Why are human organs asymmetrical? Cell chirality may provide the answer

Why do we look almost symmetrical on both sides, but our organs are not symmetrical?

By Catherine Offord

Compilation | Ji Province

Suddenly turning back: accidentally entering the field of cell chirality research

The clock goes back to one day in 2009. Scientist Leo Wan was observing the mouse cells he cultured with a microscope. As he watched, he found that there was something wrong with these mouse cells, as if they had grown "twisted". The name of this batch of cells is myoblasts, which, as the name suggests, can generate muscles and are the predecessors of muscle cells. The hundreds of myoblasts he cultured grew on a microchip. When inoculating cells on the chip, Wan used a micropatterning technology: cells treated with this technology will adhere to the culture surface and grow according to a highly regular pattern or pattern designed by the researcher in advance. (Translator's note: This technology can be used to explore important physiological processes such as cell morphology control and migration behavior.)

Figure 1. Tumor cells grow in linear regions of different widths | Source
https://www.4dcell.com/cell-culture-systems/micropatterns/

Wan, who spent part of his postdoctoral research at Columbia University perfecting the technique, originally thought the long, narrow cells would arrange themselves along the long sides of the chip. But he told The Scientist that the cells looked like they were being pulled a little to the left.

According to Wan Qun's recollection, he initially thought it was just a coincidence. However, after that, the phenomenon recurred, and the cells almost always leaned in the same direction (translator's note: always leaning to the left). After discussing with his mentor, bioengineering expert Gordana Vunjak-Novakovic, they unanimously decided to adjust the direction of his research and focus on this special phenomenon he had seen.

Wan Qun seeded muscle cells onto two chips, a rectangular chip and a ring chip, and measured the tilt and twist data of the cells on the two chips. When the cells grow on the ring chip, they continue to grow in the direction of the circle. He speculated that he might have captured some kind of intrinsic bias of the cells, that is, the cells will be arranged in a certain direction rather than other directions. Although the cells occasionally deflect to the right (i.e. clockwise) or have no obvious bias, more than 80% of the time the cells will deflect to the left (counterclockwise).

Figure 2. Cell “Donut”: Mouse muscle cells grow a ring pattern, showing counterclockwise chirality (Photo provided by Wan Qun)

When he further studied the situation and found that this intrinsic bias seemed to vary from cell to cell, “some cells were clockwise and some were counterclockwise,” he became more certain of this hypothesis. For example, human muscle cells, like mouse myoblasts, have a counterclockwise bias. His team reported this phenomenon in a 2011 article in the Proceedings of the National Academy of Sciences (PNAS) [1]. In the same article, they also mentioned that many cells, including skin, heart, and bone cells, tend to deflect clockwise; skin cancer cells are an exception, tending to deflect counterclockwise, which is exactly the opposite of normal skin cells that have not yet become cancerous. For Wan Qun, who now conducts research at Rensselaer Polytechnic Institute in New York, this discovery introduced him to an obscure area of ​​animal biology at the time: cell chirality. This is a little-understood phenomenon. In the past few decades, only a handful of researchers have recorded this phenomenon in various cells.

Chirality - a common phenomenon but unknown cause

In a broad sense, chirality is a property of an object that is not symmetrical in space—when an object cannot be completely superimposed on its mirror image no matter how it is rotated, we say that the object has chirality. In this view, asymmetric objects can be right-handed or left-handed, corresponding to clockwise or counterclockwise rotation, respectively. Although chirality is sometimes difficult to describe clearly, it is extremely common in biology—from molecules to entire organisms. For example, the helices formed by biopolymers such as DNA have a naturally chiral structure (translator's note: DNA helices are mainly right-handed helices); amino acids can also have left-handed or right-handed forms when they form complex three-dimensional structures. Researchers now know that the handedness of a molecule has a crucial role in determining its function. Moreover, organisms are very picky about the chiral form they choose: although many molecules can exist in both left-handed and right-handed forms, almost all organisms choose left-handed amino acids and right-handed sugars in their synthesis and metabolism without exception.

Figure 3. Left: Schematic diagram of chiral and achiral objects (modified from illustrations in Khan Academy online course). Right: Schematic diagram of the chirality of the DNA double helix.

Chirality is also common on a macroscopic scale. Even organisms that appear symmetrical on the plane of their bodies have chirality. In humans, for example, we are clearly asymmetrical along two axes, head to toe and back to front, and along a third axis, from left to right. In normal human development, most major organs end up on one side of the body's central axis—the liver is on the right, the stomach is always on the left, and the heart is slightly to the left. Chirality can also be seen in the organs themselves: the heart, for example, is structurally asymmetrical from left to right, and is also asymmetrical along the other two axes. When these asymmetric patterns go wrong during human development, they can lead to abnormal left-right symmetry.

For example, some people have organs that are located in the opposite position to normal people. The organs that are normally on the right side appear on the left side, and vice versa. When all organs are on the opposite side of their normal positions, we call this phenomenon situs inversus totalis (Figure 4). The probability of this happening in the population is about one in ten thousand. Since the relative position relationship between the organs has not changed, this situation is not necessarily harmful to the body. However, if only a few organs are misplaced, such as levocardia (all organs except the heart are misplaced on the opposite side), dextrocardia (only the heart appears on the opposite side), or situs ambiguus (misplaced organs and non-misplaced organs are mixed together), patients often experience medical symptoms such as heart disease because the misplaced organs cannot cooperate correctly with other organs.

Figure 4. Normal organs (left) and situs inversus (right) | Modified from the source:
https://www.istockphoto.com/vector/human-internal-organs-system-people-body-internal-organs-illustration-anatomy-organ-gm1329503303-413211534

Similar asymmetric developmental abnormalities can also be found in the animal kingdom. For example, over the past few years, researchers at the University of Nottingham have tracked the direction of the spirals in the shells of the large pond snail (Lymnaea stagnalis). They found that although most of the time the spirals in the shells of this snail are right-handed, some left-handed spirals can also be found. They are currently studying the genetic basis behind this phenomenon [2].

Michael Levin of Tufts University edited Wan’s 2011 paper. Levin said that how asymmetry at the body level is formed is a long-standing mystery in the field of animal development, and the left-right body axis is the most difficult to understand and study. The up-down and front-back axes have clear practical significance, such as following gravity or indicating the direction of movement and migration for animals or polarized cells; but the left-right axis has no obvious meaning in comparison. Levin said, "If you try to explain to an alien, you say, 'Well, my left hand...'; then the question is, what does 'left' mean? This question is really difficult to answer."

Individual cells also have chirality: in addition to the asymmetry that manifests itself in up-down and front-back directions, cells are also asymmetric in the left-right axis. For Wan Qun and others, chirality at the level of individual cells is an important part of unlocking the mystery of animal body asymmetry. Today, the phenomenon of single-cell chirality has been widely explained. For example, a well-known ciliate, Paramecium, tends to spiral to the left when swimming; neutrophils, a cell line used to study the migration of immune cells, are similar to neutrophils, and also show a leftward bias in movement. Wan Qun believes that "there is a certain commonality among cells with chiral biases." He and his colleagues such as Levin believe that this phenomenon reveals a mechanistic connection between molecular asymmetry and organ or tissue asymmetry, which may have been overlooked before.

"For me, it was a natural transition from exploring the lateralized behavior of large organisms, such as right-handedness in humans, to exploring the lateralized behavior of individual cells." Since the 1990s, Levin has been brewing hypotheses about cell chirality and body asymmetry. His ideas often contradict some of the existing "scientific consensus" in the field of developmental biology. But he believes that cell chirality and body asymmetry are just the same thing happening at different scales.

Debate: Multiple research groups explore the mechanism of establishing cell chirality

Not long after Wan Qun began studying the left-biased cells, on the other side of the world, in Alexander Bershadsky’s lab at the Institute of Mechanobiology at the National University of Singapore, postdoc Yee Han Tee began applying another microscopic patterning technique to study how cells form their internal structures. The structures she studied are called microfilament skeletons, which are important mediators of cell growth, movement, and intracellular transport. Tee laid individual fibroblasts on tiny “islands” of sticky material, which “forced” the elongated cells to grow into round shapes. Over the next few hours, she used a microscope to record the process of cytoskeleton formation inside each cell. “One day, Tee came to me and told me that these cells were behaving in a very interesting way. Specifically, they were rotating inside,” recalls Alexander Bershadsky.

Using fluorescent labeling to track the movement of individual microfilaments, they found that two groups of fibers seemed to establish a counterclockwise vortex motion within the cell [3]. The first type, the so-called radial fibers, grew from the edge of the cell inward toward the center of the cell, forming a pattern similar to the spokes on a bicycle wheel. The other type, the traverse fibers, connected to the radial fibers at multiple points and gradually formed a concentric structure as the latter moved toward the center of the cell. These fibers were initially arranged in a regular radially symmetrical pattern, but just three hours after the cells were laid, the "spokes" began to tilt, causing the entire structure to begin to swirl around the center of the cell. Finally, around 11 hours, the fibers stopped swirling and stretched out, appearing more or less parallel to the diameter of the cell.

Figure 5. Rotation phenomenon during cytoskeleton formation. One possible way for chirality to be established in cells is that the microfilaments that make up the cytoskeleton have spontaneous organization and arrangement. In the first few hours of cytoskeleton establishment, the two types of fibers form a radially symmetrical pattern (Figure 5 left), but after three hours, the radial fibers begin to tilt, pulling the transverse fibers off track, causing a vortex pattern (Figure 5 center), and around 11 hours, the vortex pattern breaks into a linear pattern, with the fibers arranged along the main axis of the cell (Figure 5 right).

ILLUSTRATION BY © SCOTT LEIGHTON; DATA FROM NAT CELL BIOL, 17:445–57, 2015.

Although the chirality of the cytoskeleton is not a new discovery, Bershadsky, Tee et al., in a 2015 research paper [3], provided early visual evidence of spontaneous chirality of molecules within cells through experimental means, and also used computer simulations to reproduce the vortex pattern of the cytoskeleton. To further explore the mechanism, they also used small molecule drugs to inhibit microfilament-associated proteins and found that drug treatment caused the cytoskeleton to lose its original chiral bias and even begin to vortex in the opposite direction. Other research groups are also exploring this mechanism. A few years ago, scientists from Japan who studied zebrafish melanocytes reported that inhibitors of microfilament assembly could block the tendency of cells to rotate counterclockwise under culture conditions [4].

Figure 6 Two states of cytoskeletal vortex - counterclockwise (left) and clockwise (right). From reference [3]

Microfilaments themselves are chiral molecules that form right-handed helices. Bershadsky, Levin and others therefore suspect that the molecular structure of microfilaments plays a central role in the establishment of cellular asymmetry. Bershadsky said, "Our view is that microfilament fibers and their helical asymmetry are the factors that form chirality." He also added that it is still unclear how this mechanism works, and it is possible that the molecular structure of microfilaments affects the response of microfilament fibers to mechanical forces and their interaction with other intracellular proteins.

In fact, microfilaments are not the only component of the cytoskeleton; microtubules are also part of the cytoskeleton. Some laboratories are also studying the role of microtubules. Microtubules are stiffer and thicker than microfilaments, and play a greater role in certain intracellular processes such as vesicle transport. In an early study on cell chirality, researchers treated neutrophils with drugs that interfered with microtubule assembly and found that although these cells could still migrate, they no longer showed a left-biased movement pattern [5]. A few years later, Levin and his colleagues found a similar phenomenon: when tubulin (the protein that makes up microtubules) was knocked out in neutrophils, the asymmetry of the cells disappeared completely.

However, some researchers have different opinions on the importance of microfilaments and microtubules in establishing cell chirality. Bershadsky mentioned that they did observe microtubules swirling along the microfilament fibers in the experiment, but destroying the microtubule function did not block the swirling process or affect the swirling direction. The Japanese research group studying melanocytes also mentioned that microtubule inhibitors would intensify cell rotation. In addition to microtubules, Levin's research group also studies microfilament-associated proteins. He believes that both microfilaments and microtubules may be involved in the establishment of cell chirality. "There is solid evidence for both, and there is no need to choose one or the other."

In order to better understand how different factors drive chirality at the macroscopic scale of organisms, researchers have tried to observe cell chirality in experimental settings that can better simulate natural conditions. For example, Wan Qun and others developed a three-dimensional micro-patterning technology (Wan Qun is also one of the inventors of patents related to micro-patterning technology in the field of cell chirality research) that can better replicate the embryonic development environment and then detect the rotational behavior of microspheres formed by epithelial cells [6]. The research team found that most microspheres rotated counterclockwise, but when drugs blocked microfilament assembly, most microspheres began to rotate clockwise, as if a switch had been pressed.

Wan Qun’s team has also developed methods to identify cell chirality from visual features, such as analyzing the distribution of organelles within cells—they hope to pave the way for in vivo observation experiments in animals. In a recent paper [7], they introduced a new method based on the relative positions of the nucleus and centrosome: since in moving cells, the nucleus tends to stay at the rear end of the moving direction, while the centrosome (translator’s note: the centrosome is the center of internal activity during cell division, located in the cytoplasm outside the nucleus) is often close to the front end, they drew a virtual line between the nucleus and the centrosome, and then recorded the position of the cell’s center of gravity relative to this virtual line. Using this new observation method, they observed that the center of gravity of endothelial cells tends to appear to the right of the anterior-posterior axis, and it was previously known that endothelial cells tend to deflect to the right (clockwise), indicating that the observation results are consistent with the known chirality. This suggests that the strategy they developed can roughly determine cell chirality.

Wan Qun and his colleagues also studied the behavior of multiple chiral cells after they aggregate. For example, in embryos, some migratory cells aggregate into a group or swirl together to form specific organs. Such studies can help scientists determine the importance of cell chirality in animal development and resolve related controversies.

From the small to the big: the role of chirality in animal body asymmetry

In the early 21st century, an important part of the puzzle of left-right asymmetry in vertebrate embryonic development was solved: after the head-tail axis and the dorsal-ventral axis were established, some cells along the ventral side of the embryo assembled tiny hairy structures called cilia at their edges. These hairs created a leftward flow of fluid by swaying. This fluid flow triggered the asymmetric gene expression of cells in the relative positions of the left-right axis, and ultimately divided the body into left and right sides. Gene knockout experiments showed that when proteins necessary for cilia assembly (such as kinesin, which transports the raw materials required for cilia assembly along microtubules) were knocked out, the fluid flow in mouse embryos would be disrupted or even reversed, resulting in the misalignment of organs in the animal body (positioning defects). Genetic screening of mice with organ symmetry positioning defects showed that dozens of cilia-related genes had mutations, and these mutations were significantly associated with developmental defects in mice [8]. Cilia seemed to be the key to breaking the symmetry of the mysterious third body axis.

This view has dominated researchers' understanding of animal development to this day. However, Levin pointed out that most studies have overlooked a key fact, that is, after knocking out cilia-related proteins, the cytoskeleton and various intracellular processes will also be affected (Translator's note: In other words, the organ symmetry positioning defects observed after knocking out cilia-related proteins cannot be simply attributed to embryonic fluid flow that depends on cilia, and processes such as cytoskeleton assembly may also be involved). In addition, left-right asymmetry also exists in animals such as chickens, pigs, and roundworms, and these animals do not have ciliated cells in their embryos to guide fluid flow [9, 10]. Even in ciliated animals such as frogs, asymmetric distribution of important development-related molecules such as RNA can be detected long before the cilia are assembled and begin to beat. The phenomenon that knocking out tubulin interferes with lateralized development (Translator's note: bilateral asymmetry during development) exists not only in animals, but also in plants. This suggests that there may be a universal mechanism for establishing asymmetry that depends on tubulin but not cilia.

“It’s totally unreasonable to say that cilia cause asymmetry… The fairest thing you can say is that cilia are involved in some intermediate step in the pathway.” Levin thinks that perhaps cilia amplify differences in left-right asymmetry that are established by mechanisms within the cell.

Although a series of evidence suggests that there is a mechanism that does not depend on cilia to establish asymmetry at the tissue and body level, the details of how it works are far from clear. Studying cell chirality requires answering the following two questions:

First, how do cells provide directional information at the tissue level? That is, what counts as left and what counts as right?

The second, and more challenging, question is how do cells encode specific location information? That is, how do cells know where the embryonic midline is and which side they are on?

Scientists have now devoted themselves to analyzing these two problems separately.

Regarding the first question, many research groups have confirmed in vitro that when cells are aligned with each other, cell chirality controls the direction of cell deflection at the group level. Similar to the left-biased mouse cells used by Wan Qun, the fibroblasts used by Tee also exhibit group behavior patterns when aligning and migrating, and this pattern can be eliminated or reversed by microfilament-disrupting drugs. The relevant results have been published on the preprint platform BioRxiv in April 2021 [11]. Other researchers have reported that this type of group behavior of cells can have an impact at the level of entire tissues and organs. Kenji Matsuno is a fruit fly researcher at Osaka University in Japan. He has been studying the asymmetry of the fruit fly embryonic hindgut (translator's note: the end of the embryonic digestive system is generally referred to as the hindgut). The fruit fly hindgut will turn 90 degrees to the left during embryonic development, and the epithelial cells that make up the hindgut duct themselves have an asymmetric shape [12]. Matsuno and his team found that interfering with actin-associated proteins could both flip the chirality of epithelial cells and reverse the direction of rotation of the hindgut (Figure 6) [13]. In a recent paper [14], Matsuno’s team proposed that chirality at the cellular level is a necessary and sufficient condition for driving the hindgut rotation phenomenon.

Figure 7. Drosophila intestinal inversion: During normal Drosophila development, the hindgut undergoes a counterclockwise rotation and finally bends to the right (left). After the researchers knocked out the protein involved in the cytoskeleton function, they reversed the rotation direction of the hindgut and obtained a hindgut pointing to the left (right). 丨Source: M. INAKI ET AL., FRONT CELL DEV BIOL, 6:34, 2018.

Wan is also studying the development of the bird heart. The heart is one of the first organs in embryonic development to break its axial symmetry—a process that begins with a special group of cells that normally form circuits that rotate to the right. Wan’s team reports[15] that cells isolated from the hearts of chicken embryos show an intrinsic right-handed bias, and that they can be reversed in culture by treating them with drugs. The drugs used are generally those known to disrupt the chirality of the microfilament cytoskeleton and intracellular structure. When chicken embryos were treated with these drugs, a large number of them developed left-handed hearts. “This gives us some evidence that cellular chirality is likely to play a role,” Wan says, adding that his team has also come across a chicken embryo that naturally developed a left-handed heart. In this particular embryo, the heart cells rotated counterclockwise, as if they had been treated with a small molecule drug. He and his colleagues have expanded this work to study the potential significance of chirality for human health and disease, including heart development, the permeability of the endothelial barrier[16], and the competition between cancer cells and normal cells[17].

However, although the above mechanisms provide a partial explanation for the development of asymmetric organs, it is still unclear how these mechanisms work at a higher level of the animal's body plane. Researchers generally believe that at some intermediate time point in development (the specific node may vary from species to species), a certain molecular barrier is formed in the midline of the embryo, which blocks the free diffusion of growth factors on both sides of the body and exacerbates the asymmetric accumulation of gene expression products. But Levin also pointed out that the existence of individuals with abnormal contralateral patterns, such as half male and half female hermaphrodites (such as the hermaphroditic butterfly in the figure below), suggests that the basic left-right separation pattern has been formed earlier in embryonic development.

Figure 8. Male-female chimeras. Although most male-female chimeras occur in insects and arachnids, they also occur in some relatively advanced animals, including crustaceans, cardinals, chickens, etc. | Image from Sina Photo Station
http://slide.tech.sina.com.cn/d/slide_5_453_35289.html#p=1

“If these genetic disorders occurred in late embryonic development, the male and female characteristics would never be clearly separated on the left and right as they are now.” Levin published a model to describe cell chirality[18], and specifically pointed out that the biased transport of specific intracellular proteins along the cytoskeleton can help the embryo form left-right asymmetry by establishing a voltage or pH gradient in the embryo. Of course, he now believes that the true mechanism is still unknown and “a complete mystery.”

The road ahead is long and arduous: the mystery of cell chirality remains to be solved

Whatever role cell chirality plays in animal development, the scientists interviewed acknowledge that there are still some key fundamental questions that need to be answered. First, it is still unclear why some cells show a clockwise bias while others are counterclockwise? In other words, how does this difference come about? Wan Qun mentioned that his colleagues have shown that muscle cells contain more microfilaments than other cells, which may explain why mammalian muscle cells are biased to the left while other cells are biased to the right or have little bias at all. In addition, endothelial cells and epithelial cells often have opposite chirality, a phenomenon that he is very interested in studying further.

Because cells and groups of cells are not 100% consistent in the direction of tilt, some researchers believe this is a limitation of the field of cell chirality, which means that related studies must rely on statistical methods to identify chiral biases. After all, in Wan Qun's 2011 paper, only 80% of muscle cells showed a counterclockwise rotation bias, which is far less than the proportion of left-handed amino acids in the human body or the proportion of right-handed deflections in the embryonic hearts of vertebrates, both of which are close to 100%. The same limitation applies to studies of single cells: most cells show chirality in one direction, but there are always cells that tilt in the opposite direction.

Molecular engineer Ding Jiandong of Fudan University and his colleagues recently called for caution in a number of papers, noting that low consistency in cell chirality studies is common and that results should be analyzed with caution [19, 20]. However, it is still unclear whether this inconsistency is due to factors such as experimental error or reflects real differences in cells.

Wan Qun believes that cell chirality is very important, and studies using cytoskeleton-interfering drugs to change the chirality of cells and entire organs have good reproducibility. Even if the chirality of some cells is not uniform, the cell population may still be sufficient to drive behavior at the tissue level, especially since cell chirality may be just one of many mechanisms involved in establishing and amplifying asymmetry during development.

Matsuno added that some researchers are beginning to shift away from the old, binary view of chirality to a more complex combination of preferences for left and right. "Cell chirality may not be a 0 or 1 switch. I now believe it is a very complex phenomenon."

Bershadsky believes that the answers to these puzzles will be part of future research in the field of cell chirality. He is collaborating with Wan Qun to organize a discussion session on cell chirality and symmetry breaking at the World Congress of Biomechanics in July 2022 [21]. “This field is still a new topic, which is why we like it,” Bershadsky said. “In fact, most animals are bilaterally symmetrical, which is also difficult for us to understand.” “Deviations in symmetry are, in a sense, nature’s change of the symmetry coding formula. The disordered coding creates the beauty of asymmetry. (The) asymmetry we see is not random, it can be well inherited by offspring and precisely regulated.”

References

[1] https://www.pnas.org/doi/10.1073/pnas.1103834108

[2] https://www.cell.com/current-biology/fulltext/S0960-9822(16)00056-7

[3] https://www.nature.com/articles/ncb3137

[4] https://onlinelibrary.wiley.com/doi/10.1111/gtc.12194

[5] https://www.pnas.org/doi/10.1073/pnas.0703153104

[6] https://www.pnas.org/doi/10.1073/pnas.1805932115

[7] https://www.tandfonline.com/doi/full/10.1080/19420889.2019.1605277

[8] https://www.nature.com/articles/nature14269

[9] https://www.sciencedirect.com/science/article/pii/S0012160613001693?via=ihub

[10] https://elifesciences.org/articles/04165

[11] https://www.biorxiv.org/content/10.1101/2021.04.22.440942v1

[12] https://www.science.org/doi/10.1126/science.1200940

[13] https://www.sciencedirect.com/science/article/pii/S0925477314000240

[14] https://www.mdpi.com/2073-8994/12/12/1991/htm

[15] https://www.pnas.org/doi/10.1073/pnas.1808052115

[16] https://www.science.org/doi/10.1126/sciadv.aat2111

[17] https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1006645

[18] https://journals.sagepub.com/doi/full/10.1177/154411130401500403

[19] https://onlinelibrary.wiley.com/doi/10.1002/cjoc.201800124

[20] https://www.sciencedirect.com/science/article/pii/S1742706121001276

[21] https://www.wcb2022.com/programTracks.asp

This article is authorized to be translated from TheScientist February cover special article
https://www.the-scientist.com/features/cell-chirality-offers-clues-to-the-mystery-of-body-asymmetry-69584

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