Abstract
During gastrulation in the mouse embryo, dynamic cell movements including epiblast invagination and mesodermal layer expansion lead to the establishment of the three-layered body plan. The precise details of these movements, however, are sometimes elusive, because of the limitations in live imaging. To overcome this problem, we developed techniques to enable observation of living mouse embryos with digital scanned light sheet microscope (DSLM). The achieved deep and high time-resolution images of GFP-expressing nuclei and following 3D tracking analysis revealed the following findings: (i) Interkinetic nuclear migration (INM) occurs in the epiblast at embryonic day (E)6 and 6.5. (ii) INM-like migration occurs in the E5.5 embryo, when the epiblast is a monolayer and not yet pseudostratified. (iii) Primary driving force for INM at E6.5 is not pressure from neighboring nuclei. (iv) Mesodermal cells migrate not as a sheet but as individual cells without coordination.
Highlights
Establishment of the three germ layers during gastrulation occurs via a highly orchestrated set of morphogenetic events that shape the early embryo [1,2,3,4,5]
Mouse embryo culture system for digital scanned light-sheet microscopy (DSLM) In order to observe a whole mouse embryo during gastrulation, we developed a series of techniques for mouse embryo culture in the chamber of DSLM
Since mouse embryos cannot be cultured in the sort of conventional agarose holder typically used for samples in light sheet-based fluorescence microscopy (LSFM), we made a specimen holder for mouse embryos (Figure 1A and B)
Summary
Establishment of the three germ layers during gastrulation occurs via a highly orchestrated set of morphogenetic events that shape the early embryo [1,2,3,4,5] In mouse embryos, this process begins as epiblast cells traverse the primitive streak at the posterior end. In order to overcome these limitations, we used digital scanned light-sheet microscopy (DSLM) [8,9], a type of light sheet-based fluorescence microscopy (LSFM) that uses planar illumination perpendicular to the detection axis (Figure S1) This method offers the advantages of a high signal-to-noise ratio (S/N), high speed, and good optical penetration [10,11]. We developed software tools for tracking of the nuclei, and report cell migration properties of the epiblast and the mesodermal cells
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