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Kenji Matsuno Osaka University

Symmetry breaking mechanisms: an evolutionary enigma

Looking at both hands allows one to get an intuitive grasp of what “chirality” means. Indeed, a (admittedly, somewhat simplified) definition states that an object is “chiral” if it is distinguishable from its mirror image and cannot be super-imposed onto it. Exactly as the left hand is a non super-imposable mirror image of the right hand; no matter how often and in which direction they are rotated: it is not possible for both hands to completely coincide across all axes. Chirality is widespread across the animal world and can be recognized at the molecular level or at the anatomical level, in features as diverse as the clockwise or anti-clockwise coiling of gastropod shells, the structure and position of internal organs, or the species-specific left or right turning of marine flatfish, prior to their roaming existence at the bottom of the ocean.

Given the fact that genetic mutations resulting in inconsistency of each organ's chirality in humans - e.g. manifested through the discordant internal positioning of the heart, spleen and/or lungs - are often connected to health problems, it is tempting to speculate that left-right asymmetry is a crucial aspect of the developmental program of metazoans. As to the reason why biological structures demonstrate a genetically determined left-right asymmetry - that is to say, involving an energy consuming decision making process, based upon an elaborate machinery of gene interplay under evolutionary pressure - one can only speculate at present.

Individual cells can have chirality

The embryonic hindgut is essentially a single layered epithelial tube, covered by visceral muscle. It is in fact the first organ in the fly to demonstrate left-right asymmetry at the anatomical level: at embryonic stage 12 the hindgut curves ventrally and subsequently rotates 90 degrees anti-clockwise, forming a rightward curving structure by stage 13 (a process that takes about 1-2 hours to complete). Based on their experimental findings, Prof. Matsuno has demonstrated that:

1) The hindgut epithelium is responsible and sufficient to induce rotation. The role of the overlaying visceral muscle cells is dispensable for this process.
2) Rotation of the hindgut is not accompanied by cell proliferation or controlled cell death (apoptosis).

Furthermore, Prof. Matsuno has postulated that the hindgut cells themselves possess left-right polarity and that cell shape changes are somehow causally connected to the rotation of the hindgut. And indeed, this seems to be a valid assumption, as his team could demonstrate that “planar cell-shape chirality” (PCC), as they term it - e.g. manifested through asymmetrical centrosome position and E-cadherin distribution in individual cells - is indeed a fundamental feature of hindgut epithelial cells. These cells have a columnar shape and are arranged like pillars in a sheet, with their apical side facing the lumen of the hindgut. Thus far, most studies dealing with epithelial morphological changes have been focusing on this apical side, as it is enriched in actin bundles and E-cadherin, essential components of the cellular machinery that could generate mechanical force to provoke tissue deformations or rearrangements. In their quest for answers, Prof. Matsuno and his co-workers however decided to look at hindgut epithelial cells from a (literally) new angle, taking the entire cell into account: from the basal to the apical side. An important and most fortunate decision this turned out to be, as it paved the way for an understanding of tissue deformations from an entirely new perspective…

Do Winding & Rewinding of individual cells provoke rotation of the hindgut?

Movie 1. A 3D model of an epithelial tube, recapitulating the directional rotation of the Drosophila hindgut. This model predicts that the axial rotation of the hindgut can be induced by twisting of each epithelial cell.

In a collaborative effort with Prof. Masakazu Akiyama (Hokkaido University) and Prof. Yasuhiro Inoue (Kyoto University), a 3D computer model was developed that recapitulates all cell and tissue rearrangements relevant to hindgut rotation. These computer simulations suggested a direct link between the rotation of the hindgut and individual cell shape changes. Consequently, a working hypothesis was formulated, stating that coordinated cell shape changes - that is to say, twisting of each cell, whereby the relative position of the basal and apical surface changes towards each other - cause rotation of the hindgut.

Movie 2. A 3D structure of the Drosophila embryonic hindgut, which is composed of tall columnar epithelial cells. Thus, 3D mechanics is required for understanding the mechanisms of hindgut rotation (apical and lateral cell boundaries are shown in green and magenta, respectively).

In an effort to prove their hypothesis, Prof. Matsuno and his team set out on an experimental journey with the aim of measuring twisting of individual epithelial cells, before and after hindgut rotation in the fly embryo. To that end, they obtained confocal images in 10 different optical planes (from basal to apical) for each columnar cell, thus enabling them to mathematically define a major and minor axis: twisting being defined as rotation of the major cell axis in a given focal plane. Such an experimental setting would allow recording of whether and in which direction an epithelial cell is twisted. The gathered data deliver evidence for hitherto unobserved collective cell behavior: epithelial cells twist clockwise before rotation of the hindgut (which itself rotates anticlockwise) and twist anticlockwise after rotation, essentially confirming the predictions of the 3D model. Given this scenario, it is tempting to speculate that torque - generated by collective winding and rewinding of cells - provides the mechanical force that drives tissue deformations, associated with hindgut rotation.

Hunting for the genes

To identify genes involved in left-right asymmetry of the hindgut, a large-scale genetic screen was performed in Drosophila. This resulted in the identification of Myo31DF, encoding the evolutionary conserved MyosinID: an unconventional myosin that shows ATP-dependent interaction with actin filaments. Prof. Matsuno and his co-workers could demonstrate that Myo31DF is required in the hindgut epithelium for normal embryonic handedness, as both PCC and the direction of the hindgut twisting are reversed in homozygous mutants. Moreover, they could show that the asymmetric distribution of DE-Cadherin in epithelial cells is controlled by the Myo31DF gene, in a cell autonomous manner. As such, Drosophila MyosinID is the first actin-based motor protein shown to be involved in left-right patterning.

A look into the Future
Science Fiction
Science or Fiction?
Prof. Matsuno, in which directions are your research projects developing?
Prof. Matsuno:
We believe that planar cell-shape chirality is responsible for left-right asymmetry, at least in Drosophila. The puzzling thing is: how do these epithelial cells - which can be loaded in either a left or a right status, to say it a bit simplified - collectively adopt the same status? How is such a process controlled? And what is the initial trigger? We have reasons to believe that some mechanical element induces a collective twisting of all cells in a given direction, prior to hindgut rotation. At present, we are searching for such a mechanism that triggers and/or coordinates collective cell shape changes.
How do you want to tackle this problem?
Prof. Matsuno:
From a genetic analysis, we know that actin is involved in this process: if actin is functionally knocked-out in these epithelial cells, the directional twisting of the hindgut is also disrupted. On the other hand, we also believe that a directional cytoplasmic flow could be involved. So, one experimental route is to try to relate dynamic changes of the actin cytoskeleton to a directional flow of the cytoplasm, in trying to answer the question: can directional flow of cytoplasm be linked to cell chirality? We already know that cells can adopt a different chirality based on the presence or absence of Myosin31DF, a protein that interacts with the actin cytoskeleton.
Does this collective cell twisting occurs at the same time in all hindgut cells, or are there “Hotspots” where the process starts and spreads out?
Prof. Matsuno:
At present we don't know. The experimental data we have gathered thus far show an average over the entire epithelial tissue and do not envision the chirality status of any particular cell. But this is certainly a legitimate question and Prof. Akiyama is developing a 3D model whereby a single cell could impose its status upon other cells. We hope to receive new insights from these mathematical simulations.
How would you describe your scientific dream?
Prof. Matsuno:
Despite the fact that most proteins are chiral, the possibility of chirality at the cellular level has long been overlooked. After we discovered cell chirality in vivo, the same phenomenon has also been observed in mammalian cells and tissues. So, I tend to believe that cell chirality is a common phenomenon in Biology. I could imagine that several morphogenetic events - such as radial versus spiral cleavage during early embryogenesis, for example - might be driven by intrinsic cell chirality, somehow linked to a biomechanical machinery, generating physical force. At this moment we do not really understand how such chirality is created and coordinated, but my scientific dream is that Myo31DF - an evolutionary conserved gene from yeast to humans - is a central regulator of chirality at the single cell level.
REFERENCES
  1. Hozumi, S., Maeda, R., Taniguchi, K., Sasamura, T., Spéder, P., Noselli, S., Aigaki, T., Murakami, R., Matsuno, K. A Drosophila unconventional myosin reverses the default handedness in visceral organs. Nature 440, 798-802, 2006. http://www.ncbi.nlm.nih.gov/pubmed/?term=hozumi+matsuno+nature
  2. Taniguchi, K., Maeda, R., Ando, T., Okumura, T., Nakazawa, N., Hatori, R., Nakamura, M., Hozumi, S., Fujiwara, H., Matsuno, K. Chirality in planar cell shape contributes to left-right asymmetric epithelial morphogenesis. Science 333, 339-341, 2011. http://www.ncbi.nlm.nih.gov/pubmed/21764746
  3. Inaki, M., Yang L. J., and Matsuno K. Cell chirality: its origin and roles in left-right asymmetric development. Phil Trans B in press 2016 (It will be open on7th November)
Who is Who?

Prof. Matsuno

Prof. Kenji Matsuno

Prof. Kenji Matsuno was born in Ogaki, known for being the most centrally located city in Japan. He obtained his doctorate in Molecular Biology from Waseda University, by virtue of his research - conducted in the laboratory of Prof. Yoshiaki Suzuki at the National Institute of Basic Biology - on the transcriptional regulation of silk fiber genes, expressed in the silk gland of the caterpillar of the silk moth (Bombyx mori). After finishing his PhD dissertation, Prof. Matsuno switched to Developmental Biology and moved to Yale University for a postdoctoral training, under supervision of Prof. Spyros Artavanis-Tsakonas. His research there dealt with unraveling the Notch signaling pathway during Drosophila development. Six years later, he returned to Japan, working first at Osaka University (at the laboratory of Prof. Hideyuki Okano) and later at the Tokyo University of Sciences (as a principal investigator), remaining in the field of Notch-Delta signaling. At present, Prof. Matsuno is leading a research team, aimed at understanding the role of left-right asymmetry during animal development, using Drosophila as a model system.

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