by Sergei SAVELYEV, Dr. Sc. (Biol.), Head of the Department of Embryology, Institute of Human Morphology, Russian Academy of Medical Sciences

Medical statistics has fixed a steady increase in the incidence of congenital abnormalities of the brain all over the world. Neural disorders are responsible for 60-65 percent embryonal and fetal mortality. What are the causes of this increase? We have no exhausting answer to this question yet, but we can assert that the problem will become more clear after deciphering of mechanisms regulating the formation of the nervous system. This was the subject of the interview of our correspondent Sergei Popov with Sergei Savelyev, a specialist in embryonal development, Doctor of Biological Sciences.

—  Sergei Vyacheslavovich, you have probably heard an opinion that genetic failures, largely caused by deterioration of ecological situation, are "responsible" for the increase in the incidence of intrauterine pathologies ?

—  This is not the only reason fixed by statistics. Of course, our notions have advanced considerably since the Middle Ages, when the time of entering of the psyche into the human embryo was an object of ardent discussions, as its presence was assumed to guarantee normal development. In late 19th and early 20th centuries embryonal death and spontaneous abortions were attributed solely to infections. Later on there predominated ideas of hormone defects. For the last decades the focus on genetic determination led to predominance of another viewpoint: the entire ontogenesis was considered as a linear realization of the genetic program, and its disorders were assumed to be responsible for deviations. However, this hypothesis was not confirmed: the actual percentage of genetic defects in human embryos with pathologies does not exceed 10 percent, 5 percent are caused by infections and toxins, while the causes of 85 percent of them, i.e. the greater part of all neural disorders, remain a mystery. According to the newest hypotheses, the majority of disorders emerge due to environmental pollution. According to these views, toxic agents do not modulate the embryonal genome directly, but act through special regulator molecules or, what is worse, through regulator genes. However, no obvious molecular defects have been revealed up to the present time.

Looking back to the history of the problem, let us remind you that modern studies of human development pathologies started simultaneously in several countries, including Russia. At the end of the 19th and beginning of the 20th century German anatomist and embryologist Wilhelm His (Foreign Corresponding Member of St. Petersburg Academy of Sciences from 1885) and Nikolai Kash-chenko, Russian zoologist, noted that embryonal abnormalities were present at early stages of development. As for

Experiments on modification of positional information coding in embryos of amphibians. Top left-neuroectoderm hyperstretching, to the right-anatomical consequences, below-histological series of brain sections (the norm is yellow-colored).

the nervous system, the scientists gradually agreed that the greatest part of these disorders (deviations) formed within the first 4-6 weeks after fertilization. These ideas became universally acknowledged by the end of the 1930s and, being confirmed by numerous facts, remain the most prevalent up to the present time. I should like to draw your attention to the following: judging by this evaluation, the embryo is the most sensitive to all kinds of negative factors during the very first month of pregnancy. Why? We shall speak about it a little bit later.

The figures confirm that the embryonal morphogenesis is fraught with numerous risks. According to common estimations, only one of a hundred of fertilized ovicells is transformed into a newborn. No doubt, this pressing problem deserves detailed studies. This is particularly so for the early postimplantation period, which is insufficiently studied as the necessary material is unavailable. Even in medical manuals, the authors have to illustrate this period on the basis of mouse embryos "at development stages similar to human". Despite the progress in genetics, molecular and cellular biology, we still know virtually nothing about the mechanisms of early embryonal formation of various systems of human organism.

—  That is, it remains unclear how this or that organ-liver, heart, brain, etc.—acquires its peculiar form, is that so?

—  Yes, it is. There is a gap between the DNA structure and the three-dimensional morphology of the organism. The genetic code is recorded in heredity molecules, which indicates the amino acid sequence for their future assembly into molecules of various proteins. But the gene is a linear structure, it has no "design" for the shape of ear, eye, nose, or length of legs. It contains only records for proteins-the basic material for future construction.

These or other proteins appear during different periods of the embryonal development. Special regulator genes "control" the time and velocity of their synthesis. These processes have nothing to do with the shape of the organ-

ism. The shape formation laws are not reduced to biochemical reactions. For example, left and right hands of the same human being differ significantly and are indispensable, though their genetic system is the same. Alas, in embryology morphological signs emerge and disappear before appearance of the first heterogeneity in the gene activities more often than we would like to.

—  What are the events that occur during the first, so much vulnerable weeks of pregnancy ?

—  Fertilized ovicell divides first into 2, then into 4, then into 8, 16 cells, and so on. The spherical single-layer embryo, consisting of about 1,000 cells, after a series of transformations becomes multi-layer. The ectoderm (surface layer) forms rudiments of the nervous system (it is the first to form—in the period from the 18th to the 27th day after fertilization), skin epithelium, organs of senses. The muscles, cartilages, bones, hemopoietic- and lymph-forming organs form later from the middle layer (mesoderm). The intestinal epithelium and glands connected with it (the pancreas and liver) as well as the lungs form from the inner layer (endoderm). By the 30th day the rudiments of limbs, body, and head become discernible.

— And how is the course of this wonderful "construction " marked? And what happens first: the unfolding of the genetic program, associated with protein synthesis with subsequent changes in the spatial cell-to-cell interactions, or these latter ones trigger the genetic cascade ?

—  I should say that the results of numerous experiments carried out in different countries can serve as an answer to these questions. They indicate that selective destruction of even a half of cells of an experimental animal embryo at the early stages of development does not lead to a loss of its viability; the losses are compensated. Hence we conclude that if the death of even many cells is not fraught with the pathology, the program of their behavior, I should like to emphasize—at the early stages of the embryo formation—is not inside them. The

hypothesis according to which the information from one cell to another is transferred by a chemical route, is not confirmed experimentally, as according to this theory, the loss of a part of these cells should block the diffusion of chemical substances and impair the signal transfer with a subsequent development of the pathology, which is not observed. The question is—what mechanism actually works here?

We presupposed that in the embryo the information is transferred from cell to cell by a biomechanical route. In order to explain this fact, I shall first remind you that a cell, similarly as an organism, is a mechanically tough construction. Under conditions of gravitation, its shape is supported by an inner carcass—a cytoskeleton, based on several specific proteins. Due to the cytoskeleton, it reacts to the "actions" of its neighbors: shrinks transversely if stretched and vice versa. As for the cell-to-cell mechanical tensions—they serve as a carrier of positional information. It informs about the place occupied by the cell in the embryo, time and program of its actions. It is not accidental that 90 percent of all receptors in our organism are mechanoreceptors. The "reading" of the data on the pressure, compression and tension is realized by millions of mechano-dependent ionic channels* located on the surface of each cell and described for the majority of animal and plant tissues (in reality the channels are opened if the cell is pressed, the membrane charge is altered, and in a minute the concentration of chlorine inside is increased—this is how it "learns" about the external pressure).

In contrast to an adult organism, the embryonal cells are arranged rather loosely, as they are suspended in a liquid. And even a slight mechanical pressure is sufficient to re-charge the ionic channels and read a positional signal. However, this process is slow, the channels have to be open during several minutes in order to rule out an error as a result of which the development can be disturbed.

—  Then what is the role of the genome in the embryonal cell?

— When an external pressure is longer than 7 minutes, the cell reads the signal as information and "triggers" the genes inside the nucleus. This leads to production of certain proteins and to a start up of genetic cascades, arresting the reading (the cell receives 10-12 signals during the entire period of embryonal development). The closer is the end of differentiation—the lesser is a role of the positional signal.

In our model the mechano-dependent ionic channels and the cell-to-cell interactions during embryonal development are stimuli determining the primary design. The genome in this case is an inhibitory system, i.e. biomechanical contacts precede a start up of genetic programs.

—  Not a new idea: the theory is verified by an experiment...

—  We showed in experiments on pregnant rats in late 1980s and early 1990s that blocking of mechano-dependent ionic channels caused development pathologies similar to those in humans. Later on we planned an experiment, in which a part of animal embryonal cells were to be exposed to conditions modifying their subsequent fate. Thus, two glass needles with 4-µ tips were inserted into the intercellular space of the embryonal neuroepithelium (the experiment was carried out on an amphibian). These needles were then slowly separated (using a special device) to a distance of virtually 1 µ, fixed for 7 minutes, after which the needles were removed and the embryo continued its normal development, except the group of cells, exposed to an unusual tension field. This resulted in programmed changes: doubling of head structures or enlargement of a

* Ionic channels are pone-forming proteins, supporting a slight difference between the potentials of the external and internal sides of the membrane of all living cells. Due to them, ions move in accordance with their electrochemical gradients.-Ed.

Experiments on embryos of amphibians to study mechanodependent ionic channels (arrows indicate directions of tension and compression). Scanning electronic microscopy.

Reactions of neuroepithelial cells in polarization, deformations of cell layers, and stretching.

Human embryo and placenta in health (6 weeks after fertilization).

certain brain compartment*. It has become clear that the morphogenesis can be literally regulated by mechanical pressure on the cells. For quantitative evaluation of the results of the experiment, the entire system during stretching was plunged in liquid nitrogen to stop ion movements. Then the embryo was examined in a scanning electron microscope with an X-ray detector to count the ions directly under the membrane. After measuring the number of ions which passed through the membrane during the above-mentioned phases of the exposure, we have found out that in amphibians the positional information is coded by chlorine mechano-dependent ionic channels, as the content of chlorine under the membrane increased, reaching the maximum during the 7th minute of exposure. Hence, we have confirmed not only the fact of sensitivity, but also the fact of modification of the subsequent fate of cells by the positional signal.

—  But how can you explain the causes of embryonal pathologies, emerging during the very first month after fertilization, by these data?

—  I have already mentioned that the rudiments of the nervous system—the brain and spine—are formed during the very first month of gestation. The initially spherical embryo, after a series of transformations acquires the shape of a strip. The neural plate is formed, which then transforms into the neural tube (this development stage is called neurulation by specialists). And then the nervous system starts its development as a more or less indepen-

* See: P. Simonov, "My Brain and Me", Science in Russia. No. 5/6, 1992: S. Medvedev, "'Universe' of the Brain", Science in Russia, No. 3, 2003: A. Ivanitsky, "Physiological Basis of Consciousness"; I. Shevelyov. "The Brain and Recognition of Visual Images", Science in Russia. No. 3, 2007.-Ed.

Human embryo, 55 days after fertilization.

Macrophotos of human embryos. In the center-neurulation stage (2 mm), to the right-in health (5.5 weeks after fertilization), to the left-pathology.

dent organ. Its size during this period is still very small, as the length of the entire embryo by the 23rd day of development is no more than 3.5 mm. The process of neural plate transformation into the neural tube is rather vulnerable, its course is normal only if there are no failures in positional information, carried (as was mentioned above) by intercellular mechanical tensions.

Our experiments on amphibians showed that the increase in the neuroblast (embryonal nerve cells) mechanical sensitivity coincided with the moments of changes in the organization of tension fields in a developing embryo. For example, before gastrulation* its mechanical sensitivity reduces: at the beginning of this stage its sensitivity to tension increases, then gradually decreases almost to zero. The scheme of events is the same in neurulation. Starting from the blastula stage, the neuroblasts grasp the direction of mechanical stretching or compression of the cell layer. The morphological organization of the embryo at subsequent stages changes, depending on the stretching/compression angle. Exposures coinciding by direction with normal tensions of the neuroectoderm (neuroblast layer) caused no appreciable changes in the development of the embryo. However, its stretching or compression at an angle of 8-12° to the axis of the layer violated the smooth course of events. If the experiment was carried out at blastula or gastrula stages, neurulation was arrested at the very beginning. In analogous experiments at the neural plate stage, the forebrain differentiation was disturbed. Various

* Gastrula is a stage of embryonal development of multi-cellular animals, following the blastula. The blastula has a single-layer structure, while the gastrula has two and then even three layers.-Ed.

Two histological sections through human embryonal head, 7.5 weeks. To the left-norm, to the right-pathology.

forms of anencephaly* in combination with brain hernias and cystic cleavage of the spine, characteristic of humans as well, were induced by modifying the angle and topology of the exposure.

Briefly, despite their great diversity, all variants of embryonal anomalies of human brain development can be classified into 3 main groups. Group 1 includes pathologies caused by disorders in the closure of the anterior terminal of the neural tube. Group 2 includes all variants of development disorders in the diencephalon and midbrain region. And group 3 includes changes in the metencephalon, medulla oblongata, spinal cord, and entero-cerebral channel.

As the terminal part of the neural tube is closed earlier than in other areas, we, as a rule, very rarely observe anomalies of the diencephalon and midbrain at the "roof level of these compartments. On the other hand, cystic cleavage of the spine (a prevalent condition) is, as a rule, associated with various types of spinal hernias and other malformations. The high incidence of anomalies of the metencephalon, medulla oblongata, and spinal cord is explained by a rather long neurulation in the embryonal spinal compartment. It is clear that the probability of toxic or other teratogenic** effects on the extremely vulnerable neurulation process is much higher for the spinal cord than at the level of the diencephalon and midbrain.

The incidence of anomalies associated with the closure of the anterior terminal of the neural tube is intermediate in comparison with the two above-mentioned groups. The results depend on the neurulation period in the zone, during which this zone is exposed to teratogenic factors. For example, if the harmful exposure occurs during the 23^rd day after fertilization, the anterior section of the neural tube will be damaged. In this case the anomaly will involve blocking up and primary differentiation of forebrain hemispheres. The most prevalent condition is alobar pros-encephalia, which is manifested by their complete inseparability. They are formed as a single protrusion, which persists in this form in fetuses and newborns.

We suppose that errors in the reading of the positional signal, leading to development pathologies, are caused by destruction of the cytoskeleton and by blocking up of the mechano-dependent ionic channels.

—  Can you enumerate risk factors associated with this pathology ?

—  Any substances, modulating the cytoskeleton, which a future mother can use, are dangerous. Just recently nobody suspected that tranquilizers of tazepam series, often used by women who are nervous during the early terms of pregnancy, depolymerize the cytoskeleton. The positional signals can no longer be read, the deflection of the neural tube is terminated, leading to the pathology formation. But we must speak here not only about tazepam, as, unfortunately, probable negative effects of drugs, used by future mothers, on fetal development are virtually not studied in Russia. In addition, I have to mention that the vitamin A excess is as dangerous as its deficit. The same is true of oxygen. One and the same substance in different concentrations can cause an anomaly. Or, say, foodstuffs containing a lot of preserving agents and other chemical additives—the list of risk factors is extremely long, it includes thousands of substances. As the information signal emerges from intercellular interactions, the embryo is extremely vulnerable.

* Anencephaly is a congenital abnormality, accompanied by the absence of the cerebral hemispheres.—Ed.

** Teratogenicity is a capacity of physical, chemical, or biological fac-tors to cause disorders in the embryogenesis process, leading to emergence of congenital teratoses.—Ed.