Nature - 2019.08.29

reSeArCH Letter

or plane addition despite rescaling and regenerating their other

tissues (Extended Data Fig. 5f–j). In summary, fission planes are pre-
established in planarians and correlate dynamically with worm size

and form.
Given the role of Wnt and TGFβ signalling in body patterning, we

tested whether genes of these signalling pathways regulate fission
planes. Worms treated with RNAi were mechanically compressed and

the quantity and relative distribution of fission planes was measured
(Fig. 3e–g). Notably, whereas RNAi of actR-1 and smad2/3 moderately

reduced the number of fission planes, RNAi of Wnt signalling com-
ponents had no effect on fission plane number or position (Fig. 3e, g,

Extended Data Fig. 6a). Even knockdown of wnt11-6 by three rounds
of amputation and regeneration did not alter fission-plane patterning

(Fig. 3f, g, Extended Data Fig. 6b). Hypomorphic RNAi knockdown of
β-catenin, actR-1 or smad2/3 revealed little or no effect on the size of

fission fragments (Extended Data Fig. 6c–e), which further supports

the conclusion that neither Wnt nor TGFβ signalling regulate fission

behaviour through the anterior–posterior patterning of fission planes.

We tested whether Wnt and TGFβ signalling instead regulated the

frequency of fission attempts. Using the automated webcam image-

capture system (Fig. 1f), we recorded fission behaviour in RNAi-

treated worms (Fig. 4a). RNAi of β-catenin, actR-1, smad2/3 and

wnt11-6 reduced fission attempts, whereas RNAi of apc increased

fission attempts (Fig. 4b–d, Extended Data Fig. 7a–l, Supplementary

Videos 4–6). RNAi of β-catenin and smad2/3, which resulted in observ-

able morphological abnormalities, also significantly reduced the

fission-success ratio (Figs. 2d, 4e, Extended Data Fig. 7k–n). dsh-B

RNAi reduced the fission success ratio without altering the number

or frequency of fission attempts (Fig. 4d, e, Extended Data Fig. 7k–n).

Finally, apc RNAi reduced the time between fission attempts by approx-

imately 50%, and worms initiated fission attempts independently of

remaining tissue, markedly reducing their success ratio (Fig. 4e,

Feed 2–3× per week

Low ssion activity

Recirculation culture

Starve

High ssion activity

Static culture

abDay 1 c Day 14

4 812

Length parent (mm)

0

1

2

3

4

5

Size rst ssion fragment

PCC = 0.1986

y = 0.03437X + 1.208

R^2 = 0.04

4 8121620

0

5

10

15

Length parent (mm)

Progeny (14 days)

PCC = 0.9187

y = 0.8727X – 4.38

R^2 = 0.84

0102030

Time (s)

Unsuccessful ssion attempt

010203040

Time (s)

Successful ssion attempt

e

h

i

f

g

0510 15

0.0

0.5

1.0

1.5

Length parent (mm)

Success ratio

PCC = –0.5798

y = –0.08225x + 1.377

R^2 = 0.3362

0510 15

0

5

10

15

Length parent (mm)

Total attempts

PCC = 0.827

y = 1.09x – 5.004

R^2 = 0.684

d

02468

Time (days)

Animal length

4 mm

5 mm 6 mm 7 mm 8 mm 9 mm

11 mm

0 min 10 min 20 min 30 min40 min0 min 10 min 20 min 30 min

Fig. 1 | Planarian fission is a size-dependent behaviour. a, Optimized
fission protocol. b, c, Representative images of 5–12-mm worms and
fission fragments less than 24  h (b) and 14  days (c) after the first fission
event (n = 46 worms from 1 experiment). Scale bars, 5  mm.
d, e, Length of the first fission fragments (d) and progeny number (e)
over 2  weeks relative to parent length (n = 46 (d) or 30 (e) worms from
one experiment). f, Webcam live-imaging schematic (left) and example

timeline depicting successful (middle) and unsuccessful (right) fission

attempts. g, Representative fission behaviour timelines from a range of

parent lengths. h, i, Total fission attempts (h) and successful attempts per

total attempts (i) relative to parent length (n = 39 (h) and 21 (i) worms).

Data are from a single experiment. Pearson correlation co-efficient (PCC),

linear regression (red line), and R^2 values are provided.

656 | NAtUre | VOL 572 | 29 AUGUSt 2019