Developmental Cell 11, 459–470, October, 2006 ª2006 Elsevier Inc.DOI 10.1016/j.devcel.2006.09.007
Multicellular Rosette Formation Links
Planar Cell Polarity to Tissue Morphogenesis
J. Todd Blankenship,1Stephanie T. Backovic,1
Justina S.P. Sanny,1Ori Weitz,1
and Jennifer A. Zallen1,*
1Developmental Biology Program
New York, New York 10021
Elongation of the body axis is accompanied by the as-
sembly of a polarized cytoarchitecture that provides
the basis for directional cell behavior. We find that pla-
nar polarity in the Drosophila embryo is established
through a sequential enrichment of actin-myosin
cables and adherens junction proteins in complemen-
tary surface domains. F-actin accumulation at AP
interfaces represents the first break in planar symme-
try and occurs independently of proper junctional
protein distribution at DV interfaces. Polarized cells
engage in a novel program of locally coordinated
behavior to generate multicellular rosette structures
that form and resolve in a directional fashion. Actin-
myosin structures align across multiple cells during
rosette formation, and adherens junction proteins
assemble in a stepwise fashion during rosette resolu-
tion. Patterning genes essential for axis elongation
selectively affect the frequency and directionality of
rosette formation. We propose that the generation of
higher-order rosette structures links local cell interac-
tions to global tissue reorganization during morpho-
Convergent extension is a conserved morphogenetic
event that generates one of the striking properties of
embryonic form—the elongated body axis. A common
mechanism for elongation of epithelial and mesenchy-
mal tissues is cell intercalation, in which oriented cell
movements cause a tissue to narrow in one dimension
and lengthen in a perpendicular dimension (Keller
et al., 2000; Wallingford et al., 2002; Nikolaidou and
Barrett, 2005; Solnica-Krezel, 2005). Cell intercalation
involves a diverse repertoire of behaviors, including
polarized protrusive activity (Hardin, 1989; Shih and Kel-
ler, 1992; Elul and Keller, 2000; Munro and Odell, 2002),
differential adhesion (Wieschaus et al., 1991; Irvine and
Wieschaus, 1994), and cell-shape changes (Fristrom,
1988; Bertet et al., 2004). These behaviors are associ-
ated with the formation of a polarized cytoarchitecture
that concentrates the Bazooka/PAR-3 scaffolding pro-
tein and the Myosin II actin motor in distinct cortical
domains in Drosophila (Bertet et al., 2004; Zallen and
Wieschaus, 2004), as well as Par-6 and atypical protein
kinase C at sites of motile activity in vertebrates
(Hyodo-Miura et al., 2006). However, it is not known
how these polarized proteins engage the cellular
machinery that provides the basis for directional cell
In an emerging theme from studies in vertebrates and
invertebrates, the spatial information that guides cell
intercalation is provided locally by contact between dif-
ferent cell types (Irvine and Wieschaus, 1994; Ninomiya
et al., 2004). In Drosophila, the eve and runt transcrip-
oping anterior-posterior (AP) axis, and axis elongation
is disrupted when either gene is absent or expressed
uniformly (Irvine and Wieschaus, 1994; Zallen and Wie-
schaus, 2004). Moreover, ectopic eve or runt expression
is sufficient to reorient the polarity of adjacent cells
(Zallen and Wieschaus, 2004), demonstrating that polar-
ity is actively modulated by local interactions. A related
mechanism operates in vertebrates, where AP differ-
ences promote intercalary behavior when Xenopus me-
sodermal cells from different axial positions are juxta-
posed in culture (Ninomiya et al., 2004). These studies
indicate a deep homology in the spatial mechanisms
that organize cell rearrangement during tissue morpho-
Diverse cellular mechanisms translate global spatial
cues into directional cell behavior in different tissues.
In vertebrates, intercalating cells characteristically elon-
gate in the direction of cell movement (Concha and
Adams, 1998; Keller et al., 2000; Topczewski et al.,
2001), while no significant cell-shape anisotropy is
observed in Drosophila (Irvine and Wieschaus, 1994).
Here, we show through live imaging studies that interca-
lating cells in the Drosophila germband locally organize
resolve in a directional fashion. Germband cells become
polarized prior to intercalation through a sequence of
events that leads to the asymmetric distribution of F-
actin and adherens junction proteins. These polarities
are dynamically remodeled during the processes of
rosette formation and resolution. Rosette frequency is
reduced in eve mutants that partially elongate the
body axis, and rosette frequency and directionality are
both disrupted in bicoid nanos torso-like mutants that
lack AP patterning and fail to elongate. These results
suggest that multicellular rosette structures, and not in-
dividual cells or cell interfaces, represent the functional
units of cell behavior during tissue elongation.
Planar Polarity Is Established Prior to Intercalation
During axis elongation in Drosophila, polarized cell
movements cause the embryonic germband to narrow
along the dorsal-ventral (DV) axis and lengthen by 2.5-
fold along the anterior-posterior (AP) axis (Hartenstein
These directional behaviors require the Bazooka/PAR-3
scaffolding protein and the Myosin II actin motor pro-
tein, which localize to complementary surface domains
along the planar axis (Bertet et al., 2004; Zallen and
Wieschaus, 2004). To ask when planar polarity is first
generated, we developed a quantitative immunofluores-
cence assay for protein localization (Experimental
Procedures). In early stage-6 embryos, Bazooka and
Myosin II were localized uniformly at the apical cell sur-
face (Figures 1A and 1E). By contrast, late stage-6 em-
bryos displayed an enrichment of Bazooka at the bor-
ders between dorsal and ventral cells (DV interfaces,
Figure 1B) and Myosin II at the borders between anterior
Figure 1. Polarized Localization of Bazooka/PAR-3 and Myosin II Prior to Intercalation
(A–L0) (A–D) Bazooka (Baz), (E–H) Myosin II (Myo), (I–L) Neurotactin (Nrt). Anterior is oriented toward the left, and dorsal is up. (A–H) Baz (A) and
Myo (E) localized uniformly at the apical surface in early stage-6 embryos.Late stage-6 embryos displayed an enrichment of Baz (B) at horizontal
DV interfaces and Myo (F) at vertical AP interfaces. Baz and Myo polarities were also present in stage 7 (C and G) and stage 8 (D and H). (I–L) Nrt
was not polarized in (I) early stage 6, (J) late stage 6, (K) stage 7, or (L) stage 8. (A0–L0) Relative edge intensities plotted over the full angular range
(2–7 embryos/histogram). The same embryos were analyzed for all three proteins. Bars represent the average relative intensity of edges within
a 15?angular range. Far-left (0?–14?) and far-right (165?–179?) bars are nearly parallel to the AP axis (DV interfaces); central bars (75?–89?and
90?–104?) are nearly normal to the AP axis (AP interfaces). The scale bar is 10 mm.
multicellular rosette structures promote rapid and
efficient tissue elongation in the Drosophila germband.
An understanding of the principles that govern such
higher-order cell behaviors will provide insight into how
erties of tissue organization.
Fly Stocks and Genetics
Flies were maintained at 25?C by standard procedures. Oregon R
was the wild-type stock. Germline clones for the DaPKCk06403
(Wodarz et al., 2000) and DmPar-6D226(Petronczki and Knoblich,
2001; Hutterer et al., 2004) alleles were generated by the FLP-DFS
system (Chou and Perrimon, 1992). Time-lapse imaging was con-
stock were imaged starting at stage 6b/7 and were genotyped by
cephalic furrow defects and partial germband extension. Maternal
bcd nos tsl mutant embryos were progeny of GFP:Resille; bcdE1
nosL7tsl146females. Bazooka:GFP embryos were the F2 progeny
of UASp:Bazooka:GFP (Benton and St. Johnston, 2003) and
mataTub-Gal4VP16 67C;15 (gift of D. St. Johnston). Ecad:GFP (Oda
and Tsukita, 2001), GFP:Moesin (Kiehart et al., 2000), and Sqh:GFP
(Royou et al., 2004) were expressed from endogenous promoters.
Embryos were fixed by heat-methanol fixation (Muller and Wie-
schaus, 1996) for staining with rabbit anti-Arm (1:200 [Riggleman
by Wodarz et al., ), rabbit anti-Myo II (Zipper heavy chain,
1:1250, gift of C. Field [Foe et al., 2000]), and mouse anti-Nrt (1:200,
Developmental Studies Hybridoma Bank, DSHB). Embryos were
fixed for 1 hr at the interface of heptane and 3.7% formaldehyde in
0.1 M sodium phosphate buffer (pH 7.4) and were manually devitelli-
nized for staining with Alexa-488 phalloidin (Molecular Probes),
guinea pig anti-Baz (1:500), rat anti-DE-cadherin (1:100 [Oda et al.,
1994], DSHB), and rabbit anti-GFP (1:100, Torrey Pines). Secondary
antibodies (1:500) were conjugated to Alexa-488, Alexa-568, or
Alexa-647 (Molecular Probes). Embryos were mounted in Prolong
Gold (Molecular Probes). Images were acquired on a Zeiss LSM510
META confocal microscope with a PlanNeo 403/1.3NA objective.
Quantitation of Polarized Protein Distribution
Embryos were staged as described by Campos-Ortega and Harten-
stein (1985); stage-6 embryos displayed no pole cell movement and
donot include embryos in6a or6b. Images weremaximum-intensity
projections of up to eight 1 mm apical slices acquired at 0.5 mm steps
to accommodate stage-specific differences in apical-basal distribu-
tion. F-actin and Myosin II were analyzed in projections of the same
and mean fluorescence intensity were measured in Object Image for
all edges in a 50 mm2region (73–276 interfaces/embryo). Absolute
fluorescence intensities were ranked and normalized to a scale
from 20.5 to 0.5 to generate relative intensity values that were neg-
ative for edges with below-average intensity and positive for edges
with above-average intensity. Distributions were scored as polar-
ized if all positive and negative bars were consecutive.
In Figures 4 and 5, Z stacks of 1 mm steps were acquired at 15 s in-
tervals on a Perkin Elmer RS5 spinning disc confocal microscope
with a Zeiss PlanNeo 403/1.3NA objective. Single optical slices
(GFP:Resille) or projections of 1–3 slices (GFP:Spider) within 3 mm
of the apical surface were analyzed. In Figures 7J–7L, Z stacks of
1 mm slices at 0.5 mm steps were acquired at 30 s intervals on a Zeiss
LSM510 META confocal microscope with a PlanNeo 403/1.3NA ob-
jective. For Bazooka:GFP and Ecad:GFP, maximum-intensity pro-
and mean interface intensity was measured in ImageJ. Interfaces
were scored as positive if they reached >50% of their final intensity
(average of ten steady-state measurements) after subtracting back-
ground intensity (average of ten cytoplasmic measurements).
Confocal images were analyzed in a semiautomated fashion by us-
ing custom software written in Matlab. A binary image representing
cell boundaries was obtained by repeated use of filters and image
operations and was used to construct a polygonal cellular lattice
whose geometric properties were calculated. Four-cell vertices
were manually tracked in 6–10 embryos for each genotype. To iden-
tify cells involved in rosette formation, all cells in a 403 field at the
onset of intercalation in stage 7 were tracked for 20–25 min. To an-
alyze interface dynamics, AP interfaces that were 3–7 mm long and
oriented at 72?–108?relative to the AP axis (20% of the angular
range) were measured in ImageJ at 1 min intervals starting 3–4
min before the increase in topological variance at stage 7.
Supplemental Data include color-coded pixel-intensity plots for
Figures 1 and 3, images of cell patterns in fixed embryos, and time-
lapse movies of wild-type and mutant embryos. These files are
available at http://www.developmentalcell.com/cgi/content/full/
We are grateful to Richard Zallen (Department of Physics, Virginia
Tech) for many valuable discussions and for advice on the statistical
analysis of cell patterns. We thank Keith Amonlirdviman for gener-
ously sharing an algorithm that we used as the basis for developing
our computer program and Chris Field for providing Myosin II anti-
bodies. We also thank Kathryn Anderson, Mary Baylies, Natalie De-
nef, Mimi Shirasu-Hiza, Eric Wieschaus, and anonymous reviewers
roughs Wellcome Fund Career Award in the Biomedical Sciences,
a March of Dimes Basil O’Connor Starter Scholar Research Award,
and a Searle Scholar Award to J.A.Z.
Received: June 13, 2006
Revised: August 9, 2006
Accepted: September 12, 2006
Published: October 2, 2006
Abdelilah-Seyfried, S., Cox, D.N., and Jan, Y.N. (2003). Bazooka is
a permissive factor for the invasive behavior of discs large tumor
cells in Drosophila ovarian follicular epithelia. Development 130,
Baum, B. (2004). Animal development: crowd control. Curr. Biol. 14,
Benton, R., and St Johnston, D. (2003). A conserved oligomerization
domain in Drosophila Bazooka/PAR-3 is important for apical locali-
zation and epithelial polarity. Curr. Biol. 13, 1330–1334.
Bertet, C., Sulak, L., and Lecuit, T. (2004). Myosin-dependent
junction remodelling controls planar cell intercalation and axis elon-
gation. Nature 429, 667–671.
Campos-Ortega, J.A., and Hartenstein, V. (1985). The Embryonic
Development of Drosophila melanogaster (Berlin: Springer-Verlag).
Chen, X., and Macara, I.G. (2005). Par-3 controls tight junction
assembly through the Rac exchange factor Tiam1. Nat. Cell Biol.
Chou, T.B., and Perrimon, N. (1992). Use of a yeast site-specific
recombinase to produce female germline chimeras in Drosophila.
Genetics 131, 643–653.
Classen, A.-K., Anderson, K.I., Marois, E., and Eaton, S. (2005). Hex-
agonal packing of Drosophila wing epithelial cells by the planar cell
polarity pathway. Dev. Cell 9, 805–817.
Concha, M.L., and Adams, R.J. (1998). Oriented cell divisions and
cellular morphogenesis in the zebrafish gastrula and neurula:
a time-lapse analysis. Development 125, 983–994.
Elul, T., and Keller, R. (2000). Monopolar protrusive activity: a new
morphogenic cell behavior in the neural plate dependent on vertical
interactions with the mesoderm in Xenopus. Dev. Biol. 224, 3–19.
Rosette Formation and Axis Elongation
Foe, V.E., Field, C.M., and Odell, G.M. (2000). Microtubules and
mitotic cycle phase modulate spatiotemporal distributions of
F-actin and myosin II in Drosophila syncytial blastoderm embryos.
Development 127, 1767–1787.
Franke, J.D., Montague, R.A., and Kiehart, D.P. (2005). Nonmuscle
in multiple tissues during dorsal closure. Curr. Biol. 15, 2208–2221.
Fristrom, D. (1988). The cellular basis of epithelial morphogenesis.
A review. Tissue Cell 20, 645–690.
Hardin, J. (1989). Local shifts in position and polarized motility drive
cell rearrangement during sea urchin gastrulation. Dev. Biol. 136,
Harris, T.J., and Peifer, M. (2004). Adherens junction-dependent
in Drosophila. J. Cell Biol. 167, 135–147.
Harris, T.J., and Peifer, M. (2005). The positioning and segregation
of apical cues during epithelial polarity establishment in Drosophila.
J. Cell Biol. 170, 813–823.
Hartenstein, V., and Campos-Ortega, J.A. (1985). The spatio-tempo-
ral pattern of embryonic cell divisions. Roux’s Arch. Dev. Biol. 194,
Hutterer, A., Betschinger, J., Petronczki, M., and Knoblich, J.A.
(2004). Sequential roles of Cdc42, Par-6, aPKC, and Lgl in the estab-
lishment of epithelial polarity during Drosophila embryogenesis.
Dev. Cell 6, 845–854.
Hyodo-Miura, J., Yamamoto, T.S., Hyodo, A.C., Iemura, S., Kusa-
kabe, M., Nishida, E., Natsume, T., and Ueno, N. (2006). XGAP, an
ArfGAP, is required for polarized localization of PAR proteins and
cell polarity in Xenopus gastrulation. Dev. Cell 11, 69–79.
Irvine, K.D., and Wieschaus, E. (1994). Cell intercalation during
Drosophila germband extension and its regulation by pair-rule
segmentation genes. Development 120, 827–841.
Jacinto, A., Wood, W., Woolner, S., Hiley, C., Turner, L., Wilson, C.,
Martinez-Arias, A., and Martin, P. (2002). Dynamic analysis of actin
cable function during Drosophila dorsal closure. Curr. Biol. 12,
Keller, R., Davidson, L., Edlund, A., Elul, T., Ezin, M., Shook, D., and
Skoglund, P. (2000). Mechanisms of convergence and extension
by cell intercalation. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355,
Kiehart, D.P., Galbraith, C.G., Edwards, K.A., Rickoll, W.L., and
Montague, R.A. (2000). Multiple forces contribute to cell sheet
morphogenesis for dorsal closure in Drosophila. J. Cell Biol. 149,
participates in adherens junctions and polymerization of linear actin
cables. Nat. Cell Biol. 6, 21–30.
Kovacs, E.M., Goodwin, M., Ali, R.G., Paterson, A.D., and Yap, A.S.
(2002). Cadherin-directed actin assembly: E-cadherin physically
associates with the Arp2/3 complex to direct actin assembly in
nascent adhesive contacts. Curr. Biol. 12, 379–382.
Lecaudey, V., and Gilmour, D. (2006). Organizing moving groups
during morphogenesis. Curr. Opin. Cell Biol. 18, 102–107.
Martin, P., and Lewis, J. (1992). Actin cables and epidermal move-
ment in embryonic wound healing. Nature 360, 179–183.
Muller, H.A., and Wieschaus, E. (1996).armadillo,bazooka,and star-
dust are critical for early stages in formation of the zonula adherens
and maintenance of the polarized blastoderm epithelium in
Drosophila. J. Cell Biol. 134, 149–163.
Munro, E.M., and Odell,G.M. (2002). Polarizedbasolateral cell motil-
ity underlies invagination and convergent extension of the ascidian
notochord. Development 129, 13–24.
Nance, J. (2005). PAR proteins and the establishment of polarity
during C. elegans development. Bioessays 27, 126–135.
Nikolaidou, K.K., and Barrett, K. (2005). Getting to know your neigh-
Ninomiya, H., Elinson, R.P., and Winklbauer, R. (2004). Antero-
posterior tissue polarity links mesoderm convergent extension to
axial patterning. Nature 430, 364–367.
Nusslein-Volhard, C., Frohnhofer, H.G., and Lehmann, R. (1987).
Determination of anteroposterior polarity in Drosophila. Science
Oda, H., and Tsukita, S. (2001). Real-time imaging of cell-cell adhe-
rens junctions reveals that Drosophila mesoderm invagination
begins with two phases of apical constriction of cells. J. Cell Sci.
Oda, H., Uemura, T., Harada, Y., Iwai, Y., and Takeichi, M. (1994). A
Drosophila homolog of cadherin associated with armadillo and
essential for embryonic cell-cell adhesion. Dev. Biol. 165, 716–726.
Petronczki, M., and Knoblich, J.A. (2001). DmPAR-6 directs epithe-
lial polarity and asymmetric cell division of neuroblasts in Drosoph-
ila. Nat. Cell Biol. 3, 43–49.
Redd, M.J., Cooper, L., Wood, W., Stramer, B., and Martin, P. (2004).
Wound healing and inflammation: embryos reveal the way to perfect
repair. Philos. Trans. R. Soc. Lond. B Biol. Sci. 359, 777–784.
Riggleman, B., Schedl, P., and Wieschaus, E. (1990). Spatial expres-
sion of the Drosophila segment polarity gene armadillo is post-
transcriptionally regulated by wingless. Cell 63, 549–560.
Royou, A., Field, C., Sisson, J.C., Sullivan, W., and Karess, R. (2004).
Reassessing the role and dynamics of nonmuscle Myosin II during
furrow formation in early Drosophila Embryos. Mol. Biol. Cell 15,
Schock, F., and Perrimon, N. (2002). Molecular mechanisms of
epithelial morphogenesis. Annu. Rev. Cell Dev. Biol. 18, 463–493.
Shih, J., and Keller, R. (1992). Cell motility driving mediolateral inter-
calation in explants of Xenopus laevis. Development 116, 901–914.
Solnica-Krezel, L. (2005). Conserved patterns of cell movements
during vertebrate gastrulation. Curr. Biol. 15, R213–R228.
Tepass, U., and Hartenstein, V. (1994). The development of cellular
junctions in the Drosophila embryo. Dev. Biol. 161, 563–596.
Topczewski, J., Sepich, D.S., Myers, D.C., Walker, C., Amores, A.,
Lele, Z., Hammerschmidt, M., Postlethwait, J., and Solnica-Krezel,
L. (2001). The zebrafish glypican knypek controls cell polarity during
gastrulation movements of convergent extension. Dev. Cell 1, 251–
Verma, S., Shewan, A.M., Scott, J.A., Helwan, F.M., den Elzen, N.R.,
Miki, H.,Takenawa,T.,and Yap, A.S.(2004).Arp2/3activity is neces-
sary for efficient formation of E-cadherin adhesive contacts. J. Biol.
Chem. 279, 34062–34070.
Wallingford, J.B., Fraser, S.E., and Harland, R.M. (2002). Convergent
extension: the molecular control of polarized cell movement during
embryonic development. Dev. Cell 2, 695–706.
Weaire, D., and Rivier,N. (1984). Soap, cells,and statistics—random
patterns in two dimensions. Contemp. Phys. 25, 59–99.
Wei, S.Y., Escudero, L.M., Yu, F., Chang, L.H., Chen, L.Y., Ho, Y.H.,
Lin, C.M., Chou, C.S., Chia, W., Modolell, J., and Hsu, J.C. (2005).
Echinoid is a component of adherens junctions that cooperates
with DE-Cadherin to mediate cell adhesion. Dev. Cell 8, 493–504.
Wieschaus, E., Sweeton, D., and Costa, M. (1991). Convergence and
extension during germband elongation in Drosophila embryos. In
Gastrulation, R. Keller, ed. (New York: Plenum Press), pp. 213–223.
Wodarz, A., Ramrath, A., Kuchinke, U., and Knust, E. (1999).
Bazooka provides an apical cue for Inscuteable localization in
Drosophila neuroblasts. Nature 402, 544–547.
Wodarz, A., Ramrath,A., Grimm, A., and Knust, E.(2000).Drosophila
atypical protein kinase C associates with Bazooka and controls
polarity of epithelia and neuroblasts. J. Cell Biol. 150, 1361–1374.
Wood, W., Jacinto, A., Grose, R., Woolner, S., Gale, J., Wilson, C.,
and Martin, P. (2002). Wound healing recapitulates morphogenesis
in Drosophila embryos. Nat. Cell Biol. 4, 907–912.
Young, P.E.,Richman,A.M.,Ketchum,A.S., andKiehart,D.P. (1993).
Morphogenesis in Drosophila requires nonmuscle myosin heavy
chain function. Genes Dev. 7, 29–41.
Zallen, J.A., and Wieschaus, E. (2004). Patterned gene expression
directs bipolar planar polarity in Drosophila. Dev. Cell 6, 343–355.
Zallen, J.A., and Zallen, R. (2004). Cell-pattern disordering during
convergent extension in Drosophila. J. Phys.: Condensed Matter