In vivo characterization of chick embryo mesoderm by optical coherence tomography-assisted microindentation.

Embryos are growing organisms with highly heterogeneous properties in space and time. Understanding the mechanical properties is a crucial prerequisite for the investigation of morphogenesis. During the last 10 years, new techniques have been developed to evaluate the mechanical properties of biological tissues in vivo. To address this need, we employed a new instrument that, via the combination of micro-indentation with Optical Coherence Tomography (OCT), allows us to determine both, the spatial distribution of mechanical properties of chick embryos, and the structural changes in real-time. We report here the stiffness measurements on the live chicken embryo, from the mesenchymal tailbud to the epithelialized somites. The storage modulus of the mesoderm increases from (176 ± 18) Pa in the tail to (716 ± 117) Pa in the somitic region (mean ± SEM, n = 12). The midline has a mean storage modulus of (947 ± 111) Pa in the caudal (PSM) presomitic mesoderm (mean ± SEM, n = 12), indicating a stiff rod along the body axis, which thereby mechanically supports the surrounding tissue. The difference in stiffness between midline and presomitic mesoderm decreases as the mesoderm forms somites. This study provides an efficient method for the biomechanical characterization of soft biological tissues in vivo and shows that the mechanical properties strongly relate to different morphological features of the investigated regions.

Somite Division and New Boundary Formation by Mechanical Strain.

Somitogenesis, the primary segmentation of the vertebrate embryo, is associated with oscillating genes that interact with a wave of cell differentiation. The necessity of cell-matrix adherence and embryonic tension, however, suggests that mechanical cues are also involved. To explicitly investigate this, we applied surplus axial strain to live chick embryos. Despite substantial deformations, the embryos developed normally and somite formation rate was unaffected. Surprisingly, however, we observed slow cellular reorganizations of the most elongated somites into two or more well-shaped daughter somites. In what appeared to be a regular process of boundary formation, somites divided and fibronectin was deposited in between. Cell counts and morphology indicated that cells from the somitocoel underwent mesenchymal-epithelial transition; this was supported by a Cellular Potts model of somite division. Thus, although somitogenesis appeared to be extremely robust, we observed new boundary formation in existing somites and conclude that mechanical strain can be morphologically instructive.

Micro-indentation and optical coherence tomography for the mechanical characterization of embryos: Experimental setup and measurements on chicken embryos.

The investigation of the mechanical properties of embryos is expected to provide valuable information on the phenomenology of morphogenesis. It is thus believed that, by mapping the viscoelastic features of an embryo at different stages of growth, it may be possible to shed light on the role of mechanics in embryonic development. To contribute to this field, we present a new instrument that can determine spatiotemporal distributions of mechanical properties of embryos over a wide area and with unprecedented accuracy. The method relies on combining ferrule-top micro-indentation, which provides local measurements of viscoelasticity, with Optical Coherence Tomography, which can reveal changes in tissue morphology and help the user identify the indentation point. To prove the working principle, we have collected viscoelasticity maps of fixed and live HH11-HH12 chicken embryos. Our study shows that the instrument can reveal correlations between tissue morphology and mechanical behavior. STATEMENT OF SIGNIFICANCE: Local mechanical properties of soft biological tissue play a crucial role in several biological processes, including cell differentiation, cell migration, and body formation; therefore, measuring tissue properties at high resolution is of great interest in biology and tissue engineering. To provide an efficient method for the biomechanical characterization of soft biological tissues, we introduce a new tool in which the combination of non-invasive Optical Coherence Tomography imaging and depth-controlled indentation measurements allows one to map the viscoelastic properties of biological tissue and investigate correlations between local mechanical features and tissue morphology with unprecedented resolution.

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos.

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture. The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).