Download When Cells Tell Their Neighbors Which Direction to Divide

DEVELOPMENTAL DYNAMICS 218:23–29 (2000)
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When Cells Tell Their Neighbors Which Direction
to Divide
BOB GOLDSTEIN*
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
ABSTRACT
Many cells must divide in specific orientations, yet for only a handful of cases
do we have some understanding of how cells
choose division orientations. We know of only a
few cases where division orientations are controlled by specific cell– cell interactions. These
cases are of interest, because they tell us something new and seemingly fundamental about how
cells can function during development. Here, the
evidence that interactions control division orientation in some cells of the early C. elegans embryo is presented, and what is known about how
contact can regulate division orientation is discussed. Whether contact-mediated division orientation is a peculiarity of C. elegans or whether
it may be more widespread is addressed. Dev Dyn
2000;218:23–29. © 2000 Wiley-Liss, Inc.
Key words: cell division orientation; cell interaction; cytoskeleton; mitotic spindle;
Wnt pathway; C. elegans
INTRODUCTION
Many cells must choose their division orientations
carefully. For example, a cell in a developing monolayer epithelium must divide in a particular plane for
its daughters to remain in the monolayer epithelium,
and a cell in which certain components are asymmetrically localized must orient its division axis if it is to
partition such components to just one daughter cell
(Fig. 1).
Embryologists have been isolating embryonic cells
for more than a century, and for scant few cases do we
know that division orientations are affected, suggesting at first glance that cell contacts may be generally
dispensable for proper division orientations. However,
in most cases, the appropriate landmarks for proper
division orientation are not apparent. Therefore, the
question of whether division orientation requires specific cell contacts has remained unaddressed in most
instances.
We have known for some time that cell shape can
play a role in division orientation, as mitotic spindles
tend to shift into the longest axis of a cell as the
growing asters run out of room (Hertwig, 1895). Hence,
© 2000 WILEY-LISS, INC.
cells, or physical barriers such as a vitelline envelope,
can influence a cell’s division axis by altering cell
shape. Only recently have we learned that cells also
can specifically orient division axes of certain neighbors; this is the topic of this review.
CELL CONTACTS ORIENT SOME CELL
DIVISION AXES IN C. ELEGANS
In the course of experiments using cultured C. elegans embryonic cells to investigate a potential role for
cell interactions in specifying certain cell fates, some
cells, when placed in contact with certain other cells,
were found to divide in predictable orientations. After
placing cells in contact in various orientations, one cell
consistently divided in an axis that left its daughters in
line with the contacting cell, suggesting that certain
cells can induce specific division orientations in certain
other cells (Goldstein, 1995).
Which Cells Can Have Their Divisions Oriented
by Cell Contacts?
By making pairwise combinations of all cells of the
four cell stage C. elegans embryo, one cell was found to
be capable of inducing division orientation, and another cell was found to be capable of responding by
orienting its division axis. The names of cells in early
C. elegans development are shown in Figure 2; the cell
capable of inducing is called P2, and the cell capable of
responding is called EMS. As most of the ensuing experiments have involved these two cells, for simplicity
P2 is often referred to here as the “inducing cell,” and
EMS as the “responding cell.” There are, however,
other cells which can induce and other cells which can
respond: similar experiments performed with selected
cells of the eight cell stage revealed that both daughters of P2 (P3 and C) could orient the divisions of both
daughters of EMS (E and MS). In the eight cell stage
embryo, neither daughter of the inducing cell touches
the MS cell, suggesting that in normal development
MS has an unrealized potential to respond. These re-
*Correspondence to: Bob Goldstein, Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280.
E-mail: [email protected]
Received 10 January 2000; Accepted 8 February 2000
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GOLDSTEIN
Fig. 1. The consequences of alternative spindle orientations for a
polarized cell (A) and for a cell in a monolayer epithelium (B). Black areas
in (A) represent asymmetrically localized components.
sults, along with cell isolation experiments in other
cells (Goldstein, 1995) and results described below
have led to the generalization that cell division orientations can be regulated in two ways in the early C.
elegans embryo— by cell interactions in certain somatic
cells, and cell-autonomously in germline precursor cells
(Fig. 2).
Fig. 2. The 1-, 2-, 4-, and 8-cell stages of C. elegans. Cell division
orientations are regulated cell autonomously in the cells marked blue
(Goldstein, 1995; Gönczy et al 1999b), and can be regulated by cell
interactions in the cells marked yellow (Goldstein, 1995). The white cells
are those in which cell division orientations are likely to be unregulated,
as divisions follow a default orthogonal pattern (Hyman and White, 1987).
HOW DO CELL CONTACTS CONTROL
DIVISION ORIENTATION?
As the plane of cell division is determined by the
positions of the mitotic asters (Rappaport, 1961), which
are nucleated by centrosomes, division orientation by
cell contact was suspected to work by affecting the
positions of the centrosomes.
Hyman and White (1987; Hyman, 1989) have shown
that centrosomes are positioned in certain cells of the
early C. elegans embryo by a mechanism involving
capture of astral microtubules at a specific site in the
cortex. This site is referred to here as the “cortical site.”
The microtubule tether between the centrosome and
the cortical site shortens, resulting in rotation of the
centrosome-nucleus complex and, consequently, alignment of the division axis with the cortical site (Fig. 3).
Evidence which suggested this model included the findings that depolymerizing microtubules prevented rotation of the centrosome-nucleus complex, and that
laser-ablating the proposed cortical site, the leading
centrosome, or at sites along the proposed tether could
prevent rotation, whereas ablating other sites in the
cell had no such effect. Additional evidence for a pulling force generated by the microtubule tether was the
Fig. 3. Model for cell-autonomous spindle orientation by a cortical site
in the P1 cell of the two cell stage (Hyman, 1989; Skop and White, 1998;
Waddle et al., 1994). The black spot represents the cortical site, and the
arrow shows the direction of rotation of the centrosome-nucleus complex.
The plus and minus ends of the tethered microtubule(s) are marked.
observation of a dimple forming around the cortical site
during rotation (Hyman, 1989; Keating and White,
1998). Cortical sites for orienting mitotic spindles have
also been reported in budding yeast (Palmer et al.,
INDUCED CELL DIVISION ORIENTATION
1992) and in embryos of sea urchins (Dan, 1979), clams
(Dan and Ito, 1984), flies (Kraut et al., 1996), ascidians
(Hibino et al., 1998; Nishikata et al., 1999), and fucoid
algae (Fowler and Quatrano, 1997), as well as for meiotic spindles in annelids (Lutz et al., 1988), suggesting
they represent a common theme in eukaryotic cells
which divide asymmetrically.
Does contact-induced division orientation work by
inducing such cortical sites? To test this, centrosome
positions were followed live in isolated inducing-responding cell pairs (Goldstein, 1995). Rotation of the
centrosome-nucleus complex occurred in the responding cell toward the site of cell– cell contact. Anti-microtubule immunofluorescence showed that one end of the
mitotic spindle in the responding cell ended up close to
the site of contact with the inducing cell, as in intact
embryos (Hyman and White, 1987). These movements
were dependent on cell contact, as in the absence of an
inducing cell, no rotation occurred, and division always
occurred in a plane perpendicular to the usual division
plane (Goldstein, 1995). Placing the inducing cell near
to but not touching the responding cell (about 1 cell
diameter apart) also failed to cause rotation (BG, unpublished). As expected, contact-induced rotation could
be prevented by treating cells with a microtubule-depolymerizing drug. The results suggest that the interaction affects spindle orientation by inducing a cortical
site which captures astral microtubules, resulting in
realignment of the mitotic spindle.
Rotation of the centrosome-nucleus complex is induced by cell interactions in only certain cells of the
early C. elegans embryo. In other cells, rotation occurs
cell-autonomously (i.e., it occurs even when these cells
are cultured in isolation), and in yet other cells, rotation does not occur at all and divisions occur in successively orthogonal planes (Fig. 2).
To test whether cell contact induces a cortical site de
novo, or alternatively whether each responding cell
might contain only a single site that translocates to the
area of contact with the inducing cell, two inducing
cells were placed on one side of a responding cell (Goldstein, 1995). Each of the two centrosomes was found to
move toward one of the two inducing cells, pulling the
nucleus to an eccentric position in the cell. The result
indicates that a responding cell is capable of forming
new cortical sites in response to cell contacts.
Contact Is Required Only Briefly, and the
Response Does Not Require New Transcription
Cell contact was found to be required at a specific
time, 10 min prior to cytokinesis in the responding cell.
Recent experiments by A. Schlesinger (personal communication) using cells of disparate ages have revealed
that this is a property of the responding cell: the inducing cell could induce at any time during its cell cycle,
but the responding cell required contact at the critical
time. Whether the responding cell requires contact
only at this time, or alternatively also for some time
afterward, is not yet known.
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Schlesinger et al. (1999) have tested whether transcription is required for contact-mediated spindle orientation in EMS by treating cell pairs with actinomycin D, a chemical inhibitor of transcription, and by
inactivating RNA polymerase II by RNA interference
(RNAi; a method of inactivating gene function by injection of double-stranded RNA; Fire et al., 1998). When
either or both of these treatments were used, a GFP
transgene used to monitor transcription level was undetectable by fluorescence microscopy, yet the spindle
in the responding cell still rotated toward the site of
contact with the inducing cell, suggesting that the interaction affects the responding cell’s cytoskeleton directly, without requiring new transcription.
The Molecular Basis for Contact-Induced
Division Orientation
A recent article has identified for the first time some
of the genes required for contact-induced division orientation— homologs of Wnt pathway components
(Schlesinger et al., 1999). The first Wnt gene was discovered in 1982 as a mouse proto-oncogene, and mutations in most of the Wnt pathway genes have since
been implicated in cancers. Wnt pathway genes function in many developmental signaling pathways in diverse organisms (see Wodarz and Nusse, 1998 for review). Wnt pathway components were previously
known to be required in C. elegans for P2 to induce
EMS to produce a gut cell lineage (see Han, 1997 for
review).
Schlesinger et al. (1999) isolated the cell (called P1)
which produces inducing and responding cells from
embryos in which each of nine genes in the gut induction pathway were inactivated by mutation or RNAi.
Each P1 cell was allowed to divide to form the inducing
and responding cells, and then the division plane of the
responding cell was followed. In wild-type embryos,
this manipulation results in the responding cell dividing in a single division plane with respect to the plane
of cell contact, whereas in several of the mutants and
RNAi-affected embryos, responding cells divided in
various orientations. Isolated cells were also combined
from mutant and wild-type embryos to determine in
which cell each gene is required. Genes found to be
required in the signaling cell included mom-1 (a homolog of fly porcupine) and mom-3 (uncloned). Genes
found to be required in the responding cell included
gsk-3 (zeste-white3) and mom-5 (frizzled). Although inactivation of any of these genes affect spindle orientation in this assay, none of the relevant alleles are
completely penetrant for loss of rotation, suggesting
that either none of the alleles used are null alleles or
that these genes might play partially redundant roles
in spindle orientation.
The signal for spindle orientation remains unidentified. One would expect a Wnt gene to be required, but
surprisingly mom-2 (wingless), a proposed signal for
gut induction, seems dispensable for spindle orientation, as it can only be shown to be defective in signaling
26
GOLDSTEIN
in an experiment where the inducing cell is aged prior
to being placed on a younger responding cell
(Schlesinger et al., 1999). The C. elegans genome has
only four other apparent wingless-related genes
(cwn-1, cwn-2, lin-44, and egl-20; C. elegans Sequencing Consortium, 1998; Herman et al., 1995; Maloof et
al., 1999; Shackleford et al., 1993); whether any of
these signal for spindle orientation, or play a redundant role with mom-2, have yet to be tested. It will be
interesting to see whether two signals are required
from the inducing cell, as this possibility has been
raised for the case of gut induction by the incomplete
penetrance of all known mutations affecting the inducing cell’s ability to signal (Rocheleau et al., 1997;
Thorpe et al., 1997).
Genes in the Wnt pathway expected to act downstream of gsk-3, such as apr-1 (APC-related), wrm-1
(␤-catenin-related), pop-1 (pan/LEF-1) and mom-4
(TAK1) appear to be involved only in gut induction and
not in spindle orientation, as mutations or RNAi for
each of these genes have little if any effect on spindle
orientation. apr-1;wrm-1 double RNAi also has no effect on spindle orientation. Another cell division in the
early C. elegans embryo, the division of ABar, has a
similar dependence on Wnt pathway genes as the EMS
cell (Rocheleau et al., 1997; Thorpe et al., 1997), suggesting that its division orientation might also be induced by cell contacts.
The results of Schlesinger et al. (1999) demonstrate
that many of the Wnt pathway genes required for gut
induction also play a role in spindle orientation, and that
the pathway for spindle orientation splits following gsk-3.
Especially in light of the fact that no new transcription is
required for the response, it will be interesting to see in
the future how this Wnt pathway links to the cytoskeleton. Together with the known roles of mutations in Wnt
pathway genes in cancer, the results additionally raise
the possibility that a failure of induced division orientation might play a role in the development of some cancers.
Cells leave epithelia in an early step of many cancers, yet
we do not know whether cells sometimes leave epithelia
in these cases because of defects in spindle orientation. In
fact, little is known about how cell divisions are properly
oriented in monolayer epithelia (see Reinsch and
Karsenti, 1994).
Additional candidates for genes involved in contactmediated spindle orientation include the genes involved in cell-autonomous spindle orientation in the
germline precursor cells. The growing list of genes required for cell-autonomous spindle orientations, or required to prevent rotation in cells in which rotation
does not normally occur, includes the six par genes,
pkc-3, let-99, nmy-2, pod-1, gpb-1, mes-1, ooc-3, ooc-5,
stu-10, stu-11, lin-5, and rot-1,2,3 (see Rose and Kemphues, 1998 for review; Basham and Rose, 1999; Gönczy et al., 1999a; Lorson et al., 2000; O’Connell et al.,
1998; Rappleye et al., 1999; Strome et al., 1995; Zwaal
et al., 1996). Mutations in most of these genes affect
early divisions dramatically, making it difficult to test
their functions in cells with induced cell division orientations, as the appropriate cells do not form normally
in the mutants. It is clear, however, that in normal
development those PAR proteins which are asymmetrically localized in cells with autonomous cell division
orientations are not asymmetrically localized in cells
with induced cell division orientations. This result suggests that the asymmetrically localized PAR proteins
do not function in these induced cell division orientations (see Kemphues and Strome, 1997 for review).
Other genes which might be expected to be necessary
for contact-mediated spindle orientation include genes
required for the normal execution of the inducing cell’s
fate; in fact, the pie-1 gene, which is necessary for
normal execution of the P2 cell’s fate (Mello et al.,
1992), is required for normal spindle orientation in
P2-EMS cell pairs (BG, unpublished communication).
What Is at the Cortical Site
Data presented thus far suggests that contact between
particular cells induces a site, in the cortex of the responding cell, which can capture astral microtubules. In
cells with autonomously orienting spindles, several components have been found to be present at cortical sites.
These include a concentration of actin (Waddle et al.,
1994) and two dynactin complex members—actin capping protein (Waddle et al., 1994), and DNC-1 (a C. elegans homolog of p150-Glued; Skop and White, 1998)—
which assemble at the site of the midbody (the persistent
remnant of cell division). These findings have led to the
hypothesis that a dynactin complex links cortical actin at
the midbody to a dynein motor, which reels in microtubules by walking in place toward the minus end of any
astral microtubules that it encounters (Skop and White,
1998; Waddle et al., 1994). Data consistent with this
model include Skop and White’s (1998) finding that RNA
interference of dnc-1 or dnc-2 (a homolog of p50/Dynamitin, another dynactin complex member) can prevent
rotation from occurring in P1, although Gönczy et al.
(1999b) have suggested that this may be a result of eccentric placement of the cortical site rather than failure of
rotation. Gönczy et al. (1999b) also found that a dynein
homolog is present at the cortical site, although its distribution is not restricted to this site: DHC-1, a dynein
heavy chain, is present throughout the cytoplasm and on
nuclei, and is enriched in the cortex, particularly along
cell boundaries. Although the hypothesis outlined here is
at least consistent with current data, other hypotheses
are clearly plausible as well. For example, it remains
possible that dynein or another microtubule binding protein at the cortical site might cause rotation by simply
holding onto the plus end of microtubules during microtubule catastrophe events (as in Lombillo et al., 1995),
rather than by walking along microtubules. A motor for
rotation might be present at the centrosome end of the
microtubule tether rather than at the cortical site, as no
INDUCED CELL DIVISION ORIENTATION
27
Fig. 4. Concentrations of actin and actin capping
protein at areas of contact between specific cells
(left). These concentrations are visible under only
certain fixation conditions; these embryos were fixed
in 3% formaldehyde and then 100% methanol. Note
that for each disc of staining at the border of two
cells, it is not clear whether the disc is in the cortex
of just one or both cells. (Figure and methods kindly
provided by Jim Waddle.)
one has yet determined which end of the tethered microtubules shorten during rotation. Consistent with this possibility, a kinesin-like immunoreactivity has been found
localized to both the cortical site and the centrosomes (S.
Hird, personal communication).
Less is known about the cortical site in cells with
induced spindle orientation. What we do know is that
these cells show a striking concentration of actin and
actin capping protein at their boundaries, in a broad
disc along the entire area of contact between P2 and
EMS, and P3 and E (Fig. 4; J. Waddle, personal communication). A disc is also found at the area of contact
between P4 and Ep, raising the possibility that these
cells might interact similarly. DNC-1 is present at the
P2-EMS border too, but as a spot localized to the midbody, rather than in a broad disc (Fig. 5; A. Skop,
personal communication).
FUTURE DIRECTIONS
How Do Cell Contacts Affect Mitotic Spindle
Orientation?
The results cited here suggest a hypothesis for how
cell contacts might affect cell division orientation: The
wnt pathway may induce a dynactin complex to form in
the cortex of a responding cell, either at the midbody or
all along the area of contact with the inducing cell, and
dynein might associate with the dynactin complex and
pull astral microtubules, resulting in rotation of the
centrosome-nucleus complex in line with the inducing
cell. Clearly, many of the issues central to this hypothesis remain unresolved. For example, the signal presented by the inducing cells for spindle orientation is
not yet known. We would expect a wingless homolog to
be required in inducing cells, but none have yet been
firmly implicated. How the Wnt pathway links to the
cytoskeleton to affect division orientation is also an
open question. The motor for rotation of the centrosome-nucleus complex has not yet been identified or
localized, for either cell-autonomous or contact-induced
spindle orientation, although the possibility that dy-
Fig. 5. A spot of DNC-1 is present at the border of P2 and EMS. EMS
is in prophase in the top figure, telophase in the bottom figure. Red is
DNA, green is DNC-1 antibody, and arrows point to spots of DNC-1 at
P2-EMS cell borders. (Figure kindly provided by Ahna Skop.)
nein may pull microtubules toward the cortical site has
been suggested (Gönczy et al., 1999b; Skop and White,
1998; Waddle et al., 1994).
Whether any of the identified cortical site components are even induced by cell contact is still unclear;
in fact, DNC-1 is still present at the P2-EMS border in
mom-5 mutants (A. Skop, personal communication)
and actin is still concentrated at the P2-EMS border in
pie-1 mutants (BG, unpublished). If these suggestivelylocalized gene products in fact do play a role in contactmediated spindle orientation, then their localization
must not be dependent on at least certain components
of the P2-EMS signaling pathway.
28
GOLDSTEIN
Is Contact-Mediated Spindle Orientation a
Peculiarity of C. elegans or Is It More
Widespread?
One case which closely resembles the cases described
in C. elegans is found in the oligochaete annelid Tubifex
(Takahashi and Shimizu, 1997). In the Tubifex embryo,
one cell (called CD) of the two cell stage divides unequally, as in C. elegans. As the CD cell goes through
mitosis, one end of the spindle ends up close to the site
of contact with the other cell. The aster at this end of
the spindle has an altered morphology: the side of the
aster close to the cortex appears flattened. Takahashi
and Shimizu have shown that both the altered astral
morphology and asymmetric division do not occur in
the absence of cell contact early in the two-cell stage.
Placing the other cell of the two cell stage back in
contact with CD, or even placing cells of other stages in
contact with CD, can rescue unequal division of CD,
although curiously, only when the inducing cell is
placed on the side of CD where the inducing cell originally lay. A bead the size of the inducing cell, used to
mimic the alteration in cell shape caused by reassociating cells, could not rescue asymmetric division, suggesting that the cells are not affecting CD via a change
in cell shape (altering cell shape in this way can in fact
rescue normal division pattern in leech CD cells; Symes
and Weisblat, 1992). These results suggest that cell
contacts induce asymmetry of division and altered astral morphology in Tubifex. As in C. elegans, there is a
concentration of actin at the area of contact between
the two cells (Shimizu et al., 1998). Depolymerizing
actin can prevent movement of the spindle toward the
area of cell contact (Takahashi and Shimizu, 1997).
Cell contacts do affect mitotic spindle orientations in
organisms other than C. elegans and Tubifex, but generally by changing cell shape (Freeman, 1983; Meshcheryakov, 1978). For embryonic cells of marine shrimp,
Wang et al. (1997) have made an alternative claim on
the basis of observations of spindle orientations in partial embryos—that mitotic spindles in these cells are
not orienting based on cell shape but instead are orienting away from areas of cell contact. It would be
interesting to see whether this hypothesis holds up in
controlled manipulations of cell contacts and cell
shapes.
Cell contacts have been proposed to orient cell divisions in plants as well, although the evidence here is
not as strong. Stebbins and co-workers (Stebbins and
Jain, 1960; Stebbins and Shah, 1960) have proposed
that stomatal cell divisions in monocotyledonous plants
are oriented by specific cell contacts, primarily on the
basis that natural variation in cell positions can be
correlated with variation in cell division orientations.
Although contact-mediated division orientation has
only rarely been shown, the more general phenomenon
in which the cytoskeleton is oriented by external cues
has been demonstrated in many types of cells. Examples include budding yeast, which orients polarized
growth toward a source of mating pheromone (Maddox
et al., 1999), the algae Fucus and Pelvetia, which orient
growth of the zygote in response to unilateral light
(Fowler and Quatrano, 1997), mouse early embryonic
cells, which polarize components in response to cell
contacts (Ziomek and Johnson, 1980), and cytotoxic
and helper T cells, which orient their cytoskeletons
with respect to the position of target cells (Geiger et al.,
1982). Chemotaxing cells from a variety of organisms
also orient their cytoskeletons with respect to a source
of chemoattractant (Devreotes and Zigmond, 1988). In
some of these cases, division plane is also affected, but
as a secondary effect of orienting the cytoskeleton for
directed secretion or migration; these cases may be
valuable in suggesting candidates for genes involved in
contact-mediated division orientation.
In the vast majority of potential cases, whether cell
divisions are oriented by cell contacts has not been
tested, often because of the inaccessibility of cells to
direct manipulation. Fortunately, as the number of
genetic tools becomes larger, the number of cases
where we can genetically alter cell fates and hence
place types of cells in new positions will likely grow.
This and the potential for directly manipulating cells in
more systems should make it possible to test whether
cells orienting their neighbors’ divisions is a common
theme in multicellular organisms.
ACKNOWLEDGMENTS
I thank Bruce Bowerman, Paul Maddox, and Jim
Waddle for comments on the manuscript; Laurie Smith
for discussions of cell division orientation in plants;
Steve Hird, Ann Schlesinger, Ahna Skop, and Jim
Waddle for sharing unpublished results; Jim Waddle
for Figure 4; and Ahna Skop for Figure 5.
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