-
DOI: 10.1126/science.1167094 , 1546 (2008); 322Science
et al.Amy McMahon,Provide Insights into Collective Cell
Migration
GastrulationDrosophilaDynamic Analyses of
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We have uncovered a negative regulationcascade that is essential
for successful cyto-kinesis. Although negative regulation has
beenproposed to be important during cytokinesis,previous models
have emphasized inhibition ofcortical contractility by astral
microtubules thatcontact the polar regions of the cell (29, 30).
Arequirement for negative regulation of an inhib-itory pathway at
the cell equator has not beenwidely considered. Our findings lead
to a modelin which inactivation of Rac by CYK-4 GAPfunctions in
parallel with activation of RhoA todrive contractile ring
constriction during cyto-kinesis (fig. S7).
References and Notes1. M. Glotzer, Science 307, 1735 (2005).2.
W. M. Bement, H. A. Benink, G. von Dassow, J. Cell Biol.
170, 91 (2005).3. Y. Nishimura, S. Yonemura, J. Cell Sci. 119,
104 (2006).4. O. Yuce, A. Piekny, M. Glotzer, J. Cell Biol. 170,
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Mishima, S. Kaitna, M. Glotzer, Dev. Cell 2, 41 (2002).7. W. G.
Somers, R. Saint, Dev. Cell 4, 29 (2003).8. V.
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Biol. Cell 18, 4992 (2007).9. W. B. Raich, A. N. Moran, J. H.
Rothman, J. Hardin, Mol.
Biol. Cell 9, 2037 (1998).10. J. Powers, O. Bossinger, D. Rose,
S. Strome, W. Saxton,
Curr. Biol. 8, 1133 (1998).11. V. Jantsch-Plunger et al., J.
Cell Biol. 149, 1391 (2000).12. A. F. Severson, D. R. Hamill, J. C.
Carter, J. Schumacher,
B. Bowerman, Curr. Biol. 10, 1162 (2000).13. Y. Minoshima et
al., Dev. Cell 4, 549 (2003).14. P. P. D’Avino, M. S. Savoian, D.
M. Glover, J. Cell Biol.
166, 61 (2004).
15. T. Yamada, M. Hikida, T. Kurosaki, Exp. Cell Res. 312,3517
(2006).
16. A. Toure et al., J. Biol. Chem. 273, 6019 (1998).
17. T. Kawashima et al., Blood 96, 2116 (2000).18. W. M. Bement,
A. L. Miller, G. von Dassow, Bioessays 28,
983 (2006).19. See supporting material on Science Online.20. S.
E. Encalada et al., Dev. Biol. 228, 225 (2000).21. S. Ahmed et al.,
J. Biol. Chem. 269, 17642 (1994).22. K. Rittinger, P. A. Walker, J.
F. Eccleston, S. J. Smerdon,
S. J. Gamblin, Nature 389, 758 (1997).23. K. Oegema, A. Desai,
S. Rybina, M. Kirkham,
A. A. Hyman, J. Cell Biol. 153, 1209 (2001).24. E. A. Lundquist,
in WormBook (www.wormbook.org/
chapters/www_smallGTPases/smallGTPases.pdf).25. H. Yoshizaki et
al., J. Biol. Chem. 279, 44756 (2004).26. T. D. Pollard, Annu. Rev.
Biophys. Biomol. Struct. 36, 451
(2007).27. A. F. Severson, D. L. Baillie, B. Bowerman, Curr.
Biol. 12,
2066 (2002).28. J. Withee, B. Galligan, N. Hawkins, G. Garriga,
Genetics
167, 1165 (2004).29. R. Rappaport, Cytokinesis in Animal Cells
(Cambridge
Univ. Press, Cambridge, 1997).30. R. Dechant, M. Glotzer, Dev.
Cell 4, 333 (2003).31. We thank all members of the Oegema, Desai,
and
Bowerman labs; A. Maddox, J. Dumont, and R. Greenfor reading
this manuscript; J. Dumont for makingdouble-stranded RNAs; Y.
Kohara for cDNA clones; andthe Caenorhabditis Genetics Center for
strains. Supportedby the Jane Coffin Childs Memorial Fund for
MedicalResearch and the Leukemia and Lymphoma Society( J.C.C.),
National Institute of General Medical Sciencesgrant T32 GM008666
and National Cancer Institutegrant T32 CA067754 (L.L.), the Ludwig
Institute forCancer Research (K.O. and A.D.), and NIH grantGM058017
(B.B.).
Supporting Online
Materialwww.sciencemag.org/cgi/content/full/322/5907/1543/DC1Materials
and MethodsFigs. S1 to S7Movies S1 and S2References
10 July 2008; accepted 24 October
200810.1126/science.1163086
Dynamic Analyses of DrosophilaGastrulation Provide Insights
intoCollective Cell MigrationAmy McMahon,1* Willy Supatto,2* Scott
E. Fraser,2 Angelike Stathopoulos1†
The concerted movement of cells from different germ layers
contributes to morphogenesisduring early embryonic development.
Using an optimized imaging approach and quantitativemethods, we
analyzed the trajectories of hundreds of ectodermal cells and
internalized mesodermalcells within Drosophila embryos over 2 hours
during gastrulation. We found a high level of cellularorganization,
with mesoderm cell movements correlating with some but not all
ectodermmovements. During migration, the mesoderm population
underwent two ordered waves of celldivision and synchronous cell
intercalation, and cells at the leading edge stably
maintainedposition. Fibroblast growth factor (FGF) signaling guides
mesodermal cell migration; however,we found some directed dorsal
migration in an FGF receptor mutant, which suggests thatadditional
signals are involved. Thus, decomposing complex cellular movements
can providedetailed insights into collective cell migration.
Anembryo is shaped by a complex combi-nation of collective cell
movements thatresult in cell diversification and tissue for-mation
(1–4). Themajority of thesemorphogeneticevents are dynamic and
involve the simultaneousexecution of different movements, with
large pop-
ulations of cells moving in three-dimensional (3D)space deep
inside the embryo (4, 5). Gastrulationis the earliest morphogenetic
event involving mas-sive cellular movements of the germ layers
(6).Because it is technically challenging to image in-dividual
cellmovements inside an embryowithout
B
An=6wve-1(RNAi)
100%0%0%0%0%
n=5wsp-1(RNAi)100%0%0%0%0%
n=9arx-2(RNAi)100%0%0%0%0%
n=22wve-1(RNAi)CYK-4 GAP(E448K);
0%0%0%100%0%
n=33CYK-4 GAP(E448K);wsp-1(RNAi)
6%33%33%61%0%
n=26CYK-4 GAP(E448K);wsp-1;wve-1(RNAi)
38%31%31%31%0%
n=23CYK-4 GAP(E448K);arx-2(RNAi)
52%22%22%26%0%
30 s % of Embryos0 50 100
Complete CytokinesisFull Ingression Followed by Furrow
Regression
Partial Ingression Followed by Furrow RegressionNo
Ingression
100%0%0%0%0%
wsp-1;wve-1(RNAi) n=9
Fig. 4. CYK-4 GAP inactivates Rac and its effectors, WASp/WAVE
and the Arp2/3 complex, topromote cytokinesis. Cytokinesis in
CYK-4GAP(E448K) embryos is rescued by (A) co-depletion ofWASpWSP-1
and WAVEWVE-1 or (B) depletion of Arp2ARX-2. Scale bar, 20 mm.
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compromising its viability, studies of mesodermcell migration
during gastrulation in Drosophilahave relied on the extrapolation
of dynamical eventsfrom observations of fixed embryos (Fig. 1, A
andB) or from in vivo descriptions of small numbersof cells
(7–9).
We used optimized two-photon excited fluo-rescence (2PEF) (10,
11) to image large domainsof Drosophila embryos ubiquitously
expressingnuclear green fluorescent protein (GFP) (Fig. 1,C andD)
(12) with sufficient spatial and temporalresolution to examine
mesoderm spreading non-invasively over 2 hours (Fig. 1E and movie
S1)(13). We extracted the complex cell movementsof the mesoderm and
ectoderm cells from eachlarge imaging data set (~3 billion voxels)
by using3D segmentation of cell positions and 3D trackingover time
(Fig. 1, F to H, and movie S2). Thisinvolved the analysis of over
100,000 cell positionsper embryo (movieS3) (13).Weused
computationalanalysis to capture the three main morphogeneticevents
of the mesoderm (Fig. 1F) and confirmed
that the ectoderm cell layer, upon which meso-derm cells are
migrating, undergoes germ-bandelongation by means of convergent
extensionmovements (Fig. 1, I and J) (14, 15).
We developed custom software tools to extractquantitative
information from the cell trajectoriesand to describe the dynamic
behavior in detail (movieS3) (13). First, we redefined the
positions of cellsin accordance with a cylindrical coordinate
system[radial (r), angular (q), and longitudinal (L)] byfitting a
cylinder on the average position of ecto-derm cells. This
coordinate system, unlike thestandard Cartesian system (x, y, and
z), is moreappropriate for the body plan of Drosophila em-bryos and
the geometry of their morphogeneticevents (Fig. 2, A to E, fig. S1,
and movie S4)(14, 15).
We determined the influence of ectoderm cellmovements on the
migratory path of the over-lying mesoderm by investigating the
coupling be-tween the motions of these two cell populations.The
ectoderm is in close physical contact with themesoderm: The
mesoderm invaginates from theectoderm, and the ectoderm serves as
the sub-stratum onwhich themesoderm cells spread duringgerm-band
elongation (15, 16). Previous qualita-tive studies suggested a
coupling of their move-ments; in mutants that fail to form
ectoderm,mesoderm cells are specified but fail to move
(14). Statistical analysis of our data revealed thatthe
trajectories of mesoderm and ectoderm cellscorrelate highly in the
anterior-posterior (AP) di-rection (the L axis) (Fig. 2H). However,
in theother directions (the r and q axes), little to nocorrelation
was found (Fig. 2, F and G). Sub-tracting axial motions of the
local ectoderm cellsfrom the motion of each mesoderm cell
resultedin no residual movement of the mesoderm in theL direction
(Fig. 2I andmovies S5 and S6), whichsuggests that the mesoderm
cells are carried bythe strongmovement of the ectoderm during
germ-band elongation in this direction. The lack of cor-relation in
the radial and angular directions suggeststhat mesoderm cells
undergo active movement,distinct from that of the ectoderm.
In the angular direction (q), mesoderm cellmovement was
symmetrical with respect to theventral midline of the embryo, as
demonstratedby a q mean value of 0 (Fig. 2D). Using a colorcode to
identify each cell track by its position oforigin in the furrow
(Fig. 3A), we revealed a sta-ble chromatic pattern of the
trajectories in the qdirection, highlighting the fact that the
spatial or-ganization of cells in this direction is preservedover
time. The straightness of the trajectories andthe limited
intermixing of cells support the viewthat cell movements are
directed. The cell trajec-tories revealed that a group of cells
originating
1Division of Biology, California Institute of Technology,
1200East California Boulevard, Pasadena, CA 91125,USA.
2BeckmanInstitute, California Institute of Technology, 1200 East
CaliforniaBoulevard, Pasadena, CA 91125, USA.
*These authors contributed equally to this work.†To whom
correspondence should be addressed. E-mail:[email protected]
Fig. 1. Two-photon mi-croscopy and analysis ofhistone2A
(H2A)–GFP ex-pressing embryos captureskey events in gastrula-tion.
(A and B) Cross-sections of wild-type (A)and htl mutant (B)
em-bryos stained with anti-body to Twist. (C and D)Confocal 1PEF
(C) fails toimage internalized meso-derm cells, whereas 2PEF(D)
captures the positionsof the internalized cells.(E) A 50-mm-deep
and10-mm-thick lateral slicethrough an H2A-GFP em-bryo demonstrates
thesignal-to-noise ratio (an-terior, left). (F) Segmenta-tion of
mesoderm nuclei(orange spheres) by theuse of Imaris
software(BitplaneAG,Zurich, Switz-erland). Each sphere wasdefined
by the fluores-cent intensity of H2A-GFP.Furrow formation,
furrowcollapse as a result of anEMT, and spreading ofthe mesoderm
to form amonolayer are illustrated from top to bottom,
respectively. (G to J)Tracking cell positions in three dimensions
over time. Shown are dorsal (G)and posterior (H) views of mesoderm
tracks (blue and yellow indicate early
and late time points, respectively) and dorsal (I) and posterior
(J) views ofmesoderm (orange) and ectoderm (gray) net displacement
vectors. Scalebars, 20 mm.
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from the upper lateral parts of the furrow (Fig. 3A)becomes
positioned at each leading edge of themesoderm cell population,
which was maintainedfor the entire course of theirmigration (movie
S7).These leading cells were neither the first nor thelast to
invaginate; instead, their locationwithin thefurrow positioned them
to land in the leading po-sition as the furrow collapsed after the
epithelial-to-mesenchymal transition (EMT).
We explored other morphogenetic events thatmight contribute to
mesoderm spreading, such ascell division pattern and cell
intercalation, basedon our cell-tracking data. Each mesoderm
celldivided twice (7, 8, 17, 18), and these divisionswere ordered
in space and time (Fig. 3B). Cellsnearest the ectoderm divided
first, followed bycells nearer to the top of the ventral furrow.
Thisorder was maintained during the second divisioncycle. Analysis
of the cell division mutants didnot uncover any of the
characteristic mesodermmigration defects observable in fixed
sections(fig. S6) (18). Our tracking data revealed that
theorientation of cell divisions within the mesodermis random and
that altering the organization ofcell divisions had no effect on
mesoderm spread-ing or embryo viability (fig. S7, A to C). Thus,
itis unlikely that these organized cell divisions playa role in
mesoderm spreading. The radial cell in-tercalation events (19) were
synchronous with thesecondwave of cell division (Fig. 3, C andD),
butthe orientation of the cell divisions did not seem toplay a
causal role in the intercalation motions.Mesoderm cell
intercalation contributes to mono-layer formation and spreading
(Fig. 3, C and D).
To facilitate comparisons between embryos,we developed a
statistical analysis characterizingthe spreading behavior of the
mesoderm cells. Assuggested by the spatial organization of the
spread-ing (Fig. 3A), the angular positions of each cell atthe
onset (qstart) and at the end (qend) of the pro-cess were highly
correlated. A plot of starting andending positions revealed a
linear relationship(fig. S4, A to C). Given this, linear
regressionsthat were applied to the qend(qstart) valuesprovided a
measure of both the strength of thespreading (as the slope of the
line, A) (fig. S4,D and E) and a quantitative measure of
col-lective behavior (the degree of correlation, R)(13).Wild-type
cells followed an ordered spread-ing behavior [qend ≈ 2(qstart)],
which is sharedby the majority of cells (R > 0.9) (fig. S5).
Com-parison of the regression analysis from five wild-type embryos
showed the consistency of cellbehaviors (n = 5 embryos and n = 596
cells)(fig. S5).
Previous studies of fixed embryos (8, 9, 20, 21)have suggested
that fibroblast growth factor (FGF)signaling is involved in
regulating mesoderm cellmigration, but its exact function has
remainedelusive. We used our methodology to study thefunction of
the FGF signaling pathway on theregulation of gastrulation by
analyzing embryosof the FGF receptor mutant heartless (htl) in
thesame way as wild-type embryos (figs. S2 and S3and movie S9). We
decomposed the cell move-
ments within htl mutant embryos into their com-ponents in r, q,
and L (fig. S3, A to C), permittingdirect comparisonswithwild-type
embryos (Fig. 2,
C to E). The ectoderm-coupled movements ofmesoderm cells in the
L direction were unaffectedin htlmutants (fig. S3F), and we
obtained no evi-
Fig. 2. Decomposition and correlative analysis of cell movements
with the use of cylindrical coordinates.(A and B) The use of
cylindrical coordinates allows the positioning of cells according
to the body plan ofthe embryo at stage 6. (C to E) Cell
trajectories (blue lines) reveal that each axis corresponds to a
mor-phogenetic movement. (C) r is the radial position over time
(for example, furrow collapse and intercalation;0 indicates the
center of the embryo). (D) q is the angular movement (for example,
mesoderm spreadingand ectoderm convergence; 0 indicates the
position of the ventral midline). (E) L corresponds to themovement
of cells along the length of the embryo (for example, germ-band
elongation). In (C) to (E),time (t) = 0 is set as the point when AP
movement begins. (F to H) Correlation of the velocity (v) of
eachmesoderm cell with its six nearest ectodermal neighbors along
the (F) radial, (G) angular, and (H) AP axes,with correlation
values of 0.21 T 0.43, 0.08 T 0.18, and 0.90 T 0.06, respectively
(n = 3 embryos). (I)Dorsal view of mesoderm cell displacement
before (orange) and after (blue) subtraction of local ectodermcell
movements.
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dence for defects in cell-division events (fig. S7D).However,
htl mutant embryos displayed meso-derm cell defects that affected
both collapse ofthe furrow (r axis) and spreading in the
angulardirection (q axis) (fig. S3, A and B). A statisticalanalysis
of cellmovement conducted on htlmutant–tracking data showed a
scattered distribution ofqend(qstart) values (figs. S4I and S5),
resulting inlow spreading and correlation values (A < 1 and
R < 0.5 to 0.7, respectively) (fig. S5C). Values ob-tained
with analysis of individual htl embryos orby pooling the cells
frommultiple htl embryos (n =3 embryos and n = 284 cells) (fig. S5,
B and C)quantitatively demonstrated that a similar dis-ruption of
spreading is present in all htl embryos.
Cell tracking analysis revealed that loss ofFGF signaling
affected the mesoderm cells non-homogenously (movie S10). In the
radial direc-
tion, cells originating from the upper half of thefurrow
(“upper-furrow” cells) in general did notcollapse, remaining far
from the ectoderm duringthe entire acquisition time (Fig. 4A, fig.
3SA, andmovies S11 and S8). The angular movement ofupper-furrow
cells was strongly affected in htlmutants (Fig. 4, B to G). In
contrast, the last cellsto invaginate in htl mutants, which make up
thelower furrow, behaved in amanner similar towild-type mesoderm
cells and could achieve the samedorsal position as the wild type
(Fig. 4G). Ourstatistical analysis of cell movements of upper-and
lower-furrow cells confirmed the presence oftwo distinct cell
behaviors in htl embryos (fig. S5,D and E). Other cell labeling
approaches, such asphotoactivatable GFP, can be used to
characterizemutant phenotypes, but the limited number of cellsthey
can follow (7) make interpretation difficult,especially when there
are multiple behaviors, suchas in htl mutant embryos.
Some cells from the upper furrow in htl mu-tants displayed
normal positions in the qend(qstart)graph, similar to those of
wild-type embryos.These cells were positioned close to the
ectodermat the end of spreading (Fig. 4, I and J, and fig.
S4J).This suggested that the distance from the ecto-derm might have
a major influence on spreadingbehavior. Indeed, the distinction
between the twomigratory behaviors observed was more clearwhen
analyzing cells that were close to or far fromthe ectoderm (Fig.
S5, D and E). We confirmedthis by plotting a qend(qstart) graph
using a colorcode for the radial position of the cells at the endof
the spreading process (Fig. 4, H and I): The htlcells that followed
wild-type behavior [qend ≈2(qstart) such that A = 2] ended up close
to theectoderm (Fig. 4I, green), whereas the cells thatstayed far
from the ectoderm (Fig. 4I, red) hadclearly disrupted behaviors,
with several cellscrossing the midline and migrating in the
wrongdirection (A < 0). All wild-type cells ended upclose to the
ectoderm (Fig. 4H).
Our analysis provides several insights into thehtlmutant
phenotype. First, the primary functionof FGF signaling must be to
help all cells withinthe furrow to collapse, directing them toward
theectoderm (Fig. 4K). Second, another as-yet un-identified signal
must guide the migration of thecells in the angular direction
toward the dorsalectoderm, becausemovement is observed even inthe
absence of FGF signaling. Third, contact withthe ectoderm is key
for the mesoderm to respond tothis guidance cue, because the
distance of the meso-dermcells from the ectodermdefines
theirmigratorycompetence. Any cell that encounters the ectodermis
capable of directed movement in the angulardirection in response to
a cue that cannot be solelyFGF-dependent. Movement of the mesoderm
cellsmight require contact with the ectoderm to makethem competent
to respond to a directional signal, asevidenced in other systems
(22–24).
This study demonstrates that stereotypicalmorphogenetic events
during embryo develop-ment can be systematically quantified,
analyzed,and compared between wild-type and mutant em-
Fig. 3. Quantitative analysis of morphogenetic events reveals a
high level of organization in wild-typeembryos. (A) A color code
marks the angular position of cells in the furrow at stage 7 and
shows thespatial organization as cells move over time. rad,
radians. Each line represents the trajectory of one cell.(B)
Position and timing of each cell division (colored circle). The
color code represents the radialposition in the furrow at stage 7.
DNA morphology during cell division in H2A-GFP embryos is shown(top
left). (C) Analysis of intercalation events within the mesoderm
over time shown as a percentage ofmesoderm cells intercalating (n =
3 embryos). (D) The position of mesoderm cells before and
afterintercalation events.
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bryos by means of the live imaging of largegroups of cells.
Complex cell movements are de-composed into particular cell
behaviors, reveal-ing a high level of organization and
permittingthe interpretation of subtle mutant phenotypes
inDrosophila. Future developments in imaging andcell tracking will
facilitate this quantitative ap-proach, enabling its application at
a larger scaleand in other model systems, to expand the
un-derstanding of collective cell migration and em-bryonic
development from the molecular level tothat of the entire organism
(25).
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comments on the manuscript, M. Liebling foradvice on Imaris and
Matlab, and the CaltechBiological Imaging Center for sharing
equipment.This work was supported by grants to A.S. from NIH(R01
GM078542), the Searle Scholars Program,and the March of Dimes
(Basil O'Conner StarterScholar Award, 5-FY06-12), and grants to
S.F. fromthe Caltech Beckman Institute and NIH (Centerfor
Excellence in Genomic Science grant P50HG004071).
Supporting Online
Materialwww.sciencemag.org/cgi/content/full/322/5907/[page]/DC1Materials
and MethodsFigs. S1 to S7Movie S1 to S11References
16 May 2008; accepted 12 November
200810.1126/science.1167094
Fig. 4. Furrow collapse and spreading of mesoderm cells are
disrupted in htlmutants. (A) Position of mesoderm cells (circles)
at stage 7 and stage 10 in wild-type and htl embryos shown with a
radial color code. (B toG) Angular movementof cells over time
analyzed in wild-type [(B) to (D)] and htl mutant [(E) to
(G)]embryos within the entire [(B) and (E)], upper [(C) and (F)],
and lower [(D) and(G)] furrow (black line indicates the
averagemesoderm displacement with respectto the midline). (H and I)
Spreading profile of wild-type (H) and htl (I) embryos.The color
code represents the distance from the ectoderm at the end of
spreading(red indicates far from ectoderm and green indicates close
to ectoderm). The grayline represents a spreading coefficient of A
= 2, where qend = A(qstart) + B [B,
constant (13)]. Cells that do not spread within the collective
are representedwithin gray regions of the graph (13). In general,
cells located close to theectoderm fall along the gray line. (J)
The radial position (r) of two particulargroups of mesoderm cells
from the upper furrow of htl mutants is depictedover time. One
group exhibits normal spreading behavior (light blue), and theother
group exhibits aberrant spreading (dark blue). (K) The furrow
collapse inhtl mutants is disrupted, resulting in cells falling
randomly to one side of theembryo. Upper-furrow cells that reach
the ectoderm (light blue) undergonormal spreading, whereas cells
that remain far from the ectoderm spreadabnormally (dark blue). Red
cells are Red indicates lower-furrow cells.
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