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X-ray phase-contrast in vivo microtomography probes new aspects of Xenopus gastrulation

Abstract

An ambitious goal in biology is to understand the behaviour of cells during development by imaging—in vivo and with subcellular resolution—changes of the embryonic structure. Important morphogenetic movements occur throughout embryogenesis, but in particular during gastrulation when a series of dramatic, coordinated cell movements drives the reorganization of a simple ball or sheet of cells into a complex multi-layered organism1. In Xenopus laevis, the South African clawed frog and also in zebrafish, cell and tissue movements have been studied in explants2,3, in fixed embryos4, in vivo using fluorescence microscopy5,6 or microscopic magnetic resonance imaging7. None of these methods allows cell behaviours to be observed with micrometre-scale resolution throughout the optically opaque, living embryo over developmental time. Here we use non-invasive in vivo, time-lapse X-ray microtomography, based on single-distance phase contrast and combined with motion analysis, to examine the course of embryonic development. We demonstrate that this powerful four-dimensional imaging technique provides high-resolution views of gastrulation processes in wild-type X. laevis embryos, including vegetal endoderm rotation, archenteron formation, changes in the volumes of cavities within the porous interstitial tissue between archenteron and blastocoel, migration/confrontation of mesendoderm and closure of the blastopore. Differential flow analysis separates collective from relative cell motion to assign propulsion mechanisms. Moreover, digitally determined volume balances confirm that early archenteron inflation occurs through the uptake of external water. A transient ectodermal ridge, formed in association with the confrontation of ventral and head mesendoderm on the blastocoel roof, is identified. When combined with perturbation experiments to investigate molecular and biomechanical underpinnings of morphogenesis, our technique should help to advance our understanding of the fundamentals of development.

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Figure 1: Experimental set-up for propagation-based phase-contrast X-ray microtomography.
Figure 2: 3D time-lapse series of X. laevis embryo during mid-gastrulation.
Figure 3: Collective versus differential flow and cavity morphogenesis. a,
Figure 4: Confrontation of head and ventral mesendoderm.

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Acknowledgements

We would like to acknowledge discussions with J. Wittbrodt, H. Steinbeisser, R. Winklbauer and D. Moss. R. Keller and D. Shook helped us with interpreting the data. D. Wedlich and M. Köhl commented on the manuscript. Discussions with M. Köhl on data analysis are gratefully acknowledged. We also would like to thank T. van de Kamp and D. Karpov for their help visualising the set-up, as well as F. de Carlo and K. Fezza for allocating beamtime at 2-BM-B station and at 32-ID, respectively, of Advanced Photon Source, Argonne National Laboratory. Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under Contract No. DE-AC02-06CH11357. E. Becker, C. Huang, A. Merks and R. Langhe helped analysing blastopore radii. J.K.’s Young Investigator Group received financial support from the ‘Concept for the Future’ programme of Karlsruhe Institute of Technology within the framework of the German Excellence Initiative. This research partially was funded by the German Federal Ministry of Education and Research under grant numbers 05K12CK2 and 05K12VH1.

Author information

Authors and Affiliations

Authors

Contributions

J.M., J.K. and R.H. conceived, organised and planned the experiments at beamlines 2-BM-B and 32-ID of Advanced Photon Source including feasibility studies, data management and testing/optimizing the set-up. V.A. established first contact between physicists and developmental biologists at Karlsruhe Institute of Technology to investigate X. laevis embryos. The physics part of the experiment was performed by J.M., X.X. and R.H., the biological parts by J.K. C.LaB. and M.S.P. helped with preparing samples. X.X. conducted heat-load measurements. R.H. investigated heat load theoretically. J.M. and A.E. developed data pre-processing routines. J.M. performed data pre-processing, phase retrieval and tomographic reconstruction. A.E. developed optical flow methods, computed velocity fields and performed image analysis. A.E. and J.M. worked on visualisations. A.E., J.M., V.A. and J.K. performed segmentation of the data. A.E., J.M. and R.H. analysed cell trajectories. J.M., A.E., T.B., X.X., J.K. and R.H. continuously discussed physics and biology problems. R.H. drafted the manuscript, A.E., J.M. and J.K. discussed and contributed text. C.LaB., M.S.P. and T.B. provided critical review. All authors contributed to the final manuscript.

Corresponding authors

Correspondence to Jubin Kashef or Ralf Hofmann.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-4 and Supplementary Text. (PDF 2322 kb)

Rendering of entire Xenopus embryo with two sets of parallel slicings through embryo containing midsagittal and midhorizontal planes at stage 12 (time 73 min)

Abbreviations: archenteron (ARC), dorsal and ventral sides (D,V), blastocoel (BLC), animal pole (AP), and vegetal pole (VP). Data taken at station 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, 15 ms exposure time per projection, and flux density 1012 photons/mm2/s). (MP4 11302 kb)

Rotating pair of midsagittally halved tomographically reconstructed embryos at 62 min (stage 12) and 114 min (stage 12.5) including schematic labelling of major tissues

Ectoderm (blue), mesoderm (orange), and endoderm (green). Abbreviations: dorsal and ventral sides (D,V), animal pole (AP), and vegetal pole (VP). Data taken at station 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, 15 ms exposure time per projection, and flux density 1012 photons/mm2/s). (MP4 10698 kb)

Time-lapse series of midsagittal (left panel) and horizontal (right panel) slices through tomographic reconstruction (10 or 20 min waiting time, 114 min duration)

Following structures are labelled: blastopore (BP), blastocoel (BLC), and archenteron (ARC). Jumps are related to fact that four (times 21, 42, 93, and 124 min) out of thirteen volumes were corrupted (software flaw in rotation-stage control during exposure) and thus had to be dismissed, resulting in a time-lapse series with variable time steps (10 or 20 minutes). Data taken at station 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, 15 ms exposure time per projection, and flux density 1012 photons/mm2/s). (MP4 2215 kb)

Time-lapse series of midsagittal (left panel) and horizontal (right panel) slices through tomographic reconstructions (initial stage 11.5, 8 min waiting time, 80 min duration, non-optimised preprocessing)

Right panel shows slices one cell layer away from blastocoel floor, crawling of cells on blastocoel floor can be seen. Following structures are labelled: blastopore (BP) and blastocoel (BLC). Data taken at beamline 32-ID of Advanced Photon Source (E=34 keV, z=70 cm, N=834, ΔE/E=10-4, 20 ms exposure time per projection, and flux density ~2˙1012 photons/mm2/s). Archenteron did not yet inflate (natural variations in archenteron morphogenesis, see [4]), it is shorter than the one shown in Supplementary Video 3 due to a higher flux density, a shorter waiting time, and a larger exposure time per projection. (MP4 2049 kb)

Time-lapse series of anterior transversal (left panel) and parasagittal (right panel) slices through reconstructions (non-optimised preprocessing) for initial stage 14 (10 or 20 min waiting time, 25 ms per projection, 62 min duration)

Left panel: dorsal at top, neural tube closure can be observed, white arrows define edges of neural folds (NF), prospective head endoderm (PHM) remains static; Right panel: dorsal at top, anterior to left, following tissues and cavities can be distinguished: anterior part of neural plate (ANP), somitogenic mesoderm (SM), remnant of blastocoel (RBLC), and archenteron (ARC). Data taken at 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, and flux density 1012 photons/mm2/s). Two tomograms were dismissed since embryo slid down the buffer cone (0 min) and due to software flaw during data acquisition (21 min). (MP4 1129 kb)

Time-lapse series of sagittal slice through reconstructions (non-optimised preprocessing) for initial stage 12.5 (10 min waiting time, 50 ms per projection, 56 min duration)

Animal at top, dorsal to left, following tissues and cavities can be distinguished: dorsal involuting mesendoderm (DIM), ventral involuting mesendoderm (VIM), blastocoel (BLC), “pipe” system of porous, endodermal tissue in between archenteron and blastocoel (PS), and archenteron (ARC), closure of the blastopore (white arrows), confrontation zone between head and ventral mesendoderm (red arrowhead). Data taken at 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, and flux density 1012 photons/mm2/s). (MP4 805 kb)

Time-lapse series of sagittal slice through reconstructions (non-optimised preprocessing) for initial stage 11 (10 or 20 min waiting time, 50 ms per projection, 56 min duration)

Animal at top, dorsal to left, following tissues and cavities can be distinguished: dorsal involuting mesendoderm (DIM), ventral involuting mesendoderm (VIM), blastocoel (BLC), closure of the blastopore (white arrows). Data taken at 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, and flux density 1012 photons/mm2/s). Two tomograms were dismissed due to software flaws during data acquisition (12 and 68 min). (MP4 776 kb)

Time-lapse series of sagittal slice through reconstructions (non-optimised preprocessing) for initial stage 11 (10 min waiting time, 50 ms per projection, 56 min duration)

Animal at top, dorsal to left, following tissues and cavities can be distinguished: dorsal involuting mesendoderm (DIM), ventral involuting mesendoderm (VIM), blastocoel (BLC), closure of blastopore (white arrows). Data taken at 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, and flux density 1012 photons/mm2/s). (MP4 922 kb)

Time-lapse series of parasagittal (left panel) and central transversal (right panel) slices through reconstructions (non-optimised preprocessing) for initial stage 16 (5 min waiting time, 50 ms per projection, 21 min duration)

Left panel: dorsal at top, anterior to left, following tissues and cavities can be distinguished: anterior part of neural plate (ANP), somitogenic mesoderm (SM), remnants of blastocoel (RBLC), and archenteron (ARC); Right panel: dorsal at top, following tissues and cavities can be distinguished: notochord (NC), neural fold (NF), somitogenic mesoderm (SM), lateral plate mesoderm (LPM), archenteron (ARC), and endodermal yolk mass (EYM). Neural tube closure can be observed (white arrows). Data taken at 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, and flux density 1012 photons/mm2/s). (MP4 943 kb)

Time-lapse series of anterior transversal (left panel) and parasagittal (right panel) slices through reconstructions (non-optimised preprocessing) for initial stage 18 (5 min waiting time, 75 ms per projection, 37 min duration)

Left panel: dorsal at top, following tissues and cavities can be distinguished: neural fold (NF), archenteron (ARC), and endodermal yolk mass (EYM); Right panel: dorsal at top, anterior to left, following tissues and cavities can be distinguished: eye anlage (EA), brain anlage (BA), somitogenic mesoderm (SM), remnant of blastocoel (RBLC), archenteron (ARC), and blastopore (BP). Data taken at 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, and flux density 1012 photons/mm2/s). (MP4 1629 kb)

Rotating velocity field calculated from volumes at 52 and 62 min

For better visibility the flow field is shown on the midsagittally halved embryo volume only. Data taken at station 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, 15 ms exposure time per projection, and flux density 1012 photons/mm2/s). (MP4 13154 kb)

Time-lapse series of 3D rendering of archenteron inflation

Abbreviations: archenteron (ARC) and blastocoel (BLC). Data taken at station 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, 15 ms exposure time per projection, and flux density 1012 photons/mm2/s). (MP4 307 kb)

Blastopore closure from stage 10.5 to 12.5 of three wild-type Xenopus embryos imaged by light microscopy

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. (MP4 2823 kb)

Rotating 3D rendering of the gastrula's cavities at 62 min (top) and 114 min (bottom)

Abbreviations: archenteron (ARC), blastocoel (BLC), and “pipe” system (PS) of porous endoderm in between ARC and BLC. Data taken at station 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, 15 ms exposure time per projection, and flux density 1012 photons/mm2/s). (MP4 17807 kb)

Rotating 3D rendering of the confrontation zone between head and ventral mesendoderm on the roof of the blastocoel at 52 min

The transient ridge of ectoderm is fully pronounced at this time. Abbreviations: dorsal and ventral leading edges (Dors-LE, Vent-LE). Data taken at station 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, 15 ms exposure time per projection, and flux density 1012 photons/mm2/s). (MP4 8590 kb)

Sagittal slicings through embryo volumes at times 114, 124, and 135 min

Left panel: embryo is intact at 114 min; Middle panel: cells ooze out of raptures in dorsal ectoderm (white arrowheads) at 124 min; Right panel: decay of embryo has progressed on the dorsal side giving rise to massive cell outflow (white arrowheads) at 135 min. Data taken at station 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, 15 ms exposure time per projection, and flux density 1012 photons/mm2/s). (MP4 35155 kb)

Sagittal slicings through blastopore at times 0, 11, 31, 52, 62, 73, 83, 104, and 114 min

Small cavities disappear during blastopore closure suggesting that blastopore becomes watertight before onset of archenteron inflation. Data taken at station 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, 15 ms exposure time per projection, and flux density 1012 photons/mm2/s). (MP4 22519 kb)

Time-lapse series of slices through confrontation zone between head and ventral mesendoderm near animal pole from 0 to 114 min

Formation of ectodermal cusp and overlap of head and ventral mesendoderm are visible at late times. Data taken at station 2-BM-B of Advanced Photon Source (E=30 keV, z=62 cm, N=1200, ΔE/E=10-2, 15 ms exposure time per projection, and flux density 1012 photons/mm2/s). (MP4 216 kb)

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Moosmann, J., Ershov, A., Altapova, V. et al. X-ray phase-contrast in vivo microtomography probes new aspects of Xenopus gastrulation. Nature 497, 374–377 (2013). https://doi.org/10.1038/nature12116

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