Moosmann J et al. (2013), X-ray phase-contrast in vivo microtomography pr...

XB-ART-47108

Nature 2013 May 16;4977449:374-7. doi: 10.1038/nature12116.

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

Moosmann J , Ershov A , Altapova V , Baumbach T , Prasad MS , LaBonne C , Xiao X , Kashef J , Hofmann R .

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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 organism. In Xenopus laevis, the South African clawed frog and also in zebrafish, cell and tissue movements have been studied in explants, in fixed embryos, in vivo using fluorescence microscopy or microscopic magnetic resonance imaging. 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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Species referenced: Xenopus laevis

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	Figure 2: 3D time-lapse series of X. laevis embryo during mid-gastrulation.a–f, Mid-sagittally (a–c) and mid-horizontally (d–f) halved embryo renderings at stages 11.5 (0 min), 12 (62 min) and 12.5 (114 min). Ectoderm (blue), mesoderm (orange), and endoderm (green). g–i, Velocity fields on a 180-µm-thick 3D slab centred about the cutting planes of (a–c). Colour bar indicates velocity magnitude representation. Animal pole (AP), Archenteron (ARC), Brachet's cleft (BC), blastocoel (BLC), blastocoel floor (BLCF), blastocoel roof (BLCR), blastopore (BP), dorsal and ventral sides (D, V), dorsal and ventral blastopore lip (DBL, VBL), ‘pipe’ system in-between archenteron and blastocoel (PS), ventral animal pole (VAP) and vegetal pole (VP).
	Figure 3: Collective versus differential flow and cavity morphogenesis. a, b, Magnitude of velocity on sagittal slice for two different times. c, d, Field G on same sagittal slice and for the same times. e, Sagittal slice at 52 min, highlighted cell pairs line the archenteron, associated trajectories (period of 30 min) (colour bar: blue, early; red, late). f, g, 3D renderings of cavities within the ‘pipe’ system in between archenteron and blastocoel for times 62 min and 114 min. h, Volume changes of ARC, BLC and gastrula from 52 min to 114 min.
	Figure 4: Confrontation of head and ventral mesendoderm. a–e, Sequence of sagittal slices through confrontation zone at 52, 73, 83, 104 and 114 min showing formation and relaxation of a cusp of ectoderm on the blastocoel roof. Dorsal and ventral leading edges (Dors-LE, Vent-LE). Ectoderm (blue), mesoderm (orange), endoderm (green). f, Sequence of shrinking contours (projections of migrating leading edge along posterior–anterior axis onto horizontal plane) starting at 0 min (blue), terminating at 73 min. g, 3D rendering of individual cells and cavities in the confrontation zone at 73 min. Dors-LE cells (shades of green), Vent-LE cells (shades of red).

References [+] :

Blanchard, Measuring the multi-scale integration of mechanical forces during morphogenesis. 2011, Pubmed