Naoto Ueno

A new actor on stage: physical force

Neural tube closure in Xenopus

Fig 1. Neural tube closure in Xenopus

Several decades of intensive academic research have led to the characterization of major molecular pathways and signaling cascades that guide embryonic development. Many of the gene regulatory networks controlling major morphogenetic events - involving dramatic cell shape changes and tissue deformations - have been identified, and a lot of research is dedicated towards understanding these processes in molecular detail. Nevertheless, during the past few years it has become apparently clear that yet other, thus far unknown mechanisms are playing a major role during development. Prof. Naoto Ueno and his co-workers are trying to understand to what extend other factors than genes and proteins - information that is not evolutionary imprinted in the genome - can influence the shaping of a biological entity. During the past few years, one such factor has found the spotlight of their scientific work: physical force. Indeed, they and others have conclusively shown that major tissue rearrangements - when cells change shape and position within a 3D field - can generate mechanical force. Consequently, one question has become at the forefront of their research: “What is the biological relevance of physical force during animal development?”

Cell non-autonomous mechanisms contribute to neural tube formation

Migration of deep cells towards the midline

Fig 2. Migration of deep cells towards the midline

Neural tube closure is an early morphogenetic process during formation of the central nervous system in vertebrates, whereby a hollow tube is formed through the bending of a flat sheet of neurectodermal tissue. For years it has been known that this highly coordinated process in space and time is driven through cell autonomous mechanisms. Only cell autonomous mechanisms? As a matter of fact: no. Other machinery is also at work. Based upon their experimental data, Prof.
Deep cells of the non-neural ectoderm migrate towards the dorsal midline

Fig 3. Deep cells of the non-neural ectoderm migrate towards the dorsal midline

Ueno and his colleagues has proposed a mechanism that involves cell non-autonomous phenomena contributing to neural tube formation in Xenopus. As it turns out, the activity of non-neurectodermal cells which does not give rise to the tube itself is also in demand. In particular, deep layer cells of the non-neural ectoderm actively migrate to the midline and create physical force, causing the neural plate to bend mediolaterally to form a hollow cylinder. Ablating this population of non-neurectodermal cells, ultimately leads to impaired neural tube formation.

Intracellular fireworks: transient Ca2+ increase precedes neural tube closure

Intracellular Ca2+ transients during neural tube closure

Fig 4. Intracellular Ca2+ transients during neural tube closure

Recently, an intriguing phenomenon has been observed in the Ueno lab: neurectodermal cells undergoing apical constriction during neural tube closure - a process whereby cells collectively minimize their apical surface, necessary to facilitate epithelial sheet bending and invagination - transiently increase their intracellular Ca2+ concentration, prior to cell shape changes.

At present, a working hypothesis is being verified, whereby this Ca2+ influx has an impact on cytoskeletal remodelling, mechanically linked to cell shape transformations in the neurectoderm. One intriguing observation still awaits further explanation: Ca2+ influx does not occur at the same time in all cells participating in neural closure. Quite on the contrary, seemingly isolated cells at different positions within the tissue transiently enhance their intracellular Ca2+ concentration. Video monitoring of living neurectodermal cells, using a fluorescent Ca2+ sensor (R-GECO), reveal an on-and-off firing in separated, individual cells, apparently without any underlying pattern. Is this dynamic Ca2+ influx truly a random process, or does it follow an internal logic? At present, this question still awaits an answer. What has become increasingly clear however is that “random” pulses appear to be far more effective for tissue deformation. Indeed, when the intracellular Ca2+ concentration is artificially increased in all neurectodermal cells, the result is aberrant neural tube formation. Recently, an algorithm has been developed by mathematician Dr. Hiroshi Koyama (working at another lab in the Institute), allowing simulations of the transient Ca2+ influx and tissue deformations. This could be helpful in answering some intriguing questions:
- Which mechanism control this intracellular Ca2+ propagation?
- Is this Ca2+ influx linked to cytoskeletal changes (e.g. through regulating F-actin dynamics)?
- How are physical forces generated and how do they contribute to the bending of the neurectoderm?

Dissecting the role of individual cells during gastrulation

Only few biologists will deny that gastrulation is a key process during embryonic development, as it involves the dynamic tissue remodeling necessary to establish the proper body plan. Setting out on a quest to characterize the nature and the biological significance of the physical forces involved, Prof. Ueno joined forces with the team of Prof. Takeo Matsumoto (Nagoya University), thereby focusing on the leading edge mesoderm (LEM): a population of cells that collectively migrate toward the future anterior side during gastrulation in Xenopus. In a series of elegant experiments, they cut out the LEM and put the tissue (+/- 500 cells) on a glass slide coated with extracellular matrix. Their aim: recapitulating the initial stages of gastrulation in vitro and measuring the physical force generated by the migrating cells (in the range of 40 nN). Meanwhile, it has become apparent that not every cell equally contributes to this force. Moreover, tracking of (histone-GFP labeled) nuclei shows that cells continuously change their relative positions within the LEM field. Are both phenomena causally linked? Unknown at present, but it seems at least legitimate to speculate on it: depending on their (changing) position, individual cells play a different role during the migration process. Recently, an experimental set up has been designed to describe the movement of each cell and measuring the associated tracking force, by means of a tracking force microscope: which cells generate a force and which cells don't? By carefully monitoring the behavior of each individual cell, Prof. Ueno hopes to unravel the physical principles underlying collective cell migration.

A look into the Future
Science Fiction
Science or Fiction?
Prof. Ueno, in which directions are your research projects developing?
Prof. Ueno:
We want to unravel the mechanisms that drive morphogenesis. Developmental processes obviously involve complex cell shape and cell identity changes, as well as dynamic tissue rearrangements, but I believe there must be one or at most a few underlying principles. So, our long term goal is to understand the basic strategies by which a multicellular organism is shaped.
How do you plan to tackle this problem? Do you want to register every cell shape change, every cell movement, every molecule involved and every force generated during amphibian gastrulation?
Prof. Ueno:
That could be one way of approaching this scientific challenge, but this would be very complex and time consuming. The major problem is that all gathered information must be extremely accurate; this is an absolute prerequisite for such a strategy to be successful. At present, such a degree of accuracy is by no means guaranteed, although many developmental biologists are trying to grasp developmental programs by characterizing the gene regulatory networks involved.
But the majority of them do not take into account mechanical force.
Prof. Ueno:
That is correct. One of our objectives is therefore to determine the in vivo position of every cell and to map every force generated during Xenopus gastrulation. In this manner we are incorporating physical force in our models, with the aim - as I just said - to unravel fundamental biological principles.
Do I hear you saying that you are trying to unravel the interaction between physical and biological responses such as chemical modifications?
Prof. Ueno:
That is certainly one of our research objectives. For example, in collaboration with Princeton University, we want to characterize the phospho-proteome of a gastrula. To that end, we are applying physical force - by means of weak centrifugation - to a gastrulating Xenopus embryo and determine changes in the phosphorylation state of proteins. As you probably know, phosphorylation of specific proteins is an important mechanism when a rapid response to a particular stimulus is required.
Did you find some protein modifications that could be linked to mechanical force?
Prof. Ueno:
It is far too early to make conclusive statements, but we have identified a set of cytoskeletal proteins whose phosphorylation state differs before and after centrifugation. That could underscore a link between cytoskeletal dynamics and physical forces, during morphogenetic processes.
Would you be willing to speculate on the nature of the general developmental principle you are seeking?
Prof. Ueno:
I am intrigued by the findings of behavioral biologist Iain Couzin, whose work aims at understanding evolved collective behavior. Making use of the artificial life program BOIDS, developed by Craig Reynolds, he found that the collective movements of individuals in a swarm - for example fish schools or bird flocks, but also human crowds - are controlled by no more than 3 parameters: separation (avoid crowding), alignment (steer towards local flockmates) and cohesion (move towards the center of mass). I am tempting to believe that the same general principles could guide the collective behavior of cells in a dynamically changing 3D tissue field: alignment is controlled through cell polarity, attraction is cell-cell adhesion and repulsion is anti-cell adhesion. I could imagine that such individual cell behavior is integrated in a program that coordinates morphogenetic events, controlled by a general sensory system that responds to stimuli - such as physical forces, let's say- and which is linked to a feed-back mechanism.
  1. Negishi, T., Miyazaki, N., Murata, K., Yasuo, H. Physical association between a novel plasma-membrane structure and centrosome orients cell division. eLife, e16550, 2016
  2. Hara, Y., Nagayama, K., Yamamoto, T.S., Matsumoto, T., Suzuki, M. and Ueno, N. Directional migration of leading-edge mesoderm generates physical forces: Implication in Xenopus notochord formation during gastrulation. Dev Biol 382, 482-495, 2013.
  3. Suzuki, M., Morita, H. and Ueno, N. Molecular mechanisms of cell shape changes that contribute to vertebrate neural tube closure. Dev Growth Differ 54, 266-276, 2012.
  4. IshimatsuMorita, H., Kajiura-Kobayashi, H., Takagi, C., Yamamoto, T. S., Nonaka, S. and Ueno, N. Cell movements of the deep layer of non-neural ectoderm underlie complete neural tube closure in Xenopus. Development 139, 1417-1426, 2012.
Who is Who?

Prof. Ueno

Prof. Naoto Ueno

Born and raised in Tokyo, Prof. Naoto Ueno was trained as a biochemist at Tsukuba University. After finishing his PhD work on rennin (a peptide hormone involved in the regulation of blood pressure) he decided to go abroad and join the laboratory of Prof. Roger Guillemin - awarded the Nobel prize for Medicine in 1977 for his pioneering work on neurohormones - at the Salk Institute. During his postdoctoral training, Prof. Ueno was involved in the purification of inhibin / activin and other peptide hormones. Following his work on the purification and characterization of fibroblast growth factor (FGF), he switched to the field of Developmental Biology, having returned at Tsukuba University. Using Xenopus as a model system, he identified bone morphogenetic protein (BMP), a ventralizing growth factor, which turned out to be an important contribution to the molecular understanding of the Spemann's organizer. At present, Prof. Ueno is leading a research group at the National Institute for Basic Biology at Okazaki. His research aims at understanding cell and tissue dynamics in early embryos, both at the molecular and the biophysical level.