1. Scheme of the experiment on DNA sequencing from extracellular chromatin.

Many laboratories in different countries are actively involved in solving these issues, and new publications appear quite regularly. Our news is devoted to one of the recent publications on this topic.

The impetus for the work was the curious appearance of very unusual mutants in a large-scale screening study on fish Danio rerio, performed at the Matthew Harris lab at Harvard Medical School. The purpose of the screening was to identify genes that are important for various aspects of morphogenesis in vertebrates. For this, mutagenesis was induced in the fish and then the arising developmental defects were analyzed.

To obtain mutants, a mutagenic substance ethyl nitrosourea was added to the aquariums where males were kept, the action of which led to an increase in the number of mutations in spermatozoa. Some of these mutations affected genes important for embryonic development – such mutations can lead to developmental abnormalities and the appearance of new phenotypic traits. Males were crossed with females that did not undergo mutagenesis, resulting in heterozygous offspring. Further series of crosses made it possible to identify new inherited phenotypes and the nature of their inheritance.

The attention of scientists was attracted by a particularly unusual mutant phenotype: in some fish, additional intercalated long bones appeared in the pectoral fins along the proximal-distal axis (Fig. 2), which were not attached directly to the shoulder girdle, but were located distally behind the radials. Such mutants appeared independently twice; the phenotype in both cases was inherited according to the dominant type.

Fig. 2. Anatomy and development of the fin skeleton in wild type fish and in mutants (rephaim). A – a photo of the skeleton of a wild-type fish fin and the corresponding structure diagram. Proximal radials are highlighted in green. B – photo and diagram of the skeleton of fins in mutants. Sirenev shows newly formed intermediate radials. B – the course of development of the skeleton of fins in wild-type fish (upper row) and in mutants (lower row). The photo shows the stages corresponding to 3, 4 and 5 weeks from fertilization of the egg (wpf – weeks post fertilization). Cartilage is colored blue, bones are reddish. It can be seen that the mutants first form a long cartilage, in the place of which two bones are then formed, between which a joint is subsequently formed (in the place of the arrow). Drawing from the discussed article in Cell

Sequential tracking of embryogenesis showed that first a solid cartilaginous “blank” is formed, inside which two foci of mineralization are formed, which turn into two long bones – proximal and intercalated radials, connected by a full-fledged joint (Fig. 2).

Inserted radials had a complex structure typical of long limb bones. At both ends there were epiphyses, on the outer surfaces of which full-fledged joints with synovial bursae were formed, articulating the intercalary bones with neighboring ones, and blood vessels grew into the periosteum. Moreover, these bones were connected to other bone elements using muscles, as shown schematically in Fig. 3. The article contains high-quality histological preparations, which, if desired, make it possible to personally verify the objectivity of all these statements.

Fig. 3. Scheme of muscle attachment in the fins of wild and mutant zebrafish, as well as in the limbs of tetrapods. Wild-type fish have no muscles attached to the proximal radials. In mutants, muscles also do not attach to the proximal radials, but the newly formed intercalated radials are connected by muscle fibers both with the shoulder girdle (shoulder) and with the fin rays (fin rays). As a result, the musculature of the fin turns out to be significantly complicated and becomes more similar to the musculature of the limb of a quadruped. Drawing from the discussed article in Cell

By sequencing (sequence analysis) of the genetic material, scientists have established the exact location of the mutations. It turned out that in both cases, the mutant phenotype was due to the replacement of one single nucleotide. However, the mutation affected two different genes: in one case, the vav2 gene was affected, in the other case, the wasl gene. Both mutations were nonsynonymous – they led to the substitution of one amino acid in the corresponding protein. Interestingly, none of these two genes were previously known to be involved in the regulation of fish fin development. Moreover, when scientists artificially turned off these genes by introducing deletions above the mutation site using the CRISPR / Cas9 system, the structure of fins in fish turned out to be completely normal. It turns out that the mutations that caused the new phenotype did not lead to the loss of functions by proteins, but to the acquisition of some new function. This is consistent with the fact that the mutations demonstrated a dominant inheritance pattern (that is, the trait manifested itself in individuals not only with a homozygous, but also with a heterozygous genotype). The authors, meanwhile, note that in fish carrying a mutation in a homozygous state, complex violations of many phenotype traits were often observed, and with varying degrees of severity, so that further they worked only with heterozygous individuals.

It is known about the intracellular functions of the aforementioned genes that the protein encoded by the vav2 gene is involved in the regulation of the work of G-proteins (this group of proteins, in turn, regulates many different intracellular processes), and the protein encoded by the wasl gene is necessary for the formation of actin filaments of the cytoskeleton … Expression of both genes was found in the fin buds of Danio rerio embryos. A separate medical marijuana argumentative essay series of experiments with directed gene shutdown (using the same CRISPR / Cas9 system) made it possible to find out that these two genes are part of the same signaling pathway in the regulation of fin development, where vav2 is an upstream regulator in relation to wasl. If the mutant vav2 is active, and the wasl gene is artificially disabled, then fish with the usual phenotype develop. If, on the contrary, the mutant wasl remains, and the vav2 gene is switched off, then an abnormal phenotype is obtained.

Based on what is known about the structure of the wasl protein, the mutation probably influenced the processes of self-inhibition and intracellular localization of this protein. Changes in intracellular localization were also shown by additional experiments carried out by the authors of the study on a model HeLa cell line: the non-mutant version of the protein was concentrated mainly in the cell nucleus, and the mutant version, in the cytoplasm.

To understand which parts of the skeleton are homologous to each other in the structure of fins and extremities, it helps to study the expression of well-known genes-conductors of ontogenesis, among which the Hox-genes are especially famous. Thus, it was found that the development of the cutaneous rays of the fin blade of teleost fish depends on the Hoxd13 gene, and the normal development of the skeleton of the hand, that is, the most distal part of the skeleton of the forelimb, depends on it. Hence, it is possible to draw a presumptive conclusion about the homology of the skeleton of the hand and the fin blade.

A logical step was to check how the expression of Hox genes in mutants changes in the part of the fin where intercalated long bones are formed. The test did not disappoint: the expression of the Hoxd11a gene was found to be increased in the mutant fin bud, and the artificial deactivation of this gene in mutants for the vav2 or wasl genes led to the restoration of the usual phenotype. And, on the other hand, experimental switching off of the wasl or Hoxd11 genes causes an anomaly in the development of the forearm in mice! Very curious conclusions suggest themselves: the mutation, as it were, reproduces the isolation of the “shoulder” and “forearm” in the structure of the Danio rerio fin skeleton.

Fig. 4. Areas of Hox gene expression in different parts of the fins in wild-type and mutant fish, as well as in the limb of a quadruped. The Hox11 gene, which “marks” the forearm in the skeleton of the limb, is also highly expressed in the insertion radials of the mutants. At the same time, the fin rays in the fish skeleton, as well as the bones of the hand, are “marked” by the expression of the Hox13 gene. Drawing from the discussed article in Cell

How closely does the evolutionary scenario reproduce the phenomenon observed here? One should not get too carried away in conclusions. The authors themselves point out that the positions affected by mutations are extremely conservative: in proteins of all vertebrates, the same amino acids are located in these positions. That is, the increased expression of wasl and hoxd11, which is necessary for the formation of the forearm, is provided by some other regulatory mechanisms.

And yet, there is reason to believe that the similarities in the mechanism of embryonic development of the limbs of tetrapods and the complex skeleton of the fin in Danio rerio mutants were not without reason. The authors interpret the transformations observed in mutants as the realization of the "latent developmental potential" contained in the system of regulation of the embryonic development of paired fish appendages, which was probably realized during the evolution of ancient vertebrates. In discussing their results, they quote from Darwin’s The Origin of Species: “If a previously lost trait reappears after many generations, then the most plausible hypothesis is not that the offspring suddenly awakens the trait of their ancient ancestors, but that they retain a tendency to develop of the considered trait, which realizes itself in some new favorable conditions "(" When a character which has been lost in a breed, reappears after a great number of generations, the most probable hypothesis is, not that the offspring suddenly takes after an ancestor some hundred generations distant, but that in each successive generation there has been a tendency to reproduce the character in question, which at last, under unknown favorable conditions, gains an ascendancy “).

This study prompts us to revisit the concept of “promising monsters” proposed by Richard Goldschmidt in the 1930s: we return to the question of whether, in some cases, a single mutation can create serious innovation (complication) through transformation of development processes and thus to give a basis for a completely new group of organisms with a fundamentally different plan of body structure from related groups (on this topic, see the review by I. Yu. Popov Monsters in evolution).

Source: M. Brent Hawkins, Katrin Henke, Matthew P. Harris. Latent developmental potential to form limb-like skeletal structures in zebrafish // Cell. 2021. DOI: 10.1016 / j.cell.2021.01.003.

See also about limb evolution:1) The rate of reduction of fingers in archosaurs depends on genes that regulate embryonic development, "Elements", 17.10.2013.2) Newly discovered fish genes helped to understand why the first tetrapods were multi-fingered, "Elements", 07/09/2010.3) Long fins of rays – the result of adding a new growth point, "Elements", 12/22/2015.

Tatiana Romanovskaya

Fig. 1. Scheme of the experiment on DNA sequencing from extracellular chromatin. The cells of the body differ in the activity of their genes. For example, the ALB gene, which encodes the albumin protein, is active in liver cells (yellow), but remains inactive in heart cells (heart cells). Blood samples were collected from healthy people and patients with various diseases and blood plasma was isolated. The blood plasma contains extracellular chromatin (cell-free chromatin), consisting of extracellular DNA and histone proteins with appropriate modifications (red dots – modifications of inactive genes, green dots – modifications of active genes). This chromatin belongs to cells from a variety of sources. Then, using the chromatin immunoprecipitation method (ChIP), chromatin fragments containing the histone modifications of interest were isolated. Then the DNA sequences in the isolated chromatin were determined using next-gen sequencing and, finally, the results were compared with published data on the results of DNA sequencing of immunoprecipitated chromatin in various cell types. Illustration from a discussed article in Nature Biotechnology

There is a small amount of extracellular DNA in the blood plasma of each person. Its amount increases in patients with various diseases, such as tumors or infections. The main source of extracellular DNA is decaying cells. However, for a long time there were no reliable methods to determine exactly which cells this DNA belonged to for a long time. Researchers in Israel have taken advantage of the fact that extracellular DNA is associated with histones and other chromatin proteins and developed such a method based on careful analysis of epigenetic tags. The method has been tested on blood plasma samples from over 250 volunteers. It turned out that in healthy people the main source of extracellular DNA is bone marrow megakaryocytes, and in patients – different groups of cells from damaged organs (and in the case of cancer, tumor cells). In addition, the method shows which parts of the DNA of the original cells were working at the time of the appearance of extracellular DNA in the blood plasma. The developed approach can become the main one for new minimally invasive methods for diagnosing diseases.

In a milliliter of blood plasma of a healthy person, approximately 1-10 ng of extracellular DNA (Circulating free DNA) can be found. Its main source is human cells, which have completed their work and have been destroyed through apoptosis, necrosis, or other cell death processes. In patients with cancer, the concentration of extracellular DNA in the blood plasma is higher than in healthy people due to the contribution of tumor cells.

New DNA sequencing techniques have turned extracellular DNA into a diagnostic tool. In this way, non-invasive diagnostics of oncological diseases can be carried out (see. Based on tumor DNA circulating in the blood, it is possible to diagnose relapses of lung cancer very early, "Elements", 08/22/2017). To do this, patients extract extracellular DNA from blood plasma, and then test it for the presence of mutations specific to various types of tumors (for more details about these methods, see the review by S. Fiala, E. P. Diamantis, 2018. Utility of circulating tumor DNA in cancer diagnostics with emphasis on early detection). However, the most famous diagnostic method based on the study of extracellular DNA is non-invasive prenatal tests. They are based on the analysis of fetal extracellular DNA circulating in the mother’s blood (for more details on the development of the method, see Breakthrough Prize awarded for protein design, pheromone receptors, mitochondrial utilization and extracellular DNA, Elements, 09/23/2020).

But sequencing is not the only way to study extracellular DNA. In addition to the nucleotide sequence itself, DNA fragments also have epigenetic modifications that reflect the work of genes or, in general, all processes associated with DNA and do not affect the nucleotide sequence. A detailed story about epigenetics can be found in the article by Alexei Rzheshevsky and Alexander Vayserman Epigenetics: Genes and Something Above.

The most studied of these modifications is the methylation of the cytosine bases of DNA, that is, the addition of a methyl group to the cytosine located next to guanine (CpG, where C is cytosine, p is a phosphodiester bond, G is guanine).