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3.3.1 Analyzing rounding dynamics

Analysis procedure

After placing dissected explants in culture, we observed that they were round- ing over time with different dynamics depending on the region they were dissected from. Posterior explants rounded faster than anterior explants (see Figure3.3). To quantify the rounding dynamics, we automatically segmented each explant using the CellProfiler software (Carpenter et al., 2006) and fitted the shape with an ellipse using the normalized central moments calculation. The major and minor axes, a and b, were measured to compute the aspect ratio a/b over time. As expected from the relation3.1, the aspect ratio follows an exponential decay over time (see Figure3.4

A). The typical time of this decay τ was computed for all explants by fitting the slope of the semilog plots (with a0and b0kept fixed):

log   a(t) b(t) −1 a0 b0 −1   = −t τ (3.4)

Three typical rounding experiments of explants along the AP axis are presented Fig- ure3.4B and Movies 1, 2 and 3. The rounding dynamics is graded along the axis with a faster dynamics towards the posterior of the body. On average, τ=456±234 min in anterior, τ=149±114 min in medial and τ =68±26 min in posterior. Special case of anterior explants

A phenomenon complicated the analysis in anterior-most explants. Inter-somitic clefts are prepatterned in the anterior PSM up to 4 future somites (Palmeirim et al., 1997). Therefore, for a majority of anterior explants (n = 32/36) somite formation took place during the rounding process. In this case, cleavage was initiated during 2-3 hours, then failed and the explants subsequently rounded up. A typical sequence of snapshots of this process is presented in Figure3.5A. For these peculiar situations, the aspect ratio was either constant or increased during the cleavage process. We, therefore, fitted the exponential decay only after the end of the cleavage phase (see Figure3.5B).

3.3.2 A graded viscocapillary velocity along the anteroposterior axis

In order to verify the relation 3.2, we plotted τ against the final radius of the rounded explant Rf. We were limited by the accessible range of Rf, because the

starting explants could not be too small as they would be already almost round. They could not be longer than a third of the PSM as they would encompass more

anteroposterior axis

B

A _{0min 360min 720min}

0min 60min 120min

a b C ant med pos

FIGURE3.3 – Explant rounding dynamics. A: Schematic of explant

dissection. Snapshots during anterior (B) and posterior (C) explant rounding. Green lines show segmented contour. Blue line shows the major axis of length a of the fitted ellipse. Red line shows the minor axis of length b of the fitted ellipse. Scale bars: 100 µm.

B A

FIGURE3.4 – Measurement of the rounding dynamics along the an-

teroposterior axis. A: Typical exponential decay of the aspect ratio a/b for a medial explant. B: Semilog plots of a/b along the antero- posterior axis (ant: anterior, med: medial, pos: posterior). Solid lines: linear fits with a0/b0fixed.

A

B

0min

380min

830min

FIGURE3.5 – Somitogenesis in anterior explants. A: Snapshots of a time-lapse of an anterior explant. The explant starts cleaving (arrow- heads indicate the cleavage plan), then fuses again and rounds up. Scale bar: 100 µm. B: Quantification of the rounding dynamics. The aspect ratio a/b increases during the first 400 min (extension), and then exhibits an exponential decay (rounding).

anteroposterior axis than one region. For this reason, Rf was limited to less than a 2-fold range (from 70

µm to 130 µm). Despite data variability, we could see that there was a general trend of increasing τ with respect to Rf (see Figure3.6A, B). Thus, we considered that the

linear dependency is verified.

We next investigated how viscocapillary velocity varied along the AP axis. Fig- ure3.6C shows that the posterior region rounds up nearly 8 times faster than the an- terior region (vp=0.21±0.08 µm·min−1in anterior and vp=1.57±0.53 µm·min−1

in posterior, see Table3.1). We observed intermediate rounding dynamics in the me- dial region, with a wide dispersion: some explants were already anterior-like, some were still posterior-like (vp = 0.92±0.44 µm·min−1). This decreasing viscocapil-

lary velocity as the PSM matures can be explained by two non-mutually exclusive hypotheses:

• surface tension decreases from posterior to anterior. Since the PSM epithelial- izes during its maturation, this possibility seems unlikely;

• viscosity increases from posterior to anterior. As the ECM is deposited along the AP axis and cellular density increases, this hypothesis seems more plausi- ble.

Therefore, if we assume that surface tension stays at least constant, or even increases during PSM maturation, viscosity needs to increase to an even greater extent to ex- plain the graded dynamics of rounding.

3.3.3 Effect of cell motility

As motility of PSM cells is a key driver of elongation, we next investigated the effect of motility on rounding dynamics. Cell motility is likely to impact tissular viscosity as cell-cell rearrangement is an important determinant of tissue fluidity. We, therefore, investigated the rounding dynamics of explants incubated with two different drugs known to inhibit cell motility in vivo: blebbistatin and the MAPK in- hibitor PD0325901 (Bénazéraf et al., 2010).

Blebbistatin is an inhibitor of myosin-II phosphorylation which decreases actomyosin contractility. Consequently, cell motility is impaired, but so is cortical tension. There- fore, we could expect that the explant surface tension would be lowered by blebbis- tatin, and thus viscocapillary velocity (if viscosity stays constant). We incubated explants with 20 µM blebbistatin. Surprisingly, viscocapillary speed showed a weak but significant increase in anterior explants (Student t-test’s p-value: 0.039) and no change in medial and posterior explants (Student t-test’s p-values respectively: 0.81 and 0.19). We verified that the blebbistatin treatment was effective. First, we ob- served that cell protrusive activity was blocked and thus explants exhibited a char- acteristic smooth surface. Second, as it will be shown in Chapter4, we measured that surface tension was significantly lowered upon blebbistatin treatment, also con- firming blebbistatin activity.

One likely hypothesis to explain these results is that inhibiting actomyosin contrac- tility decreases viscosity and surface tension to similar extents in medial and poste- rior regions. However, viscosity might be decreased to a greater extent than surface tension in the anterior region, hence vp increase. We also noticed that the somitic

cleft formation was always absent in blebbistatin-treated explants. As such, another potential explanation for a higher vp is that somitic cleft formation in control ex-

plants slows down the rounding process.

Next, we studied the effect of the FGF/MAPK gradient. 2 µM PD0325901 treat- ments also led to a significant increase of viscocapillary velocity in anterior (+45%,

*

*

**

A

B

C

FIGURE3.6 – Viscocapillary velocity along the anteroposterior axis.

A: Semilog plots of a/b for posterior explants of various final radii.

Solid lines: linear fits with a0/b0fixed. B: Decay time τ with respect

to the final radius Rf. C: Viscocapillary velocity along the axis (ant:

anterior, med: medial, pos: posterior), and for different conditions (blebbi: 20 µM blebbistatin, PD03: 2 µM PD0325901). Student’s t-test p-value p: n.s: p>_0.05,⋆_{: p}<_0.05,⋆⋆_{: p}<_0.01.

anteroposterior axis

TABLE 3.1 – Viscocapillary velocity along the anteroposterior axis measured by rounding dynamics (mean and standard deviation, in µm/ min).

Anterior Medial Posterior Control 0.21±0.08 0.91±0.44 1.56±0.53 Blebbistatin 0.28±0.07 0.96±0.25 1.36±0.32

PD0325901 0.30±0.06 1.13±0.21

Student t-test’s p-value: 0.014). In posterior explants, we noticed an important re- duction of vp upon FGF/MAPK inhibition (-28%, Student t-test’s p-value: 0.005).

FGF/MAPK inhibition should have a greater effect on cell motility in posterior ex- plants. Therefore, the posterior reduction of vp suggests that cell motility inhibition

increases viscosity (assuming the effect on surface tension is low). However, it is difficult to anticipate the impact of the FGF/MAPK inhibition on surface tension, therefore the interpretation of these results remains unclear while we do not have independent measurements of surface tension and viscosity.

3.3.4 Fusion dynamics yields a similar viscocapillary velocity

In order to confirm the values of viscocapillary velocity obtained by rounding experiments, we carried out a fusion experiment. We dissected out explants of simi- lar sizes with aspect ratios close to 1, so they could rapidly round up. We incubated them in DMEM-F12 enriched with 10% FBS at 37◦_{C with 7.5% CO}

2for an hour until

they were spheroids. We then brought them in contact by pairs and imaged their fusion in same culture conditions (see Figure3.7 and Movies 4, 5). The neck of fu- sion was manually measured using the Fiji software (Schindelin et al., 2012). We fitted the square of the neck radius x2_{with respect to time during the linear regime}

(the first half of the experiment) and extracted viscocapillary speed. Figure 3.8 C shows that fusion experiments gives similar AP gradient to rounding experiments (vp = 0.16±0.05 µm·min−1in anterior and vp = 0.99±0.15 µm·min−1 in poste-

rior). We also monitored the fusion of anterior with posterior explants, as potential differences in surface tension might lead to the engulfment of one region by the other. Surprisingly, these explants from different regions failed to fuse.