For decades, the development of the early embryo and patterning of tissues has been studied with the help of a workhorse of developmental biology, the frog embryo. Xenopus embryos are large and undergo clear morphological changes throughout their development that make them very quick and easy to work with in answering questions surrounding the formation of early germ layers and processes such as gastrulation.
However, a major disadvantage to working with Xenopus embryos is the amount of yolk contained within them. Unlike in organisms such as zebrafish embryos, which have a separate yolk cell from which to draw their nutrition, the fertilised Xenopus embryo contains large yolk platelets, predominantly in the lower (vegetal) half of the embryo. This yolk is primarily made up of the protein vitellogenin and can account for half of the protein content of the cell – a problem that has contributed to the somewhat lagging development of Xenopus mass spectrometry proteomics. But the yolk also results in an opaque embryo that limits the options for imaging live embryos with light microscopy. Mouse embryos are also opaque and so a method of imaging these embryos would be of great interest to many fields of developmental biology.
One approach to overcome this problem – published recently in Nature – has been developed at the Karlsruhe Institute of Technology by Jubin Kashev and Ralf Hofmann, with contributions from Xenopus embryos from the lab of Carole LaBonne. This technique is phase-contrast X-ray tomography, using synchrotron radiation.
That sounds more complex than it is! Regular X-ray imaging is typically carried out by firing X-rays at a subject and building up an image of tissues depending on how much they absorb X-rays. However this is not at a good enough resolution for small samples such as Xenopus embryos, and requires a high blast of radiation and additionally injection of some sort of reagent to add contrast to the image. This is where the synchrotron comes in. This provides a coherent wave of radiation that is slowed through different tissues in a distinctive pattern at high resolution. A series of 2D images is produced whilst the embryo is rotated and these 2D images are used to build up a 3D picture – this is the tomography part.
The researchers were able to control timing and duration of X-ray blasts to minimize damage to the embryo whilst capturing as much of the process of gastrulation as possible between stages 11.5 and 12.5 (the authors comment that a 2 hour window of development is reasonable to capture before damage to the embryo becomes a significant problem). They found further evidence for the idea that the archenteron expands by uptake of external water; and also suggest previously unseen adhesive interactions occur between mesoendodermal cells, forming a ridge of contracted ectoderm at the point where dorsal and ventral mesendoderm meet and ectodermal cells begin to spread over the surface of the gastrula. They suggest this structure may be destroyed in the traditional process of taking explants for further study, hence why it has not been observed prior to these studies.
The obvious limitation in this approach is the current availability of synchrotrons (in the Research Highlight in Nature, it is suggested that there are only 8 facilities in the world!). But it is exactly this sort of innovation, at the frontiers of developmental biology and biophysics, which can help us overcome the traditional limitations to studies in developmental biology.
Moosmann, J. et al. X-ray phase-contrast in vivo microtomography probes new aspects of Xenopus gastrulation. Nature 497, 374–377 (2013).
Nawy, T. Embryos under the X-ray. Nature Methods 10, 603