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First published online 25 July 2007
doi: 10.1242/dev.004911
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Research Report |
Medical Research Council, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK.
* Author for correspondence (e-mail: ssilva{at}nimr.mrc.ac.uk)
Accepted 18 June 2007
SUMMARY
Tissue elongation is a general feature of morphogenesis. One example is the extension of the germband, which occurs during early embryogenesis in Drosophila. In the anterior part of the embryo, elongation follows from a process of cell intercalation. In this study, we follow cell behaviour at the posterior of the extending germband. We find that, in this region, cell divisions are mostly oriented longitudinally during the fast phase of elongation. Inhibiting cell divisions prevents longitudinal deformation of the posterior region and leads to an overall reduction in the rate and extent of elongation. Thus, as in zebrafish embryos, cell intercalation and oriented cell division together contribute to tissue elongation. We also show that the proportion of longitudinal divisions is reduced when segmental patterning is compromised, as, for example, in even skipped (eve) mutants. Because polarised cell intercalation at the anterior germband also requires segmental patterning, a common polarising cue might be used for both processes. Even though, in fish embryos, both mechanisms require the classical planar cell polarity (PCP) pathway, germband extension and oriented cell divisions proceed normally in embryos lacking dishevelled (dsh), a key component of the PCP pathway. An alternative means of planar polarisation must therefore be at work in the embryonic epidermis.
Key words: Tissue elongation, Planar cell polarity, Pair rule, string, Mitosis, Drosophila
INTRODUCTION
The best-known example of tissue elongation is the extension of the
vertebrate axis following gastrulation. In Xenopus, elongation of the
prospective notochord is driven by convergence and extension, a process that
requires cell intercalation. In fish embryos, cell intercalation also
contributes to axis elongation during gastrulation
(Wallingford et al., 2002
). In
these embryos, oriented cell division is a key additional contributory factor.
Both processes appear to be controlled by the planar cell polarity (PCP)
pathway (Gong et al., 2004;
Wallingford et al., 2000).
Drosophila embryos also undergo dramatic tissue elongation: during
early embryogenesis, the germband, which gives rise to the segmented trunk of
the larva, doubles in length while thinning commensurately. In this case, the
elongating tissue is constrained by external membranes and the germband folds
over itself as it elongates. At the end of elongation, the posterior half of
the germband (segments A3-A9) ends up on the dorsal side of the egg, while the
anterior half (segments T1-A2) remains on the ventral side throughout
(Sonnenblick, 1950)
(Fig. 1A). Upon completion of
germband extension (GBE), the posterior tip of the germband has travelled over
70% of the egg length towards the head region. Therefore, the displacement of
the posterior tip provides a quantitative measure of the progression of GBE.
Using this simple assay, two phases can be distinguished during GBE
(Hartenstein and Campos-Ortega,
1985). Most of the elongation takes place during the fast phase,
which lasts approximately 25 minutes. Extension is completed during the
following 70 minutes, which make up the slow phase.
What are the cellular behaviours that drive GBE? Early morphological
studies suggested that the contraction of an actin network underlying the
forming epidermis could be responsible
(Rice and Garen, 1975;
Rickoll and Counce, 1980).
Subsequent work assessed the role of cell rearrangements
(Bertet et al., 2004;
Irvine and Wieschaus, 1994;
Zallen and Wieschaus, 2004).
These observations were focused on the anterior of the germband because it
remains within the same field of view throughout extension (remaining at the
anterior of the fold). In this region, no cell division occurs during the
first 15 minutes of GBE (Foe,
1989; Hartenstein and
Campos-Ortega, 1985), excluding the possibility that oriented cell
divisions could contribute to elongation, at least at these early times and in
this part of the germband. Instead, early extension of the anterior region of
the germband is powered by an orderly process of cell intercalation driven by
junctional remodelling (Bertet et al.,
2004; Blankenship et al.,
2006; Irvine and Wieschaus,
1994). Polarisation of this intercalary behaviour requires cues
from the genetic cascade that patterns the anteroposterior axis. Indeed, no
extension is seen in embryos laid by triple-mutant bicoid nanos
torso-like (bcd nos tsl) mothers, hence lacking all
anteroposterior information (Irvine and
Wieschaus, 1994). GBE is reduced in embryos lacking the pair-rule
transcription factor encoded by even skipped (eve). These
embryos are partly segmented, and this is correlated with a strong decrease in
intercalary behaviour (Blankenship et al.,
2006; Irvine and Wieschaus,
1994). Little attention has been given to the posterior of the
germband (the region that ends up posterior to the fold during extension; see
diagram in Fig. 1). This region
is known to undergo mitoses shortly after the beginning of GBE. Indeed,
so-called mitotic domain 4 is recognisable at the posterior tip of the
extending germband shortly after the onset of extension
(Foe, 1989). Are these
divisions oriented and do they play a role in the extension of the posterior
region of the germband? We found that, in the posterior region of the
germband, mitoses are oriented along the axis of elongation. Moreover, in the
absence of mitosis, germband elongation is reduced and no tissue deformation
is seen in the posterior region. We also present evidence that segmental
patterning provides a cue for the orientation of cell division.
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Fly strains
Df(2R)eveR13 was obtained from the Tubingen Centre. The
string alleles used were stgAR2, which causes a
deletion of the coding region (Edgar et
al., 1994) and stg7B, an amorphic point
mutation. Flies carrying His2AvDGFP were kindly provided by Rob Saint
(Australian National University, Canberra, Australia). Embryos lacking all
segmental patterning were obtained from bicoid nanos torso-like
triple-mutant females (Nusslein-Volhard et
al., 1987) that also carried His2AvDmRFP
(Schuh et al., 2007). Embryos
deficient in PCP-specific dsh activity were obtained from
dsh1/dshV26 females crossed to
dsh1 males. The dshV26 allele results
from a frame shift after amino acid residue 94 and is presumed to be a null.
The dsh1 mutation results from a single amino acid
replacement that specifically affects PCP
(Penton et al., 2002). Note
that the zygotes could be either
dsh1/dshV26 or
dsh1/dsh1. In both cases, no
PCP-specific dsh activity is expected.
Image capture
All images were acquired at 25°C. Five embryos were analysed for each
genotype (except for the embryos from bicoid nanos torso-like triple
mutants). Embryos were dechorionated in 10% bleach for 3 minutes and mounted
in Voltalef oil on a coverslip. The images were acquired with a Perkin-Elmer
UltraVIEW spinning disc confocal scanner mounted on an Olympus IX70 inverted
microscope with a 20x (0.5 NA) or a 40x (0.8 NA) objective lens.
Ten z-sections (covering 10 µm) were collected every 30 seconds.
The time-lapse series was assembled and analysed with Volocity (Improvision)
and ImageJ [National Institutes of Health (NIH)]. Angles and lengths were
measured with the angle and measure tools on ImageJ. Polynomial regression
curves (Fig. 2Q) were
determined with Excel (Microsoft) as:
y=(-3x10-8x6)+(4x10-6x5)-0.0003x4+0.0085x3-0.1249x2+0.5932x+2.6912
(R2=0.9558) for wild-type embryos;
y=(4x10-8x6)-(6x10-6x5)+0.0003x4-0.007x3+0.0738x2-0.3718x+2.9431
(R2=0.9483) for stg embryos; and
y=(4x10-8x6)-(6x10-6x5)+0.0003x4-0.0062x3+0.0453x2-0.0232x+2.1822
(R2=0.9905) for eve embryos.
RESULTS AND DISCUSSION
Cell divisions are oriented during the fast phase of germband extension
We monitored the timing and orientation of cell divisions in the posterior
of the Drosophila germband as it comes into view when the egg is
observed from its dorsal side (posterior to the fold,
Fig. 1A). Embryos uniformly
expressing a histone-green fluorescence protein (His-GFP) fusion protein
(Clarkson and Saint, 1999) were
imaged by 4D confocal microscopy. By virtue of marking chromatin, His-GFP
reports on the various stages of the cell cycle in live embryos. We confirmed
that mitoses take place as soon as the posterior tip of the germband comes
into view, early during germband elongation. Although easily monitored, the
orientation of the metaphase plate did not provide a reliable measure of the
angle of division because it often rotates before anaphase (data not shown).
Orientation of division was therefore assessed at telophase as the angle
between the spindle and the ventral midline. This was measured for dividing
cells within view (up to 10-cell diameter from either side of the midline)
(Fig. 1B). The data was
compiled in two separate histograms, one for the fast phase and one for the
slow phase (Fig. 1C-H).
Distinct distributions can be seen. From a total of 250 divisions (five
embryos) during the fast phase, 73% occurred at an angle of less than 30°
from the midline, whereas 21% were oriented between 30° and 60°. Only
6% of all observed fast-phase mitoses took place at an angle between 60°
and 90°. These numbers show that cell divisions have a longitudinal bias
during the fast phase of elongation. When the process of GBE slowed down (slow
phase), out of 500 divisions counted, 40% occurred at an angle below 30°,
whereas 25% divided at an angle between 30° and 60°, and 36% between
60° and 90°. These data indicate that the orientation of cell
divisions becomes randomised during the slow phase of elongation. Our data
confirm the existence of an early mitotic domain at the posterior end of the
germband, as shown originally by Foe (Foe,
1989). In addition, our data show that numerous mitoses occur
throughout the germband without being associated to a defined mitotic domain
(Fig. 1 and see Movie 1 in the
supplementary material). Importantly, mitoses within the posterior half of the
germband tended to be longitudinally oriented during the fast phase of GBE.
Therefore, oriented cell divisions could play a role in tissue elongation
during this period.
|
),
the rate and extent of elongation was not assessed. We tracked the posterior
tip of the germband in homozygous string embryos carrying the His-GFP
transgene. For comparison, extension was monitored in parallel in wild-type
(Fig. 2A-D,M) and eve
mutant (Fig. 2I-L,O) embryos.
In these various genetic backgrounds, the onset of elongation was measured
relative to the time when the ventral furrow appeared. Using this temporal
reference, we found that, in eve mutants, the posterior germband
appears into view on the dorsal side with a 3-minute delay as compared with
the situation in wild-type or string mutant embryos
(Fig. 2O,P; t=0
corresponds to the time when the posterior tip appeared on the dorsal side of
wild-type embryos). Initial elongation appeared to be slower in
string mutant embryos than in wild-type embryos
(Fig. 2P). After 15 minutes of
displacement, the posterior tip had moved to approximately 40% egg length in
wild type but had only reached around 30% of egg length in string
mutants. Therefore, lack of cell division leads to a reduction in the
elongation rate. The absence of cell division also caused increased
variability in the rate of elongation. Perhaps mitoses help synchronise the
mechanical behaviour of the cell. Beyond the initial phase, the germband
continued to extend at a relatively slow rate in string mutants and
never extended as much as in wild-type embryos. GBE in string mutants
reached a maximum of approximately 55% egg length (compared with 70% for
wild-type embryos). Velocity measurements
(Fig. 2Q) show that
string embryos (and to a lesser extent eve embryos) were
specifically defective during the fast phase of elongation, whereas they
exhibited a near wild-type elongation rate during the slow phase. It is
unlikely that cell intercalation (or junctional remodelling) is affected on
the ventral side of string mutants
(Bertet et al., 2004;
Irvine and Wieschaus, 1994;
Zallen and Wieschaus, 2004).
We therefore conclude that the reduction of extension seen in string
mutants is a consequence of the lack of cell divisions. One interpretation is
that mitoses could provide a driving force for elongation. Alternatively, as
cells turn around the fold, they might encounter reduced resistance, which
would allow longitudinal mitoses and tissue expansion.
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Segmental patterning polarises the orientation of cell divisions
Embryos mutant for eve are defective in GBE and this is correlated
with a strong reduction in cell intercalation
(Irvine and Wieschaus, 1994).
It is thought that the segmentation cascade could provide cells with a
polarising signal that orients junctional remodelling and intercalatory
behaviour (Bertet et al., 2004;
Zallen and Wieschaus, 2004).
Weakening of this signal in eve mutants would deprive cells of the
cue that polarises intercalary behaviour and thus would reduce GBE. In order
to find out whether eve activity also affects the orientation of cell
divisions, we imaged mitoses at the posterior germband of eve mutants
and, as before, compiled separate data for the first 25 minutes and the
subsequent 70 minutes (Fig. 4
and see Movie 3 in the supplementary material). During the first period, the
longitudinal bias of cell divisions is much reduced compared with the
situation in wild-type embryos. Out of 260 mitoses, 38% occurred between
0° and 30°, 41% between 30° and 60°, and 21% between 60°
and 90° (Fig. 4E). In the
subsequent period, no longitudinal bias could be seen. Out of 150 mitoses, 32%
were oriented along the elongation axis (0°-30°), 27% were in the
30°-60° bracket, and 41% occurred between 60° and 90°
(Fig. 4G). While performing
this analysis, we noticed that the longitudinal divisions tended to take place
near the midline. To verify this apparent bias, we monitored separately the
orientation of cell divisions in the medial half of the germband
(Fig. 4F, diagram) and in the
more lateral half. Out of 130 mitoses near the midline, 52% occurred between
0° and 30°, 36% between 30° and 60°, and 12% between 60°
and 90°. Out of 130 mitoses counted in the more lateral region, 23% were
oriented along the elongation axis (0°-30°), 45% between 30°and
60°, and 32% between 60° and 90°
(Fig. 4F). Therefore, in
eve mutants, longitudinal orientation of cell divisions is
preferentially lost in more-lateral cells. The residual longitudinal bias near
the midline of eve embryos suggests that an eve-independent
orientation cue might be present near the midline. Residual polarisation in
eve mutants must come from residual segmental patterning in these
embryos, because, in embryos lacking all segmental information (obtained from
bicoid nanos torso-like triple-mutant females), cell divisions
appeared to be completely randomised (Fig.
4H-K; see Movie 4 in the supplementary material). In conclusion,
we suggest that segmental patterning is required for elongation throughout the
germband. Anterior to the fold, lack of segmental information caused a loss in
cell intercalation, whereas, at the posterior, it led to a reduction in the
orientation of cell divisions.
|
). As we have shown, GBE in Drosophila also follows
from a combination of oriented cell divisions (posterior to the fold) and cell
intercalations (anterior to the fold). However, in Drosophila,
mutations in frizzled or dishevelled, two genes involved in
the classical PCP pathway, have no noticeable effect on extension
(Perrimon and Mahowald, 1987;
Zallen and Wieschaus, 2004)
(Fig. 5A-D). We assessed the
orientation of cell divisions in embryos carrying a combination of
dsh alleles known to cause PCP defects in the adult wing
(Penton et al., 2002)
(Fig. 5A). From a total of 250
fast-phase divisions (five embryos), 58% occurred at an angle of less than
30°, while 36% were between 30° and 60° from the midline. Only 6%
of fast-phase mitoses took place at an angle between 60° and 90°. This
suggests that, during the fast phase, divisions in dsh mutants are
polarised, as they are in wild type (Fig.
5C,D; Fig. 1G,H;
and see Movie 5 in the supplementary material). In order to compare the extent
of polarisation in the two genetic backgrounds, we devised a simple index of
longitudinal bias (as described in the legend of
Fig. 5) and found this index to
be similar in dsh and wild-type embryos. We conclude that the
dsh-dependent PCP pathway is neither required for oriented cell
divisions nor for GBE in Drosophila embryos. Note, however, that
apico-basal polarity seems necessary for elongation, because mutations in
bazooka (Zallen and Wieschaus,
2004) cause a reduction in GBE. It is conceivable that, in early
Drosophila embryos, a dsh-independent mechanism imparts PCP
to epidermal cells. In fact, an additional polarising signal must exist,
because most denticles are properly oriented in embryos lacking maternal and
zygotic frizzled (Price et al.,
2006), or lacking maternal and zygotic PCP-competent dsh
(Fig. 5B). Work in the adult
fly abdomen suggests that the so-called dachsous/fat system
could act as an independent polarising source in epithelia
(Casal et al., 2006). It
remains to be seen whether this system contributes to the polarisation of
denticles, cell intercalation and/or mitoses in embryos. Another outstanding
issue concerns how the segmentation cascade controls dsh-independent
PCP.
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ACKNOWLEDGMENTS
We are grateful to Pierre-Luc Bardet, Bill Crum and Mark Miodownik for helpful suggestions, and to the NIMR's confocal and image analysis laboratory for help and advice with the time-lapse recordings. This work was supported by the Medical Research Council, UK.
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