Cells Communicate With Each Other Through Signals And Receptors

To reiterate the central point of this chapter, development can be viewed as a sequence of events in which initially equivalent cells acquire distinct patterns of gene expression. These differences in gene expression define different developmental potentials, such as whether a cell will become part of a muscle or part of the nervous system. As development proceeds, the potential of a given cell becomes progressively more limited until its identity is unambiguously established. This view of development as a progressively hardening plastic leads to two key questions: (1) How are differences in gene expression generated during development? (2) How do differences in gene expression alter the developmental potential of cells?

One important mechanism by which cells acquire and maintain distinct developmental potentials is by communicating with one another. Cellular communication may create a difference between initially equivalent cells, or it may exaggerate subtle preexisting differences between two cells. Communication between cells is mediated by two types of molecules referred to as "signals" and "receptors." When a cell sends a message, it liberates a signal, which is sensed by receptors present on neighboring cells (Fig. 1.8). A "receiving" cell senses a signal by virtue of the signal sticking to receptors on its surface. When a signal sticks to its receptor (or "binds" to its receptor, in the jargon), the receptor changes shape and becomes activated. Activation of the receptor alters gene expression in the responding cell, thereby defining the developmental potential of that cell. Signals and receptors are exquisitely monogamous molecules. A signal typically binds to only one receptor, and the receptor likewise is faithful to its signal.

Some signals are tethered to the surface of the signaling cell. Tethered signals can only be sensed by receptors on neighboring cells in direct contact with the signaling cell. This highly restricted form of communication is akin to carrying on a confidential conversation with a single person in a low voice. Other kinds of signals are secreted from cells, travel or "diffuse" some distance, and bind to receptors present on the surface of cells several cell diameters away. This more public form of communication is similar to broadcasting or widescale publication of a message. A developmentally important type of secreted sig-

FIGURE 1.8. Cell-to-cell communication—signals and receptors.

nal is called a morphogen (Fig. 1.9). The term morphogen reflects the fact that such molecules determine the morphology or proportions of an organism or structure. The intriguing characteristic about morphogens is that they mean different things to different cells based on how much of the morphogen a cell receives. To be considered a morphogen, a molecule ideally should satisfy three criteria: (1) The molecule should be synthesized in some but not all cells, (2) the molecule should diffuse from its site of synthesis to become progressively less concentrated farther from the source of synthesis, and (3) cells should respond to different concentrations of the morphogen by activating expression of distinct sets of genes.

A typical morphogen signal is synthesized by a small group of cells. The morphogen is secreted from these cells and diffuses over several cell diameters to reach cells that do not synthesize the morphogen themselves. The combination of localized production of the mor-phogen signal and its subsequent diffusion away from its source creates a graded concentration of the morphogen (Fig. 1.9). To create and maintain a graded distribution of the morphogen, it also is necessary to destroy or inactivate the morphogen at a rate that balances its rate of synthesis. Cells synthesizing the morphogen, and their immediate neighbors, experience high concentrations of the morphogen; cells a small distance away sense intermediate levels of the morphogen; and cells farther from the morphogen source detect little if any signal.

Morphogen signals bind to and activate specific receptors, which then alter gene expression in receiving cells. The key defining characteristic of morphogens is that different concentrations of a morphogen activate expression of different subsets of genes. Cells close to the source of the morphogen receive high concentrations of the mor-

Morphogens

Graded concentration of morpho,

'9en

Morphogen

Morphogen

FIGURE 1.9. Graded concentrations of morphogens.

phogen and respond by activating expression of one set of genes to define cell state A (Fig. 1.9). Cells a short distance away from the mor-phogen source receive intermediate levels of the signal and respond by activating a different group of genes to define cell state B, and cells far away from the source activate yet another subset of genes to define cell state C. Because the response of cells to a morphogen is dosage dependent, the set of genes activated in a given cell is determined by the distance between that cell and the source of morphogen. There are examples where morphogens can elicit as many as five distinct responses depending on the concentration of morphogen. In such cases, distance from the morphogen source can be measured in five discrete increments. Although most examples of morphogens are secreted signals, other types of molecules can also satisfy the conditions for being morphogens. For example, in Chapter 3, we show that transcription factors can behave as morphogens under certain circumstances.

Another important type of cellular communication takes place when two equivalent cells communicate to determine which of two alternative identities each cell will adopt (i.e., cell type A versus cell type B). In these cases, it may be completely random whether a particular cell assumes the A or B identity. Often, however, one of the two communicating cells is biased toward one of the two identities. What is important in such binary decisions is that one A cell and one B cell always are produced. A common type of communication assuring this binary result is known as mutual inhibition (Fig. 1.10). In cases of mutual inhibition, one cell state (say A) is the default or preferred state. This means that in the absence of communication, both cells would become A cells (Fig. 1.10A). When the two cells communicate, however, both of them attempt to prevent the other from becoming the A cell type. This mutually inhibitory interaction is very unstable. As soon as one cell sends a stronger inhibitory signal to its neighbor than it receives, it assumes the default A state and forces the other cell to adopt the alternative B cell state (Fig. 1.10B).

Mutual inhibition

A. Equivalent cells signal reciprocally B. Resolution into sending vs. receiving cells

A. Equivalent cells signal reciprocally B. Resolution into sending vs. receiving cells

FIGURE 1.10. Cell signaling and mutual inhibition.

ORGANIZING CENTERS ORCHESTRATE EMBRYONIC DEVELOPMENT

In the early part of this century, Hilde Mangold, a talented graduate student of the prominent developmental biologist Hans Spemann, performed a very important experiment (Fig. 1.11). She cut out small regions from an early frog embryo (the donor embryo) and grafted them into various positions in a second embryo (the host embryo). The goal of these transplantation experiments was to identify regions of the embryo that might influence the developmental potential of neighboring regions. These tedious experiments paid off when Mangold transplanted a small piece of future dorsal tissue from a donor embryo into a ventral position in a host embryo. The dorsal region of vertebrate embryos normally gives rise to the brain, spinal cord, and backbone, whereas ventral regions give rise to nonneural structures such as skin, muscle, and blood. When Mangold grafted dorsal donor cells into a ventral position in a host embryo, she obtained a monstrous two-headed tadpole.

In these grafting experiments, two different species of amphibian embryos with morphologically distinguishable cells were used as donor and host. Because cells derived from these two different embryos could be told apart, it was possible to determine whether the second neural axis (brain and spinal cord) of a two-headed frog embryo was composed of donor or host cells. This analysis determined that the second neural axis was formed entirely from host cells. Because donor cells themselves did not contribute to the second neural axis, it could be inferred that they acted by organizing nearby host cells and "inducing" them to change developmental course by generating neural structures rather than skin. This inductive event, initiated by the dorsally derived donor "organizing" cells (now referred to as the Spemann organizer), was proposed to be mediated by secreted signals referred to as neural inducing factors. It was hypothesized that such neural inducing factors were liberated by the Spemann organizer and received by surrounding host cells. Cells exposed to sufficient concentrations of the neural in-ducer responded by developing as nervous system instead of skin. These experiments ultimately earned Hans Spemann a Nobel Prize for providing the first clear evidence for the existence of embryonic organizing centers that alter the developmental potential of neighboring cells.

Since the seminal experiments of Mangold and Spemann, organizing centers have been identified in various developing organisms. A common property of organizing centers is that they emit morphogen signals influencing the developmental potential of cells over considerable distances. This action at a distance permits a relatively small

1. How the early frog embryo maps onto the tadpole

Frog embryo

Muscle, heart blood

Surface view

Surface view

Internal view

Internal view

Tadpole

Frog embryo

Tadpole

2. Mangold's organizer transplantation experiment

Cut out small [-region

2. Mangold's organizer transplantation experiment

Cut out small [-region

Spemann Organizer emits signals

Donor embryo

Host embryo

Spemann Organizer emits signals

Grafted embryo

Donor embryo

Host embryo

Grafted embryo

3. Organizer grafts induce a second axis

Normal tadpole

Dorsal view

Grafted tadpole

Normal axis

Internal view

Normal axis

Internal view

Graft induced axis

.y -Graft derived

Graft induced axis

FIGURE 1.11. The presence of organizing centers—the Mangold-Spemann experiment.

group of organizing cells to orchestrate the developmental paths of large numbers of surrounding cells. The molecular identities of signals mediating the activity of several organizing centers, including the Spemann organizer, have been determined in recent years and are discussed further in subsequent chapters.

Hilde Proescholdt Mangold's doctoral dissertation won a Nobel Prize for Hans Spemann in 1935 and spawned a search for the inductive, or organizer, factor—often dubbed the "mystery of the century" in embryology and developmental biology—that continues to this day. Mangold's studies with Spemann derived from a line of research focusing on induction that began in the early 19th century with the work of Karl Ernst von Baer and continued with Caspar Friedrich Wolff and others during the 19th century. The term "Auslosung," which could be translated as "permissive induction," was used in 1901 by Curt Herbst to describe his evidence for interactions between embryonic parts in the sea urchin—a stimulus triggering an already-present response.

In this atmosphere of "the first golden age of developmental biology," Spemann performed his groundbreaking eye lens ablation and induction experiments published in 1901. In 1918, he focused on experiments in which small clumps of cells were removed from gastrula-stage amphibian embryos and grafted into different locations in other amphibian embryos at the same stage. He found that these transplanted cells followed the same development as the cells already in the new location.

Hilde Proescholdt came to Hans Spemann's lab at the Zoological Institute in Freiburg in the spring of 1920 from the University of Frankfurt. Her benchmates in the lab included the developmental biologist Johannes (Hans) Hoftfreter and Viktor Hamburger. Hamburger described her as "...open, frank, and cheerful. She had a penetrating intellect " Spemann encouraged Mangold to transplant the various parts of an early unpigmented newt gastrula into different positions in a pigmented newt gastrula. These were technically very difficult experiments performed using a low-power binocular microscope, glass needles, and hair loops. Almost immediately in May, 1921, Proescholdt obtained an embryo that had a large secondary neural tube. This pigmented embryo had received a graft from the upper lip of the blastopore of an unpigmented donor embryo. Because Proescholdt could distinguish between pigmentation of the donor and host cells, she was able to make the critical observation that the secondary axis was formed entirely of host cells (i.e., pigmented). This experiment therefore provided strong evidence for a secreted factor produced in the host cells which redirected the developmental course of host cells surrounding the implant. Over two breeding seasons, she had only six embryos that she thought could be presented in the famous 1924 organizer paper. In all, she made 275 chimeras, of which 55 survived, and 28 had prominent secondary neural axes. Spemann proposed that the donor material had induced the ectoderm of the recipient embryo to become neural tissue. She and Spemann submitted their paper to Roux's Archiv in June 1923, and it was published in 1924. They described the organizer: "A piece of the upper blastoporal lip of an amphibian embryo undergoing gastrulation exerts an organizing effect on its environment in such a way that, if transplanted to an indifferent region of another embryo, it causes there the formation of a secondary embryonic anlage. Such a piece can there for be designated as an organizer."

Hilde Proescholdt Mangold (ca. 1898-1924)

Possibly indicative of women's status in the world of science in the early 20th century, Spemann included his name as author on Mangold's thesis, a procedure not followed with the male students in his lab. Viktor Hamburger, in his remembrance of Mangold, says that Spemann was correct in doing this because "...she apparently did not fully realize the significance of her results." Coincidentally, Ethel Browne Harvey, a graduate student in another lab, performed analogous experiments on hydra 12 years earlier in 1909. She showed induction by transplanted tissue of a secondary axis of polarity in the host hydra. Spemann seemed to know of her work, but he never cited it. It was said that Harvey, too, did not understand the significance of her findings. However, in later years, Harvey told a colleague, "You know that it was I who first discovered the organizer."

While finishing up her doctoral studies, Proescholdt married Otto Mangold, who was also in Spemann's lab, and they moved to Dahlem-Berlin in 1924, where Otto Mangold took up a position at the then Kaiser Wilhelm Institute for Biology. Tragically, Hilde died from severe burns in an explosion of a gasoline heater in her kitchen in September, 1924, at the age of 26. Otto Mangold wrote up his wife's findings under her name and published them in 1929 in a Festschrift for Spemann. Sadly, in Hamburger's words, "It was not granted for her to live to see the great impact her experiment had on the course of experimental biology."

Subsequent work on the inductive factor included discoveries in the 1930s that dead organizer tissue could induce neural plates, and the organizer elicited inherent capabilities of cells but did not provide detailed instructions. The organizer was linked to growth factors in the 1950s, and the inducer was found to be diffusible in the 1960s. Organizer molecules could finally be identified by the newly developing technology of the 1980s, and in the 1990s, noggin, chordin, and follistatin were identified as three likely neural inducers. Thus, the inducing principle, or organizer, is likely to be several factors and not just one. And "the mystery of the century" finally appears to be reaching some resolution.

In this chapter, we have seen that the units of genetic information, genes, which consist of DNA, comprise two parts, a coding region and a regulatory region. All cells in an organism contain the same DNA, which is a double-stranded molecule made up of subunits called bases (A,C,G,T). Different genes are active or expressed, however, in different cell types. When a gene is expressed, the coding region of the gene directs the synthesis of a single-stranded RNA copy of itself. This RNA in turn directs the synthesis of a protein according to the universal genetic code, which converts the sequence of triplets of RNA bases into a sequence of amino acids comprising protein. Proteins are molecular machines that carry out the myriad of functions required for life. The hierarchical relationship of DNA^RNA^protein is known as the central dogma of biology. Mutations are changes in the coding region of a gene that lead to the production of abnormal proteins. Most mutations reduce or eliminate the function of the protein product of a gene. In some rare cases, however, mutant forms of proteins have new activities that can render them dangerous and capable of causing diseases such as cancer.

The regulatory region of a gene functions as an on/off switch to determine where and when a gene will be expressed as RNA and ultimately as protein (i.e., according to the central dogma). Proteins known as transcription factors bind to DNA in the regulatory region of a gene in a sequence-specific fashion and interact with RNA polymerase either to increase the rate of transcription (activators) or to block transcription (repressors). The combination of activator and repressor transcription-factor-binding sites in the regulatory region of a given gene determines which transcription factors can bind to that regulatory region and influence expression of that gene. This combination of activator- and repressor-binding sites thereby defines a regulatory code for when and where the gene can be expressed.

During development, cells communicate with one another to determine which genes will be activated in different regions of the embryo. Cell-cell communication is based on cells sending secreted signals that are received by receptors on other cells. Signals can act over varying distances. Some signals are tethered to the cell surface of the producing cell and therefore only activate receptors in immediate neighboring cells. Other signals travel (or diffuse) varying distances to activate receptors on cells that are separated from those producing the signal. When signals bind to their receptors, they initiate a chain of events that lead to changes in transcription factor activity in the receiving cell, which in turn alters the pattern of gene expression in that cell.

A special type of signal known as a morphogen can cause cells to activate different subsets of genes, depending on the level of that morphogen. Morphogens are often produced in a restricted region of a developing embryo and diffuse into surrounding regions. Because cells close to the source of morphogen experience high levels of the signal, whereas cells farther way sense lower levels, morphogens can activate different patterns of gene expression at different distances from their source. In this way, morphogens can organize the expression of genes and hence the development of large territories in the embryo. Morphogens that can mediate the effect of organizing tissues defined by classic cell transplantation experiments, such as those of Mangold and Spemann, have been identified. In subsequent chapters, we show how development relies on the interplay between signaling morphogens and transcription factors.

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