RESEARCH ARTICLE SUMMARY
◥CELL BIOLOGY
p53 directs leader cell behavior, migration, and
clearance during epithelial repair
Kasia Kozyrska†, Giulia Pilia†, Medhavi Vishwakarma‡, Laura Wagstaff‡, Maja Goschorska,
Silvia Cirillo, Saad Mohamad, Kelli Gallacher, Rafael E. Carazo Salas, Eugenia Piddini*
INTRODUCTION:Layers of epithelial cells pro-
tect animals from environmental insults. When
the integrity of these tissues is compromised by
injury, epithelial cells migrate as a cohesive
unit over the exposed area to seal the breach. In
some epithelia, collective migration is guided
by cells that acquire leader behavior. Leader
cells activate specific migratory pathways and
drive directed migration of the remaining
epithelial cells, which act as follower cells
to enable wound closure. How leader cells arise
from a seemingly homogeneous population
remains unresolved, particularly because only
a few cells at the wound edge develop into
leader cells. It is also unclear what happens
to leader cells once epithelial monolayers are
repaired. Understanding the basis of leader cell
specification will shed light on the fundamental
cellular processes that drive wound healing and
will help to identify interventions that could
accelerate and improve wound repair.
RATIONALE:Madin-Darbycaninekidney(MDCK)
epithelial cells are a well-characterized model
for investigating epithelial repair and leader
cell migration. The genetic tractability of these
cells and their ease of use for imaging assays
allow for in-depth molecular dissection of epi-
thelial cell biology. Prior work has shown that
leader cells have a characteristic flattened mor-
phology and distinct cytoskeletal properties and
that they activate specific migratory pathways.
In this study, we sought to identify how the
leader program is activated in epithelial cells.
We took advantage of the fact that in untreated
MDCK cultures, a few cells spontaneously dis-
play a morphology reminiscent of leader cells
and, on contact with neighbor cells with epi-
thelial morphology, lead directed migration.
These“spontaneous leaders”behave similarly
to leaders observed at the edge of injured MDCK
epithelial sheets. We investigated the mecha-
nisms that drive spontaneous leader cell behav-
ior, as an entry point to identify what invokes
leader cell specification upon epithelial injury.RESULTS :We found that spontaneous leaders
exhibited elevated cellular tumor antigen p53
levels. Indeed, inducing p53 activation, either
by inducing binucleation (which is a common
feature among leader cells) or with the DNA-
damaging agent mitomycin C (MMC) or the
Mdm2 inhibitor nutlin-3, was sufficient to
instruct leader behavior. Ablation of p53 by
CRISPR mutagenesis strongly inhibited the
emergence of leader cells upon MMC treatment,
indicating that p53 is necessary for cells to
be leaders. Working downstream of p53, wefound that its target gene p21WAF1/CIP1(p21)
was also elevated in spontaneous leaders. In-
deed, p21 elevation and its functional output,
cyclin-dependent kinase (CDK) activity inhibi-
tion, were sufficient and necessary to induce
leader cell behavior. Up-regulating p21 was
sufficient to elevate integrinb1 and phos-
phoinositide 3-kinase (PI3K), which are known
markers of spontaneous leader cells and are
required for their migration.
Next, we investigated whether the p53-p21-
CDK inhibition pathway that we identified in
spontaneous leaders was also responsible for
leader behavior upon epithelial injury. We found
that scratch-induced leader cells experienced
cell cycle delay, consistent with CDK inhibi-
tion, and showed high levels of both p53 and
p21. Using a live reporter of p53 activity, we
showed that injury itself induced p53 elevation
at the edge of the damaged epithelium. This
induction likely resulted from the mechanical
insult, as it was dependent on the stress kinase
p38, which activates p53 in response to me-
chanical stress. We then found that p53 and
p21 both promoted cell migration in mono-
layers undergoing repair. Indeed, activating
p53 at the migration front by laser-induced
DNA damage accelerated cell migration, an
effect that could be rescued by p53 inhibition.
Conversely, inhibiting p53 or p21 slowed down
migration.
We then followed the fate of leader cells as
the injury resolved itself. Prior work has shown
that cells with moderate p53 activation are
eliminated by mechanical cell competition. Ac-
cordingly, we found that, once the epithelium
was repaired, leader cells with high p53 activity
were cleared by cell competition, undergoing
extrusion or apoptosis. Failure to remove leader
cells compromised the regular cobblestone-like
morphology of the epithelium.CONCLUSION:We have identified p53 as a key
determinant of leader-driven cell migration in
epithelial repair. p53 activation appeared to
instruct leader cell specification and accelerate
cell migration by modulating p21 and CDK
activity. Upon epithelial repair, p53 induced
leader cell elimination by mechanical cell
competition, reinstating epithelial integrity.
Nonproliferative cells leading collective migration
have been previously observed in vivo in various
physiological and pathological contexts. The p53-
p21-CDK pathway could therefore have broader
relevance in leader-driven cell migration.
▪RESEARCH
628 11 FEBRUARY 2022•VOL 375 ISSUE 6581 science.orgSCIENCE
The list of author affiliations is available in the full article online.
*Corresponding author. Email: [email protected]
†These authors contributed equally to this work.
‡These authors contributed equally to this work.
Cite this article as K. Kozyrskaet al.,Science 375 , eabl8876
(2022). DOI: 10.1126/science.abl8876READ THE FULL ARTICLE AT
https://doi.org/10.1126/science.abl8876p53 and p21 induce leader cell behavior and drive migration in epithelial repair.(Left panel) p21-
overexpressing cells [green fluorescent protein (GFP)–negative] lead a colony of wild-type cells (GFP-
positive) and direct their collective migration. Cells are immunostained with the leader marker PI3K (white),
which is elevated in p21-expressing leaders. Nuclei are counterstained with DAPI (4′,6-diamidino-2-
phenylindole); phalloidin marks the actin cytoskeleton. (Middle and right panels) Model summarizing the
role of p53 in epithelial repair. Upon injury, mechanical damage of epithelial cells induces p53, which,
through p21 elevation and CDK inhibition, instructs leader cells to drive cell migration, accelerating epithelial
closure. High-p53 leader cells are also“mechanical losers”and are cleared by means of cell competition
once the epithelium closes. Removal of leaders contributes to the restoration of tissue integrity.