Nature - USA (2020-08-20)

Nature | Vol 584 | 20 August 2020 | 417

To confirm the source of endogenous ecdysone, we used
ovary-specific Gal4 drivers^3 ,^7 to express RNAi transgenes that tar-
get the ecdysone synthesis enzymes Dib or Spo. This suppressed
mating-induced ISC divisions and midgut growth, both of which
could be restored by exogenous 20HE (Fig. 2h, j, Extended Data
Fig. 5n–p). spo mutants^16 also failed to resize the midgut after mat-
ing (Fig. 2i, Extended Data Fig. 5m), confirming these results. To

learn how the gut grows in mated females, we investigated the effects

on cell size and number. Depleting EcR in ECs did not reduce EC

size (Extended Data Fig. 5q), but mating caused a large 20HE- and

EcR-dependent increase in female ISC numbers (Fig. 2k, Extended

Data Fig. 5r–t). This expansion of the stem-cell pool could cause an

increase in the total number of midgut cells. These results indicate

that mating-dependent ISC division, ISC expansion and gut growth

esg-Gal4ts(8 days)

Mated 37 h

mated 37 h

42 h

Mated 72 h

Control

EcR

RNAi

Virgins

a Control iesmated b

48 h72 h

c esg-Gal4ts(8 days)

72 h

3 days

EcR

RNAi

3 days

yy

Area = 0.46 mm^2

Length = 3.61 mm

Area = 1.1 mm^2

Length = 5.6 mm

Area = 0.47 mm^2

Length = 4.44 mm

esg-Gal4ts(9 days) f

d

e esgts Su(H)-Gal80 (8 days)

37 h

12 days

traRNAi

traRNAi

48 h 72 h

ghC587-Gal4ts^ (8 days)

i j

Su(H)-Gal4ts (8 days)

72 h

kl

m n o p

Whole-body mutant

20HE feeding

EBs (Su(H)-Gal4tsGFP+), ISCs (delta lacZ+)

R4 region of the midgut

6 h feeding versus

different lengths of mating (n = 4)

Vehicle20HEHaem

esg-FOts (6 days) ,16 h feeding esg-FOts (3 days) esg-FO16 h feedingts(16 days),

5 mM 20HE

Averages: control: total = 411 ± 50 eggs

EcRRNAi: total = 260 ± 44 eggs, ****P < 0.0001

Eip75BRNAi: total = 246 ± 36 eggs, ****P < 0.0001

RNAi in progenitors esg-Gal4ts

C587-Gal4ts

(14 days)

0

10

20

30

(^10050)
150
200
ISC mitoses per midgut

---

ControlEcR
RNAi
Eip75B
RNAi
ControlEcR
RNAi
Eip75B
RNAi
0.4
0.6
0.81.0
1.2
1.41.6
1.8
Total midgut area (mm
2 ) ****

---

Controltra
RNAi
Controltra
RNAi
Controltra
RNAi
0.20.4
0.6
0.81.0
1.21.4
1.6
Total midgut area (mm
2 )
** NS
ControlRNAi#2EcRControlRNAi#2EcRControlRNAi#2EcR
05
1015
20
4080
120160
ISC mitoses per midgut
**** NS****
ControlRNAi#1EcRRNAi#2EcRControlRNAi#1EcRRNAi#2EcRControlRNAi#1EcRRNAi#2EcR
05
1015
2025
4060
(^10080)
ISC mitoses per midgut

---

---

---

NS****

---

---

Control dib
RNAi
spo
RNAi#1
spo
RNAi#2Control RNAidib
spo
RNAi#1
spo
RNAi#2
02
46
(^108)
30
60
ISC mitoses per midgut
NS****
spo
Z339
/Df
spo
Z339
/+
spo
Z339
/Df
spo
Z339
0.4 /+
0.6
0.8
1.0
1.2
1.4
Total midgut area (mm
2 )
NS

---

Controldib
RNAi
0
100
200
300
400
500
No. of delta
+cells
per area in R4

---

24 h42 hO/N7 days
Raised 7 days
0
20
40
6080
Positive cells perR4 region (%)
SuH+
delta+

---

Vehicle
24 h48 h
0.25 mM0.5 mM
1 mM2 mM5 mM10 mM
0.0
2.5
5.0
68
1015
30
mRNA fold change
(RT–qPCR)
Eip75B
br
Control
Eip75B
RNAi
Control
Eip75B
RNAi
Control
Eip75B
RNAi
0
1020
50
100100
200
ISC mitoses per midgut

---

---

---

ControlEip75B
0
20
40
60
ISC mitoses per midgut

---

Control
Eip75B
RNAi
Hr3
RNAi
Eip75B
RNAi
,
Hr3
RNAi
0
10
20
4060
(^10080)
ISC mitoses per midgut
NS

---

(^0061218243036)
200
400
600
Days after mating at 29 °C
Cumulative eggs laid
DAPIGFP DAPIGFP DAPIGFP
DAPIGFP
DAPIGFP
DAPIGFP
delta-lacZDAPIGFP
delta-lacZDAPIGFP
DAPIGFP DAPIGFP
Control, n = 111
EcRRNAi, n = 96
Eip75BRNAi, n = 82
48 h 72 h
Fig. 2 | Ovary-derived 20HE drives mating-induced midgut growth through
Eip75B. a, Midgut mitotic counts from control w^1118 f lies before mating and 48
and 72 h thereafter. b, Representative virgin and mated midguts, with their ISCs
and EBs marked by GFP, with or without EcR depletion using EcRRNAi. c, Midgut
size measurements of virgin and mated females, with or without EcRRNAi or
Eip75BRNAi expressed in progenitors. d, Midgut images with or without
EcR-depleted progenitors. e, Midgut size measurements and images of virgin
and mated females with masculinized traRNAi-treated ISCs and EBs. f, ISC
mitoses of virgin female midguts before and 48 or 72 h after mating expressing
EcRRNAi specifically in ISCs using the esgts Su(H)-Gal80 Gal4 driver. g, ISC
mitoses of virgin female midguts and 48 h or 72 h after mating expressing
EcRRNAi in EBs using the EB-specific Su(H)-Gal4ts dr i ve r. h, Midgut mitoses after
depletion of ecdysone synthesis enzymes Dib or Spo by RNAi using the
ovary-specific driver C587-Gal4ts. i, Midgut areas of whole-body spo mutants
(spoZ339/Df) rescued to adulthood by an exogenous 20HE pulse given to
embryos, and controls (spoZ339/+). j, Ovary-derived 20HE promotes gut ISC
number. k, Images (left) and counts (right) of the percentage of delta+ cells or
EBs as a fraction of the total cells per region in R4. O/N, overnight.
l, Quantitative PCR with reverse transcription (qRT–PCR) analysis of Eip75B and
br mRNA in whole, 20HE-fed virgin or mated female midguts. m, Mitoses of
Eip75BRNAi ISC clones with or without haem or 20HE treatment. n, Mitotic
counts of ISC clones overexpressing Eip75B. o, Epistasis tests assaying
interactions between Eip75B and Hr3. p, Cumulative eggs laid by mated females
with progenitor-specific EcRRNAi or Eip75BRNAi, and controls. Data are mean ± s.d.
P ≤ 0.05, P ≤ 0.01, P < 0.001, ****P < 0.0001, ordinary ANOVA test, followed
by Bonferroni’s multiple comparisons test (c, e), Mann–Whitney (a, f–o), or
general linear models (GLMs) with binomial errors (p). Representative images
are shown, n ≥ 3 independent experiments. Scale bars, 100 μm (b, e, k) or
1 mm (d). Exact n values and P values are in the Source Data.