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Fig. 1. Active RhoA localizes to the apical domain of emerging MCCs. (A) Schematic of MCC morphogenesis. Upon specification of an MCC progenitor (1),
a nascent MCC radially intercalates (2, 3) and docks at the tricellular junction to finally integrate with the pre-existing epithelium by expanding its apical
surface (3, 4, apical emergence). (B) Image sequence of an active RhoA biosensor (rGBD, green) throughout apical emergence of a single unmanipulated cell.
(C)Corresponding image of an apicallyemergingMCC(visualized using an actin marker, utrophin,GFP–UtrCH, gray, driven by anMCC-specific α-tubulin promoter).
(D) Representative plot of medial actin and active RhoA dynamics during apical emergence. (E) Values of the intensity of active RhoA for consecutive categories of
apical domain sizes, categorized by the binnedmean radius in controls. Data representmean and s.e.m., n=4 cells fromfour embryos. Actin and rGDB intensities were
normalized by dividing the mean intensities of medial actin and/or rGBD by the mean intensities of actin and/or rGBD within the cortical region. Scale bars: 10 μm.
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Fig. 2. RhoA controls the dynamics of MCC apical emergence. (A) Image sequence of an apically emerging MCC (visualized using an actin marker, utrophin,
GFP–UtrCH, gray, driven by an MCC-specific α-tubulin promoter) in controls, (B) upon expression of a constitutively active RhoA construct (CA-RhoA) and
(C) upon expression of dominant negative RhoA (DN-RhoA). CA-RhoA and DN-RhoA were expressed in MCCs under an MCC-specific α-tubulin promoter, see
Fig. S1. Quantification of MCC apical surface area (e.g. dotted yellow line in A) is indicated in μm2 in the bottom left corner of each panel. Note that for a given
apical domain surface, an apical domain of an MCC expressing DN-RhoA has a more polygonal shape compared to that of controls and to an MCC expressing
CA-RhoA. (Note that an image in Fig. 2A is replicated in Fig. 4B; see Fig. 4 legend for details). The time after the start of imaging is indicated. (D) Dynamics of the
apical domain area of the MCCs shown in A–C; controls, black; MCC expressing CA-RhoA, green; MCC expressing DN-RhoA, pink. (E) Expansion rate of an
MCC apical domain in controls (black) and upon expression of CA-RhoA (green) or of DN-RhoA (magenta). The expansion ratewas determined by measuring the
slope of the linear fit of the surface area data between 50 μm2 and 150 μm2. (F) The final apical domain areas of MCCs under the conditions described in E. Boxes
extend from the 25th to 75th percentiles, with a line at the median. Whiskers represent the minimum and maximum values. ***P<0.001; n.s., not significant
(Mann–Whitney U test). n>5 embryos; n values on the graph represent the number of cells analyzed. Scale bar: 10 μm.
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Fig. 3. RhoA controls force balance in the apical surface during MCC emergence. (A) Schematic of forces acting on an MCC apical domain. Effective 2D
pressure, δP (orange arrows), and junctional pulling forces, Π(blue arrows), acting against cortical tension, γ (black arrows), and elasticity from the surrounding
cells, E (red arrows). (B) Kurtosis values for the consecutive categories of apical domain sizes, categorized by binned mean radius, in controls and cells
expressing DN-RhoA or CA-RhoA. Data represent mean±s.e.m. ***P<0.0005; n.s., not significant (two-tailed unpaired Studentâs t-test). n=10 cells, n>5 embryos.
(C) Simulations of apical domain shapes upon expression of DN-RhoA or CA-RhoA. The simulations assumed that only pressure or both pressure and cortical
tension were impacted upon expression of the different RhoA constructs. The final shapes of the apical domains are each represented by a different color.
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Fig. 4. RhoA controls the rate of MCC apical
actin network assembly. (A) Schematic of the
medial and cortical regions within an apical domain
of an MCC. (B) A representative image sequence
of an automatically segmented cortical and medial
region within an apically expanding MCC
(visualized using α-tubulin-promoter-driven GFP–
UtrCH, gray). Note that the image here is replicated
from Fig. 2A as an example. The time after the start
of imaging is indicated. Scale bar: 10 μm.
(C) Apical actin concentration (ratio of mean
medial to mean cortical actin) dynamics in a control
cell (black), an MCC expressing CA-RhoA (green)
and an MCC expressing DN-RhoA (magenta).
Circles, data points; lines, data smoothed with a
moving average of 5. (D) Apical actin concentration
as a function of apical area in control cells, in
MCCs expressing CA-RhoA and MCCs
expressing DN-RhoA; n>10 cells from n>5
embryos. Data represent mean and s.e.m. The
slope for MCCs expressing DN-RhoA (pink dotted
line) is significantly higher than that for control
(P<0.05, Z-test for correlation coefficients),
whereas the slope for MCCs expressing CA-RhoA
is not significantly different from that of control.
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Fig. 5. RhoA, FMN1 and Arp2/3 control the rate of turnover of actin in the MCC apical actin network. (A) Image sequence of an MCC apical domain in a control cell and (B) in a cell expressing DN-RhoA, visualized using monomeric YFP–actin upon photobleaching of a region of interest (dotted yellow line) within the medial portion of the apical domain. 0 s indicates the time of photobleaching. (C) Normalized actin recovery after photobleaching (FRAP) of monomeric actin within the medial portion of an apical domain of MCCs: in control cells (black), cells expressing CA-RhoA (green), cells expressing DN-RhoA (red), FMN1-knockdown cells (FMN1 KD; blue) and upon Arp2/3 inhibition with CK666 (orange). Data are mean±s.d. (D) Fluorescence half-time recovery after photobleaching of medial actin in controls (black), MCCs expressing CA-RhoA (green), MCCs expressing DN-RhoA (magenta), FMN1-knockdown cells (blue) and upon Arp2/3 inhibition with CK666 (orange). (E) The mobile fraction measured upon FRAP analysis of medial actin under the conditions described in D. Boxes extend from the 25th to 75th percentiles, with a line at the median. Whiskers indicate minimum and maximum values. *P<0.05; ***P<0.001; ns, not significant (Mann–Whitney U
test). n>5 embryos. Scale bars: 10 μm.
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Fig. 6. Different modes of apical
emergence depend on RhoA
activity. (A) Upon knockdown of
Fmn1 (FMN1 KD), the apical domain
collapses as a consequence of low
pushing forces and high cortical
tension. (B) Expression of DN-RhoA
decreases both pushing forces and
cortical tension. Depending on the net
contribution of these forces, the
apical domain (i) expands in an
angular manner to an overall smaller
size, (ii) undergoes non-periodic
oscillations, (iii) collapses.
(C) Expression of CA-RhoA does not
change the dynamics of apical
emergence.
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Figure S1. RhoA regulates apical emergence dynamics. (A) Image sequence of an apically emerging MCC upon
expression of CA-RhoA-RFP specifically in MCC using a MCC-specific -tubulin promoter. (B) Image sequence of
an apically emerging MCC upon expression of DN-RhoA-RFP specifically in MCC using a MCC-specific -tubulin
promoter. Scale bar, 10 μm.
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Figure S2. Expression of DN-RhoA impairs the localization and dynamics of formin 1. (A) Image sequence
of fluorescently labelled Fmn1 (green) during apical emergence in a control cell. (B) Image sequence of an apically
emerging control cell visualized with an actin marker, UtrCH (grey). (C) Image sequence of fluorescently labelled
Fmn1 (green) during apical emergence upon expression of DN-RhoA. (D) Image sequence of an apically emerging
cell visualized with actin marker, UtrCH (grey) upon expression of DN-RhoA. (E) Kymograph of FMN1 signal during
apical expansion of a control cell. (F) Kymograph of actin signal (visualized by UtrCH) in a control cell. (G) Kymograph
of FMN1 signal upon expression of DN-RhoA. (H) Kymograph of actin signal (visualized by UtrCH) upon
expression of DN-RhoA. (I) Formin 1 and actin (visualized by UtrCH) fluorescence intensity profile along the apical
domain diameter (yellow dotted line, starts at 0 and ends at 1) in controls and upon expression of DN-RhoA (J). (K)
Dynamics of Fmn1 and actin in controls and upon expression of DN-RhoA (L). Scale bar, 10 μm.
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