Science - USA (2022-01-07)

therefore inactivation, was observed (Fig. 3,
E and G). This geometry-dependent region-
alization of YAP activity was again lost at later
time points (>36 hours) when cells throughout
thetissuebecameuniformlypacked(fig.S4A).
To test whether differential cell spreading (and,
conversely, cell crowding) is sufficient to drive
differences in YAP activity, we cultured ISCs at
equal cell-seeding density in microcavities of

small (50mm) and large (100mm) diameter,

resulting in a packed or a spread system (fig.

S4B). YAP activity strongly correlated with

cell spreading: Nuclear translocation was sig-

nificantly more frequent within 100-mm wells

than in 50-mm wells, which suggests that tissue

geometry per se controls the spatially patterned

activation of YAP through differential cell

spreading (fig. S4, C and D).

Several recent studies have implicated YAP

activation in the repression of canonical ISC

signatures, includingLgr5,Olfm4, andEphB3,

during intestinal regeneration and cancer

( 24 , 27 , 30 , 31 ). Given the spatial and temporal

correlation between YAP induction and Lgr5

suppression, we hypothesized that the pattern-

ing of this system is driven by geometrically

and mechanically established gradients in

Gjorevskiet al.,Science 375 , eaaw9021 (2022) 7 January 2022 4of9

24 h

Nuclei E-cad Nuclei YAP

<24 h 24 h 36 h 72 h

YAP Lgr5 YAP Lgr5 YAP DLL1

ISCs

Paneth cells

Enterocytes

Mechanical

patterning

Symmetry breaking

Crypt domain appearance

Differentiation

Villus domain appearance

E

2 h 12 h 24 h 36 h 48 h 60 h 72 h 96 h

Brightlield

Lgr5-eGFP

48 h

Nuclei E-cad YAP Nuclei AldoB Nuclei DLL Lys

72 h

YAP E-cad Lys Nuclei AldoB

average internuclear distance% cells with nuclear YAP

F

TipsSides

G

Tips Sides

A

DLL

+ cells per location

I

Tips Sides

JK

YAP

Nuclei DLL Lys

L

Tips

Sides

C time, hours

B 2 h 12 h 24 h 36 h 48 h 60 h 72 h 96 h 100 200 300 400

Lgr5-eGFP frequency

min

max

) (^03)
2.5
2
1.5
1
0.5
10 20 30 40 50 60 70 80 90
D
Time, hours
0

---

---

---

50
45
40
35
30
25
20
15
10
100
80
60
40
20
0 8 6 4 2 0
length of the organoid, μm
Average Lg
r5-eGFP intensity
96
H 36 h
Nuclei E-cad Nuclei DLL YAP
Fig. 3. Tissue geometry controls organoid
patterning through cell shapeÐmediated
regulation of YAP and Notch signaling.
(AandB) Bright-field and Lgr5-eGFP time-lapse
imaging of the representative organoid development
(A) and frequency maps showing average Lgr5
expression over ~80 tissues (B). (CandD) Relative
changes in the Lgr5-eGFP expression in curved
ends and flat sides of the organoids over time
(C) and Lgr5-eGFP localization along length of the
averaged tissue over time (D). (E) Immuno-
fluorescence images showing the difference in
internuclear distance, cell shape, and subcellular
distribution of YAP between cells of the end
and the side regions, 24 hours after cell loading.
(F) Quantification of internuclear distance within
the end and side regions of the tissues. Individual
points, which represent the distance between
neighboring nuclei, and means are shown.
(G) Quantification of the nuclear localization of
YAP within cells of the different organoid regions.
Individual points and means are shown.
(H) Immunofluorescence images showing the
difference in the subcellular distribution of YAP cells
between cells of the end and the side regions
and appearance of the first DLL+, 36 hours after
cell loading. Arrowheads denote adjacent pairs of
YAP-ON/OFF cells; the YAP-ON cell expresses DLL1.
(I) Quantification of DLL+cell localization. (Jand
K) Immunofluorescence images showing YAP
expression and localization of the enterocytes
(AldoB), Paneth cells (Lys), and DLL+cells in the
representative organoids. (L) Schematic illustration
summarizing the proposed mechanism of the
geometry-driven organoid patterning. Scale bars,
25 mm. ****P< 0.0001 [(F), (G), (I)].
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