SUPPLEMENTARY INFORMATION

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1 doi: /nature12116 Supplementary Figure 1 Theoretical prediction of temperature profiles. a, sketch of sample, immersed into X-ray beam, its environment (agarose and buffer solution are not distinguished), and choice of coordinates. The axis of tomographic rotation is along. b, computed, radial ( ) and c, angular ( ) temperature profiles for kev, cm, photons/s/mm 2, subject to a Gaussian beam profile of mm 2, and assuming cm (height) of an approximately cylindrical polyethylene tube. 1

2 Supplementary Figure 2 Temperature measurements. a, sequence of 24 cycles (waiting time ~10 min) of temperature ( ) measurements (central: red; peripheral: blue) in polyethylene tube filled with water. For explanation of the measurement protocol within a cycle, see Supplementary Information. The central and peripheral thermistors are identical and not calibrated. b, temperature measurements resolved within one cycle (central: red, peripheral: blue). First series is before, second series during, and final series after exposure. Notice equilibration towards the end of after-exposure measurements. c, difference between central and peripheral temperatures at the end of afterexposure measurements over all 24 cycles. 2

3 Supplementary Figure 3 Comparison of blastopore closure between X-rayed and light-microscopy imaged embryos. a, time-lapse plots of blastopore radii. The 3

4 blastopore radii of three embryos were measured from stage 10.5 (t=-150 min) until stage 12.5 (240 min) at 22.5 C under a light microscope. They decrease from 301 µm (average over three embryos, t=0) to 219 µm within four hours. The alternating behaviour in the curves is due to the fact that embryos prefer to turn to their vegetal sides necessitating a manual swap to re-orientate them properly during the time-lapse measurements. This leads to slightly different viewing angles of the blastopores. As a consequence, projection errors occur in the measurements of the blastopore radii. The blastopore radii of the three X-rayed embryos were calculated from midsagittal slices. They decrease from 326 µm (t=0 min) to 180 µm (t=114 min) for X-rayed embryo 1 (Supplementary Video 3), from 151 µm (t=190 min) to 111 µm (t=246 min) for X-rayed embryo 2 (Supplementary Video 6), and from 175 µm (t=145 min) to 96 µm (t=225 min) for X-rayed embryo 3 (Supplementary Video 4) at ~25 C. (White arrowheads indicate the blastopore lips.) The curve of X-rayed embryo 1 was brought to intersect the curve of embryo 3 imaged by light microscopy while the curve of X-rayed embryo 2 initially intersects that of embryo 1, and the curve of X- rayed embryo 3 starts at the intersection with the curve of embryo 3. The temperature of 22.5 C during light microscopy did not match the 2-BM-B or 32-ID hutch temperature of ~25 C during X-ray imaging. This likely explains the steeper average slope of the plots belonging to X-rayed embryos in comparison to the curves obtained from light microscopy. b, images taken from Supplementary Video 13 at indicated times. c,d, and e mid-sagittal slices of X-rayed embryos 1, 2, and 3 taken from Supplementary Videos 3, 6, and 4, respectively, at indicated times. Supplementary Figure 4 Trajectories and velocities of single cells lining the archenteron. a, parasagittal slice at 52 min with highlighted cell pairs lining the region along the archenteron. b-e, trajectories of these cell pairs (top row) and associated velocity components in x-y (parasagittal) plane (bottom row; left cell: red, right cell: blue; solid: x- component, dashed: y-component). 4

5 Heat load: estimate of temperature rise after one tomographic recording and comparison with experiment We model heat flow in water with a thermal conductivity of J/s/cm/K, a heat capacity at atmospheric pressure of J/g/K, and a mass density of g/cm 3. At an X-ray energy of E=30 kev the absorption length of water is cm. To estimate an upper bound on the temperature rise after one tomographic exposure of s it is assumed that the entire absorbed photon energy is converted into heat. With a central, monochromatic photon flux density of photons/s/mm 2, a Gaussian beam cross section of mm 2, and the dimensions cm (radius), cm (height) of an approximately cylindrical polyethylene tube we obtain a central temperature rise of K by solving the heat equation. Here is the change in heat density induced by the (time dependent) heat source, represented by a central intersection of the rotating beam with the polyethylene cylinder:. denotes the central intensity (energy per time and area), (tomographic exposure), and, is the period of rotation the 3D Laplacian. Moreover, we use the free heat kernel to construct an approximate solution subject to the initial condition. One has. The approximate solution is good if where denotes the global temperature rise in the limit of instantaneous heat propagation: In the horizontal slice of maximum heat load little heat flows out of the cylinder in comparison with instantaneous heat propagation that is confined to the cylinder. In turn, this implies that heat conductivity is sufficiently low in relation to the speed of heat entry for the selfconsistency of assuming boundary conditions at spatial infinity only. According to Supplementary Fig. 1b this condition is satisfied. With temperature rises of a small fraction of a Kelvin after recording of one tomogram and a number of 14 tomograms we conclude that heat load is excluded as a potential cause for abnormal development or even apoptosis during X-ray exposure. The above estimate was performed prior to the life-cell experiment to gain confidence in its feasibility. In a dedicated experiment at station 2-BM-B of Advanced Photon Source 5

6 (Argonne National Laboratory), performed under conditions identical to the actual life-cell scan (polyethylene tube filled with water), we have verified this estimate, compare Supplementary Fig. 1b and Supplementary Fig. 2c, where the predicted value of differs from the value measured in the X-ray experiment by about 30 % only. (Mixing and self-capacitance effects cancel out in the difference between central and peripheral temperatures.) In this experiment two identical thermistors (Thermometrics, RTD type sensor) were moved into and out of the beam region (4 mm vertical shift) to measure the central temperature and the peripheral temperature. More precisely, 100 temperature measurements were performed within 20 seconds before X-ray exposure with the thermistors in the beam region (beam shut off), 100 temperature measurements within 20 seconds during X-ray exposure with the thermistors out of the beam region (beam on), and 100 temperature measurements within 20 seconds after X- ray exposure with the thermistors in the beam region (beam shut off). This cycle was repeated 24 times with a waiting time of ~10 min. In Supplementary Fig. 2a-c each point in the plots corresponds to an average within a group of five adjacent temperature values. Shorter time-lapse sequences from gastrulation to neurulation To demonstrate that X-ray phase-contrast in vivo microtomography is applicable to various developmental processes we have recorded and reconstructed several shorter time-lapse series covering early gastrulation up to neurulation. These shorter sequences represent major morphogenetic movements during gastrulation like blastopore closure and involution of dorsal and ventral mesendoderm (Supplementary Videos 6, 7, 8) as well as archenteron formation and confrontation of dorsal and ventral mesendoderm (Supplementary Video 6). At neurula stages closure of the neural tube (Supplementary Videos 5, 9, 10) can be observed. Discussion of the trajectories of single cells in association with archenteron inflation Here we discuss in detail the single-cell trajectories of Fig. 3e and Supplementary Fig. 4a. The two cell pairs most proximal to the animal pole (Supplementary Fig. 4b) and the blastopore (Supplementary Fig. 4e) display near-parallel trajectories in the direction of mesendodermal mantle movement, representing collective motion. On the contrary, distinct directions occur for cell pairs occupying more medial positions along the posterioranterior extent of elongation (Supplementary Fig. 4c,d). For the more anterior of these 6

7 pairs (Supplementary Fig. 4c) this is expressed by a large difference between radial (maximum difference in x-components: ~2 µm/min) and tangential (maximum difference in y-component: ~0.8 µm/min) displacement, the former being a consequence of the onset of archenteron inflation, the latter arising due to continuing push by the vegetal cell mass. An analogous but less pronounced displacement with a shear-motion component is seen for the cell on the blastocoel side of the archenteron in the pair shown in Supplementary Fig. 4d, indicating the onset of tissue separation on both sides of the archenteron. Uptake of external water during archenteron inflation Here we investigate in detail the cause for early archenteron inflation: uptake of external water. In [28] an analysis of water uptake and fluid distribution during early Xenopus embryogenesis was performed by independently measuring the reduced weight in water ( ) and the density of intact and open embryos. Embryos are opened for substitution of the fluid of the cellular cavities (archenteron and blastocoel including pipe system) by the gradient fluid of the density measurement. Combining reduced weight and density, yields a precise determination of embryo volume (closed embryos) and cell-mass volume (open embryos). The density of cavity fluid was found to be equal to that of water [28]. Thus, the cell-mass volume and the of the intact embryo imply the weight of the cell mass (dry weight in Eq. (3) of [28]) which does not vary. Up to 20 hours of development, hence including gastrulation, changed by less than 3% subject to an error of comparable magnitude (Table 3 of [28]). From (differential of Eq. (3) of [28]), being the increase in cell-mass volume and g cm -3 the mass density of water, and taking µg (from Table 3 of [28]), we obtain µm 3. This is small in magnitude compared to the changes in cavity volumes (archenteron and pipe system, blastocoel volume remains constant, see Fig. 3h and below) and total embryo volume during early archenteron inflation (times 52 to 114 min), measured in vivo by segmentation of our 3D data. Thus essentially is attributed to the uptake of external water by the embryo. We proceed by investigating how this water is distributed over the main embryonic cavities: blastocoel, archenteron, and pipe system. Small cavities present within the blastopore at times 0 and 11 min have disappeared well before 52 min, see Supplementary Video 17, and so do not have to be accounted for in the fluid balance. Comparing three independent segmentations of the outer boundary of the 7

8 embryo, the error in the rendering of total embryonic volume estimates as 0.2%. At 52 min we have 10 6 µm, and at 104 min we have 10 6 µm 3. Thus the (maximum) change of total embryonic volume during early archenteron inflation is 10 6 µm 3 an increase by ~1.8%. For the according change in archenteron volume we have 10 6 µm 3 (no error due to clearly defined walls ) while the volume of the pipe system increases as 10 6 µm µm 3. (Since it is not possible to unambiguously define the boundaries of the pipe system the relative volume error is guessed to be not more than 30 %.) Within variations of ~ µm 3 the blastocoel volume is found to be constant (Fig. 3h). Comparing six alternative segmentations of the blastocoel, the error of estimates as 0.5% or ~ µm 3 which is ~11 % of maximum archenteron volume. Thus 10 6 µm 3 which, within errors, is compatible with, the volume change of the archenteron being the dominant component. To summarise, archenteron inflation affirmatively is attributed to the uptake of external water. 8