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Showing content from https://pubmed.ncbi.nlm.nih.gov/32382445/ below:

Force microscopy of the Caenorhabditis elegans embryonic eggshell

doi: 10.1038/s41378-020-0137-3. eCollection 2020. Force microscopy of the Caenorhabditis elegans embryonic eggshell

Affiliations

¹ 1Laboratory of Microsystems, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.
² 2Multi-Scale Robotics Laboratory, ETH Zurich, Zürich, 8092 Switzerland.

PMID: 32382445
PMCID: PMC7196560
DOI: 10.1038/s41378-020-0137-3

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Force microscopy of the Caenorhabditis elegans embryonic eggshell

Roger Krenger et al. Microsyst Nanoeng. 2020.

doi: 10.1038/s41378-020-0137-3. eCollection 2020. Affiliations

¹ 1Laboratory of Microsystems, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.
² 2Multi-Scale Robotics Laboratory, ETH Zurich, Zürich, 8092 Switzerland.

PMID: 32382445
PMCID: PMC7196560
DOI: 10.1038/s41378-020-0137-3

Item in Clipboard

Abstract

Assays focusing on emerging biological phenomena in an animal's life can be performed during embryogenesis. While the embryo of Caenorhabditis elegans has been extensively studied, its biomechanical properties are largely unknown. Here, we demonstrate that cellular force microscopy (CFM), a recently developed technique that combines micro-indentation with high resolution force sensing approaching that of atomic force microscopy, can be successfully applied to C. elegans embryos. We performed, for the first time, a quantitative study of the mechanical properties of the eggshell of living C. elegans embryos and demonstrate the capability of the system to detect alterations of its mechanical parameters and shell defects upon chemical treatments. In addition to investigating natural eggshells, we applied different eggshell treatments, i.e., exposure to sodium hypochlorite and chitinase solutions, respectively, that selectively modified the multilayer eggshell structure, in order to evaluate the impact of the different layers on the mechanical integrity of the embryo. Finite element method simulations based on a simple embryo model were used to extract characteristic eggshell parameters from the experimental micro-indentation force-displacement curves. We found a strong correlation between the severity of the chemical treatment and the rigidity of the shell. Furthermore, our results showed, in contrast to previous assumptions, that short bleach treatments not only selectively remove the outermost vitelline layer of the eggshell, but also significantly degenerate the underlying chitin layer, which is primarily responsible for the mechanical stability of the egg.

Keywords: Engineering; Physics.

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Conflict of interest statement

Conflict of interestThe authors declare that they have no conflict of interest.

Figures
Fig. 1. System for micro-indentation measurements on…

Fig. 1. System for micro-indentation measurements on C. elegans embryos.

a Photograph of the cellular…

Fig. 1. System for micro-indentation measurements on C. elegans embryos.

a Photograph of the cellular force microscope (CFM), integrated with an inverted microscope for high-resolution imaging. The configuration, combining xyz precision linear positioners and a piezo system, allows manipulation of the MEMS-based capacitive force sensor with an attached micro-indenter (blue) with nanometer resolution and centimeter travel range. b Schematics of the CFM. Once a suitable embryo has been selected by means of the xy coarse stage of the inverted microscope, the xyz positioner centers the micro-indenter over the egg and moves it in z direction close to the shell. c Indentation Δz of the eggshell is performed by further downward motion of the micro-indenter, controlled via the xyz piezo stage. The indenter tip position and the resulting force F_z are recorded simultaneously

Fig. 2. Morphological changes of C. elegans…

Fig. 2. Morphological changes of C. elegans embryos upon indentation for different eggshell treatments.

a…

Fig. 2. Morphological changes of C. elegans embryos upon indentation for different eggshell treatments.

a Schematic cross-sectional views of the eggshell in its untreated and degraded forms. The untreated eggshell has a trilaminar structure, consisting of an outer vitelline layer, a middle chitin layer and an inner chondroitin proteoglycan layer (ai). Treatment with sodium hypochlorite solution (bleach) selectively removes the VL, but may also attack the underlying CL (aii). Chitinase treatment is applied after bleach treatment to strip away the CL from the embryo shell (aiii). b Frontal and lateral schematic cross-sections of an embryo, illustrating the shape changes in width Δw and length Δl upon indentation with a depth Δz. c Bright field microscopy images in relaxed and poked states of a live, bleach-treated embryo in the twitching stage. A lateral extension Δw upon indentation with respect to the relaxed egg width w is well visible (F_z = 5 µN). d Scatter plot of embryo elongation Δl and widening Δw as derived from microscopy images upon indentation. Widening is more prominent than the elongation changes for all eggs and conditions. Paired t-test was applied to calculate p between l and l + Δl and w & w + Δw, respectively (**p ≤ 0.01; ****p ≤ 0.0001; ns not significant). Force values are F_z = 5.0 µN for untreated and bleached eggs and F_z = 0.5 µN for chitinase-treated eggs. e Scatter plot of the embryo ellipticity (ratio l/w) in relaxed and poked states for treated and untreated conditions. Very soft chitinase-treated eggs tend to shorten slightly and show stronger widening, but also larger deviation from mean values than untreated and bleached eggs

Fig. 3. Force-displacement measurements of C. elegans…

Fig. 3. Force-displacement measurements of C. elegans embryos.

a Representative micro-indentation loading curves (after baseline…

Fig. 3. Force-displacement measurements of C. elegans embryos.

a Representative micro-indentation loading curves (after baseline correction), i.e. measured force response F_z of the eggshell vs sensor tip indentation depth Δz, for untreated and treated eggs, including displacements beyond eggshell rupture. F_z increases in a nonlinear way with respect to Δz, indicating increasing an effective stiffness of the eggshell up to the puncture point. Force response to loading is strongest for untreated eggs (dark blue) and becomes very weak for bleach + chitinase-treated eggshells (light blue), where complete removal of the vitelline layer and the chitin layer is expected. Shell puncture events are marked as dotted circles in the plots. b The critical force value F_z^punct where eggshell puncture occurs is plotted vs the corresponding indentation depth Δz^punct, for different treatments and for all embryonic stages (gastrulation, bean and twitching stage). F_z^punct decreases drastically with the severity of the treatment. Very low values in the range of 0.5 μN are found after bleach + chitinase treatment, where only the innermost chondroitin proteoglycan layer of the eggshell remains. Untreated eggs can sustain the highest indentation depth Δz^punct, whereas the limit of eggshell deformation is lower but comparable after different treatments

Fig. 4. FEM modeling of C. elegans…

Fig. 4. FEM modeling of C. elegans embryo indentation experiments.

a A spheroidal, fluid-filled elastic…

Fig. 4. FEM modeling of C. elegans embryo indentation experiments.

a A spheroidal, fluid-filled elastic shell is used as model for indentation simulations and data fitting. Eggshell deformation and resulting membrane stress upon indentation by a spherical indenter are shown for Δz = 15 µm (tip radius r_ind = 1 µm, shell thickness t = 300 nm, shell modulus E_shell = 0.12 GPa, p_int = 1.6 × 10⁵ Pa). The initial embryo shape in the relaxed state is indicated by gray lines (egg length l = 50 µm, width w = 35 µm). b 3-D representation of the simulated p_int-Δz-E_shell parameter space. p_int, the internal egg pressure that builds as a function of the indentation depth Δz, is an important parameter required for accurate fitting of the experimental F_z(Δz) curves. c Simulated and interpolated F_z^sim-Δz-E_shell parameter space. The total force F_z^sim exerted on the micro-indenter by the eggshell comprises an elastic membrane component and the force related to the internal egg pressure p_int

Fig. 5. Evaluation of specific mechanical properties…

Fig. 5. Evaluation of specific mechanical properties of C. elegans embryos.

a Representative force-indentation measurement…

Fig. 5. Evaluation of specific mechanical properties of C. elegans embryos.

a Representative force-indentation measurement F_z(Δz) of an untreated embryo and the best-fitting simulated F_z^sim(Δz) curve. Good agreement of both curves is achieved by using a value of E_shell = 0.12 GPa for the eggshell modulus. b The full set of experimental F_z(Δz) loading curves (solid lines) for all treatments and embryo development stages is superposed to the simulated F_z^sim-Δz-E_shell parameter space. F_z(Δz) curves, displayed up to the puncture point, are arranged with respect to the corresponding E_shell value derived from the best-fitting simulated curve. c E_shell values extracted from the experimental data by applying the described model for all embryonic stages, separated with respect to the corresponding eggshell treatment. The mean value of the Young’s modulus of the shell decreases significantly after bleaching and becomes extremely low after additional chitinase exposure. d Maximum values p^max_int of the internal egg pressure just before shell rupture for all treatments and embryonic stages