418 | Nature | Vol 584 | 20 August 2020
Article
are driven by 20HE signalling from the ovaries to progenitor cells
in the gut.
Gut growth after mating is expected to increase the absorption of
nutrients by the intestine and the supply of nutrients to other organs.
Because egg production is limited by nutrient availability to the ovaries,
we tested whether 20HE-dependent gut growth affected female fecun-
dity. When we blocked gut resizing by expressing EcR RNAi (EcRRNAi)
in midgut ISCs, or in both ISCs and EBs, egg production was reduced
by approximately 40% (Fig. 2p, Extended Data Fig. 6b–d; see also
ref.^7 ). This suggests that 20HE-dependent gut remodelling maximizes
female reproductive fitness. However, we also noticed that our Gal4
drivers were active in a small number of escort cells in the germarium
of the ovary (Extended Data Fig. 6a, e–l), which raises the possibility
that these fecundity defects were due in part to a requirement for EcR
in those cells.
A study of Drosophila juvenile hormone ( JH), a sesquiterpenoid,
came to conclusions similar to ours—namely that JH promotes
mating-dependent gut growth and fecundity in females^15. We there-
fore investigated the relative roles of 20HE and JH. We found that the
JH receptors Gce and Met are essential for ISC divisions in response
to not only the JH receptor agonist, methoprene^15 , but also 20HE and
infection (Extended Data Fig. 7a–c). We confirmed the mitogenic effects
of methoprene, but these were weaker than those of 20HE (Extended
Data Figs. 5a, 7a–g) or mating (Fig. 2a). Furthermore, we discovered that
methoprene-stimulated divisions require 20HE (Extended Data Fig. 7g),
and that JH or methoprene could suppress ISC divisions driven by 20HE
or other stimuli (Extended Data Fig. 7a, d–f ). Although these results
indicate interaction between 20HE and JH, further work is required to
determine their precise physiological relationship (see Supplementary
Discussion).
To better understand how ecdysone activates ISCs, we tested two
known EcR targets: the transcription factor Broad, and the nuclear
receptor Eip75B, a homologue of human PPARγ and REV-ERB^17. Eip75B
and broad (br) mRNA were induced in midguts by 20HE or mating
(Fig. 2l, Extended Data Fig. 8a), and progenitor-cell-specific depletion
of either factor suppressed 20HE-induced mitoses (Fig. 2m, Extended
Data Fig. 8b–e). ISC clonal growth, however, required Eip75B but not
br (Extended Data Figs. 2b, c, 8e, f ), highlighting that Eip75B is a more
essential effector. Overexpression of Eip75B was sufficient to promote
ISC division and gut epithelial turnover (Fig. 2n, Extended Data Fig. 8g),
whereas Eip75B loss impaired both ISC mitoses and maintenance
(Extended Data Fig. 5s, 8c, f, h, i). Progenitor-specific loss of Eip75B also
blocked gut growth after mating (Fig. 2c), and compromised egg pro-
duction (Fig. 2p, Extended Data Fig. 6b–d), phenocopying the effects
of EcR loss. Eip75B binds DNA to repress target genes, and also binds
the nuclear receptor Hr3 to inhibit Hr3-mediated transcriptional acti-
vation^18. Consistent with this mechanism, overexpression of Eip75B or
20HE feeding suppressed an Hr3 activity reporter, and Hr3 overexpres-
sion suppressed ISC proliferation (Extended Data Fig. 8j–l). Moreover,
depletion of Hr3 counteracted losses in ISC proliferation caused by
Eip75B depletion (Fig. 2o, Extended Data Fig. 8n). Although these results
indicate that Hr3 is a crucial Eip75B effector, Hr3 loss was not sufficient
to activate ISC division (Extended Data Fig. 8m, n), which indicates that
Eip75B has other targets. Further tests revealed that Eip75B and Hr3
mediate 20HE-independent ISC responses to stress. Enteric infection
strongly induced levels of Eip75B mRNA (Extended Data Fig. 8a) andControl #1Control #2EcRADN#1
EcRADN#2
EcRRNAi
UspRNAi
Eip75BRNAi050100Dysplasia frequency (%)Control#1Control#2EcRADN#1
EcRADN#2 RNAiEcRRNAiUsp
Eip75BRNAi(^100)
100
200
ISC mitoses per midgut
NS ****
Controldib
RNAi
spo
RNAi
Control dib
RNAi
spo
RNAi
Controldib
RNAi
spo
RNAi
0
50
100
150
200
250
300
ISC mitoses per midgut
NS
14 days14 days14 days14 days
0
25
(^10050)
200300
400500
ISC mitoses per midgut
Control Control
7 days old 35 days old
EcRADN EcRRNAi UspRNAi Eip75BRNAi
esg-Gal4
ts
abcd
Raised on
vehicle
Raised on
vehicle
Raised on
1 mM 20HE
C587-Gal4ts
20 days 42 days
esg-Gal4(14 days) tsNRNAi
f
ControlNRNAi
e Tu mour induction esg-Gal4ts gh i
NRNAi(14 days)
Control NRNAi
Vir
gins
Mated
EcRADN;NRNAi
N (14 days) RNAiphenotypes NRNAiinitiation phenotypes
RNAiNRNAiNEcRAVehicle1 mM20HE
DN
n = 13n = 13
0
20
40
60
80
100
Tumour frequency (%
)
0
20
40
60
80
100
Tumour frequency (%
)
n = 25n = 36
Vehicle1 mM20HEvehicle1 mM20HE
n = 30n = 13n = 20n = 11
NRNAiEcRANRNAiDN
1 mM 20HE (7 days)NRNAi (^)
then (7 days)
n = 25n = 42n = 62n = 11n = 46n = 67n = 69
35-day-old mated females
esg-Gal4ts
35-day-old mated females
esg-Gal4ts
Reproductive
tness
Eggs
Ovary growth
Nutrient uptake Mating
Stem-cell division
Food
Dysplasia Gut growth
Mortality
Ecdysone
H
HO
HO
O
H OH
HO
H
OH
OH
Non-dysplastic Dysplastic
GFP
Pdm-1
DAPI GFP
Pdm-1
DAPI GFP
Pdm-1
DAPI GFP
Pdm-1
GFP DAPI
Pdm-1
GFP DAPI
GFP GFP GFPGFP GFPGFP
Pdm-1
Pdm-1 Pdm-1 Pdm-1 Pdm-1 Pdm-1 Pdm-1
DAPI
StroModerateng
Mild
DAPIGFP DAPIGFP DAPIGFP
DAPIGFP DAPIGFP DAPIGFP
NRNAi
Fig. 3 | Exogenous and mating-dependent 20HE promote intestinal
dysplasia and tumorigenesis. a, In aged, control females, mis-differentiated
cells accumulate that are positive for both esg (GFP, green) and the EC marker
Pdm-1 (red; thick white arrows), and can divide (as measured by positive
staining for phospho-histone H3 (PH3+); thin white arrows). Intestinal dysplasia
is blocked by reduced EcR (using EcR ADN), Usp or Eip75B (using UspRNAi or
Eip75BRNAi), such that progenitors express GFP only. b, The percentage of
midguts classified as dysplastic (purple) or non-dysplastic (green) in region R4.
Guts with at least 10 Pdm1+ GFP+ progenitors were scored as dysplastic.
c, Mitotic counts of midguts as in a. d, Midgut mitoses in females expressing
ovary-targeted RNAi against the ecdysteroidogenic enzymes Dib or Spo.
e, NotchRNAi (NRNAi)-expressing ISC tumours (green cells) are induced in mated
females by esg-Gal4ts, but not in age-matched virgins. Tumour progression is
blocked by EcR ADN. f, Mitotic counts as in e. g, Distributions of esg-Gal4ts
UAS-NRNAi tumour types under conditions as classified in Extended Data
Fig. 10d. h, Tumour distributions of virgin females fed 1 mM 20HE for 7 days
before NRNAi induction by esg-Gal4ts. Representative images are shown from
three independent experiments. Scale bars, 100 μm. i, Model summarizing our
conclusions. The R4 region is in red. Data are mean ± s.d. ****P < 0.0001,
Mann–Whitney test. n ≥ 3 independent experiments. Exact n values and
P values are in the Source Data.