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Takeo Matsumoto

Complex force fields exist in an embryo

During the past years, substantial insight has been accumulated on the role of mechanical forces that act during morphogenetic processes and it is by no means an exaggeration to state that, at the turn of the 21st century, Developmental Biology has entered a new era. Indeed, accumulating evidence suggests that during early animal development - associated with dramatic cell movements and tissue rearrangements - complex force fields exist in the embryo that have essential functions during the 3D shaping of the organism. Identifying and characterizing such forces will become a major challenge for the future. Such is the scientific objective of Prof. Takeo Matsumoto. Together with his research team, he aims at analyzing and measuring mechanical forces during vertebrate development, as a decisive step towards a full appreciation of their physiological role.

Now, characterizing such scientific endeavor as “heroic” seems quite exaggerated and, as a matter of fact, it probably is. But not very much. It might even be a close call, given the immense task to accomplish: “How can one look inside an embryo and identify all physical forces associated with complex tissue deformations at a given embryological stage?” To that end, the team of Prof. Matsumoto has been developing a method that actually enables them to visualize the mechanical stress and strain distribution inside the embryo.

Measuring mechanical force during 3D morphogenesis

Measuring mechanical force during 3D morphogenesis

Fig 1. Movie of sectioning of a fresh Xenopus laevis embryo. Embryos are mounted in an agar gel topped with a float and cut with a fine electrified Pt wire.

The approach towards realizing their objective - establishing a method for mechanical force estimation in an embryo - is to … cut the embryo in half. Cutting the embryo? To what end? Yes, cutting the embryo. As a matter of fact, an early embryo can be considered an elastic body with residual mechanical stress (i.e. the force acting on a unit area that resides in a body after all external loads were removed).This means that upon sectioning the embryo, bulges and dents appear at the cut surface, as a result of this residual stress (if there would be no residual stress in the embryo, the cut surface would be flat). Or in other words: the observed topography at the cut surface is a reflection of the internal stress and strain distribution within the different tissues.

Measurement of the surface topography of the cut surface of Xenopus laevis embryos at different stages.

Fig 2. Measurement of the surface topography of the cut surface of Xenopus laevis embryos at different stages.

To make things clear, there are 3 major players on the field: strain, elastic modulus and mechanical stress.

* The strain distribution at the cut surface of the embryo is determined through topography measurements.
* The elastic modulus is determined through an indentation test, which measures the relationship between a force applied at a given point on the embryo's surface - such a mechanical force can be applied by either an intender probe or a small particle - and measuring the resultant deformation at that particular point.
* A quantitative value of mechanical stress is (roughly speaking) obtained by multiplying strain and elastic modulus.

So, in other words, by accurately measuring the topography and distribution of the elastic modulus of the section, information can be obtained about the physical forces at work during a given embryological stage.

Climbing Hills & Valley's during gastrulation

In a collaborative effort with Prof. Naoto Ueno (National Institute for Basic Biology, Nagoya), Prof. Matsumoto has started to analyze the mechanical forces that are associated with the complex tissue rearrangements during Xenopus gastrulation. To that end, staged embryos were either fixed with formalin and sectioned, or alternatively cryosectioned and thawed afterwards. Using a VHX-100 Keyence digital upright microscope, the topographical landscape of the cut surface along a median plane was accurately measured, revealing the strain distribution in the respective tissues. The results can be summarized as follows:

* No significant topographical differences could be observed between the different preparation conditions.
* It became immediately apparent that the size of the observed bulges and dents encompass a broad range (0 μm - 120 μm), being indicative of the fact that mechanical strain and possibly stress are unevenly distributed within the gastrulating embryo.
* Moreover, topographic measurements taken at different stages reveal that the strength of the physical forces changes during tissue rearrangements. For example, the height difference between the ectoderm and the meso-endoderm changes from 17μm (st.10) to 7μm (st.11 and st. 12) and again to 17μm (st.13). The high mechanical strain observed at stage 10, is most likely involved in triggering the gastrulation event.
* The dorsal marginal zone (DMZ) is a key embryonic tissue region that contains precursors of all germ-layers (ectoderm, endoderm, mesoderm) and is involved in body axis formation and neural induction. During early gastrulation (st. 10, st.11) the DMZ region elongates and invaginates. Given these dramatic tissue rearrangements, it could be anticipated that a complicated force field exists within this region. This is indeed the case: comparison of topographic measurements between the core and the outer layer of the embryo consistently reveal strong height differences. This suggests that a strong mechanical stress field exists in the outer layer close to the DMZ, consistent with its highly active role during early embryogenesis.

No time for a coffee break...

Measurement of Young's modulus with a parallel indentation method. Glass beads (diameter: 200 µm) were sprayed on the cut surface of a Xenopus laevis embryo at St 14. Young's moduli were obtained by measuring the indentation of each bead.

Fig 3.Measurement of Young's modulus with a parallel indentation method. Glass beads (diameter: 200 µm) were sprayed on the cut surface of a Xenopus laevis embryo at St 14. Young's moduli were obtained by measuring the indentation of each bead.

Although the obtained results did not differ between both experimental set-ups (formalin fixation or cryosectioning and thawing), the ultimate goal of Prof. Matsumoto's team is nevertheless to obtain strain and elastic moduli measurements of various tissues from a fresh cut embryo as to minimize preparation artefacts (mechanical properties should be measured in a fresh embryo, as they may be influenced by fixation, freezing and thawing procedures). Nothing short of an immense experimental challenge that is, given the fact that a fresh cut Xenopus embryo rapidly shrinks (1.38 mm @ 0 min versus 1.01 mm @ 10 min) or breaks apart, as an active response to wound infliction. This means that the tests have to be carried out very quick. Obvious question: “How quick?” Answer: “Very quick.“ Indeed, pilot experiments have made it clear that an imploding gastrula would not grant the researchers more than 30 seconds to obtain accurate and reliable topographic and indentation measurements. Thirty seconds. Consequently, methods were developed allowing for a quick cut and observation of the surface topography and for parallel indentation measurements at different positions along the cut surface (as opposed to conventional sequential measurements with prepared embryos) within the given time span. The first topography results are qualitatively similar with fixed or frozen embryos, but quantitatively different. For example, the height difference at the cut surface between the (lower) ectoderm and the (higher) meso-endoderm was +/- 50 μm with fresh embryos, as compared to 5-15 μm in fixed specimens. The parallel indentation technique used gave elastic modulus measurements that were similar to reported values. As such, both methodological approaches demonstrate that the outer layer of the early embryo is under tension, whilst the core is in compression - underscoring the consistency of the data - but the absolute values differ. At present, the team is exploring new methodological routes for determining the physical forces existing within a fresh Xenopus embryo.

A look into the Future
Science Fiction
Science or Fiction?
Prof. Matsumoto, in which directions are your research projects developing?
Prof. Matsumoto:
We want to accumulate data on the stress distribution inside a developing embryo and to that end we are trying to improve our methods. For example, at the moment we are measuring the elastic modulus with a conventional indentation tester similar to an atomic force microscope (AFM). On the other hand, we are also exploring alternative routes.
Alternative routes?
Prof. Matsumoto:
We are establishing a technique whereby micro gold beads are evenly sprayed over a section; by measuring the surface topography with a 3D laser scanner microscope, the indentation of the particles and the elastic modulus can be calculated. This parameter gives us information on the stiffness of embryonic tissues and - by using an appropriate algorithm - on stress distribution. Preliminary data show that the elastic modulus in the early embryo, occupies a broad range (from 7 - 37 Pa). At present we are trying to improve this technique: both the spraying method and the size of the beads have to be optimized.
You want to understand the stress distribution at the single cell level?
Prof. Matsumoto:
That is correct. Obviously, obtaining such level of resolution imposes an enormous technical problem at the moment.
How would you define your ultimate objective?
Prof. Matsumoto:
We want to calculate the stress field at every position in the embryo and thus understand how stress distribution changes during development. Our ultimate goal is to construct a model - based on these experimental data - that simulates the 3D morphogenetic processes in a developing embryo, from a biomechanics perspective.
A model of development that is driven by mechanical force?
Prof. Matsumoto:
Whereby the role of mechanical force is integrated in our understanding of development. Mechanical force is probably not the only parameter that drives morphogenesis: presumably there is a dynamic interplay with biological parameters. I can imagine that a physical force could stimulate in a highly selective way certain gene expression programs, which in their turn may stimulate other mechanical adaptations.
Do you believe that there is one universal principle guiding development?
Prof. Matsumoto:
Well, I am certainly inclined to think that there exist only a few basic principles that control morphogenesis. It is my scientific dream to develop an algorithm - integrating all mechanical and biological forces - that recapitulates the entire developmental program of a multicellular organism.
REFERENCES
  1. Nagayama K, Hamaji Y, Sato Y, Matsumoto T. Mechanical trapping of the nucleus on micropillared surfaces inhibits the proliferation of vascular smooth muscle cells but not cervical cancer HeLa cells. J Biomechanics, 48, 1796-1803, 2015.
    Pubmed: http://www.ncbi.nlm.nih.gov/pubmed/26054426
  2. Matsumoto T, Nagayama K. Tensile properties of vascular smooth muscle cells: Bridging vascular and cellular biomechanics (Review). J Biomechanics, 45, 745-55, 2012.
    Pubmed: http://www.ncbi.nlm.nih.gov/pubmed/22177671
  3. Matsumoto T, Furukawa T, Nagayama K. Microscopic Analysis of Residual Stress and Strain in the Aortic Media Considering Anisotropy of Smooth Muscle Layer. Mechanics of Biological Tissue, Springer-Verlag 257-268, 2006.
    Pubmed: N.A.
Who is Who?

Prof. Matsumoto

Prof. Takeo Matsumoto

Prof. Takeo Matsumoto was born in Sapporo and studied biomedical engineering at the University of Tokyo, where he was involved in the development of control elements (such as electrical circuits and microprocessor systems) for artificial organs. After that, he returned to Hokkaido University, where he obtained his PhD in the field of biomedical engineering. During his doctoral work, Prof. Matsumoto was involved in the development and improvement of a left ventricular assist device (LVAD), an artificial organ that temporarily can assist the function of the heart. As a Research Associate at Hokkaido University, he has been doing research in the field of blood vessel mechanics, more specifically on the adaptation of blood vessel walls to hypertension. Having gained experience on the micromanipulation of cells at the Georgia Institute of Technology in Atlanta, Prof. Matsumoto returned to Japan and became Associate Professor at Tohoku University, entering the field of cellular biomechanics. Having accepted a professorship at the Nagoya Institute of Technology and later at Nagoya University, Prof. Matsumoto is presently performing research in the field of cellular mechanics and biomechanics.

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