Nature - 2019.08.29

Letter reSeArCH

Pare

nt days 0 and 14

d

wnt11-6 wnt11-3

tsh

sfrp-2

evi

ptc

wnt2-1 follistatin

wnt5

apc

activin smo hh wnt11-1/2 wnt11-2 sfrp-1

wnt11-4

Ctrl 2

dsh-B

sufu

actR-1smad2/3 fz-4 wnt11-5

Dsh-A

Ctrl 1 E-cat notumwnt-1

gli-1

b

a

(^01)
(^23)
(^45)
(^67)
(^89)
(^1011)
(^1213)
AV^14 G
Days
(^01)
(^23)
(^45)
(^67)
(^89)
(^1011)
(^1213)
AV^14 G
Days
0 Normalized ssions
2
1
0 Normalized ssions
2
1
Primary RNAi ssion screening
Ctrl 2
Ctrl 2
Ctrl 1
Ctrl 1
c
(^01)
(^23)
(^45)
(^67)
(^89)
(^1011)
(^1213)
AV^14 G
Days
0 Normalized ssions
2
1
Secondary RNAi ssion screening
Phase 1
Phase 2
actR-1
actR-1
P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P = 0.0088 P < 0.0001
smad2/3
smad2/3
E-cat
E-cat
dsh-B
dsh-B
wnt11-6
wnt11-6
tsh
tsh
apc
apc
Record no. of
ssions daily
Measure
initial length
Recirculation
system
RNAi
feedings
Normalize and
colour
P = 0.004
Fig. 2 | Wnt signalling and TGFβ signalling
components modulate fission activity.
a, RNAi screen workflow (see also Extended
Data Fig. 3). b, c, Heat maps depicting fission
activity after RNAi treatment for both the two-
phase primary (b) and secondary (c) RNAi
screens. Normalized cumulative fissions over
time are displayed for individual worms from
each RNAi condition (n = 10 worms for phase
I, n = 12 worms for phase II and secondary
screen). Targets in secondary screening
(independently repeated three times) depicted
in green (activators) and red (inhibitors).
P values determined by two-way analysis of
variance (ANOVA) interaction factor. Ctrl,
control. d, Representative parent images on
days 0 and 14 of the fission assay (n = 10–12,
independently repeated 3 times). Scale bars,
1  mm.
a
Compress
Ventral side up
1st 2nd 3nd 4th5th 6th7th
0
2
4
6
8
10
Fragment
Distance to posterior (mm)
Fissions
Compression
c d
PCC = 0.74
y = 0.4678X – 0.002
R^2 = 0.55
0510 15
0
2
4
6
8
Length (mm)
Compression planes
1 mm
Pre-compression Post-compression
0
+1
1st
2nd
3rd
4th
5th
+2 6th
–2
–1
Relative postionFragment order
0
0 0
0
+1
+1
+1
+2
+2
–1
Control E-catenin dsh-B apc actR-1
3 × RNAi feedings
smad2/3
Control wnt11-6
ControlE-catenindsh-B
apc
actR-1smad2/3
Number of planes per length
23 × RNAi feedings, 3× regeneration
Controlwnt11-6
e
fg
NS
P = 1.0P = 0.6
NS
P =
0.08
NS P < 0.02P < 0.05
0.0
0.4
0.8
1.2
b
NS
P = 0.
9
Fig. 3 | Pre-established fission planes determine progeny size
independently of Wnt and TGFβ signalling. a, Schematic of compression
assay (Supplementary Video 3). b, Pre- and post-compression
worm (inset) and compression planes revealed in 3–6-mm worms
(independently repeated 5 times). c, Compression plane number relative
to worm length (n =  117 worms). PCC, linear regression (red lines),
R^2 values and 95% confidence interval (black lines) are shown. d, Fission
(n = 196 fission progeny from 50 worms) and compression plane (n =  173
planes from 30 worms) overlap along the anterior–posterior axis of the
worm. e, f, Representative images of post-compression worms after
knockdown of Wnt and TGFβ signalling components using the specified
number of RNAi feedings and rounds of regeneration (n depicted by dot
plot quantification; experiment performed three times (e) or once (f)).
Scale bars, 1  mm. g, Plot of the number of fission planes per worms length
after RNAi treatment of 2 experiments (n = 20 and 10 worms to the
left and right of the dotted line, respectively). P values determined
by two-sided t-test. NS, not significant. Data are mean ± s.d. (d) or
mean ± s.e .m. (g).
29 AUGUSt 2019 | VOL 572 | NAtUre | 657