Yoichi Gondo

Behavioral phenotypes of Disc1 missense mutations in mice

To support the role of DISC1 in human psychiatric disorders, we identified and analyzed two independently derived ENU-induced mutations in Exon 2 of mouse Disc1. Mice with mutation Q31L showed depressive-like behavior with deficits in the forced swim test and other measures that were reversed by the antidepressant bupropion, but not by rolipram, a phosphodiesterase-4 (PDE4) inhibitor. In contrast, L100P mutant mice exhibited schizophrenic-like behavior, with profound deficits in prepulse inhibition and latent inhibition that were reversed by antipsychotic treatment. Both mutant DISC1 proteins exhibited reduced binding to the known DISC1 binding partner PDE4B. Q31L mutants had lower PDE4B activity, consistent with their resistance to rolipram, suggesting decreased PDE4 activity as a contributory factor in depression. This study demonstrates that Disc1 missense mutations in mice give rise to phenotypes related to depression and schizophrenia, thus supporting the role of DISC1 in major mental illness.

Phylogenetic conservation of a limb-specific, cis-acting regulator of Sonic hedgehog ( Shh).

Polarized expression of the Sonic hedgehog (Shh) gene in the posterior mesenchyme is essential for pattern formation in the appendages of higher vertebrates, from teleost fins to tetrapod limb buds. We report on a sequence in intron 5 of the Lmbr1 gene, which resides approximately 1 Mb from the Shh coding region in the mouse genome and is highly conserved among teleost fishes and throughout the tetrapod lineage. Positional cloning revealed that two mouse mutations, Hx and M100081, characterized by mirror-image digit duplication and ectopic anterior Shh expression, have base substitutions in this sequence. Absence of the conserved sequence in limbless reptiles and amphibians and a cis-trans test using the Hx and Shh KO alleles suggest that the sequence is a cis-acting regulator that controls the polarized expression of Shh.

Implementation of the modified-SHIRPA protocol for screening of dominant phenotypes in a large-scale ENU mutagenesis program

SHIRPA is a three-stage protocol for the comprehensive assessment of primarily mouse behavior. The first stage consists of high-throughput phenotyping of 33 behavioral observations and 7 metabolic or disease observations. We modified this part of the protocol by integrating new morphologic observations into the initial phenotype assay of behavior and dysmorphology. Behavioral observations assessed by this protocol, now referred to as the “modified-SHIRPA,” are compatible with the original “SHIRPA” protocol. Using modified-SHIRPA, we screened dominant phenotypes of more than 10,000 G1 progeny generated by crossing DBA/2J females with ENU-treated C57BL/6J males. To date, we have obtained 136 hereditary-confirmed mutants that exhibit behavioral and morphologic defects. Some independent mutant lines exhibited similar phenotypes, suggesting that they may represent alleles of the same gene or mutations in the same genetic pathway. They could hold great potential for the unraveling of the molecular mechanisms of certain phenotypes.

Development and implementation of a database system to manage a large-scale mouse ENU-mutagenesis program.

A mouse ENU-mutagenesis program at RIKEN GSC has been initiated to conduct a large-scale, genome-wide, early- and late-onset phenotypic screen of mutant mice. We screened about a hundred mice every week with a comprehensive set of phenotype assays including behavioral tests based on a modified SHIRPA protocol, blood tests (both clinical biochemical testing and hemogram), and measurement of locomotor activity in their home cages. To manage the entire program, we developed a client/server architecture database system and named it MUSDB (Mutagenesis Universal Support DataBase). It manages mouse husbandry, mating protocols, procedures for ENU injection and phenotypic screens, phenotype inheritance tests, preservation of sperm and organs, and other materials generated during the program. We have implemented MUSDB in quite a large-scale system that includes 150 client computers. It has, helped reduce typographical errors and provided simple and efficient operation via its front-end user interface. It significantly contributed to the communication within and between workgroups in the program and in the accumulation of various phenotypic and inheritance data.

Thep locus is closely linked to the mouse homolog of a gene from the Prader-Willi chromosomal region

Prader-Willi Syndrome (PWS) and Angelman Syndrome (AS) are genetic diseases associated with human chromosome (chr) 15q aberrations. PWS and AS localize to the Prader-Willi chromosomal region (PWCR), 15ql 1-13 (Knoll et al. 1989; Knoll et al. 1990; Williams et al. 1990). PWS involves a loss of the paternal component of 15q11-13, demonstrated by the absence of paternal 15q11-13 information, with or without maternal disomy for 15q (Nicholls et al. 1989). The opposite inheritance pattern is seen for sporadic (but not familial) cases of AS, as it is associated with a deletion of the maternal copy of 15q11-13, with or without a paternal 15q (Knoll et al. 1989; Knoll et al. 1990; Williams et al. 1990). These results demonstrate a strong imprinting effect of sequences on 15q. In addition, there is evidence for genetic instability (Donlon et al. 1989) and high recombination frequencies (Kirkilionis et al. 1991) in the PWCR. Among the common clinical features of the PWS are hyperphagia leading to obesity, mental retardation, behavioral problems, craniofacial abnormalities, and hypogonadism (especially in males) leading to infertility (Bray et al. 1983). The clinical features of AS include severe mental retardation, microcephaly, seizures, hypotonia, ataxia, and craniofacial abnormalities (Magenis et al. 1990; Willems et al. 1987). Both syndromes are associated with hypopigmentation, as many of these patients have much lighter skin, eye and hair color than other family members (Apkarian et al. 1989; Butler 1989; Creel et al. 1989; Fryburg et al. 1991; Hittner et al. 1982; Magenis et al. 1989; Wiesner et al. 1987; Willems et al. 1987). The hypopigmentation is observed in most patients with deletions of 15q. In addition, several cases of PWS and AS are associated with tyrosinase-positive occulocutaneous albinism, or

MUTANT MOUSE: bona fide Biosimulator for the Functional Annotation of Gene and Genome Networks

The advancements of genomics and genome projects led to the current paradigm that the blueprint of life is depicted in the genome sequences. To decipher the life system, deductive methods have been applied from genome sequences to genes, transcripts, proteins, organelles, cells, tissues, organs, organisms, and populations. As a result we encountered an astronomical scale of complicated molecular and cellular networks in the life system. There is a way, however, to directly connect the function of a single base pair (bp) in genome sequences to the life system by bypassing all the molecular and cellular labyrinths. “MUTANT” provides the ultimate tool as a bona fide biosimulator for the functional annotation of gene and genome networks. Genetics, with mutations and mutants, is revealing the life system. Mendel deduced the concept of “gene” from a large dataset of the pea phenome. Snell discovered the mouse H2 locus by graft rejection that led to the identification and understanding of the major histocompatibility complex. Many other mouse mutants (i.e., nu, scid, lpr, gld, Sl, and W) provided model systems for the functional characterization of key genes in immunological networks. In this context, “reverse genetics” methods have been developed since the 1980s to systematically produce mutant mice carrying a particular gene of interest, for example, transgenic mice, knockout mice, and gene targeting. Recently, more versatile, large-scale, and high-throughput methods such as ENU mutagenesis and insertional mutagenesis are being used to generate mutant mice. This chapter offers a review of the history and current status of mouse mutagenesis and discusses the value of mouse model systems.