Development 142: doi:10.1242/dev.117150: Supplementary Material
Movie 1. Lumen formation in the notochord of a control embryo. The movie was taken every three
minutes using differential contrast interference (DIC). This is one the movies that contribute to Fig.
1C,D. Frame rate is 10 fps. Scale bar = 10 μm.
Movie 2. Lumen formation in the notochord of an embryo treated with 100 µM blebbistatin. The
movie was taken every three minutes using DIC. This is one of the movies that contributed to Fig. 3BD. Frame rate is 10 fps. Scale bar = 10 μm.
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Movie 3. Expression MRLC T18E-S19E changes lumen expansion dynamics and causes a
constriction of lumen at the level of apical/lateral junction. Notochord cells are labelled with
MRLC T18E-S19E-mCherry and Lifeact-tGFP. Frame rate is 3.7 fps. Scale bar = 10 μm.
Movie 4. Treatment of jasplakinolide before lumen formation causes a conversion of
extracellular lumens to intracellular vacuoles. (A) Notochord cells are labelled with Slc26a mCherry for apical luminal domain. (B) Notochord cells are labelled with Slc26a -mCherry for apical
luminal domain and Par6-tGFP for tight junctions. This movie is used to make Fig. 5Ah,i. Frame rate is
6.4 fps. Scale bar = 10 μm.
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Development 142: doi:10.1242/dev.117150: Supplementary Material
Movie 5. Treatment of jasplakinolide at the extracellular lumen expansion stage causes a
conversion of lumens to intracellular vacuoles. DIC time-lapse video of notochord cells converting
large extracellular lumens to intracellular vacuoles. Frame rate is 6.4 fps. Scale bar = 10 μm.
Movie 6. Extracellular lumens form initially then are converted by jasplakinolide to intracellular
vacuoles when the drug is applied before lumen formation begins. Notochord cells are labelled
with DE-Cad-mCherry for lateral domain and Par6-tGFP for apical/lateral junction. This movie is used
to make Fig. S3Ca. At the beginning of the treatment, the transverse diameter continues to increase. It
then starts to decrease, and finally contracts to a point at the lateral surface. Frame rate is 6.4 fps. Scale
bar = 10 μm.
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Development 142: doi:10.1242/dev.117150: Supplementary Material
Movie 7. Treatment with jasplakinolide at the lumen expansion stage results in a conversion of
pre-existing lumens to intracellular vacuoles. Notochord cells are labelled with Slc26a -mCherry
and ZO-1-tGFP. The junctional opening contracts to form a single focus. This movie is used to make
Fig. S3Cb1. Frame rate is 6.4 fps. Scale bar = 10 μm.
Movie 8. Treatment with jasplakinolide at the lumen expansion stage results in a conversion of
lumens to intracellular vacuoles. Notochord cells are labelled with DE-Cad-mCherry and LifeacttGFP. As the junctional opening closes, lateral actin filaments are concentrated to a single cluster that
juxtaposes the lateral surface and the intracellular vacuole. Frames of the right cell in this movie are
used to make Fig. S3Cb2. Frame rate is 10 fps. Scale bar = 10 μm.
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Development 142: doi:10.1242/dev.117150: Supplementary Material
Movie 9. Treatment of Jasplakinolide at the lumen expansion stage results in a conversion of
lumens to intracellular vacuoles. Notochord cells are labelled with Slc26a -mCherry and LifeacttGFP. As vacuoles separate from the lateral surfaces and plunge into cytoplasm the actin filaments
remain at the surface of the vacuoles. This movie is used to make Fig. S3Cb3. Frame rate is 6.4 fps.
Scale bar = 10 μm.
Movie 10. Rescue of jasplakinolide-induced lumen-vacuole conversion phenotype by
simultaneous blebbistatin treatment. Notochord cells (labelled with Slc26a -mCherry for
apical/luminal domains and Lifeact-tGFP for actin) form extracellular lumens instead of intracellular
vacuoles. Frame rate is 4.8 fps. Scale bar = 10 μm.
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Movie 11. TGF RI inhibitor SB431245 treatment causes a constriction of lumen at the level of
apical/lateral junction. Notochord cells are labelled with MRLC-mCherry for myosin and LifeacttGFP for actin. This movie is used to make Fig. 6Cc. Frame rate is 5.5 fps. Scale bar = 10 μm.
Movie 12. Overexpression of Slc26a removes phase 2 in the lumen formation process. Notochord
cells express wild type Slc26a fused to mCherry. Frame rate is 10 fps. Scale bar = 10 μm.
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Fig. S1. Parameters used in the study and statistical analyses. (A) 3D projections of single lumen
pockets, shown at different angles. Notochord cells simultaneously express apical membrane marker
Slc26a (red) and tight junction marker ZO1 (green). (a-e) the longitudinal plane (xy), (a’-e’) the 3D
projection is rotated 90° to show the junction ring (xz, in transverse plane, visualized by ZO-1). These
images reveal the circular characteristic of the junctional ring, i.e., the transverse diameter of the
junctional ring at any angle is constant at a given time point. When the longitudinal radius and the
radius of the junction ring (1/2 transverse diameter) is unequal, the lumen takes the shape of spheroid.
(B) Rate of lumen expansion during different phases. For statistical evaluation, linear regression is
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performed for each phase and the slopes of the regression lines are compared. (a) The longitudinal
radius increase is not significantly different between phase 1 and 2 (P = 0.7169). (b) The transverse
diameter increase is significantly faster in phase 3 than in phase 1 (P < 0.0001).
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Fig. S2. Anti-pT18pS19 MRLC staining reveals lateral localization of activated myosin. Embryos
were doubly stained for pT18pS19 MRLC and actin using phallacidin before lumen formation (A), at
phases 1 (B), 2 (C) , and 3 (D) of lumen formation. Yellow arrows and white arrows point to myosin
and actin respectively. Scale bars = 5 μm.
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Fig. S3. Supplemental data on blebbistatin and jasplakinolide treatments. (A) Blebbistatin
treatment impairs the morphology of notochord tube. Embryos were treated with DMSO or
blebbistatin just after lumen opening. (a and c) show the beginning of the treatment; (b and d) show the
morphology of fusing notochord tube 294 min later, under continuous treatment. In the control, lumen
pockets are tilted and widely fused (yellow arrows), whereas in blebbistatin-treated embryos lumen
pockets are not tilted and the fused pockets encompass maximum two cells (red arrows). Scale bars =
10 μm. (B) Treatment of jasplakinolide results in the formation of intracellular vacuoles in
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notochord cells. (a) Embryos at lumen expansion stage were treated with 1 µM jasplakinolide for one
hour, then disassociated with protease E. Isolated notochord cells after jasplakinolide treatment
contained intracellular vacuoles. (b-b') Intracellular vacuoles formed after jasplakinolide treatment
were positive for anion transporter Slc26a
(b'), which is normally present at the apical/luminal
domain of notochord cells (b). Scale bars = 5 μm. (C) Conversion of extracellular lumens to
intracellular vacuoles by jasplakinolide treatment. (a) Conversion at the beginning of lumen
formation. Frames of supplementary material Movie 6 of notochord cells labelled with DE-CadmCherry and Par6-tGFP. Arrows indicate the junctional opening marked by Par6. (b) Conversion
during the lumen expansion stage. (b1) Frames from supplementary material Movie 7. Notochord cells
are labelled with Slc26a -mCherry and ZO1-tGFP. Arrows indicates the junctional opening that
undergoes contraction and closure. (b2) Frames from supplementary material Movie 8. Notochord cells
are labelled with DE-Cad-mCherry and Lifeact-tGFP. Arrows indicates the junction-associated actin
(arrows) cluster as the junctional opening contracts. (b3) Frames from supplementary material Movie
9. Notochord cells are labelled with Slc26a -mCherry and Lifeact-tGFP. Actin filament cluster (arrow)
originating from the lateral membrane remains at the vacuole surface as the vacuoles (asterisks) move
inside the cells. Scale bars = 5 μm. (c). Changes of transverse diameter after jasplakinolide treatment.
The embryos were treated with the drug either before (c1) or during (c2) lumen opening. The 0 time
point corresponds to the beginning of the closing phase. “exp” refers to the experiment number and
“cell” to individual cell that was measured.
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Fig. S4. Localization of phosphorylated non-muscle myosin II regulatory chain (pS19 MRLC)
with respect to cortical actin under different drug treatments. (A) Blebbistatin treatment. (B)
Jasplakinolide treatment. Arrows point at the apical/lateral junctions prior to (Bb-b”) and just after
collapsing of lumen pockets (Bc-c”). Arrowhead points at vacuolar accumulation of myosin. (C)
Combined blebbistatin/jasplakinolide treatment. Arrows point to the apical/lateral junction. Scale bars
= 5 μm.
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Fig. S5. A regulatory network mediating appropriate actomyosin contractility at the
apical/lateral junction to ensure correct lumen expansion kinetics. A correct amount of TGF
signalling results in an inhibition of ROCK pathway. This inhibition leads to a reduced myosin II
activity at the apical/lateral junction, which promotes lumen opening. Inhibition of TGF
receptor
activation, freezing of actin dynamics with jasplakinolide, or overexpression of a constitutively active
myosin II form leads to a heightened contractility, which constricts lumen pockets or even converts
them to vacuoles. Conversely, inhibition of ROCK activity, of myosin activity, or a nonphosphorylatable myosin II form, causes an insufficient contractility that leads to accelerated lumen
opening and abnormal lumen morphology. As a conclusion, a correct amount of TGF
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signalling
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creates a balance in actomyosin activation that ensures a correct lumen opening during phases 1 and 3.
At phase 2, we hypothesize that a reduction of TGF signalling increases contractility to trigger the
lag-phase that secures a correct kinetics. We hypothesize based on literature that the effect of TGF on
ROCK is mediated by rhoA-GTP (transparent box and dashed gray lines) and that rhoA-GTP inhibition
causes down-regulation of actin.
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Fig. S6. Stronger posterior TGFb-RI expression may functionally correlate with a stronger
pMLC accumulation. (A) phase 1, (B) and (C) phase 2. The embryo in (C) was also stained for actin
using phallacidin. Anterior to the left.
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Fig. S7. Laplace law dictates the presence of the maximal surface tension at the apical/lateral
junction. To withstand such a high wall tension and prevent the cells from being torn apart at the
junction, notochord cells have recruited an elaborated system centred at actomyosin contractility.
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