Imaging the onset of oscillatory signaling dynamics during mouse embryo gastrulation

ABSTRACT A fundamental requirement for embryonic development is the coordination of signaling activities in space and time. A notable example in vertebrate embryos is found during somitogenesis, where gene expression oscillations linked to the segmentation clock are synchronized across cells in the presomitic mesoderm (PSM) and result in tissue-level wave patterns. To examine their onset during mouse embryo development, we studied the dynamics of the segmentation clock gene Lfng during gastrulation. To this end, we established an imaging setup using selective plane illumination microscopy (SPIM) that enables culture and simultaneous imaging of up to four embryos (‘SPIM- for-4’). Using SPIM-for-4, combined with genetically encoded signaling reporters, we detected the onset of Lfng oscillations within newly formed mesoderm at presomite stages. Functionally, we found that initial synchrony and the first ∼6-8 oscillation cycles occurred even when Notch signaling was impaired, revealing similarities to previous findings made in zebrafish embryos. Finally, we show that a spatial period gradient is present at the onset of oscillatory activity, providing a potential mechanism accounting for our observation that wave patterns build up gradually over the first oscillation cycles.

Fig. S1. Medium perfusion system for live embryo imaging on SPIM. Schematic section view through the Z.1 sample chamber with inserted embryo culture chamber and attached closedcycle perfusion system. The embryo culture chamber sits inside the Z.1 sample chamber which is filled with PBS. Only the embryo culture chamber and the perfusion system are filled with culture medium. The medium in the culture chamber is covered with mineral oil to reduce evaporation. Medium is continuously pumped through the chamber and the perfusion system that is positioned outside of the front system cavity of the microscope. In the gas equilibration chamber the medium is saturated with a defined gas mixture before it is reintroduced into the culture chamber. Thinwalled, gas permeable silicon tubes are used inside the equilibration chamber, thick PVC tubes are used outside. The entire system is heated (indicated in red). , SPIM-imaging of nuclear marker H2B-mCherry (mean:6.5, n=4), cultured on the microscope but not imaged (mean:6.2, n=9), and cultured in roller culture (mean:6.6, n=29). Embryos were dissected at E7.5 and cultured for 24 hours. Bar plot shows mean value of samples within each group, error bars show standard deviation.  . Image stability test with multi sample imaging. An E7.5 mouse embryo expressing R26-H2BmCherry was imaged in a multi-sample imaging routine. Three consecutive time-frames of the same imaging location are shown (left). In between these frames, three other embryos were imaged (not shown). Frames were color coded in red, green, and blue and overlaid (right) to visualize positional stability. Pixels with the same intensity in all three images appear white. The visible shift is due to morphological changes of the embryo. Scale bar: 100 µm.
pulse/wave1 wave 1/2 wave 2/3 peak-to-peak interval (min) 240 180 120 300 Fig. S4. Timing of pulse and the first waves. Boxplot showing the peak-to-peak intervals between the pulse and the first three waves measured in profiles as shown in Fig. 2G for di↵erent embryos. Time intervals (given as median with IQR) from pulse peak to wave 1: 267 min (46.5 min, n=12), from wave 1 to wave 2: 144 min (17.7 min, n=24), from wave 2 to wave 3: 147 min (25.2 min, n=30). Sample sizes di↵er between measurements because the start of the imaging experiment varied in respect to the developmental stage of the embryos.
Consequently, some embryos were already too far developed to capture e.g. the pulse.     Fig. 1D. The movie shows the bright-field signal of an embryo developing on the Z.1 light-sheet microscope using the customized culture and mounting setup. The field of view is readjusted at 16:20 h to account for the embryo growth. The embryo was imaged every 20 min but is shown at ˜1 h intervals because acquisition failed for a number of frames due to a software problem of the microscope. Time is indicated in hr:min.
Movie 2. Imaging of R26-H2BmCherry +/embryos prior to RNA in situ hybridization chain reaction (HCR) Related to Fig. 1E(ii). An embryo expressing the nuclear marker H2BmCherry (red) was light-sheet imaged from E7.5 for 24 hrs, with Z-stacks with 7.5 µm spacing, 120 slices per sample, 200 ms exposure time with 1.2% 20mW 561nm laser and 10 min imaging interval. The movie shows the embryos as MIP from posterior (top) and distal orientation (bottom). Time is indicated in hr:min.
Movie 3. Four embryos imaged simultaneously in a single experiment using SPIM-for-4. Related to Fig. 1F,G. Four embryos expressing the dynamic Notch signaling reporter LuVeLu (cyan) was simultaneously light-sheet imaged from E7.5 for 28 hrs using SPIM-for-4. The movie shows the embryos as MIP from posterior view. Time is indicated in hr:min. The m ovie s tarts 48 min prior t o t he peak of t he pulse, but DAPT t reatment had s tarted already 12 h earlier and was continued during i maging. Since embryos s hare t he s ame culture m edium on t he l ightsheet m icroscope, t he control embryo ( Movie 5) was i maged i n a di↵erent experiment.
Movie 11. Somite boundary formation in relation to the first LuVeLu waves. Related to Fig. 4A-D. An embryo expressing the dynamic Notch signaling reporter LuVeLu (cyan) and nucleus marker R26-H2BmCherry (red) was imaged from late allantois bud stage onwards. To monitor the formation of somite boundaries, visible as clefts between nuclei, the embryo was mounted with the distal aspect toward the detection objective (posterior up). The same embryo with only the R26-H2BmCherry signal (red) is shown on the right, for better visualization of clefts. Time is indicated in hr:min.
Movie 12. Line of interest used to generate surface kymographs from LuVeLu signal. Related to Fig. 5. LuVeLu (cyan) fluorescence in an embryo imaged from the early bud stage is shown from posterior (top) and side orientation (bottom). On the right, the line of interest used to generate a surface kymograph is overlaid. Time is indicated in hr:min. Development: doi:10.1242/dev.200083: Supplementary information Movie 13. Transferred line of interest on cell tracking datasets. Related to Fig. 5C, Fig. S9. Cell locations in the cell tracking datasets are represented as octahedrons colorcoded with the following cell fate annotations : neural tube, somitic mesoderm, lateral plate mesoderm, anterior paraaxial mesoderm, and heart field/cardiogenic mesoderm. The transferred line of interest used to generate cell track/flow plots in Fig. 5C and Fig. S9 are represented as a line of white spheres. Cells within 30 µm of the line of interest are colored red. Time is indicated in hr:min, and the datasets are aligned so that 00:00 is set to the timepoint which corresponds to the start of the first wave. Scale bar: 200 µm.