The acquisition of higher-order cognitive functions in primates required the enlargement of the functional area of the cerebral cortex, addition of new areas, and complex organization of neural circuits, along with the anatomical enlargement of the cerebral cortex. To clarify the mechanism underlying the acquisition of these higher-order cognitive functions it is important to understand the mechanism underlying the formation of the functional areas in the cerebral cortex (e.g., the prefrontal area) and the areas that form direct connections to these functional areas (e.g., the pulvinar and hippocampus) during embryonic development. These functional and associated areas are unique to primates and not present in common experimental animal such as the mouse, therefore, we used the small New World monkey, the common marmoset, as a primate model organism. Our aims are to, first, characterize the expression patterns of genes in the marmoset brain whose functions have already been established experimentally in the mouse brain. Additionally, we aim to analyze the effects that genes that are unique to marmosets have on mouse brain development when expressed ectopically in the mouse brain. Finally, we aim to improve the method of introducing genes (transfection) in utero into the embryonic marmoset brain to establish an experimental system with which functions of these genes can be directly analyzed to clarify the mechanism underlying the evolution of the cerebral cortex that is unique to primates.
(1) Comparative analysis of gene expression in the marmoset and mouse brains
Throughout embryo development, gene expression patterns are often spatially restricted and dynamic. This dynamic expression plays an important role in stage-specific events, such as pattern formation in the brain and connection of neural circuits. Information on gene expression patterns in the mouse brain is widely available and has been compiled into databases, such as the Allen Brain Atlas. However, information on stage- and area-specific gene expression patterns in the marmoset brain is limited. Therefore, the expression patterns of genes in the marmoset brain need to be characterized first, in order to compare to mouse genes with spatially restricted expression patterns in the mouse brain whose functions have already been described. In particular, we will focus our analysis on the prefrontal cortex and the areas that form direct connections to the prefrontal cortex, which are considered to be associated with higher-order cognitive functions in the primate brain.
(2) Functional analysis of genes unique to marmoset in the mouse brain
On the basis of the results obtained in (1), we are analyzing the functions of genes that are expressed in a stage- and area-specific manner in the marmoset cerebral cortex. We are focusing on genes that are expressed in specific layers of the marmoset brain, unlike those in the mouse brain (e.g., subplate neurons and Cajal-Retzius cells), in addition to genes that are expressed in a spatially restricted manner in the marmoset cerebral cortex. Once characterized, we functionally test these genes by locally overexpressing them in the mouse cerebral cortex. The marmoset genes are introduced into the embryonic mouse brain in utero and the effects of the transfection on the mouse cerebral cortex and the formation of neural circuits are examined. Furthermore, the genes that are specifically expressed in the marmoset thalamus are overexpressed in the mouse brain using the same method in order to determine the roles of the thalamus, which forms many connections to the cerebral cortex, and to analyze the effects of the genes on the development of the connection between the cerebral cortex and the thalamus.
(3) Establishing a method of transfecting genes into the marmoset brain in utero
In order to analyze in more detail the roles of the above-mentioned genes on brain evolution that is unique to primates it is necessary to manipulate the dose, timing, and location of expression of these genes. To this end, the generation of transgenic marmosets is advantageous, although it takes a long time to generate such marmosets. In this study, we aim to establish a system for precise and efficient transfection of genes into the embryonic marmoset brain in utero. Thus far, various plasmids and electrodes that can be used for this method have been developed. These plasmids and electrodes can be applied to marmosets in a creative way. To advance this technique the following are required: development of a safe method of open abdominal surgery of pregnant marmosets, development of a technique to visualize the fetus through the uterine wall, development of an efficient technique of gene transfection into a targeted area, and designing plasmids that have an efficient transfection rate into marmoset neuronal cells. After this technique is developed, candidate genes will be transfected into the marmoset brain one by one on the basis of the findings obtained in (1) and (2). The effects of gene transfection on the changes in the functional areas of the cerebral cortex and formation of neural circuits will be assessed using genetic markers developed in (1) and other methods.
The findings of this study will provide insight not only into the mechanism underlying the evolution of higher-order cognitive functions in primates but will also demonstrate the usefulness of marmosets as a primate animal model, which will further promote research using marmosets.
