CELL BIOLOGY
Active cell migration is critical
for steady-state epithelial turnover
in the gut
Denis Krndija^1 *, Fatima El Marjou^1 , Boris Guirao^2 , Sophie Richon^1 , Olivier Leroy^2 ,
Yohanns Bellaiche^2 , Edouard Hannezo^3 †, Danijela Matic Vignjevic^1 †
Steady-state turnover is a hallmark of epithelial tissues throughout adult life. Intestinal
epithelial turnover is marked by continuous cell migration, which is assumed to be driven by
mitotic pressure from the crypts. However, the balance of forces in renewal remains ill-defined.
Combining biophysical modeling and quantitative three-dimensional tissue imaging with
genetic and physical manipulations, we revealed the existence of an actin-related protein
2/3 complex–dependent active migratory force, which explains quantitatively the profiles of
cell speed, density, and tissue tension along the villi. Cells migrate collectively with minimal
rearrangements while displaying dual—apicobasal and front-back—polarity characterized
by actin-rich basal protrusions oriented in the direction of migration. We propose that active
migration is a critical component of gut epithelial turnover.
T
he gut epithelium is the largest mucosal
surface of the body ( 1 )andoneofthemost
rapidly renewing tissues (3 to 5 days) in
adult mammals ( 2 ). A single layer of colum-
nar cells lines the villi (finger-like evagina-
tions that project into the gut lumen) and the
crypts (small invaginations into the connective
tissue). Stem cells in the crypts give rise to transit-
amplifying cells, which divide four to five times
before terminal differentiation ( 2 ). After exiting
the crypt, differentiated epithelial cells migrate
for ~3 days, until they reach the top of the villus,
where they get extruded ( 2 ). Continuous migra-
tion of cells between the spatially distant func-
tional compartments of celldivisionandlossisa
major component in gut epithelial renewal. The
predominant theory is that cells migrate pas-
sively, driven by a pushing force resulting from
cell division (i.e., mitotic pressure) ( 3 , 4 ). However,
irradiation and mitotic inhibitor treatment do
notblockgutepithelialmigration,despitecausing
substantial cell loss in the crypts ( 5 , 6 ), raising
the possibility of active migration. In this study,
we investigated the mechanism(s) of epithelial
cell migration during homeostatic renewal in the
small intestine.
To quantitatively test the contribution of mi-
totic pressure in epithelial cell migration along
the villi, we performed 5-ethynyl-2′-deoxyuridine
(EdU) pulse-chase assays in mice injected with
hydroxyurea (HU), a specific S-phase inhibitor ( 7 , 8 ).
A low dose of HU (HUlo) efficiently inhibited
mitosis, without affecting cell migration along
the villi (Fig. 1, A to C, and figs. S1, A to D, and S2,
A to C). This suggests that cell division per se is
not the principal driving force for cell migration.
High concentrations of HU (HUhi) were shown
to inhibit cell migration but are proapoptotic ( 5 ).
As expected, HUhireduced crypt cellularity (fig. S1,
A to D), but cell migration was decreased only in
the lower villus region (Fig. 1, A to C, and fig. S2,
A, B, and D). Together, these data suggest that
the acting range of mitotic pressure is limited to
crypts and the lower villus region.
We next investigated the alternative possibility
that cells could migrate actively. Because of the
potentially complex spatial interplay between
passive and active migratory forces, we developed
a biophysical model for gut epithelium renewal.
This minimal continuum theory for migrating
and proliferating cells ( 9 , 10 ) (Fig. 1D, fig. S3, and
theory note in the supplementary materials) pre-
dicted that proliferation forces alone would result
in a gradual decrease in cell density toward the
villus, whereas active migration would cause a
density increase because of cell overcrowding
near the villus top (Fig. 1E). We measured one-
dimensional (1D) densityprofiles along the villus
axis [as well as 2D density (fig. S4)] and observed
that cell density first gradually decreased toward
the middle of the villus and then increased in the
top region of the villus (Fig. 1, F to H), in good
qualitative agreement with the active migration
model (Fig. 1E). We thus hypothesized that cell
migration along the villus operates according to
a two-tier migration system: proliferation forces
dominate close to the crypt, whereas active mi-
gration forces become predominant in the upper
part of the villus. By fitting the active migration
force in our model, we obtained a good quantita-
tive prediction for the nonmonotonous cell den-
sity profile along the villus (Fig. 1H and fig. S3G).
Moreover, simulating a short-term mitotic pres-
sure inhibition predicted a density decrease only
at the villus bottom, which was quantitatively
confirmed, with HUhitreatment decreasing celldensity only at the bottom 10% of the villus (Fig.
1,ItoK,andfig.S1,FandG).Thesedatastrongly
suggested the existence of an active migratory
mechanism, in addition to mitotic pressure, which
contributes to cell migration along the villi.
The model also predicted an increase in cell
speed along the villus axis, dependent on the
strength of the active migration force (Fig. 2A).
Using EdU pulse-chase assays, we found greater
cell displacements in the middle villus compared
with the villus bottom (Fig. 2B). To measure cell
speed directly, we performed live imaging of gut
explants derived from Villin:CreERT2/mTmG re-
porter mouse ( 11 – 13 ), where membrane-targeted
green fluorescent protein (GFP) is expressed
mosaically in the epithelium (Fig. 2, C to E, and
fig. S5A). Cells accelerated as they progressed
along the villus axis (Fig. 2D and movie S1). Ex
vivo speed profiles, together with in vivo EdU
pulse-chase data, correlated well with model
predictions for an active migratory mechanism
(Fig. 2E).
We then characterized collective cell dynamics,
with the tracked patch of cells retaining cohesive-
ness during migration and extending ~10% along
the villus axis without simultaneously shrinking
laterally (Fig. 2F, fig. S5B, and movie S4). This
could be almost entirely accounted for by cell
extension along the villus axis; contributions due
to junction remodeling from cell rearrangements
and delamination were limited, and, as expected,
no divisions were observed (Fig. 2F). The observed
cohesiveness, consistent with the clonal ribbons
observed previously ( 14 ), could be well explained
by a minimal 2D model of cells performing biased
random walks toward the villus top, with cell-cell
interactions minimizing cell dispersion and allow-
ing collective migration (fig. S5, C to F).
Forces driving epithelial migration have
been best characterized quantitatively in culture
( 15 – 19 ), where active migration results in a build-
up of tension in the colony center ( 16 ). Similarly,
an active migration model predicts a buildup of
mechanical tension in the villus bottom, driven by
cell traction forces toward the top. Conversely, if
proliferation forces acted alone, pressure should
be highest (and tension lowest) close to the crypt,
while dissipating along the crypt-villus axis as a
result of friction with the basement membrane.
Spatial differences in tissue tension can thus be
used to infer how and where forces act during
cell migration ( 16 , 20 – 23 ). We imaged mice with
GFP-tagged endogenous nonmuscle myosin IIA
heavy chain ( 24 ) to highlight the apical actomy-
osin belt associated with E-cadherin–containing
adherens junctions ( 25 ) (fig. S5G). To map tensile
forces, we made circular laser cuts of epithelial
adherens junctions at the lower and upper villus
regions (fig. S5H). The epithelium recoiled iso-
tropically after the laser cut, indicating that it was
always under tension (Fig. 2, G and H, and fig.
S5H). The recoil speed was significantly higher
in the lower villus region (Fig. 2H, fig. S5I, and
movies S2 and S3). This further supported a model
of active migration forcescausingacrowdingor
increased pressure effect at the top and leaving
the villus bottom under higher tension.RESEARCH
Krndijaet al.,Science 365 , 705–710 (2019) 16 August 2019 1of6
(^1) Institut Curie, PSL Research University, CNRS UMR 144,
F-75005 Paris, France.^2 Institut Curie, PSL Research
University, U934/UMR3215, F-75005 Paris, France.^3 Institute
of Science and Technology Austria (IST Austria), Am
Campus 1, 3400 Klosterneuburg, Austria.
*Corresponding author. Email: [email protected]
†These authors contributed equally to this work.