We next asked how ectodermal mitotic
rounding facilitates macrophage entry. First,
we tested whether the increased cortical stiff-
ness that causes rounding, or the free space
that results from it, might help macrophages
to advance ( 14 , 16 ). We temporarily increased
cortex contractility through optogenetic recruit-
ment of the Rho1 exchange factor RhoGEF2 to
theplasmamembrane(fig.S10A)( 17 ), inducing
rounding of ectodermal cells in a small region
(50mmby40mmby20mm) at the entry site
~40 min before normal macrophage entry (fig.
S10, B to D) without causing de-adhesion of
these cells (fig. S10, E to H). This approach,
distinct from the long-lasting widespread
increase in ectodermal tension that impedes
macrophage invasion ( 8 ), caused no change
in the timing of macrophage entry (fig. S10,
I to K). Next, using fluorescent dextran, we
examined the intercellular space that mitotic
rounding opens at the entry point in wild-type
embryos. This distance increased by only 30%,
producing a gap narrower than the diameter
of the macrophage nucleus when squeezed
between tissues and much narrower than
the space observed between the tissues during
invasion (fig. S11). These results suggest that
ectodermal cell rounding alone is not suffi-
cient to promote macrophage entry.
The ectoderm faces the mesoderm with its
basal side, forming focal adhesions (FAs)
that bind throughaPS1- andbPS-integrins to
laminin, which could thus impede macrophage
entry (Fig. 3, A and B) ( 8 ). FAs have been ob-
served to disassemble during vertebrate mitotic
rounding in vitro ( 18 ). We examined their in vivo
temporal and spatial dynamics in the GB. We
first visualized these FAs by staining against
b-integrin, which localized to dot-like adhesive
structures at the ectodermal–mesodermal inter-
face(fig.S12,AtoA′′). Focal adhesion com-
ponents vinculin and talin ( 19 ) also showed
a dotted pattern along this interface (fig. S12, B
to D). Once an ectodermal cell started to round,
these FAs (visualized live by vinculin::mCherry)
gradually disappeared, leaving only one peak
remaining in the middle of the basal side (fig.
S12E and Fig. 3, C and D), which gradually
flattened (Fig. 3, C to E). FA disassembly did
not depend on the presence of macrophages
and took place in every basally dividing cell in
the ectoderm (fig. S13, A to E, and movie S7);
FAs reassembled after daughter cells acquired
a polygonal shape (fig. S13, F and G, and
movie S8). Imaging ectodermal mitotic cells
at the entry point revealed that macrophages
always entered after disassembly of the last
adhesion spot (Fig. 3, C to F; movie S9; and
fig. S14, A to F). The macrophage nucleus
moved farther into the tissue between other
FAs, which do not disassemble (fig. S14, B′to F′
and G to H′). In cases where entry occurred
next to the rounded daughter cells produced
by division, mitosis was completed faster than
macrophage nuclear translocation; however,
new FAs were not established before entry (fig.
S13, F and G). We conclude that FA disassembly
at the entry point correlates with the macro-
phage nucleus’s penetration between tissues.
Finally, we reduced FA componentsb-integrin,
talin, or vinculin in the ectoderm by RNAi knock-
down (fig. S15, A to F) without affecting devel-
opment (fig. S15, G and H). This resulted in
higher macrophage numbers in the GB (Fig. 4A
and fig. S16, A to C) that entered earlier but
without a change in speed compared with the
control (fig. S16, D to G). In contrast,b-integrin
knockdown in the mesoderm had no such
effect (fig. S16H), confirming that mesoderm
adhesiveness does not hinder invasion. The
first macrophage could enter without ecto-
dermal rounding or division inb-integrin or
vinculin knockdown embryos, unlike in the
wild type (Fig. 4B, fig. S16I, and movie S10).
Even in dinaciclib-injected embryos, ectoder-
malb-integrin knockdown resulted in macro-
phages entering in the absence of rounding or
dividing cells at the entry point, further sug-
gesting that rounding itself is not necessary
for invasion (fig. S16J). Thus, although we can-
not exclude a contribution from other effects,
we reveal the disassembly during mitosis of
the focal adhesions that attach a cell to its
environment as the main mechanism by which
division opens the door for macrophage infil-
tration (fig. S17).
We have pinpointed surrounding tissue di-
vision as the crucial variable affecting the rate
of entry of the firstDrosophilamacrophage.
Vertebrate mitosis also leads to diminishment
of focal adhesions ( 18 ). Our findings suggest
that regulation of division during develop-ment, inflammation, or tumor growth could
affect the number and placement of immune
cells in tissues in a wide range of normal and
disease contexts.REFERENCES AND NOTES- P. Friedl, B. Weigelin,Nat. Immunol. 9 , 960–969 (2008).
- P. Friedl, S. Alexander,Cell 147 , 992–1009 (2011).
- P. M. Kulesa, L. S. Gammill,Dev. Biol. 344 , 566– 568
(2010). - K. Kierdorf, M. Prinz, F. Geissmann, E. Gomez Perdiguero,
Semin. Immunol. 27 , 369–378 (2015). - T. A. Stewart, K. Hughes, D. A. Hume, F. M. Davis,Front. Cell
Dev. Biol. 7 , 250 (2019). - S. Epelman, K. J. Lavine, G. J. Randolph,Immunity 41 , 21– 35
(2014). - A. Ratheesh, V. Belyaeva, D. E. Siekhaus,Curr. Opin. Cell Biol.
36 , 71–79 (2015). - A. Ratheeshet al.,Dev. Cell 45 , 331–346.e7 (2018).
- K. Comberet al.,J. Cell Sci. 126 , 3475–3484 (2013).
- D. Siekhaus, M. Haesemeyer, O. Moffitt, R. Lehmann,Nat. Cell
Biol. 12 , 605–610 (2010). - K. Valoskovaet al.,eLife 8 , e41801 (2019).
- V. Belyaevaet al.,PLOS Biol. 20 , e3001494 (2022).
- S. Emtenaniet al.,EMBO J.10.15252/embj.2021109049
(2022). - A. V. Taubenberger, B. Baum, H. K. Matthews,Front. Cell Dev. Biol.
8 , 687 (2020). - Y. Matsubayashiet al.,Curr. Biol. 27 , 3526–3534.e4 (2017).
- W. Daiet al.,Science 370 , 987–990 (2020).
- E. Izquierdo, T. Quinkler, S. De Renzis,Nat. Commun. 9 , 2366
(2018). - C. L. Dixet al.,Dev. Cell 45 , 132–145.e3 (2018).
- B.Klapholz,N.H.Brown,J. Cell Sci. 130 , 2435–2446 (2017).
ACKNOWLEDGMENTS
We thank C. Guet, T. Hurd, J. Friml, M. Fendrych, and laboratory
members for comments on the manuscript. We also thank
the Bioimaging Facility of IST Austria for excellent support and
T. Lecuit, E. Hafen, R. Levayer, and A. Martin for fly strains.
All the confocal images and movies obtained during this study
are archived on a server at IST Austria and can be obtained
upon request. Stocks used in this study were obtained from the
BloomingtonDrosophilaStock Center (NIH P40OD018537).
Funding:M.A. was supported by an Austrian Science Fund FWF:
Lise Meitner Fellowship M2379-B28 to M.A. and D.E.S. S.E.,
M.G., and A.G. were supported by internal funding from IST Austria
to D.E.S. D.K. was supported by internal funding from EMBL to
S.D.R.Author contributions:M.A. and D.E.S. conceived of
the study, designed the experiments, and interpreted the results.
M.A. discovered the phenomena with valuable input from A.R. M.A.,
S.E., A.G., and M.G. performed fly crossing, fixation, and imaging
and analyzed the experiments. A.G. generated thesrpHemo-
mCerulean::H2B::Dendraline. M.A. performed live imaging and drug
injection experiments. M.V., F.V., and A.A. performed fly work
and crossing. A.A. wrote the data analysis script. A.R. provided
PH3 staining images. D.K. and S.D.R. provided optogenetic setup
and flies, and guided M.A. during optogenetic experiments.
M.A., S.D.R., and D.E.S. acquired funding. M.A. and D.K. made
illustrations. M.A. and D.E.S. wrote the manuscript, with
contributions from S.D.R., D.K., S.E., M.G., and A.R.Competing
interests:The authors declare that they have no competing
interests.Data and materials availability:All data are available
in the main text or the supplementary materials. Image analysis
scripts are publicly available at https://github.com/Axmasha/
Image_analysis_scripts and https://github.com/andrei-akopian/
Data_alignment_script. All materials are available upon request.SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abj0425
Materials and Methods
Figs. S1 to S17
Exact genotypes ofDrosophilalines used in the figures
References ( 20 – 41 )
MDAR Reproducibility Checklist
Movies S1 to S10
Data S116 April 2021; accepted 25 March 2022
10.1126/science.abj0425396 22 APRIL 2022•VOL 376 ISSUE 6591 science.orgSCIENCE
Fig. 4. Reducing ectoder-
mal FA components
enables macrophage
entry without mitotic
rounding.(A) Percent of
all macrophages that
were found inside the GB in
the indicated knockdown
embryos. Mean ± SEM.
(Left to right) P= 0.0038, P= 0.0052, *P= 0.018, *P= 0.0005, P= 0.0041, unpairedttests.
(B) Quantification of macrophage entry timing.
RESEARCH | REPORTS