Hiroshi Sasaki
Team Leader
Laboratory for Embryonic Induction
RIKEN Center for Developmental Biology
Our bodies start growing from a single fertilized egg. The fertilized egg undergoes repeated cleavage to increase the number of cells, but at the earliest stages of embryogenesis the cells form only a cylindrical mass. This mass then changes dynamically into a complicated human body. What is the mechanism that controls the growth of living organisms? Hiroshi Sasaki, Team Leader, is attempting to probe the mechanism by focusing on special groups of cells called ‘signaling centers,’ which control the differentiation, proliferation, and migration of surrounding cells. It has recently been proved that embryogenesis is linked to signals arising from the contact between cells. This article describes recent studies on the growth of living organisms (Fig. 1).
Signaling center, a playmaker to control the growth of living organisms
“In a high-school biology class, I heard a story of how Spemann discovered the ‘organizer.’ The story was so interesting that I really wanted to study embryogenesis,” says Sasaki. “If it had not been for that talk, I might not have come here to this laboratory.”
A newly fertilized amphibian egg cleaves repeatedly to become the gastrula, developing an indentation called the blastopore. Then the surface cells start to move into the inner portion of the embryo. This process is called gastrulation, which is the biggest event in the growth of a living organism. In 1924, a German embryologist, Hans Spemann, conducted a unique experiment in which he cut out a dorsal portion, the dorsal lip, of the blastopore from the gastrula of a newt embryo, and transplanted it into the embryo of another newt. The embryo developed into a newt with two bodies, showing that the dorsal lip of the blastopore has a guidance function that enables surrounding cells to differentiate into the tissues that form the body structure. He called the dorsal lip of the blastopore the ‘organizer.’
“A fertilized egg cleaves again and again, and dynamically changes into a complex shape from a simple one, gradually forming a body structure. We aim to explore the mechanism of the growth of living organisms,” says Sasaki. As he mainly uses mice in research, let’s follow how a mouse develops and grows from a fertilized egg (Fig. 1). Fertilization occurs when a sperm cell combines with an egg cell. On day 1, the fertilized cell divides into two cells. On day 2 it continues to divide into four and eight cells. On day 3 it passes through the morula stage, which is a solid ball of cells, to become a fluid-filled sphere called a blastocyst. On day 4 the fertilized egg implants in the wall of the uterus and starts its full-scale growth. “On day 5 we cannot observe the parts of the body such as head or legs. The embryo is only a cylindrical mass of cells,” says Sasaki. On day 6 it develops into the gastrula stage. Then, gastrulation forms three major “germ layers”: ectoderm, mesoderm, and endoderm; this phase is in turn followed by the appearance of the axis and the segment that divide the embryo into anterior and posterior portions. The major parts including the head, trunk, legs, and tail are created in this sequence 7–10 days after fertilization. Later, around day 15, various organs are created. The growth continues until day 20, when the baby mouse is born.
But how does the cylindrical mass of cells develop into a more complicated body structure? To probe the mechanism of this, Sasaki is currently focusing on the ‘signaling centers’ that appear in early embryos 5–10 days after fertilization. “The process of embryogenesis involves cell differentiation, cell proliferation, and cell migration. They do not occur in a disordered manner but are controlled temporally and spatially. Special groups of cells called ‘signaling centers’ maintain the harmony of the process.” Signaling centers secrete various molecules, affecting surrounding cells to induce cell differentiation, cell proliferation, and cell migration.
Spemann discovered the organizer, which is a typical signaling center. In the embryo of a mouse, the organizer appears on day 6.5 after fertilization, guiding the creation of the head (Fig. 2). On day 7.5 the organizer turns into a different type of signaling center called the node, inducing the creation of the trunk. On day 8.5 the node turns into a signaling center called the notochord, and the posterior end of the notochord induces the creation of the tail.
“In seeking to understand the growth mechanism of living organisms, we are studying how signaling centers are formed and maintained and how they control the behavior of surrounding cells. We have made some interesting discoveries. However, let me focus on two recent studies.”
Selection control of communication pathways
To understand how signaling centers control the behavior of surrounding cells, the Laboratory for Embryonic Induction is strategically attempting to find many genes that are expressed in signaling centers. Cthrc1 is one of these genes, to which the laboratory has paid special attention (Fig. 2). “We proceeded with this analysis in view of our firm belief that Cthrc1 is capable of affecting surrounding cells because it is specifically expressed in the node and the notochord and creates secretory proteins,” said Sasaki. “The results showed that Cthrc1 is important in selecting the communication pathways (Wnt pathways) from the outside to the inside of a cell through molecules called Wnt.”
Wnt pathways are known to be important in embryogenetic processes such as cell proliferation and morphology formation; they are classified into ‘classical Wnt pathways’ and ‘Wnt/PCP pathways.’ Here, PCP (‘planar cell polarity’) means a unidirectional alignment of cells along a planar axis. It is known that classical Wnt pathways participate in cell differentiation and cell proliferation by controlling the expression of genes, whereas Wnt/PCP pathways induce the cell cytoskeleton to change cell forms. It is unknown, however, how Wnt signaling molecules distinguish between the two pathways.
Sasaki, together with his colleague Shinji Yamamoto, created knockout mice deficient in the Cthrc1 gene, investigating the resulting abnormalities. They found evidence that Cthrc1 is related to planar cell polarity. The organ of Corti, which is located in the cochlea of the inner ear, is used to detect sound. In the normal organ of Corti, four rows of hair cells are arranged in the same direction. However, it was found that the hair cells of a Cthrc1-deactivated knockout mouse were arranged in a disordered manner. “This fact shows that there is some abnormality in planar cell polarity. In other words, Cthrc1 is related to the Wnt/PCP pathways. We have also learned that Cthrc1 stabilizes the pathways by connecting the three molecules (Wnt, Frizzled, and Ror2) related to the Wnt/PCP pathways into a line, taking part in a pathway of these molecules, thereby promoting the activation of the pathways. Thus, Cthrc1 makes it possible to select one of the two pathways by selectively activating the Wnt/PCP ones.” This result is attracting a great deal of attention because Cthrc1 is the world’s first extracellular molecule related to the divergence of the Wnt pathways.
The Laboratory for Embryonic Induction temporarily suspended its study of Cthrc1 when more interesting phenomena were found.
Contact signals between cells during embryogenesis
It is known that cell proliferation is accelerated when the density of cells in the culture is low, whereas cell proliferation stops when the density is so high that adjacent cells are touching. This is because there is a signal informing cells that they are in contact, and this stops them from proliferating. This is called ‘contact inhibition.’ If an organism develops abnormalities in contact inhibition, its cells overproliferate and form tumors. Thus the thought arose that the cell–cell contact signal also has some role in the growth of living organisms.
Sasaki is focusing on two genes: Tead and Yap1. “I have been working with Tead long enough,” says Sasaki with a smile. To clarify how signaling centers are formed, Sasaki once investigated genes that are specifically expressed in the node and the notochord. In 1993, he discovered a gene with a very important role in the formation of the node and the notochord. “The gene is Foxa2 (also known as HNF3β). Neither the node nor the notochord can be created without the gene. Even the body of an organism cannot be created normally. The protein encoded by Tead binds to a region of Foxa2 (called the ‘enhancer’, which has a special sequence), inducing the expression of Foxa2.” It has also been clarified that the Yap protein binds to Tead to increase its activity.
In fact, the discovery that Tead and Yap are related to cell–cell contact signaling was accidental. While Sasaki’s colleague Mitsunori Ota was investigating the expression of Yap, he found by chance that the cell density dynamically affected the gene’s expression level and expression distribution pattern. When the cell density is low, Yap protein accumulates in the nucleus, whereas when the cell density is high, no Yap occurs in the nucleus. “The cell density does not affect the distribution of Tead protein. In other words, when the cell density is low, Yap increases the Tead activity because it is accumulating in the nucleus. In contrast, when the cell density is high, Tead activity is decreased because there is no Yap in the nucleus. Thus, we can draw up a scenario that the cell–cell contact signal controls the transcription activity of Tead by way of Yap.”
At that time, a different group was advancing a study on pathways of communication through a molecule called Hippo with the use of the fruitfly Drosophila. Hippo pathways are important in controlling cell proliferation and in deciding the sizes of tissues and organs. Investigation revealed that Tead and Yap are regulated by Hippo pathways, proving that the scenario drawn up by Sasaki is right.
“Abnormalities in Hippo pathways can lead to the development of cancer. Thus, attention has been focused on how to control cell proliferation. However, our investigation into embryogenesis revealed that Hippo pathways are significantly related to the control of cell differentiation as exemplified by the formation of the trophectoderm in the outer part of the blastocyst at the blastocyst stage, when Tead is deficient (Fig. 3). We are the only group of scientists in the world studying the relationship between Hippo pathways and embryogenesis in mice.”
There are therefore some factors other than signaling centers that can control cell behavior during embryogenesis; normal embryogenesis cannot be guided only by the molecules secreted from signaling centers. Adjacent cells that would be expected to behave in the same way might behave differently if there is a fluctuation in the concentration of molecules secreted by cells around them. If this situation were to continue, abnormalities would occur. Thus, it can be predicted that there is a system in which adjacent cells communicate directly with each other to correct the fluctuation in the concentration of these molecules so as to function in harmony.
In seeking to clarify the relationship between the contact signal between cells and developmental control, the laboratory is planning to create a mosaic knockout mouse in which normal cells and mutant cells have been mixed together and to investigate the behavior of the mouse in detail. “Developing the technology is a challenge, but I am sure that we will be able to observe interesting phenomena that no one has ever seen before. We can understand the mechanism of the growth of living organisms only by understanding both the signal secreted from signaling centers and the signal based on cell contact.”
Communication between researchers
Sasaki explains the reason for using mice in his research as follows: “Our goal is to understand how human beings develop. The basic principle is now largely understood thanks to the advancement of research on the growth of living organisms such as Drosophila and frogs. In human beings, however, a fertilized egg starts its full-scale growth when it implants in the uterus. In that point, human beings are quite different from Drosophila and frogs. Thus, to understand the embryogenesis of human beings, we need to use a mouse in which its embryos implant in its uterus during embryogenesis, just as human embryos implant in the uterus.”
In fact, however, ‘implantation’ is an obstacle to study. “We cannot observe the inside of the uterus. The use of our newly developed technique allows us to remove an embryo after implantation and to observe it for a day. However, we want to observe live embryogenesis from beginning to end. We need a technique that allows us to observe the inside of the uterus during embryogenesis.”
Six years have passed since Sasaki joined the RIKEN Center for Developmental Biology (CDB). “In CDB, there are many experts from various fields who are involved in embryogenesis. They can give us shrewd advice that we would never have thought of. Thus this is a good environment for us because we can reach the right decision immediately without taking a long route.” As cells communicate with each other during the process of growth, the researchers in CDB communicate with each other to understand the growth of human beings.
About the researcher
Hiroshi Sasaki was born in Toyama, Japan, in 1962. He received his BSc in zoology from the University of Tokyo in 1985, and his PhD in developmental biology from the same institution in 1990. He worked as a research associate at Tohoku University and then at Vanderbilt University, USA, where he became interested in mouse embryogenesis. In 1995 he returned to Japan and extended his research as an assistant professor at Osaka University. He became a Team Leader of the RIKEN Center for Developmental Biology in 2002 and started his own laboratory. His research focuses on the mechanism of mouse embryogenesis.
