Two projects are ongoing in our laboratory. The first project is focusing on the development of the cerebral cortex, and the second project is about stem cell aging. To carry out these project, we basically analyze mutant mice (knockout mice and transgenic mice) employing histologically techniques, biochemical experiments, molecular techniques including genome analyses, cell culture systems, and examination of the animal behaviors.

 

(1) Development of the cerebral cortex

Dorsal part of the mammalian telencephalon gives rise to the cerebral cortex. The cerebral cortex is six layered structure, and layered arrangement of the cortical neurons is essential for the function of neuronal circuits. To make this laminated morphology, cell proliferation of the stem cells, differentiation of neurons, migration of the differentiating neurons, and formation of axon, dendrites, and synapses should be precisely regulated. We want to know how such complex developmental events works and what kind molecules are involved here. Because developmental disorders of the cerebral cortex are involved in psychiatric diseases, obtaining insights into the cortical development will contribute to understanding of pathogenesis of the diseases. In facts, human genome analyses suggested that some molecules that we study are involved in schizophrenia, dementia, autism, and normal development of human brain functions.

 

Analyses of three lines of knockout mouse with disrupted cerebral cortex.

 

1. Dab1 conditional knockout mouse

Dab1 functions in downstream of Reelin, the large extracellular glycoprotein. Morphological abnormalities in the brain of Dab1 mutant and reeler (Reelin mutant) is indistinguishable, suggesting that Reelin and Dab1 are components of a linear signaling pathway. In reeler cerebral cortex, the six-layered structure is highly disrupted because of dysregulated radial migration of the differentiating neurons during embryogenesis (Dekimoto et al., 2011). Yotari mutant (null allele of Dab1) exhibits cerebellar ataxia. Thus, Yotari is not suitable for behavioral analysis to know brain function because of this motor dysfunction. In Our conditional knockout (cKO), Dab1 gene is deleted only in the cerebral cortex, and motor functions of the cKO are normal. We found cognitive function of this cKO mice become weak earlier than that of control mice in the time course of aging (Imai et al., 2017; Fig. 2). We examined developmental abnormalities in the Dab1 cKO hippocampus (Blume et al., 2017; Fig. 3). We are examining relationships of Dab1 and dementia using this cKO. 

2. The mice without the cerebral cortex

Based on microarray screening, we found Sno gene is downregulated by malformation of the cerebral cortex. GWAS study of human genome suggested that this gene is essential for normal brain development and suggested to be involved in intelligence, but function of the gene in the cortical development have not been experimentally studied (Baba et al., 2007). We examined developmental function of the gene employing zebrafish embryos (Takano et al., 2011). Knockdown of the gene reduced size of the head in zebrafish (Fig. 4A,B). We constructed knockout mouse lines of this gene, and found that the knockout mice can be born, but the knockout mice did not have the cerebral cortex (Fig. 4C,D). We are employing biochemical experiments to know the functions of Sno protein.

 

3. The mice with thin cerebral cortex

We found a single nucleotide polymorphism in the genome of a patient with mild craniofacial dysmorphism and mental retardation. Protein product of the mutated gene is expressed specifically in the cortical plate of the developing cerebral cortex. We recently constructed knockout mouse line of this gene, and found that the mutant exhibits spastic paralysis in hindlimbs. The cerebral cortex was much thinner than the control cortex (Fig. 5). We are performing detailed analyses of the phenotypes of the mutant mice. We are also trying to reveal significance of the mutation, which was found in the patient, in functions of the protein employing CRISPER/CAS6 gene editing technique.