1
Fluid dynamics alter Caenorhabditis elegans body length via neuromuscular
2
signaling with TGF-/DBL-1
3
4
Shunsuke Harada1*+, Toko Hashizume2, 3*, Kanako Nemoto1, Zhenhua Shao1,
5
Nahoko Higashitani1, Timothy Etheridge4, Nathaniel J. Szewczyk5, Keiji
6
Fukui6, Akira Higashibata3, 7, and Atsushi Higashitani1†
7
8
1
Graduate School of Life Sciences, Tohoku University, Sendai, Japan
9
2
Advanced Engineering Services Co., Ltd., Tsukuba, Japan
10
3
Graduate School of Medical and Dental Sciences, Kagoshima University,
11
Japan
12
4
13
United Kingdom
14
5
15
Hospital, University of Nottingham, Derby, United Kingdom
16
6
Japan Space Forum, Kanda Surugadai, Chiyoda-ku, Japan
17
7
Human Spaceflight Technology Directorate, Japan Aerospace Exploration
18
Agency, 2-1-1, Sengen, Tsukuba, Ibaraki, 305-8505, Japan
College of Life and Environmental Sciences, University of Exeter, Exeter,
MRC/ARUK Centre for Musculoskeletal Ageing Research, Royal Derby
19
20
*These authors contributed equally to this work.
21
†To whom correspondence should be addressed.
22
Postal address: 2-1-1, Katahira, Aoba-ku, Sendai, Miyagi 980-8577 JAPAN
23
Telephone Number: +81-22-217-5715
24
E-mail: [email protected]
25
26
+
Present address: School of Medicine, Kobe University, Kobe, Japan
27
28
Running title
29
C. elegans physique altered by fluid dynamics
30
31
32
33
Abstract
34
35
Skeletal muscle wasting is a major obstacle for long-term space exploration.
36
Similar to astronauts, the nematode Caenorhabditis elegans displays
37
negative muscular and physical effects grown in space microgravity.
38
However, it is still unclear what signal molecules and behavior affect the
39
negative alterations. We here studied key signaling molecules involved in
40
alterations of C. elegans physique in response to fluid-dynamics on the
41
ground-based experiments. Like as spaceflight experiment with 1G
42
accelerator onboard, a myosin heavy chain myo-3 and a TGF- dbl-1 gene
43
expression altered increasing the fluid dynamic parameters viscosity/drag
44
resistance or depth of liquid culture. These gene expression also drastically
45
increased grown liquid medium as compared with moist agar surface. In
46
addition, body length enhanced in WT and body-wall cuticle collagen
47
mutants, rol-6 roller and dpy-5 dumpy, grown in liquid culture. On the other
48
hand, in a TGF- gene dbl-1 and its signaling pathway sma-4/Smad mutants,
49
their body lengths did not alter in liquid. Similarly, a D1-like dopamine
50
receptor DOP-4 and a mechanosensory channel UNC-8 were required for
51
altered physique in which DBL-1 signaling did not upregulated in liquid.
52
Since C. elegans contraction rates are much higher in swimming mode in
53
liquid than clawing mode on agar surface, we studied the relationship
54
between
55
significantly reduced contraction rate commonly show smaller size, although
56
the rate in dop-4, dbl-1 and sma-4 mutants still increased in liquid. These
57
results suggest that neuromuscular signaling via TGF-/DBL-1 to alter body
58
physique in response to environmental conditions including fluid dynamics.
59
(250 words)
body-length
enhancement
and
contraction
rate.
Mutants
60
61
Key words
62
Degenerin,
63
TGF-signaling
64
65
Introduction
liquid
culture,
mechanosensor,
muscle,
spaceflight,
66
67
An individual’s physique is shaped over long periods by both external
68
stimuli and locomotory gaits. Bone and muscle wasting are inevitable
69
pathophysiological adaptations in microgravity, for example spaceflight, and
70
with inactivity, for example in the bedridden1-4. This wasting is a major
71
obstacle for long-tern space exploration. Microgravity in particular
72
dramatically decreases mechanical loading and also results in drastic
73
changes in fluid dynamics including hydrostatic forces. However, it is still
74
unclear what signal molecules and behavior affect the pathophysiological
75
adaptations.
76
Aquatic exercise is one of the best ways to achieve optimal body
77
strength and to improve vigor. Such exercise involves the physical
78
application of fluid dynamics, particularly hydrostatic forces and drag
79
resistance accompanying liquid viscosity, and is effective not only in healthy
80
individuals, but also in bedridden patients5-10. Although many recent studies
81
have
82
mechanisms for perception of these stimuli and signal transduction from
83
these stimuli to bone and skeletal muscle formation, enhanced physique and
84
strength remain unclear.
evaluated
flow-dynamic
parameters
as
physical
stimuli,
the
85
Caenorhabditis elegans is a free-living nematode that is also a widely
86
used laboratory animal. The body length can be altered via a highly
87
conserved transforming growth factor (TGF)-DBL-1 Smad transcription
88
factor signaling pathway23-28. C. elegans has at least two different locomotory
89
gaits, one is displayed when swimming in liquid and the other when crawling
90
on a surface11-15. The transition from the swimming gait to the crawling gait
91
and vice versa is controlled by biogenic amines as a short-term adaptive
92
response15. C. elegans also makes short-term adaptations to locomotion in
93
response to gentle mechanical stimuli through a mechanosensory complex
94
composed of the degenerin ion channels, MEC-4 and MEC-10, found in
95
touch-sensitive neurons19-22. Worms also make long-term adaptive responses
96
also occur. For example, we reproducibly found that space-flight induces
97
reduced expression of some muscle genes16-18+Akira
98
protein expression levels for muscular thick filaments, cytoskeletal elements,
npj Microgravity,
specific
99
100
and
mitochondrial
metabolic
enzymes,
body
length
and
fat
accumulation+Akira npj Microgravity.
101
This study investigated alteration of muscular myosin and TGF-
102
gene expression in response to fluid-dynamic properties (microgravity,
103
viscosity/drag resistance and depth of liquid culture). We also compared
104
relationship between the body physique established and different moving
105
behaviors, swimming and clawing cultured in liquid and on moisture agar
106
surface. We finally explored the hypothesis that neuromuscular signaling via
107
TGF-/DBL-1 modulates altered physique in response to fluid-dynamic
108
properties.
109
110
Ref 28+Akira npj MCG まで
111
112
Results
113
114
Fluid-dynamic parameters modulate myo-3 and dbl-1 gene expression.
115
In our C. elegans RNA Interference in Space Experiment, L1 larvae stage
116
animals were synchronously cultured to adulthood in liquid media for four
117
days either in microgravity or 1G centrifuge conditions onboard the
118
Japanese Experiment Module of the International Space Station (ISS)5, 6.
119
The nematode L1 larvae were launched to the ISS onboard the Space Shuttle
120
Atlantis STS-129 on November 16, 2009, cultures were initiated on
121
November 20, 2009, and the post cultivation frozen samples were returned
122
by the Space Shuttle Endeavour STS-130 on February 21, 2010. The
123
microarray expression analyses indicate that levels for muscular thick
124
filaments, cytoskeletal elements, and mitochondrial metabolic enzymes
125
decreased relative to parallel cultures on the 1G centrifuge (95% confidence
126
interval (p≤0.05): accession number GSE71770 on GEO) ref Higashibata et
127
al npj MCG. In this study, we confirmed that a myosin heavy chain myo-3
128
and a TGF- dbl-1 gene expression reduced to 60 and 70 % in microgravity
129
versus on a 1G centrifuge onboard the International Space Station (Fig. 1A).
130
On the ground-based experiments, in order to study the effect of a
131
fluid-dynamic parameter, drag resistance, on myo-3 and dbl-1 gene
132
expression, wild-type worms were cultured for 4 days after the L1 larval
133
stage under different liquid viscosities (1.0 cSt (0% methylcellulose), 36.1 cSt
134
(1.0% methylcellulose), and 123.3 cSt (1.5% methylcellulose). myo-3 gene
135
expression was significantly increased at 36.1 cSt viscosity and moderately
136
increased at 123.3 cSt. dbl-1 gene expression increased significantly but not
137
much about 20% at 123.3 cSt (Fig. 1B). To study the effects of altering the
138
depth of liquid cultivation, OP50 NGM-agar was covered with additional
139
OP50 NGM-liquid medium (0.6 mm, 1.2 mm, or 1.8 mm in depth). In gravid
140
adults (day 4), both myo-3 and dbl-1 gene expression increased with
141
increased depth of liquid culture (Fig. 1C), with maximal observed
142
expression achieved at 1.2 mm. These results indicate that the nematode C.
143
elegans can alter the muscular and TGF- gene expression in response to
144
flow-dynamic parameters including gravity force.
145
146
5. Etheridge T, Nemoto K, Hashizume T, Mori C, Sugimoto T, Suzuki H et al. The effectiveness of RNAi
147
in Caenorhabditis elegans is maintained during spaceflight. PLoS One 2011; 6: e20459.
148
6. Higashitani A, Hashizume T, Sugimoto T, Mori C, Nemoto K, Etheridge T et al. C. elegans RNAi
149
space experiment (CERISE) in Japanese Experiment Module KIBO. Biol Sci Space 2009; 23: 183-187.
150
151
152
C. elegans body length and muscle gene expression increase in liquid
153
culture.
154
Although the flow-dynamic parameters could increase myo-3 and dbl-1
155
expression (Fig. 1), it is not so much extend. In order to confirm whether C.
156
elegans alters these expression and body physique in response to different
157
environmental stimuli and moving behavior, we measured expression levels
158
and the body length of wild-type worms grown either in liquid medium or on
159
a moist agar surface was measured as described in Methods. Culturing
160
occurred in parallel with L1 larvae hatched on agar plates and then cultured
161
in liquid or on agar. The growth rate and developing timings were not
162
significantly different between the culture conditions, with all of the larvae
163
having developed into young adult hermaphrodites 3 days after onset of
164
culturing, as evidenced by the onset of egg production. However, the body
165
length at day 4 (gravid adult stage) was significantly longer for animals
166
cultured in liquid medium vs. on moist agar (Fig. 2A, B); this difference
167
persisted at the later time points (5 and 6 days) (Fig. 2C). The expression
168
levels of a myosin heavy chain gene, myo-3, and its upstream transcription
169
factor, hlh-1, were also significantly higher in animals cultured in liquid vs.
170
on agar (Fig. 2D, E). Myosin protein expression levels were similarly
171
increased by 1.6-fold for animals cultured in liquid vs. on agar (1.24-fold in
172
liquid and 0.78-fold on agar as compared with the relative ratio of a
173
ribosomal protein).
174
175
Liquid culture alters C. elegans physique through TGF-signaling. DBL-1 is
176
a member of the transforming growth factor  (TGF-protein family. It,
177
along with its signaling pathway, is a known regulator of C. elegans body
178
length23-28. To determine if DBL-1 was required for the observed body length
179
alteration, we measured the length of dbl-1(wk70) and (nk3) mutants after
180
culturing in liquid or on agar. We also measured sma-4(e729) mutants, as
181
SMA-4 is a known downstream transcription factor component of the DBL-1
182
signaling cascade that controls body size. In contrast to wild-type (Fig. 2 and
183
3), these mutants’ body lengths did not increase in liquid culture (Fig. 3),
184
implying that dbl-1 and sma-4 are required for the body length alteration in
185
response to liquid culture.
186
Because previous work reported that DBL-1 controls the expression
187
of several genes, including those encoding extracellular matrix-associated
188
collagens, we next investigated whether liquid culture suppresses dumpy
189
and roller phenotypes in collagen mutants. The body lengths of dumpy
190
dpy-5(e907) and rol-6(su1006)) mutants were markedly increased in liquid vs.
191
agar culture (Fig. 4A–D). Additionally, the frequency of the right-handed
192
roller phenotype induced by rol-6(su1006) was reduced by liquid culture (Fig.
193
4B). Taken together, the results displayed in Figures 1–3 suggest that liquid
194
culture alters C. elegans physique via activation of TGF-/DBL-1 signaling
195
(Fig. 3), not only in terms of muscle myosin expression (Fig. 2), but also in
196
terms of extracellular matrix/collagen deposition (Fig. 4).
197
198
UNC-8 degenerin channel and D1-like dopamine receptor DOP-4 are
199
required for body length increase in liquid culture.
200
In C. elegans, a degenerin/epithelial sodium ion channel composed of MEC-4
201
and MEC-10 functions as a mechanosensor for physical stimuli (e.g.,
202
touch)19-22. To determine whether degenerin family members act as
203
mechanosensors in response to fluid-dynamic parameters, body length
204
alterations in response to liquid culture was assessed in degenerin channel
205
mutants (mec-4(e1611), mec-10(e1515), and unc-8(e15)).
206
mec-4(e1611) and mec-10(e1515) displayed increased body length in response
207
to liquid culture, unc-8(e15) showed no significant increase in body length
208
(Fig. 5).
While both
209
The C. elegans transition between distinctive crawling and
210
swimming gaits is controlled by the biogenic amines, dopamine and
211
serotonin15. Therefore, we measured body length alterations in animals with
212
mutations
213
tph-1(mg280), a serotonin/octopamine receptor family member, ser-5(ok3087),
214
and the D1-like dopamine receptors, dop-1(vs101) and dop-4(tm1329).
215
dop-4(tm1329) showed no culture-dependent alterations in body length (Fig.
216
6), suggesting that DOP-4 is also required for physique alteration. In
217
contrast, body lengths were significantly increased in liquid vs. agar culture
218
for tph-1(mg280), ser-5(ok3087), and dop-1(vs101) (Fig. 6). We next
219
investigated the impact of other factors on body length in liquid culture
220
system, starting with oxygen and nutrient sensing. To do this, we used
221
animals with mutations in genes encoding hypoxia response factor, hif-1(ia4),
222
an insulin/IGF-1-like peptide, ins-7(ok1573), and a key transcriptional
223
regulator that acts downstream of insulin/IGF-1-mediated signaling,
224
daf-16(mu86). In all cases body length increased in response to liquid culture
225
in these mutants (Fig. 6). A ryanodine receptor unc-68(r1161)32 mutant,
226
which is sluggish and flaccid, also increased in liquid culture (Fig. 6). These
227
results suggesting that alteration of C. elegans physique in response to
228
liquid culturing is essential for some neuromuscular signaling, but is largely
229
distinct from oxygen and nutrient sensing.
230
in
genes
encoding
the
serotonin
biosynthetic
enzyme,
231
232
TGF-/DBL-1 signaling is essential and swimming behavior is necessary but
233
not sufficient for increasing body length.
234
Finally, we assessed whether increasing contraction rates in
235
swimming mode is involved in body-length enhancement, The Dorsal and
236
ventral (DV) head bending cycle were counted in degenerin channels,
237
TGF-signaling, dop-4(tm1329) and a ryanodine receptor unc-68(r1161)32
238
mutants. unc-8(e15) often abnormally curled and did not alter the DV cycle
239
in either liquid or agar plate, indicating completely lose normal swimming
240
behavior (Table 1). In contrast, other degenerin mutants mec-4 and mec-10 a
241
little but not significantly reduced in either behaviors. unc-68(r1161) showed
242
sluggish behavior in both crawling and swimming but increased the DV cycle
243
with normal swimming C-form in liquid. TGF- signaling dbl-1 and sma-4
244
mutants reduce the DV cycle to 70-90% both in liquid and on agar.
245
dop-4(tm1392) DV cycle a little decreased to 90% as compared with WT
246
(Table 1).
247
The quantitative gene expression analyses of a TGF-signaling,
248
dbl-1 and its downstream wrt-4 indicate that, both expression were
249
significantly unregulated in the liquid culture of the mutants, mec-4(e1611),
250
mec-10(e1515), and unc-68(r1161) increased body length (Table 1, Figs. 5 and
251
6). On the other hand, either expression did not induced in unc-8(e15) and
252
dop-4(tm1392) whose body length were constant in different culture
253
conditions. The TGF-signaling, dbl-1(wk70) and sma-4(e792) mutants did
254
unregulated dbl-1 gene but not wrt-4 in liquid. These results suggest that to
255
extend body length, (1) TGF-DBL-1 signaling is essential, but (2)
256
increasing the DV cycle by swimming behavior would be necessary but not
257
sufficient.
258
259
Table 1: Crawling and swimming behaviors and dbl-1 and wrt-4 gene
260
expression in dbl-1, sma-4, unc-8, dop-4 and unc-68 mutants.
Strains
Crawling (Hz)
Swimming (Hz)
Fold change
Fold change
dbl-1 (liquid
wrt-4
vs agar)
(liquid vs
agar)
N2
LT121 dbl-1
0.65±0.18
1.66±0.18
2.22±0.08
9.24±1.40
0.41±0.10 **
1.40±0.25 *
1.42±0.02
0.36±0.05
0.46±0.11**
1.57±0.15
1.36±0.07
0.47±0.03
0.53±0.15
1.56±0.19
nd
nd
0.53±0.19
1.53±0.13
nd
nd
0.23±0.07 ***
0.27±0.16 ***
1.11±0.01
0.60±0.06
0.54±0.17
1.48±0.21 *
0.89±0.01
0.66±0.03
0.22±0.07 ***
0.46±0.15 ***
1.48±0.04
1.56±0.11
(wk70)
DR1369 sma-4
(e729)
CB1611 mec-4
(e1611)
CB1515 mec-10
(e1515)
CB15 unc-8
(e15)
FG58 dop-4
(tm1392)
TR2170 unc-68
(r1161)
261
262
263
Discussion
264
C. elegans is a free-living nematode found in soil and decaying
265
vegetation, it persists on particles with moist surfaces and in aquatic
266
conditions. C. elegans exhibits at least two distinct locomotory gaits,
267
swimming when in liquid and crawling when on surfaces11-15. Surface tension
268
is required to retain C. elegans on moist surfaces and is predicted to be on
269
the order of 10,000 × G18, 33, 34. Thus, the crawling gait on surfaces likely
270
results from the larger forces caused by surface tension verses the flow
271
dynamics in liquid14. The crawling gait is triggered by large external loads,
272
and substantial muscle power is used to counter the external load and move
273
the body. In support of this speculation, a continuous gait transition between
274
undulations that resemble either swimming or crawling has previously been
275
observed with increasing liquid viscosity14.
276
These observations, however, do not fully explain why C. elegans
277
physique is significantly altered in liquid vs. on agar. Gait transition, by
278
necessity, must be a short term adaptive response whereas alteration of
279
physique is usually a long term adaptive response.
280
Crawling and swimming are quite different behaviors. C. elegans
281
crawling on moist agar exhibits dorsoventral bends of an S-shaped posture
282
with an average amplitude of 135° at a frequency of 0.5 to 0.8 Hz 12, 15. In
283
contrast, swimming C. elegans display dorsoventral bends of a C-shaped
284
posture with an average amplitude of 45° at a frequency of 1.7 to 2.1 Hz12, 15.
285
Moreover, C. elegans swims continuously for extended periods, 45 min
286
more15. These quantitatively distinct behaviors in frequency, amplitude, and
287
propagation of dorsoventral bends may elicit distinct long term adaptive
288
responses that alter gene expression to shape a physique most suited to the
289
environment.
290
When cultured in liquid, body length and expression levels of the
291
myosin heavy chain gene myo-3 and its transcriptional activator, hlh-1
292
(MyoD) increased versus when cultured on agar. These would appear to be a
293
long term adaptive response to growth in the liquid environment. A key
294
signaling pathway already known to modulate C. elegans body length, the
295
DBL-1/TGF- signaling pathway23-28 was both required for physique
296
alteration in response to liquid cultivation and transcriptionally induced in
297
response to liquid cultivation. The induction of dbl-1 expression in response
298
to liquid culture appears to have been due to fluid dynamics as dbl-1
299
expression was induced both by increasing viscosity and depth of liquid
300
culture (Fig. 1). Intriguingly, dbl-1 expression was induced by liquid culture
301
on a 1G centrifuge onboard the International Space Station vs. in space
302
microgravity (Fig. 1). It is possible that increased hydrostatic pressure on the
303
worms cultured with a 1G acceleration in space has impacted dbl-1
304
expression. As we have not measured hydrostatic pressure, future
305
experiments
306
hydrostatic pressure are needed.
examining
dbl-1 expression alteration in response to
307
In C. elegans, degenerin/epithelial Na+ channel (DEG/ENaC) family
308
members act as mechanosensors for assorted physical stimuli. Thus, we were
309
curious if they might be sensing fluid dynamics and possibly effecting dbl-1
310
expression. MEC-4 and MEC-10 form a heterocomplex of ion pore-forming
311
subunits in touch-sensitive neurons19-21 and this complex is essential for
312
response to hyper-gravity22. However, our finding that body size increases in
313
liquid culture even in the absence of the MEC-4/10 heterocomplex, suggests
314
that this complex is not required for altering physique in response to fluid
315
dynamics. Instead, we found that other DEG/ENaC family members,
316
specifically UNC-8 is required for altered physique in response to fluid
317
dynamics (Fig. 1). This suggests that they may be acting as fluid-responsive
318
mechanoreceptors. UNC-8 is coexpressed in ventral cord motor neurons38-40.
319
In addition, TGF-/DBL-1 which dose-dependently regulates C. elegans body
320
size, is primarily expressed in motor neurons and the nerve ring24; the
321
known site of dbl-1 expression closely matches the known site of UNC-8
322
action. Therefore, the UNC-8 degenerin complex might sense physical
323
stimuli from culture conditions and/or body posture alterations and/or
324
tension generated by varying gaits, leading to the upregulation of DBL-1.
325
The upregulated DBL-1 ligand then acts, via known mechanisms, on
326
muscular and hypodermal cells to control body physique by facilitating the
327
expression of muscle myosin and cuticle collagen.
328
As another possibility, swimming behavior would be necessary to
329
increase body physique because unc-8(e15) mutant complete losses normal
330
moving behaviors in both liquid and agar surface (Table 1). Lastly, as fluid
331
dynamics, particularly hydrostatic forces and drag resistance accompanying
332
liquid viscosity enhance bone and skeletal muscle formation in less active
333
people5-10 we were curious if this was also true of less active worms. Indeed, a
334
sluggish and flaccid mutant, unc-68 displayed body length increase in
335
response to liquid culture (Fig.
336
independent fluid dynamics effect, including easier movable by buoyancy,
337
appears to be evolutionarily conserved between C. elegans and man but also
338
that C. elegans might be a suitable model for studying and combating the
339
impact of inactivity on human muscle.
). This suggests not only that an activity
340
Recent work has elucidated that C. elegans employs biogenic
341
amines (dopamine and serotonin) to control gait transition between crawling
342
and swimming15; a short term adaptation. Dopamine is necessary to initiate
343
and maintain crawling on land after swimming in water, and serotonin is
344
necessary to transition from crawling to swimming behavior. We were
345
curious if this short term adaptation also had a role in long term adaptation
346
of physique. While mutations in dop-1, ser-5, and tph-1 displayed wild-type
347
body length increase in response to liquid culture, dop-4 mutants did not
348
display a length increase. This suggests that DOP-4 is required not just for
349
the short term gait adaptation but also for the long term physique
350
adaptation to liquid culture. DOP-4 is a D1-like dopamine receptor that is
351
known to be involved in alcohol-induced disinhibition of certain behaviors,
352
including foraging and crawling postures in water37. Thus, DOP-4 may
353
function as a key component for C. elegans adaptation to aquatic
354
environments participating in both short term and long term adaptive
355
responses.
356
C. elegans growing in liquid, as in the wild, can be subject to hypoxia
357
and limited nutrition. We therefore investigated if these factors might
358
contribute to increased body length in liquid culture. However, mutations in
359
hif-1, ins-7, and daf-16 all responded like wild-type suggesting that neither
360
oxygen sensing nor nutrition sensing was contributing to the increased body
361
length. This may not be surprising given that UNC-8 and/or DOP-4 appear
362
to be sensing the fluid dynamics and that wild-type and all other mutants
363
tested developed at the same rate in either liquid or agar culture.
364
In conclusion, our results suggest that UNC-8 and/or DOP-4 may
365
function as a neuronal stimuli that senses fluid-dynamic properties
366
including viscosity/drag resistance and possibly hydrostatic pressure. It
367
appears that activation of these neurotransmitters by fluid-dynamic
368
properties increase expression of dbl-1 to increase DBL-1 signaling causing
369
an increase in body size and expression of muscle proteins.
370
371
Methods ref 29, 30, 31
372
373
Nematode strains. C. elegans N2 Bristol strain was used as wild-type. The
374
mutant strains were as follows: BC15777 derivative: dpy-5(e907); RW1596:
375
myo-3(st386), stEx30 [myo-3p::GFP + rol-6(su1006)]; LT121: dbl-1(wk70);
376
NU3: dbl-1(nk3); DR1369: sma-4(e729); CB1611: mec-4(e1611); CB1515:
377
mec-10(e1515); CB15: unc-8(e15); NC279: del-1(ok150); ZG31: hif-1(ia4);
378
CF1038: daf-16(mu86); RB1388: ins-7(ok1573); GR1321: tpn-1(mg280);
379
RB2277: ser-5(ok3087); LX636: dop-1(vs101); FG58: dop-4(tm1392); and
380
TR2170:
381
Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN,
382
USA).
unc-68(r1161).
These
strains
were
obtained
from
the
383
384
Culture conditions.
385
Thirty to fifty wild-type or mutant adult hermaphrodites were
386
transferred onto a freshly prepared nematode growth medium (NGM)-agar
387
plate (φ6 cm plastic culture dish) with Escherichia coli strain OP50 spread
388
over the surface as a food source. Adults were allowed to lay eggs for four
389
hours at 20°C; this yielded at least 500 eggs on each plate. Adults and the
390
bacterial food source were washed off the plate by gentle pipetting with 2 ml
391
M9 buffer three times. The remaining eggs were left for an additional 12
392
hours at 20°C at which point the hatched L1 larvae were collected in 500 l
393
M9 buffer. Sixty L1 larvae per condition were simultaneously cultured at
394
20°C on either an OP50 NGM agar plate or in a 2 ml liquid NGM culture
395
system containing E. coli OP50 (OD600 = 1.0; liquid depth = 0.8 mm) in a φ6
396
cm plastic culture dish. Three days after cultivation, wild-type and all other
397
mutants tested in this study had grown to young adulthood, as evidenced by
398
the onset of egg production; this was observed under both culture conditions.
399
To prevent starvation, adult animals were picked using aφ0.2 mm platinum
400
wire and transferred to new medium each day; transfers occurred for both
401
culture conditions.
402
To study the effects of liquid viscosity on gene expression (see below),
403
final concentrations of 1.0% and 1.5% methylcellulose in 2.0 ml of
404
NGM-liquid medium containing E. coli OP50 (OD600 = 1.0) were used. The
405
kinetic viscosities, as measured by a viscometer Visoboy2 (LAUDA,
406
Germany), were 1.0 cSt (mm2/s) for the control OP50 NGM liquid, 36.1 cSt
407
for the 1.0% methylcellulose, and 123.3 cSt for the 1.5% methylcelluose.
408
To study the effects of the depth of liquid culturing, OP50
409
NGM-agar in φ6 cm plates were additionally covered with 1.5 ml (~0.6 mm
410
in depth), 3.0 ml (~1.2 mm in depth), or 4.5 ml (~1.8 mm in depth) of
411
NGM-liquid medium containing E. coli OP50 (OD600 = 1.0). From L1 to
412
adulthood, all animals are fully covered and showed swimming behavior
413
even in the shallowest condition.
414
415
Measurements of body lengths. C. elegans body length was evaluated at
416
young adulthood (3 days after starting as a L1 larvae) and the subsequent
417
three days. Each day a subset of cultured animals were fixed with 1%
418
paraformaldehyde for 30 min at room temperature, and were imaged using a
419
BX51 microscope and a DP71 camera (Olympus Optical Co., Ltd., Tokyo,
420
Japan). Body lengths were measured using CellSens image analysis software
421
(Olympus). Each
422
experiment was performed in triplicate with three independent samples
423
(total n = 60 worms per time point). Statistical analysis was performed in MS
424
Excel. Statistical significance was set at p < 0.05, using a Student's
425
two-tailed t-test.
426
427
Measurements of contraction rate of moving behavior. Each assay was
428
conducted on 10, never-starved, adult worms at 4 days from L1 larvae
429
cultured in liquid or on moisture agar plate. Dorsal and ventral (DV) head
430
bending cycle were counted for 30 sec under stereo microscopy as contraction
431
rate of swimming or crawling, just after tapping each culture plate.
432
433
Gene expression analysis. Total RNA was isolated on the indicated day of
434
cultivation from approximately 300 adult hermaphrodites using TRIzol
435
(Invitrogen Corp., Carlsbad, CA, USA). Quantitative real-time polymerase
436
chain reaction (RT-PCR) analysis was performed with a CFX96 Touch™
437
Real-Time PCR System (Bio-Rad Laboratories Inc., Hercules, CA) and a
438
SYBER ExScript™ RT-PCR Kit (TaKaRa Bio Inc., Shiga, Japan). The
439
expression level of elongation factor eef-2 was used as an internal standard,
440
and the relative ratio of gene expression for each gene was calculated as
441
described29. The following primer sets were used to amplify eef-2, hlh-1,
442
myo-3, dbl-1, and wrt-4: eef-2 (forward) 5’-GAC GCT ATC CAC AGA GGA GG
443
and (reverse) 5’-TTC CTG TGA CCT GAG ACT CC; hlh-1 (forward) 5’-GCT
444
CGG GAA CGC GGT CGA and (reverse) 5’-GGA ATG CTC GCA ACG ATC
445
CGC GA; myo-3 (forward) 5’-ACT CTC GAA GCC GAA ACC AAG and
446
(reverse) 5’-TGG CAT GGT CCA AAG CAA TC; dbl-1 (forward) 5’-CAG TTT
447
GGC TTC GAT TGC TC and (reverse) 5’-TGA AGC TGG TCC TCT GTC TG;
448
and wrt-4 (forward) 5’- TGG ATG AGC TCG CAG TGG and (reverse) 5’- CTC
449
CGT TGT CAA GTG TGA ATT CTA C. Real-time PCR experiments were
450
performed in triplicate for each biological sample.
451
452
Spaceflight experiment. We also measured expression levels of some genes in
453
spaceflown wild-type 4-day old adults from the C. elegans RNAi space
454
experiment (CERISE)30, 31.
455
456
457
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Acknowledgements
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We are grateful to the entire crew of the CERISE for their work on STS-129,
626
STS-130, and the ISS. The CERISE was organized with the support of the
627
JAXA. We also thank the Caenorhabditis elegans Genetic Center for kindly
628
supplying the mutant strains.
629
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Competing interests
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The authors have no competing interest.
633
634
Contributions
635
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Higashitani A designed research. Harada S, Hashizume T, Nemoto K, Shao Z,
637
Higashitani N and Higashibata A performed gene expression analyses and
638
microscopic observation. Fukui K, and Higashibata A coordinated the
639
CERISE flight experiment. Harada S, Higashibata A, Hashizume T, Nemoto
640
K, Etheridge T, Szewczyk NJ, and Higashitani A analyzed data, and wrote
641
the paper.
642
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Funding
644
645
This work was supported by grants from the MEXT, the JSPS (15H05937,
646
26506029), the Cross-ministerial Strategic Innovation Promotion Program
647
(J150000592), and the Cell Biology Experiment Project conducted by the
648
Institute of Space and Astronautical Science in JAXA. TE was supported by
649
the Medical Research Council of UK (G0801271). NJS was supported by the
650
National Institutes of Health (NIH NIAMS ARO54342).
651
652
653
654
Figure legends
655
Figure 1. Microgravity on the ISS reduces myo-3 and dbl-1 gene expression,
656
but on the ground based experiments, increasing liquid viscosity and depth
657
oppositely induce these expression. myo-3 and dbl-1 gene expression levels
658
were monitored in liquid cultured, spaceflown wild-type animals (4-days
659
adult) with or without 1G acceleration during the C. elegans RNAi space
660
experiment (CERISE) in Japanese Experiment Module KIBO30,
661
Wild-type animals were grown from the L1 larval stage for 4 days in
662
different liquid viscosities with 1.0% (36.1 cSt) and 1.5% (123.3 cSt)
663
methylcellulose (B). Wild-type animals were grown from the L1 larval stage
664
for 4 days on OP50 NGM-agar submerged in indicated depth of OP50 NGM
665
liquid medium (C). Alterations in dbl-1 and myo-3 gene expression were
666
monitored by quantitative RT-PCR.
31
(A).
667
668
669
670
671
Figure 2 Alterations in body length and gene expression levels of myo-3 and
672
hlh-1 for C. elegans grown under different culture conditions. Wild-type
673
gravid adult hermaphrodite grown on moist agar plate (A, indicated in pink)
674
or in liquid culture (B, indicated in blue), for 4 days starting from the L1
675
larval stage. (C) Body lengths were significantly increased by liquid vs. agar
676
culture. Alterations in myo-3 (D) and hlh-1 (E) gene expression levels were
677
monitored by quantitative RT-PCR. Data points and error bars indicate the
678
means ± the standard deviation (SD) (n = 60 worms per time point, **P <
679
0.01).
680
681
682
683
Figure 3. DBL-1 and its signaling pathway are required for C. elegans body
684
length alterations in response to liquid culture conditions. dbl-1(wk70)
685
gravid adult hermaphrodite grown on moist agar plates (A) or in liquid
686
culture (B) for 4 days, starting from the L1 larval stage. (C) Body lengths of
687
wild-type, dbl-1(wk70), dbl-1(nk3), and sma-4(e729) grown in liquid or on
688
agar for 4 days were measured. Bars and error bars indicate the means and
689
SD (n = 60 worms per condition; **P < 0.01).
690
691
692
693
Figure 4. Dumpy and roller phenotypes are mitigated by liquid culture.
694
dpy-5(e907) (A, B), and RW1596 rol-6 (su1006) and Pmyo-3::GFP::myo-3 (C,
695
D), animals were grown on moist agar (A, C) or in liquid culture (B, D) for 4
696
days, starting from the L1 larval stage. (E) Their body lengths were
697
measured. (F) The frequency of the right-handed roller phenotype in
698
RW1596 was counted. Bars and error bars indicate the means and SD (n = 60
699
worms per condition; **P < 0.01, *P < 0.05).
700
701
702
703
Figure 5. UNC-8 degenerin is required for C. elegans body length alterations.
704
mec-4(e1611), mec-10(e1515), and unc-8(e15) were grown for 4 days in liquid
705
or agar culture, starting from the L1 larval stage. Body lengths were
706
measured (A). Bars and error bars indicate the means and SD (n = 60 worms
707
per condition; **P < 0.01).
708
709
710
Figure 6. Several C. elegans mutants show wild-type body length increases
711
in liquid vs. agar culture. Of all the mutants tested, only dop-4(tm1329)
712
failed to increase its body length after 4 days in liquid culture (n = 60 worms
713
per condition; **P < 0.01).
714
715
716
717
718