CELL BIOLOGY
Cell division in tissues enables
macrophage infiltration
Maria Akhmanova^1 , Shamsi Emtenani^1 †, Daniel Krueger^2 ‡§, Attila Gyoergy^1 ‡, Mariana Guarda^1 ,
Mikhail Vlasov^3 , Fedor Vlasov^3 , Andrei Akopian^3 , Aparna Ratheesh^1 ¶,
Stefano De Renzis^2 , Daria E. Siekhaus^1
Cells migrate through crowded microenvironments within tissues during normal development, immune
response, and cancer metastasis. Although migration through pores and tracks in the extracellular
matrix (ECM) has been well studied, little is known about cellular traversal into confining cell-dense
tissues. We find that embryonic tissue invasion byDrosophilamacrophages requires division of an
epithelial ectodermal cell at the site of entry. Dividing ectodermal cells disassemble ECM attachment
formed by integrin-mediated focal adhesions next to mesodermal cells, allowing macrophages to move
their nuclei ahead and invade between two immediately adjacent tissues. Invasion efficiency depends
on division frequency, but reduction of adhesion strength allows macrophage entry independently of
division. This work demonstrates that tissue dynamics can regulate cellular infiltration.
C
ell dissemination into tissues is funda-
mental for the formation and main-
tenance of complex organisms ( 1 – 3 ).
During vertebrate development, neural
crest cells move into tissues to form
different tissue types ( 3 ); immune cells enter
organs to regulate tissue function and com-
bat infection ( 4 ); cancer cells traverse into
other organs during metastasis ( 2 ). These
various embryonic and adult environments
contain closely packed cells adherent to one
another or to the extracellular matrix (ECM)
that lies between them ( 1 , 5 ). Despite its im-
portance, invasion into such cell-dense tissues
is poorly understood.
Macrophages are scavenger cells that invade
tissues early on in development to establish
residency and patrol organs ( 6 ). In the early
Drosophilaembryo, macrophages follow guid-
ance cues ( 7 ) and invade the germ band (GB)
at an entry point (EP) in the acute angle be-
tween the basal side of the ectoderm and the
mesoderm surface (Fig. 1A and fig. S1A). They
progress farther between the ectoderm and
the mesoderm, which are juxtaposed across
a thin layer of ECM ( 8 ). During this invasion,
macrophages move as a chain, maintaining
the separation of the two tissues established
by the pioneer (Fig. 1A′;fig.S1,A′to D; and
movie S1). This first macrophage requires
~20 min to enter through the tissue barrier,
usingaPS2-andbPS-integrins that bind to
laminin ( 9 , 10 ). Matrix metalloproteolytic ECM
degradation does not affect the efficiency of
entry ( 10 ) but macrophage-specific programs
do ( 11 – 13 ). How the dynamics and properties
of surrounding cells influence macrophage
tissue invasion remains unclear.
We tracked macrophage nuclei (Fig. 1B and
fig. S1E), the stiffest of the organelles. We
plotted the velocity of the first macrophage’s
nucleus relative to the entry point over time
and defined the moment of entry between the
two tissues by its velocity jump when close to
the entry point (fig. S1, F and G). As the first
macrophage entered, one or two ectodermal
cells adjacent to the entry point had become
round, a shape change that starts at the be-
ginning of mitosis ( 14 ), or had progressedto become two connected smaller rounded
daughters, formed by the completion of divi-
sion(Fig.1,CandD;fig.S1,HandI;and
movie S2). By analyzing the division profiles
of 20 time-lapse movies, we observed that
macrophage entry always occurred during
the division (fig. S2, A and C) or rounding
(fig. S2, B and D) of a flanking ectodermal
cell (fig. S2E). We quantified how much time
the ectodermal cells at the entry point spent
in three categories: (i) at least one of the two
cells is dividing; (ii) at least one cell is round-
ing; or (iii) the interdivision phase, during
which none of these cells are mitotic (fig. S2F).
If macrophage invasion occurred randomly,
macrophages would enter during the inter-
division phase in more than half of the em-
bryos; however, we never observed entry at
this time (Fig. 1E). Within an embryo (when
viewed in a dorsal orientation), there are two
bilaterally symmetrical entry points next to
the hindgut. Macrophages arrived at these
two locations almost simultaneously; how-
ever, they did not enter at the same time if
ectodermal divisions at both points occurred
asynchronously (fig. S3). We also found no
divisions of mesodermal cells at the entry site
during invasion (fig. S4), perhaps because the
mesodermal division frequency is much lower
than in the ectoderm (fig. S5). Hence, in wild-
type embryos, macrophages enter the GB tissue
only when an adjacent ectodermal cell is divid-
ing, suggesting that this is a permissive event.
To investigate the ectoderm–mesoderm in-
terface structure, we stained embryos for the
ECM component laminin, secreted there pre-
viously by the mesoderm and also expressed
within entering macrophages ( 15 ). Quanti-
fication shows that more laminin remains394 22 APRIL 2022•VOL 376 ISSUE 6591 science.orgSCIENCE
(^1) Institute of Science and Technology Austria (IST Austria),
Klosterneuburg, Austria.^2 Developmental Biology Unit,
European Molecular Biology Laboratory (EMBL), Heidelberg,
Germany.^3 Bundesgymnasium Klosterneuburg,
Klosterneuburg, Austria.
*Corresponding author. Email: [email protected]
(M.A.); [email protected] (D.E.S.)
†Present address: Institute of Molecular Biotechnology, Austrian
Academy of Sciences, Vienna Biocenter, Vienna, Austria.
‡These authors contributed equally to this work.
§Present address: Hubrecht Institute, Royal Netherlands Academy of
Arts and Sciences, Utrecht, Netherlands.
¶Present address: Centre for Mechanochemical Cell Biology,
Warwick Medical School, University of Warwick, Coventry, UK.
Fig. 1. Macrophage invasion correlates with ectodermal cell division at the entry point.(AandA′) Still
images of cytoplasm-labeled macrophages at the entry point (EP, white triangle) and after invading as a chain.
ubiq-membrane, ubiquitous membrane marker. (BandC) Single plane (B) still images and (C) corresponding
maximum projections showing a nuclear-labeled macrophage (yellow arrowhead) at the EP with ectodermal
cell 1 (dashed outline) adjacent to, and cell 2 (solid outline) above, this entering macrophage. The outlined
ectodermal cells mitotically round and divide. Scale bars, 10mm. (D) Cell 1 and 2 division profiles in time. Peak
indicates division; middle level, rounded cell; and lower level, polygonal shape. Time of macrophage entry,
t= 0. Shading indicates macrophage at EP. (E) Quantification of macrophages entering together with ectodermal
cell cycle phases. (Left) Theoretical estimate assuming random entry proportional to average duration of each
phase, presented in fig. S2F. (Right) In vivo determined entry for 20 embryos (fig. S2, A and B).
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