Science - USA (2022-04-15)

binding is specific for polarity-deficient elim-
ination; it is seen inscribmutant clones but not
loser cells outcompeted by Myc-overexpressing
neighbors (fig. S1, G to J). Egr binding, like cell
elimination, depends on Grnd (Figs. 1, D to F,
and2Iandfigs.S1CandS3,CtoE).Weused
patched-GAL4(ptc-GAL4)to conditionally deplete
dlg, generating a consistent stripe of apoptotic
cells that accumulate EgrV (Fig. 1, G to L, and
figs. S1, D and E, and S3, A and B) ( 9 , 10 ). As in
clones, Grnd depletion blocked elimination
and led to overgrowth (Fig. 1K and fig. S1F).
Mechanical wounding also activates JNK sig-
naling ( 11 ),andwoundsitesbindsecretedEgr
in a Grnd-dependent manner (fig. S2, A to I).
Increased Egr-Grnd binding is thus associated
with malignant cell elimination and physical
wounding, both of which disrupt epithelial
integrity.
Because JNK activation in both cases above
is associated with Egr binding, we investigated

the underlying mechanism. Data argue against

increased Grnd levels (fig. S5, A to R), changes in

Grnd N-glycosylation (fig. S5S) ( 8 ), altered

endocytic dynamics (fig. S4, A to L) ( 12 ), or

increased Egr levels (fig. S6, A to Q) as media-

tors ofdlg-depleted cell apoptosis. In func-

tional experiments, neither autocrine nor

paracrine epithelial Egr was required (Fig. 2,

A, D to F, H, and L, and fig. S3, F and J).

Because polarity-deficient clones survive in

an animal completely devoid of Egr (Fig. 2, G

and L) ( 10 , 12 , 13 ), the Egr required for eli-

mination must come from another source.

Both fat body and hemocytes (innate im-

mune cells) produce Egr ( 14 – 17 ). We codepleted

egranddlgsimultaneously from both the disc

stripe and these tissues. Hemocytes did not

associate with Dlg-deficient cells, and code-

pletion of hemocyte Egr had no impact on

elimination (Fig. 2, B and D, and figs. S3G and

S6, R and S). Codepletion of fat body Egr

prevented apoptosis in the stripe; Dlg-deficient

cells persisted and overgrew (Fig. 2, C and D,

and fig. S3, H to J).scribmutant disc clones

persisted upon Egr depletion in fat body and

hemocytes (Fig. 2, J and L) but not when Egr

was depleted in hemocytes alone (Fig. 2, K

and L). Wound healing was also perturbed

by depletion of fat body Egr (fig. S2, J and K).

Together, these data indicate that circulat-

ing Egr is essential for full activation of Grnd

and JNK signaling in response to epithelial

interruptions.

The above results prompt consideration of

ligand and receptor localization in this sig-

naling axis. Fat body–produced Egr is secreted

into hemolymph (circulatory fluid), which

bathes the disc basolateral surface (Fig. 3A)

( 15 ). However, steady-state Grnd is apically

polarized (Fig. 3, B and C) ( 7 ). Coculture ex-

periments revealed that EgrV binds only basally,

suggesting limited access of circulating Egr to

298 15 APRIL 2022•VOL 376 ISSUE 6590 science.orgSCIENCE

DCP-1 DCP-1 DCP-1

ctrl clones

RFP

scrib clones

ACB

E

I

EFF

J

F

scrib clones

FB+Hem>

egr KD

I J

scrib clones

en> egr KD

A P

H

RFP

scrib clones

en> grnd KD

A P

scrib clones

egr -/-

G

D

RFP

K scrib clones

Hem>egr KD

ptcts> dlg KD

egr KD

ptcts> Hem ts>

dlg KD

egr KD

ptcts> FBts >

dlg KD

egr KD

RFP

RFP

RFP

RFP

***

n.s.

***

n.s.***

Central DCP-1 enrichment

(Ptc stripe / WT)

RFP- / sibling RFP+

clone area clone area

n.s.

***

**

L

Fig. 2. Egr required for cell elimination derives from circulation.(Ato
D)dlgcell elimination in the stripe (DCP-1, red dotted lines) is not prevented
by codepletion of autonomousegr(A) noregrcodepletion in hemocytes (B).
Depletion ofegrfrom fat body and stripe preventsdlgcell apoptosis (C).
Quantitation of apoptosis shows requirement for autonomous Grnd and fat body-
produced Egr (D). (EtoL) Although WT mitotic clones survive (E),scribclones are

eliminated (F).scribclones survive in entirelyegr-mutant animal (G), but are

eliminated in a field ofegr-depleted cells (H). As with autonomous depletion

ofgrnd(I),scribclone elimination is blocked whenegris depleted in fat body

and hemocytes (J) but not in hemocytes alone (K). Quantitation is shown in (L).

Dotted lines indicate clone boundaries or posterior (P) compartment gene

depletion. Scale bars, 100mm in (A); 25mm in (E).

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