In vitro whole-organ imaging: 4D quantification of growing mouse limb buds

Abstract

Quantitative mapping of the normal tissue dynamics of an entire developing mammalian organ has not been achieved so far but is essential to understand developmental processes and to provide quantitative data for computational modeling. We developed a four-dimensional (4D) imaging technique that can be used to quantitatively image tissue movements and dynamic GFP expression domains in a growing transgenic mouse limb by time-lapse optical projection tomography (OPT).

Live optical projection tomography

“…7 The organ chosen was the developing limb bud of the mouse embryo since it is visually accessible (unlike the heart for example, which is concealed by other tissues) and is about 1 mm in size, therefore allowing reasonable transmission of photons.…”

“…Live OPT was able to monitor a dynamically changing 3D gene expression pattern throughout the volume of a living tissue. 7 The same model system as above was used, the mouse limb bud developing in culture, but this time the signal tracked over time was the signal emitted by the green fluorescent protein (GFP) reporting for transcriptional activities of the gene Scleraxis deep inside the mesenchyme of the organ rudiment. 25 Live OPT imaging of the GFP fluorescence revealed how the expression domain of Scleraxis changes its shape and size in 3D over time (reviewed in ref.…”

“…25 Live OPT imaging of the GFP fluorescence revealed how the expression domain of Scleraxis changes its shape and size in 3D over time (reviewed in ref. 7). It was the first direct 4D observation of dynamic spatial patterning of the mesenchymal tissue in a mammalian organ.…”

“…6 After summarizing the current applications of OPT 3D imaging (especially on fixed and cleared mouse embryonic specimens), the bulk of the review will be devoted to the examination of the most recent successes in extending OPT technology into the field of live imaging of organogenesis. 7 Live OPT, in which the OPT set up and ex vivo organ culture were made compatible, has exciting potential as a molecular imaging approach to track tissue movements and dynamic gene expression in 3D over time. We will end discussing challenges ahead, in particular for approaching le milieu intérieur 8 inside the tomograph.…”

Three-Dimensional (3D) Visualisation of the Cardiovascular System of Mouse Embryos and Fetus

“…Very recently the spectrum of OPT was expanded to in vivo 3D and four-dimensional (4D) imaging of the limbs of explanted and cultured mouse embryos [81].…”