F.GENETIC STRAINS RESEARCH CENTER
F-a. Mammalian Genetics Laboratory - Toshihiko Shiroishi Group

RESEARCH ACTIVITIES

(1) Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb

Tomoko Sagai, Masaki Hosoya, Youichi Mizushina, Masaru Tamura and Toshihiko Shiroishi

--Point mutations in a conserved non-coding region in intron 5 of the Lmbr1 locus, which is 1 Mb away from the sonic hedgehog (Shh) coding sequence, are responsible for mouse and human preaxial polydactyly with mirror-image digit duplications. In the mouse mutants, ectopic Shh expression is observed in the anterior mesenchyme of limb buds. Previously we showed that the conserved intronic sequence functions as a cis-acting regulator for limb specific expression of Shh. The phylogenetic studies have also shown that this sequence is highly conserved among tetrapods, and even in teleost fishes. Paired fins of teleost fishes and tetrapod limbs have evolved from common ancestral appendages, and polarized Shh expression is commonly observed in fins and limbs. Recently we found that this conserved sequence motif is also physically linked to the Shh coding sequence in teleost fish, medaka, by homology search of a newly available genomic sequence database. On the other hand, this sequence has been lost in certain limbless species of reptiles and amphibians, such as snakes and a limbless newt.
--For the precise functional analysis of the intronic sequence, we eliminated it from mouse genome by means of ES targeting. The targeted mutant mice showed complete loss of Shh expression in the limb buds and truncation of skeletal elements distal to the stylopod/zeugopod junction. The result revealed that the sequence contains a major limb-specific Shh enhancer necessary for distal limb development. All results suggested that the conserved intronic sequence evolved in a common ancestor of fishes and tetrapods to control polarized expression of Shh in fins and limbs. It is also possible that loss of the conserved intronic sequence represents one way by which limblessness may have evolved in vertebrate species.
--In addition, in order to find other putative cis-regulators in the intervening sequence between the Shh and Lmbr1 genes, we conducted comparative genomics to search for conserved sequence in the genomes of medaka fish and mammals. We found three clustering blocks of conserved non-coding sequences between medaka fish and mammals, and the medaka sequences are located in the same scaffold as the Shh coding sequence. The ordering of the three blocks is conserved between medaka fish and mammals, with the most distant block found in intron 5 of Lmbr1. The function analysis for these conserved non-coding sequences is also underway.

(2) Genetic incompatibility between X-linked loci and two autosomal regions causes hybrid breakdown between two mouse subspecies

Ayako Oka, Nobuo Takagi¹, Kiyotaka Toshimori², Toshiyuki Takano³, Akihiko Mita, Yoichi Mizushina, Noriko Yamatani, Hiromi Yamamoto, Kazuo Moriwaki⁴ and Toshihiko Shiroishi (¹Department of economics, Hokusei Gakuen University, ²Department of Anatomy and Developmental Biology (G1) Graduate School of Medicine, Chiba University, ³Division of Population Genetics, NIG, ⁴RIKEN, BRC)

--Hybrid breakdown is a type of reproductive failure that appears afterF₂ generation of crosses between different species or subspecies. It is caused by incompatibility between alleles of interacting genes. We have studied the hybrid breakdown using a consomic strain, C57BL/ 6J-X^MSM, in which the X chromosome of C57BL/6J (derived mostly from Mus musculus domesticus) is substituted by the X chromosome of the MSM/Ms strain (M. m. molossinus). Males of this consomic strain are sterile, whereasF¹ hybrids between C57BL/6J and MSM/Ms are completely fertile. This finding implies that incompatibility between X-linked gene(s) and other chromosomal gene(s) causes the hybrid breakdown observed in C57BL/6J- X^MSM. The previous our study revealed that at least three X-linked QTLs are responsible for the sperm head abnormality and male sterility of C57BL/6J- X^MSM.
--In this study, we conducted the whole-genome QTL analysis to detect loci that interact with the X-linked genes. The result suggested that one locus in Chr 1 and two loci in Chr 11 interact with the X-linked genes for proper male reproduction in parental strains, and disruption of this interaction causes the hybrid breakdown. Phenotype characterization showed that sperms of C57BL/6J- X^MSM fail to penetrate the zona pellucida of egg, and is responsible for disability to fertilize. This result implies that the causative genes located in X-chr, Chr 1 and Chr 11 orchestrate sperm function to penetrate zona pellucida.

(3) Comparison of BAC-end genome sequences of Japanese wild mice-derived MSM/Ms strain with the whole genome sequence of standard laboratory strain C57BL/6J detected vast amount of SNPs

Kuniya Abe¹, Hideki Noguchi², Keiko Tagawa³, Misako Yuzuriha¹, Atsushi Toyoda², Toshio Kojima², Kiyoshi Ezawa⁴, Naruya Saitou⁴, Masahira Hattori², Yoshiyuki Sakaki², Kazuo Moriwaki¹ and Toshihiko Shiroishi (¹RIKEN BRC, ²RIKEN GSC, ³Inst. Mol. Embryology, Kumamoto Univ., ⁴Div. of Population Genet., NIG)

--MSM/Ms is an inbred strain established from the Japanese wild mouse, Mus musculus molossinus in National Institute of Genetics, Mishima. Inbreeding generation numbers of this strain reached to F73 as of the end of 2004. It is believed that subspecies molossinus has substantially contributed to the genome constitution of common laboratory strains of mice, although the majority of their genome is derived from the west European M. m. domesticus. Information on the molossinus genome is thus essential not only for genetic studies involving molossinus but also for characterization of common laboratory strains. We constructed an arrayed BAC library from male MSM/Ms genomic DNA, covering ~11× genome equivalent. Both ends of 176,256 BAC clone inserts were sequenced, and 62,988 BAC end-sequence (BES) pairs were mapped onto the C57BL/6J genome (NCBI mouse Build 30), covering 2,228,164 kbp or 89% of the total genome. Taking advantage of the BES map data, we established a computer-based clone screening system. Comparison of the MSM/Ms and C57BL/6J sequences revealed 489,200 candidate SNPs in 51,137,941 bp sequenced. The overall nucleotide substitution rate was as high as 0.0096. The distribution of SNPs along the C57BL/6J genome was not uniform: the majority of the genome showed a high SNP rate, and only 5.2% of the genome showed an extremely low SNP rate (percentage identity=0.9997); these sequences are likely derived from the molossinus genome. This result indicated usefulness of the MSM/Ms strain in genetical analysis particularly in cross expreiments with domesticus derived standard inbred strains.

(4) Characterization of M00745 mutants resembling Rim3 mutants

Shigekazu Tanaka, Hiroshi Masuya¹, Shigeru Wakana¹, Masaru Tamura and Toshihiko Shiroishi (¹RIKEN Genomic Sciences Center)

--A dominant skin mutant M00745 was generated by the ENU-mutagenesis project of RIKEN Genomic Sciences Center, and it exhibits scarring alopecia resembling the phenotype of Rim3 and Re^den. First, we carried out linkage analysis based on totally 70 backcross progeny generated from a backcross of (M00745/+ x JF1)F₁ to JF1. This inter-subspecific backcross showed tight linkage of M00745 to a marker D11Mit124 and the GsdmA cluster. Sequencing of the M00745 mutant revealed a point mutation in the C-terminus of GsdmA-3, which is the causative gene for Rim3 and Re^den. This single base substitution leads to a nonsense mutation at the amino acid residue conserved among all members of the Gsdm family. It suggested that the conserved C-terminus of GsdmA-3 is important for regulation of proliferation and differentiation of epidermal cells.
--To elucidate phenotypic difference of two mutations, Rim3 and M00745, we employed histological analysis of the two mutant mice. Degenerated hair follicles, atrophy of sebaceous glands, hyperproliferation of epidermis and hair follicles were commonly observed in the both mutants, but epidermis of M00745/+ mice was much thicker than that of the Rim3/+ mouse. Another difference observed between Rim3/+ and M00745/+ mice was late-onset corneal opacity. Rim3/+ mice exhibited corneal opacity after 3 months of age, whereas M00745/+ mice did not even after 10 months of age. We found that meibomian glands of the Rim3/+ mouse were cystic and seemed to be dysfunctional, but M00745/+ mouse has cells containing meibum. It indicated that meibum lipid is depleted in Rim3/+, but not in M00745/+. The affected structure of the meibomian glands may explain the late-onset corneal opacity specifically observed in Rim3/+ mice. Difference of the phenotypes between Rim3/+ and M00745/+ mice is possibly attributable to difference of the mutation type in the C-terminus of GsdmA-3, although we can not exclude another possibility that different genetic background of the Rim3/+ and M00745/+ is responsible for the difference.

PUBLICATIONS

Papers
1. Abe, K., Noguchi, H., Tagawa, K., Yuzuriha, M., Toyoda, A., Kojima, T., Ezawa, K., Saitou, N., Hattori, M., Sakaki, Y., Moriwaki, K. and Shiroishi, T. (2004). Contribution of Asian mouse subspecies Mus musculus molossinus to genomic constitution of strain C57BL/6J, as defined by BAC-end sequence-SNP analysis. Genome Res. 14, 2439-2447.
2. Oka, A., Mita, A., Sakurai-Yamatani, N., Yamamoto, H., Takagi, N., Takano-Shimizu, T., Toshimori, K., Moriwaki, K. and Shiroishi, T. (2004). Hybrid breakdown caused by substitution of the X chromosome between two mouse subspecies. Genetics. 166, 913-924.
3. Nemoto, M., Morita, Y., Mishima, Y., Takahashi, S., Nomura, T., Ushiki, T., Shiroishi, T., Kikkawa, Y., Yonekawa, H. and Kominami, R. (2004). Ahl3, a third locus on mouse chromosome 17 affecting age-related hearing loss. Biochem Biophys Res Commun. 324, 1283-1238.
4. Sagai, T., Masuya, H., Tamura, M., Shimizu, K., Yada, Y., Wakana, S., Gondo, Y., Noda, T. and Shiroishi, T. (2004). Phylogenetic conservation of a limb-specific, cis-acting regulator of Sonic hedgehog (Shh). Mamm Genome. 15, 23-34.
5. Sagai, T., Hosoya, M., Mizushina, Y., Tamura, M. and Shiroishi, T.(2005). Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb. Development 132, 797-803.
6. Sakai, T., Miura, I., Yamada-Ishibashi, S., Wakita, Y., Kohara, Y., Yamazaki, Y., Inoue, T., Kominami, R., Moriwaki, K., Shiroishi, T., Yonekawa, H. and Kikkawa, Y. (2004). Update of mouse microsatellite database of Japan (MMDBJ). Exp Anim. 53, 151-154.

Books
7. 城石俊彦（2004）「亜種マウスのゲノム比較―どうして表現型が違うのか」ヒトゲノム・生命システムの理解と医学への展開,Molecular Medicine Vol.41臨時増刊.

ORAL PRESENTATIONS
1. 田中 成和、田村 勝、桝屋 啓志、若菜 茂晴、城石 俊彦「GsdmA-3はマイボーム腺形成に関与する」日本遺伝学会第76回大会、大阪市、2004年9月
2. 小宮山 博光、田村 勝、青木 彩、前川 博、田中 成和、藤井 智明、鎌野 俊紀、前川 武男、城石 俊彦「新規Gsdm/GSDM family遺伝子GSDMBの同定とその発現解析」日本遺伝学会弟76回大会、大阪市、2004年9月
3. 嵯峨井 知子「1Mbを超えて機能する四肢特異的なshh遺伝子発現のエンハンサーについて」日本遺伝学会弟76回大会（シンポジウム:発生遺伝学再考-脊椎動物編）、大阪市、2004年9月
4. 城石 俊彦「交配後生殖隔離によるマウス亜種分化」日本遺伝学会弟76回大会（シンポジウム:種分化における生殖隔離にかかる遺伝機構）、大阪市、2004年9月
5. 城石 俊彦「A long-range cis-regulatory element for limb-specific expression of the Sonic hedgehog (Shh) gene.日本分子生物学会第27回年会（ワークショップ:シス配列からみた脊椎動物の多様化）、神戸市、2004年12月
6. Shiroishi, T. Phylogenic conservation of a cis-acting regulator that controls polarized expression of Sonic hedgehog (Shh) in limb buds. 37th Annual Meeting of The Japanese Society of Developmental Biology, Nagoya, May, 2004.
7. Sagai, T. Phylogenic conservation of an intronic sequence of the Lmbr1 gene as limb specific cis-acting regulator of Shh. 8th International conference Limb Development & Regeneration, Dundee, Scotland UK, July, 2004.
8. Shiroishi, T. Phylogenetic conservation of a long-range limb-specific, cis-acting regulator of Sonic hedgehog (Shh). International Symposium about the Development of Limbs and Epithelial Appendages, Tokyo, September, 2004.

POSTER PRESENTATION

1. 岡 綾子、三田 旻彦、山谷 宣子、山本 博美、高野 敏行、高木 信夫、年森 清隆、森脇 和郎、城石 俊彦「精子形成異常により引き起こされるマウス生殖隔離」日本発生生物学会第37回大会、名古屋市、2004年6月
2. Hosoya, M., Yada, Y., Sagai, T., Masuya, H., Makino, S. and Shiroishi, T. Genetic dissection of the network of limb A-P axis formation with preaxial polydactylous mouse mutants. 8th International conference Limb Development & Regeneration, Dundee, Scotland UK, July, 2004.

EDUCATION

1. 城石俊彦「野生マウスを利用したゲノム機能解析」ゲノムひろば2004 in福岡,福岡,8月,2004.（in Japanese）.
2. Dr. T. Shiroishi organized a satellite symposium at the 36th Annual Meeting of The Japanese Society of Genetics, Osaka, May, 2004.
3. Dr. T. Shiroishi gave a lecture at the Tohoku University, Sendai, September, 2004 (in Japanese).
4. Dr. T. Shiroishi was invited to give a seminar on “Mouse Limb development" at the Tohoku University, Sendai, September, 2004 (in Japanese).
5. Dr. T. Shiroishi organized a satellite symposium at the 27th Annual Meeting of the Molecular Biology Society of Japan, Kobe, December, 2004.

SOCIAL CONTRIBUTION AND OTHERS

Dr. T. Shiroishi served as an editor for Mammalian Genome.
Dr. T. Shiroishi served as an editor for Genes and Genetic Systems.
Dr. T. Shiroishi served as a member of the council of The Genetics Society of Japan.
Dr. T. Shiroishi served as a member of the council of Japanese Association for Laboratory Animal Science.
Dr. T. Shiroishi is a project director of Mouse Functional Genomics Research Group, Genome Science Center, RIKEN.