What is the Mechanism by Which a "Species" is Formed?

A “mule,” the offspring of a male donkey and a female horse, cannot reproduce, and therefore cannot leave any descendants. The phenomenon where a cross between two species is a threat to life or reproduction is known as “reproductive isolation” and has attracted the interest of biologists since long. In this context, it is important to understand the fundamental question: ‘What is the mechanism by which a “species” is formed?’ Appropriate bio-resources such as genetically identical inbred strains, which allow accurate gene analysis and gene manipulation, are necessary to address this difficult problem. The National Institute of Genetics maintains various “model animal” strains for experimental use. Among these, a “consomic” mouse strain, wherein one chromosome has been replaced with another from an inbred strain, was used. The results of this research, published in April 2014, shed light on the long-standing mystery of “reproductive isolation.” Below, we outline the research activities of Project Researcher Ayako Oka (Transdisciplinary Research Integration Center) from the “Gene Function (life) System” project.

Overview of global distribution of mouse strains

The rodent in the photograph, with a panda-like fur pattern, is a “fancy mouse” strain and has roots in a pet shop in Denmark. The Edo-period document Chingan Sodategusa (a rough guide to breeding mice for rare tastes) mentions that the strain was originally bred in Japan as pets for collectors, and was reported in Europe towards the end of the Tokugawa Shogunate. Currently there are two main subspecies within the global distribution of mouse subspecies: the “domesticus” found from Africa to America and western Europe, and themusculus” distributed across a wide zone from the Eurasian land mass to eastern Europe. It is believed that these two subspecies emerged from a common ancestry approximately 0.5-1 million years ago and have accumulated mutations over a long period. These species met several thousand years ago at a line arching from Germany to the eastern tip of Greece, called the “hybrid zone.” It is known that the two subspecies developed reproductive isolation at this hybrid zone and culminated in genetically separate populations. This raises an important question: What would we discover if we compare their genomes?

Repatriation of the “Bean-Spotted” Fancy Mouse

The Dobzhansky-Muller model

The famous Dobzhansky-Muller model can provide an answer to the above question. Supposing that within Group A separated from a common ancestor possessing genes “AABB,” a new genotype “a” arises and “aaBB” becomes entrenched. Similarly, in Group B, “AAbb” becomes entrenched. Reproductive isolation will occur if Group A happens to meet Group B, and if genotypes “a” and “b” are incompatible. Various studies carried out in this regard have suggested that the rationale underlying the origin of this phenomenon probably lies in a protein made by the gene AABB. However, until date, almost no genes have been discovered which may explain the separation of species in mammals. In 2009, the Prdm9 causal gene was reported for mice, but its specific separation mechanism was not explained. Since long, I have been engaged in the task of searching for causal genes. However, the more genes are limited the more the phenomenon of reproductive isolation disappears. Our current research was prompted by the assumption that a causal gene truly exists might be mistaken.

Replicating reproductive isolation accurately in a lab

The phenomenon of reproductive isolation that occurs in the hybrid zone is possible to replicate in laboratory settings. The genetic background of the current and most standard experimental strain, B6, is 90% European-derived and 10% Japanese-derived. Therefore, it can be treated as ‘domesticus’. An investigation into a B6 consomic strain, particularly in the X chromosome of an MSM strain equivalent to a molossinus subspecies genetically close to musculus, has confirmed abnormal sperm formation in this strain. This abnormality did not appear in the Y chromosome of consomic strains or in the 19 autosome pairs possessed by the mouse subspecies. Further investigation on the testicular gene expression of the X chromosome in the consomic strain revealed that the expression was lower for 15% of all X chromosomal genes and higher for 5%. A fixed pattern was however not revealed. Based on this, we carried out further statistical analysis while focusing on the cis-regulatory element that controls the level of expression near a gene. We found that the reduction in gene expression was due to the genetic incompatibility of the X chromosome in the MSM strain cis-regulatory sequence, and the autosome in the B6 strain transcription-regulation factor. In addition, many of the genes with reduced expression were those that bore function as germ stem cells related to reproduction. It is generally accepted that the evolution of genes related to reproduction is fast in those organisms exposed to harsh survival conditions. This latest finding might be significant in providing an explanation for the reproductive isolation between subspecies in the process of differentiating, because the transcription regulator itself has evolved faster than the resulting gene expression.

Confirmation of a hypothesis not only by human observation but also by statistical analysis

Repeated examination of a graph explaining the distribution of gene expression data indicated that a possible fluctuation in expression might not actually be the primary cause of reproductive isolation. This gave me the feeling that the cause of reproductive isolation as previously thought did not lie in the differences in specific protein molecules between subspecies. The X chromosomal distribution was wide enough to be clearly noticed after human observation. Thus, we investigated further whether this wide distribution bore any statistical significance or if it was simply stochastic. This problem was solved as a part of the “Genetic Function (life) System” project, in joint research with Professor Hironori Fujisawa of the Institute of Statistical Mathematics. An important basis of our proposed model is Professor Fujisawa’s statistically difficult “test for statistical significance of distribution.” We are currently accumulating further expression data via large scale RNA-sequencing. In joint research with Professor Shinji Kondo of the National Institution of Polar Research (Transdisciplinary Research Integration Center), we are now set to use this data to explain the molecular mechanism of reproductive isolation in greater details.

Besides the maintenance of strains by breeding, the National Institute of Genetics has a department responsible for maintaining a variety of laboratory mouse subspecies by freezing. Early embryos produced by artificial insemination are frozen, and the stored embryos are thawed at the time of use and transplanted into a mouse’s womb. The formal names of the two subspecies’ are Mus musculus and Mus musculus domesticus.

(Text in Japanese: Ayako Oka, Rue Ikeya. Photographs: Mitsuru Mizutani. Published: July 10, 2014)