reSeArCH Letter
Extended Data Fig. 7i–n, Supplementary Video 6). These findings
demonstrate that Wnt and TGFβ signalling regulate the frequency of
fission behaviour.
We proposed that components of the Wnt and TGFβ signalling
pathways might regulate fission behaviour through the planarian
CNS. Double fluorescent in situ hybridization (FISH) with the CNS
marker pc2 confirmed that Wnt and TGFβ fission regulators were
detected in pc2-positive cells in the anterior CNS (Extended Data
Fig. 8a, b). Removal of anterior tissue that contains the cephalic
ganglia delayed the onset of fission behaviour (Extended Data
Fig. 8c–f). Restoration of fission activity coincided with regenera-
tion and re-establishment of anterior, pc2 co-localized, tsh expression
(Extended Data Fig. 8g). Notably, removal of anterior tissue that
contained just one cephalic ganglion did not alter the total num-
ber of fission progeny produced (Extended Data Fig. 8c–f), which
indicates that half of the CNS is sufficient to initiate fission. Finally,
RNAi against coe, a transcription factor essential for the patterning
of the CNS^22 ,^23 , markedly reduced planarian fission (Extended Data
Fig. 8h, i). Together, these data support a model in which an anterior
CNS expressing Wnt and TGFβ signalling components regulates
fission initiation.
We tested whether polarity genes could modulate size-dependent
behaviour via size-dependent patterning of the CNS. To identify
neuronal subpopulations that regulate fission downstream of Wnt
and TGFβ, we analysed 17 neuronal markers^24 –^29 in small, medium
and large planaria and 10 markers in worms treated with smad2/3
RNAi (Fig. 4f, Extended Data Fig. 9a, b). Patterning of pkd1L-2+,
gabrg3L-2+ and sargasso-1+ mechanosensory neurons exhibited the
clearest changes in worms of increasing size and after smad2/3 RNAi
treatment (Extended Data Fig. 9a, b). In large worms, mechanosensory
neurons are tightly restricted to the anterior and knockdown of either
smad2/3 or wnt11-6 broadened their distribution akin to that of smaller
worms (Fig. 4g–l). RNAi against pkd1L-2 and gabrg3L-2 (homologous
to cation and chloride channel genes, respectively) increased planarian
fission activity (Fig. 4m, n), and live imaging of gabrg3L-2 RNAi worms
confirmed an increase in fission attempts without a reduction in fission
success (Fig. 4o, p, Extended Data Fig. 10, Supplementary Video 7).
These results indicate that mechanosensory neurons are differentially
patterned during growth, inhibit fission behaviour and require Wnt and
TGFβ for their appropriate patterning in accordance with worm size.
Therefore, we conclude that Wnt and TGFβ signalling coordinates worm
size and behaviour via size-dependent patterning in the adult CNS.θtrla02468
Time (days)Ctrl 1024 68actR-102468smad2/302468
Time (days)E-catenin0 2468dsh-B02468wnt11-602468
Time (days)apcbcde02468
Time (days)Ctrl 2Live imagingData visualizationCNS in situ
stainingRNAi feedingsTimeSuccessful ssion
RNAiUnsuccessful ssion1131
3pkd1L-2+
Angle
Width3222
3gabrg3L-2+
Range
Width
o pqnm Ctrl pkd1L-2 gabrg3L-2P < 0.0001 P < 0.0001(^01)
(^23)
(^45)
(^67)
(^89)
(^1011)
(^1213)
AV^14 G
Days
0.0 Normalized ssions
3.0
1.5
Pare
nt days 0 and 14
Ctrl pkd1L-2gabrg3L-2
02468
Time (days)
gabrg3L-2
0 2468
Time (days)
Control
RNAi
RNAi
NS
Control
gabrg3L-2
0.0
0.5
1.0
1.5
Successful/total attempts
Control
gabrg3L-2
0
5
10
15
20
25
To tal attempts
pkd1L-2
gabrg3L-2
Small Medium Large
pkd1L-2
gabrg3L-2
Ctrl 1 smad2/3 Ctrl 2 wnt11-6
gj
kl
f i
h
SmallMediumLargeCtrl 1
smad2/3
Ctrl 2wnt11-6
0.5
0.6
0.7
0.8
0.9
1.0
gabrg3L-2
range per width
(^50) P = 0.0004
SmallMediumLargeCtrl 1
smad2/3
Ctrl 2wnt11-6
0
10
20
30
40
pkd2L-1
angle per width
P < 0.004P < 0.0001P = 0.0002
P = 0.0007
P < 0.02
P < 0.009P < 0.003
P = 0.1547
P = 0.0031
0
5
10
15
20
25
Total attempts
0.0
0.5
1.0
1.5
Success ratio
Ctrl 1E-catactR-1
smad2/3
DshBapc Ctrl 2wnt11-6
P P = 0.0002
< 0.0001
P < 0.0001
P < 0.003P < 0.0004
P < 0.009
N/A
P < 0.0001
NS
P = 0.
2
NS
P = 0.9
P = 0.005
P < 0.006
Ctrl 1E-catactR-1
smad2/3
dsh-Bapc Ctrl 2wnt11-6
Fig. 4 | Wnt and TGFβ signalling regulates fission frequency by size-
dependent patterning of mechanosensory neurons in the CNS.
a, Schematic depicting RNAi treatment, live imaging and data analysis.
b, c, Representative activity timelines. d, e, Total fission attempts (d) and
successful attempts per total attempts (e) for RNAi-treated worms (n = 16
(d) and 10 (e) worms, from 1 (d) or 2 (e) independent experiments).
f, Schematic depicting in situ staining strategy. g, h, Representative images
of pkd1L-2+ and gabrg3L-2+ neurons in worms of increasing size (g), or
after RNAi treatment (h). Scale bars, 0.5 mm. i, k, Diagrams depicting
quantification of the angle of pkd1L-2+ cells (i) or the range of gabrg3L-2+
cells (k). j, l, Staining quantification of pkd1L-2 (j) and gabrg3L-2 (l) in
worms of increasing size or after RNAi treatment (n = 3–6, exact n depicted
in dot plot quantification). m, Fission activity heat maps after treatment
with pkd1L-2 and gabrg3L-2 RNAi (n = 12; Fig. 2 ). n, Representative
parent images on days 0 and 14 of fission assay (n = 12, 2 independent
experiments). Scale bars, 1 mm. o, Representative fission activity timelines
of worms treated with gabrg3L-2 RNAi. p, q, Total fission attempts (p) and
successful attempts per total attempts (q) for worms treated with gabrg3L-2
RNAi (n = 10 worms). P values determined by two-sided t-test (j, l, p, q) or
two-way ANOVA (d, e). Data are mean ± s.e.m. (d, e, j, l, p, q).
658 | NAtUre | VOL 572 | 29 AUGUSt 2019