanol series and
embedded following manufacturers instructions in Immuno-Bed a
Methoxy-Methylmethacrylate solution, (Polysciences). Serial sections (7 µm)
were cut on a Leica rotation microtome, stained with Ehrlich's Haematoxylin
and mounted in DPX (BDH Laboratory Supplies).
Immunohistochemistry (wholemount and cryosections)
Albino embryos were fixed at the required stage in Dent's fix with the
vitelline membrane removed, unless stated otherwise. Embryos to be wholemount
stained were washed in BBT (PBS+1% BSA+0.1% Triton X-100), then BBT+5%
heat-treated lamb serum, incubated with the primary antibody overnight at
4°C in BBT+serum, washed four times for 1 hour in BBT and blocked for 1
hour in BBT+serum. They were then incubated overnight in the secondary
antibody and washed four times for 1 hour in PBT (PBS+0.1% Tween 20). Embryos
that were Cytox Green stained were washed in TBST (TBS+0.1% Tween 20) then
incubated overnight in TBST+Cytox Green (1:1000, Molecular Probes), washed
four times for 30 minutes in TBST. Embryos were then dehydrated in methanol
and cleared in benzylbenzoate:benzyl alcohol (2:1). Embryos that were imaged
from the side were cut twice to give two flat surfaces prior to imaging.
Cryosections were prepared as previously described
(Fagotto and Gumbiner, 1994).
Sections were then washed in acetone, PBS and blocked in PBS+1% BSA+5% serum
for 30 minutes incubated with primary antibody for 2 hours, washed three times
in PBS, blocked in PBS+1% BSA+5% serum, incubated with the secondary antibody
for 1 hour, washed three times in PBS and mounted in Vectashield (Vector
Laboratories). Embryos were imaged on a BioRad Radiance confocal
microscope.
The following antibody combinations were used: anti -tubulin 1/2000
(Sigma, DM1A, T9026) with anti-mouse TRITC 1/250 (Sigma, T7782) and Cytox
Green (1/1000) (Molecular Probes) (Qian et
al., 1998
); anti occludin 1/1000 (kind gift from Dr S. Citi)
(Cordenonsi et al., 1997
) with
anti-rabbit cy3 1/1000 (Amersham Pharmacia, PA 43003); anti aPKC 1/200 [Santa
Cruz, nPKC
(C-20) sc-216] with anti-rabbit cy3 or anti-rabbit Alexa 488
1/1000 (Molecular Probes, A-11008) (the aPKC antibody is the same as used
previously (Nakaya et al.,
2000
) and recognises both
and
atypical PKC
isoforms, so the staining represents the combined aPKC distribution and is
referred to as aPKC); and anti ß1 integrin 1/200 (Developmental Studies
Hybridoma Bank, 8C8) (Gawantka et al.,
1992
) with anti-mouse Alexa 488 1/1000 (Molecular Probes,
A-11001).
Timelapse video microscopy
Ten embryos from different batches with a clear dorsoventral difference
(lighter pigment, smaller blastomeres on dorsal side) were filmed from the
eight-cell until the 4096-cell embryo using a Leica MZFL111 microscope, a
coolsnap camera (Photometrics) and Openlab software. After filming, each
embryo was left to gastrulate to confirm that is was developing normally.
Division type was established for each cell by scrolling the movie back and
forth, and was marked on a still image of the respective division stage. In
the examples presented, the embryo was traced and each cell shaded with a
colour representing the type division it is about to undergo in the next
cleavage (see Movie at
http://dev.biologists.org/supplemental).
For analysis of dorsoventral differences and analysis of the division orientation after an oblique or perpendicular division, embryos were labelled with Nile Blue at the four-cell stage on the pole that contained the two smaller, lighter pigmented blastomeres and filmed until the 1024-cell stage. They were left to develop to stage 10, when the formation of the blastopore lip indicates the dorsal side, and fixed. Thus, the future dorsoventral axis could be unequivocally identified in the five movies analysed. Lineage diagrams were produced for all descendants of each of the four animal blastomeres at the eight-cell stage up to the 1024-cell stage. The division type that each cell is about to undergo was recorded on the diagram. For dorsoventral differences, the division type from all the daughters of each of the four blastomeres was calculated as percentages for each blastomere per embryo and averaged for five embryos analysed. For analysis of division orientation after an oblique or perpendicular division, each division of that type (e.g. oblique) in the five movies (eight- to 1024-cell stage) was identified and the division types that followed the division was scored and used to calculate percentages for each orientation of division.
Quantitative real time RT-PCR (QT RT-PCR)
Isolated blastomeres (see above) were allowed to divide. After a
perpendicular division the deep and superficial daughters (scored by presence
of pigment) were separated and cultured. At stage 10, 20 deep and 20
superficial clones were pooled (only where both daughters of a division
survived) and snap frozen for QT-RT PCR. Fifteen stage 10 caps were also
frozen for establishing standard expression levels. Quantitative real-time PCR
was carried out using the lightcycler system (Roche) as per instructions and
described previously (includes primers and conditions)
(Chalmers et al., 2002
) except
the 1 step RNA kit (Cat no 2 015 137) was used for two-thirds of the
independent experiments. When using the RNA kit 6mM magnesium was used. The
stage 10 cap sample was used to generate a standard curve and the expression
of each experimental sample compared with this sample where 100 is the level
in the control cap. The results presented are the mean of three independent
experiments.
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RESULTS |
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In order to establish whether oriented cell divisions are responsible for generating the deep cells, we stained the mitotic spindle and the chromosomes of embryos from a range of cleavage stages (Figs 2, 3). This showed three classes of divisions, which were named parallel, perpendicular and oblique, according to the orientation of the spindle and the arrangement of the resulting daughter cells. Parallel divisions had their spindle oriented in a plane parallel to the surface of the embryo (Fig. 2A,B, blue arrows) and could assume any orientation within this plane (Fig. 2A). A parallel spindle is predicted to lead to a cleavage plane perpendicular to the surface and so give rise to two, parallel, superficial cells (Fig. 2E). Perpendicular divisions had their spindle orientated perpendicular to the surface (Fig. 2A,B; red arrow). These cells are predicted to have a cleavage plane parallel to the surface and so give rise to a superficial and deep cell arranged perpendicularly to the pigmented surface (Fig. 2E). Finally, oblique divisions had the spindle orientated between these two (Fig. 2B, green arrow), giving rise to two superficial cells, one with a small and one with a big external surface (Fig. 2E). To exclude the possibility that the spindles rotate to line up with the plane of the external surface at a later point of cell cycle the spindle orientation in anaphase was examined. The three different types of spindle orientation were still present in anaphase (Fig. 2C+D).
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Perpendicular divisions start at 32 cell stage and continue up to the
start of gastrulation
Perpendicular divisions, which generate the deep cells, may take place
during one or several cell divisions and either a few or the majority of the
cells may be involved. To distinguish between these possibilities, we examined
sequential divisions during early development. At the fourth cleavage (eight-
to 16-cell stage) (data not shown) and the fifth cleavage (16- to 32-cell
stage) the orientation of the spindle in most cells was parallel or
occasionally oblique (Fig.
3B,C).
Perpendicular spindles, which generate inner cells, were observed from the
sixth cleavage (32- to 64-cell stage) up to stage 9
(Fig. 3). The perpendicular
divisions could be seen in metaphase and anaphase spindles
(Fig. 3). At stage 10, they
were very rare (1/16 embryos) and were not found at stage 11 (0/5 embryos),
where all spindles were parallel to the surface (data not shown). Throughout
these stages, deep cells continue dividing (dividing deep cells can be seen in
Fig. 2B), thereby increasing
further the number of inner cells, but no attempt was made to follow their
orientation of division. Perpendicularly oriented spindles were also observed
in the marginal zone and the vegetal pole but were not characterised
further.
The three orientations of division can be identified using timelapse
video microscopy
To verify these findings using an independent method and to follow these
divisions in vivo, we took advantage of the external development of the frog
to analyse timelapse movies of early development. In this analysis, we made
the assumption that any cell that appears not to divide when viewed from the
outside, while its neighbours divide, must be generating an internal cell and
was scored as a perpendicular division. This is based on previous observations
that up to the 13th cleavage cell divisions are synchronous in the
Xenopus embryos (Clute and Masui,
1992; Newport and Kirschner,
1982
; Sato, 1977
;
Wang et al., 2000
). Cell
counts in other amphibia (Hara,
1977
) and the antibody staining shown above also provide
independent verification that all early blastomeres divide synchronously. A
division that gave rise to two superficial cells of equal external surface was
scored as a parallel division, and a division that gave rise to daughters of
unequal external surface (more than 1:2 difference) was scored as an oblique
one. An example of each kind of division
(Fig. 4), the results from one
movie (see Movie at
http://dev.biologists.org/supplemental/;
Fig. 5A) and a quantitation of
each division type over 10 embryos (Fig.
5B) are shown. The diagrams presented are tracings of still images
from the movies labelled with the type of division the cells are about to
undergo (see Materials and Methods). A timelapse movie is also available (see
Movie at
http://dev.biologists.org/supplemental/).
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|
The perpendicular divisions do not follow a set spatial pattern
To understand how the choice of division plane might be regulated in the
embryo, we examined a number of parameters that might influence it.
Perpendicular divisions could preferentially take place in one area of the
embryo, perhaps revealing the influence of signalling from that area of the
embryo. For example, perpendicular divisions could be biased towards the
dorsal or ventral side. Therefore, we calculated the number of perpendicular
divisions for the daughters of each of the four animal blastomeres of an
eight-cell stage embryo (see Materials and Methods). Equal numbers of deep
cells were generated from the four animal blastomeres and therefore there is
no bias across the prospective dorsoventral and left/right sides of the embryo
(Fig. 5D).
Despite the constant average numbers of divisions seen at different divisions and in different regions of the embryos, different movies showed that the perpendicular divisions were not arranged in a reproducible pattern. Instead there was a different distribution in each embryo (the pattern of division is shown for four embryos at 128-cell stage; Fig. 5C).
Another way of controlling number of perpendicular divisions would be by a process similar to lateral inhibition, i.e. once a cell has been `selected' to divide perpendicular it may inhibit its neighbours from doing so. However, this was not found to be the case as analysis of the timelapse movies showed that cells divide perpendicular in small clusters, doublets or single cells (Fig. 5). Finally, it was possible that once a cell starts to divide perpendicularly, it continues to divide in this orientation. The timelapse data showed that cells can divide perpendicular more than once in a row (29%, n=219 cells); however, they are not fixed in this orientation as they also often divide in parallel after a perpendicular division (57%, n=219 cells). In summary, the distribution of perpendicular divisions appears to vary between embryos and is not fixed through sequential divisions of an individual cell. However, the average numbers of divisions is fairly constant over several divisions and in each quarter of the embryo.
The orientation of division correlates with cell shape
What mechanism could produce this pattern of divisions? A potential answer
comes from an observation that cells with a small apical surface had a high
probability of dividing in a perpendicular orientation. These cells were
usually the result of a previous oblique division that generates two cells
with unequal apical surfaces (example of this is shown in
Fig. 4B). To confirm and
quantitate this observation, the orientation of division for each daughter of
an oblique division was analysed (Fig.
6A). The daughter cells with the small external surface (oblique
small) were found to divide perpendicular in 88% of cases and parallel in just
6% of cases, while the daughter cells with the big external surface (oblique
big) divided perpendicular in only 11% of cases and parallel in 68% of
cases.
|
aPKC is asymmetrically localised during the perpendicular
divisions
One could argue that deep and superficial cells differ simply in their
location in the embryo (inner or outer). Cells may have initially equal
developmental potential but they may follow different fates by virtue of their
exposure to different environments. Alternatively, differences in the
development of deep and superficial cells could be directly linked to the
oriented cell divisions that generated them, perhaps by virtue of differential
inheritance of cell fate determinants. In Drosophila and C.
elegans a conserved complex of proteins, consisting of Par3, Par6 and
aPKC, is asymmetrically localised during oriented cell divisions (reviewed by
Knoblich, 2001;
Doe, 2001
). As these proteins
are also present in early Xenopus embryos
(Choi et al., 2000
;
Nakaya et al., 2000
) they may
be asymmetrically localised during the oriented divisions.
In order to test the subcellular localisation of aPKC, cryosections of
cleavage stage Xenopus embryos were analysed by antibody staining. In
blastula embryos, aPKC showed a striking localisation, with high levels in the
apical membrane of the superficial cells (Fig.
7A,B,E).
As a comparison, embryos were stained for occludin, a component of vertebrate
tight junctions. Occludin was localised along the basolateral membrane domains
of the superficial cells and all around the deep cells with equal intensity
(Fig.
7C,F).
The occludin staining along the basolateral membrane is consistent with
previous reports of a gradient of occludin protein localisation with a high
point at the apical junctions. However, we observed less of a gradient along
the basolateral membrane than previously reported
(Fesenko et al., 2000) and
additional staining around the deep cells. This is most probably due to the
use of cryosections for immunohistochemistry in our work. The role of
basolateral occludin is unclear but it has been postulated that it might
provide Ca2+-independent adhesion or a temporal store of the
protein (Fesenko et al.,
2000
).
|
Interestingly, in one- and four-cell embryos the animal hemisphere showed
enrichment of aPKC compared to the vegetal hemisphere (Fig.
7J,L),
consistent with previous reports using whole-mount embryos
(Nakaya et al., 2000).
However, in sections it was clear that the animal enrichment is due to
cytoplasmic aPKC (Fig.
7J,L).
By contrast, the apical membrane localisation described here is present in
both the animal and vegetal hemispheres.
The apical localisation of aPKC suggests that it would only be inherited by
the superficial cells after a perpendicular division. Double staining of
blastula embryos with aPKC and tubulin showed that the apical localisation of
aPKC is maintained in cells undergoing parallel and perpendicular divisions
(Fig.
7G,H).
Thus, the superficial but not the deep daughter cell would inherit membrane
localised aPKC after a perpendicular division. To confirm that aPKC is
asymmetrically inherited after the divisions the localisation of aPKC was
established in isolated blastomeres dividing in culture. The isolated
blastomeres were double stained with anti aPKC and ß1 integrin [which is
localised to the basolateral membrane domains
(Gawantka et al., 1992)]. The
apical localisation of aPKC was found to be present in isolated blastomeres
(Fig. 8A) and so does not
require cell contact. This is consistent with previous results showing that
isolated blastomeres maintain epithelial polarity
(Muller and Hausen, 1995
;
Cardellini et al., 1996
;
Fesenko et al., 2000
). In
isolated blastomeres fixed in telophase and immediately after cytokenesis, the
division could be seen to generate one polarised daughter with apical aPKC and
one non-polarised without any membrane localised aPKC (Fig.
8B,C).
Therefore, the oriented cell divisions generate cells with different molecular
components.
|
Together with the aPKC results, these findings show that the deep and superficial cells are molecularly distinct from the outset of their generation and at the start of zygotic transcription, they show at least one differentially expressed gene, in the absence of any other signals from the embryo.
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DISCUSSION |
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In this study, we have investigated the origin of these two cell populations in order to understand how this difference in development potential is established. We show that deep and superficial cells are generated by a series of divisions oriented perpendicularly to the surface of the embryo, that take place between the 32-cell stage and the start of gastrulation.
Oriented cell divisions are widely encountered in the development of
metazoa as a means of generating cell fate diversity. To date, most of our
understanding of oriented divisions comes for work in invertebrate systems,
particularly Drosophila neuroblasts and C. elegans early
development (Knoblich, 2001).
The model that has emerged from these systems has two main components: the
localisation of cell fate determinants along an axis of polarity and the
precise alignment of the spindle with this axis. How similar is early
Xenopus to those other well characterised systems? We argue that
early oriented divisions in Xenopus show similarities but also some
differences from these other systems.
aPKC is a marker of blastomere polarity and is differentially
inherited
Similar to other systems that rely on oriented divisions to generate cell
diversity, early frog blastomeres are very clearly polarised, along the
apicobasal axis. The basolateral domain is inserted during cleavage and
expresses a number of known proteins such as B1-integrin, U-cadherin and a
catenin proteins (Angres et al.,
1991; Gawantka et al.,
1992
; Schneider et al.,
1993
). By contrast, the composition of the apical membrane, which
is inherited from the egg, has been largely unknown with the exception of the
tight-junction protein cingulin, which is recruited from the apical cortex
into junctions during early embryogenesis
(Cardellini et al., 1996
;
Fesenko et al., 2000
). We have
shown here that aPKC localises to the membrane of the fertilised egg and later
to the apical side of the blastula stage superficial cells. During
perpendicular divisions, membrane-localised aPKC is preferentially inherited
by the superficial cells. In many other polarised cells, aPKC is also
distributed asymmetrically, often found in the apical domain, and in the cases
where it has been mutated, polarity has been disrupted
(Horne-Badovinac et al., 2001
;
Izumi et al., 1998
;
Tabuse et al., 1998
;
Wodarz et al., 2000
). Thus,
the first epithelium in frog development shows molecular features of polarity
that are present in later epithelia and are conserved across species.
Previous work on the localisation of aPKC in Xenopus, using
whole-mount specimens, showed that aPKC is enriched in the animal hemisphere,
similar to the localisation in the C. elegans embryo
(Nakaya et al., 2000). We have
confirmed the animal pole enrichment of aPKC but we have found that this
enrichment is mainly cytoplasmic. By contrast, we have found
membrane-localised aPKC that is present all around the egg and is subsequently
inherited by the apical side of blastomeres. The role of the cytoplasmic aPKC
is not clear but only the membrane-localised aPKC is clearly differentially
inherited during the perpendicular (apicobasal) blastomere divisions.
Therefore, we propose that (apical) membrane enrichment, rather than the
animal cytoplasmic enrichment, may be more comparable in its potential cell
fate determining role to the anterior membrane localisation seen in C.
elegans.
Oriented divisions diversify deep and superficial cells
An important question is whether the perpendicular divisions that we
describe here generate cells that are non-equivalent in their developmental
potential. We observed that a single isolated 64-cell stage blastomere can
divide perpendicularly in culture and generate one polarised cell (normally a
superficial cell) that goes on to express the bHLH gene ESR6e and one
apolar cell (normally a deep cell) that does not. This finding suggests that
the perpendicular division, rather than any external influence, diversifies
the progeny of this division. In the embryo, perpendicular divisions precede
neural induction; therefore restricted expression of ESR6e in outer,
polarised, ectodermal cells may simply bias their response to inducing signals
later in development.
Differences from other systems
The first four divisions in Xenopus show a high degree of
reproducibility between embryos. However, perpendicular divisions, which start
at the sixth cleavage (32-cell stage) and continue until the blastula stage,
do not follow a stereotyped pattern either between embryos or from one
division to the next. They show no dorsoventral bias in their distribution and
can occur in individual cells or in clusters of cells. Furthermore, cells are
not committed to a particular mode of division and can flip between different
orientations in consecutive divisions. These are fundamental differences from
the situation in early C. elegans development
(Guo and Kemphues, 1996) where
the division pattern is invariant and from Drosophila neuroblasts
that, once specified to delaminate, are committed to an asymmetric mode of
division (Knoblich, 2001
). The
lack of stereotyped pattern in the Xenopus embryo implies an element
of randomness in the selection of cells that divide perpendicularly which may
be lacking in the Drosophila or C. elegans systems. The lack
of a stereotyped pattern sharply contrasts with the near constant percentage
(25%) of perpendicular divisions, over several consecutive stages, which
ensures that each embryo goes into gastrulation with roughly the same number
of inner versus outer cells.
How do frog blastomeres decide which way to divide? We have found that the
orientation of division for both the parallel and perpendicular divisions
appears to correlate with the shape of the cells. In the case of perpendicular
divisions, we have shown that cells that are elongated along the apicobasal
axis have a very high probability of dividing as to generate an inner cell.
These observations provide a model where the orientation of division during
early cleavage stages is influenced by cell shape and may be subject to simple
geometrical rules. The longest axis model would also be self-regulating, as
each division would reduce the length of the long axis and so make the next
division less likely to be along the same axis. This situation changes in the
late blastula, when both and deep and superficial cells are oriented within
the plane of the epithelium (this work)
(Marsden and DeSimone, 2001),
which, in the deep cells, depends on the presence of a fibronectin-rich
extracellular matrix (Marsden and
DeSimone, 2001
).
In this work, we propose that, in Xenopus, the choice of spindle
orientation correlates with cell shape, which may also be the case for the
mouse blastula stage divisions (Johnson
and Ziomek, 1983). The influence of cell shape on the orientation
of division has been noted in earlier embryological studies [as for example in
Hertwig's rules of cell division (see
Wilson, 1987
)] but has not
received much attention in recent years, presumably because in many instances
in development it can be overridden by other cues
(Concha and Adams, 1998
;
Goldstein, 2000
). What is
interesting here is that a system of oriented divisions that is guided by cell
shape is associated with the generation of cell fate diversity. Such a system
may be inherently more flexible than the systems found in Drosophila
and C. elegans and may in fact be better suited for early vertebrate
development, where the cell fate choices are less hard-wired. Indeed, for
early vertebrate development, it may be important to generate sufficient inner
and outer cells and to segregate molecules that will bias their developmental
potential, but it may be less important to do this with single cell accuracy.
This would explain why, as we describe here, the localisation of molecules
along an axis of polarity appears to be a highly conserved feature of oriented
divisions but the precise alignment of the spindle with this axis of polarity
appears not to be so.
Oriented divisions occur during early development of other
vertebrates
Oriented divisions that segregate an external polarised cell from an
internal non polarised cell, are observed in other vertebrates such as the
mouse and the zebrafish. Similar to the situation in Xenopus, in
these other cases, the outer cells follow different developmental pathways
from the inner cells (enveloping layer and embryo proper in zebrafish;
trophectoderm versus inner cells mass in the mouse)
(Johnson and Ziomek, 1981;
Pedersen et al., 1986
;
Fleming, 1987
;
Sutherland et al., 1990
;
Kimmel et al., 1995
). However,
neither the existence of determinants nor the spatial pattern of these
divisions has been established for the mouse or zebrafish early embryo.
Oriented divisions have also been reported in the vertebrate cerebral cortex
and retina and are thought to asymmetrically localise cell fate determinants
(reviewed by Lu et al., 2000
;
Cayouette et al., 2001
).
However, the existence of perpendicular (apicobasal) divisions in the
vertebrate retina has been disputed and the significance of asymmetric
divisions in this system is unclear (Silva
et al., 2002
; Das et al.,
2003
)
We would like to suggest that the early oriented divisions in Xenopus represent a new system for the study of differentiative cell divisions in vertebrates that is easily tractable and where the two daughters of the division can be experimentally manipulated in vivo and in vitro. Finally, these early oriented divisions in the frog are relevant to the development of the nervous system as they generate cells that are inherently different in molecular terms and in their ability to undergo primary neuronal differentiation.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Joint first authors (listed alphabetically)
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REFERENCES |
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