F.GENETIC STRAINS RESEARCH CENTER
F-b. Mammalian Development Laboratory - Yumiko Saga 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

Mitsuru Morimoto, Yu Takahashi¹ and Yumiko Saga (¹National Institute of Health Sciences)

--The somite is the first morphologically distinct segmental unit formed in a vertebrate embryo and gives rise to metameric structures such as vertebrae, ribs and skeletal muscles. A 'clock and wavefront' model has been proposed to explain the underlying mechanism, in which the periodicity is generated by a 'clock' in the posterior PSM and this temporal periodicity is then translated into the segmental units in the 'wavefront'. The wavefront is thought to exist in the anterior PSM and progress backwards at a constant rate. The majority of the oscillating genes are related to Notch-signaling pathway. However, an important question is whether the level of Notch activity really oscillates and how such oscillation is translated into a segmental pattern in the anterior PSM. We have succeeded in visualizing the levels of Notch1-activity in mice, by using an antibody against an activated form of Notch1, and show that it oscillates in the posterior PSM but is arrested in the anterior PSM. Detailed analyses of the distribution of an activated form of Notch1 and Mesp2 protein in the anterior PSM demonstrate that somite boundaries are formed at the interface between Notch1-activated and -repressed domains and that Mesp2 protein localizes in the Notch1-respressed domain. Thus, Mesp2 plays a crucial role in translation of the temporal oscillation of Notch activity into the formation of regularly-spaced somites.
--Somitogenesis is not only an attractive example of metameric pattern formation but is also a good model system for studies of morphogenesis, particularly epithelial-mesenchymal interconversion in vertebrate embryos. Mesp1 and Mesp2 are homologous bHLH transcription factors that are co-expressed in the anterior presomitic mesoderm (PSM) just prior to somite formation. Analysis of possible functional redundancy of Mesp1 and Mesp2 has been prevented by the early developmental arrest of Mesp1/Mesp2 double-null embryos. We performed chimera analysis using either Mesp2-null cells or Mesp1/Mesp2 double-null cells, to clarify (1) possible functional redundancy and the relative contributions of both Mesp1 and Mesp2 in somitogenesis and (2) the cell autonomy of Mesp functions in several aspects of somitogenesis. Both Mesp2-null and Mesp1/Mesp2 double-null cells fail to form initial segment borders and to acquire rostral properties, confirming that the contribution of Mesp1 is trivial in these aspects. In contrast, Mesp1/Mesp2 double-null cells contribute to neither epithelial somite nor dermomyotome formation while Mesp2-null cells partially contribute to incomplete somites and dermomyotome. This indicates that Mesp1 has a significant role in the epithelialization of somitic mesoderm. We have found that the roles of the Mesp genes in epithelialization and establishing rostral properties are cell autonomous. However, we also found that epithelial somite formation with normal rostro-caudal patterning by wild-type cells was severely disrupted by the presence of Mesp mutant cells, showing non-cell autonomous effects and supporting our previous hypothesis that Mesp2 is responsible for the rostro-caudal patterning process itself in the anterior PSM via cellular interaction.

(2) Regulation of Mesp1 and Mesp2 expression

Masayuki Oginuma, Yukuto Yasuhiko¹ and Yumiko Saga (¹National Institute of Health Sciences)

--Mesp1 and Mesp2 are both expressed in the early mesoderm and presomitic mesoderm (PSM) just before segmentation. In order to analyze global transcriptional regulation of both Mesp1 and Mesp2, we started to use BAC transgenic strategy. Mesp1 and Mesp2 are located in head to head orientation and separated only by 16 kb. Since the expression pattern is very similar, we have introduced ALP gene in the Mesp1 locus and LacZ gene in the Mesp2 locus of a BAC clone using homologous recombination in bacteria. By introducing this BAC transgene, we were able to reproduce both gene expressions in a single embryo. We have generated several BAC constructs that have deletions in the possible regulatory region. Using those transgenes, we will find regulatory regions required for faithful expression of Mesp1 and Mesp2 in vivo.
--For Mesp2 specific enhancer, we have already determined the minimum sequence required for PSM expression. Further analyses revealed that the expression was regulated by both Tbx6 and Notch signaling. Although we have identified the direct binding of Tbx6, no RBP-Jk-binding is found, indicating that another mechanism is involved in the Mesp2 regulation, which is mediated via Notch signaling.

(3) Search for target genes of Mesp2 transcription factor

Kaoru Mitsui, Yoshiro Nakajima and Yumiko Saga

--Mesp2 transcription factor is critically important for both segment border formation and establishing rostro-caudal patterning of somites. However, the direct target genes are not identified yet. In order to obtain information of the target sequences, we have employed SELEX method. Random nucleotide oligomers with PCR primers were mixed with Mesp2 protein and the possible binding sequences were identified. However, the sequence is different from so-called E-box or N-box sequences that are known to be consensus sequences for bHLH-type transcription factor. However, the similar sequence is found in the promoter region of Delta-like 1 gene that is known to be negatively regulated by Mesp2. We are currently examining the sequence by generating transgenic mice.
--In addition, the analyses of Mesp2-knockout mouse and the genetic studies have identified several genes affected by the Mesp2 during somitogenesis. One of genes that are downregulated in the Mesp2-knockout mouse is EphA4. We are expecting that the EphA4 might be a direct target of Mesp2 protein since the expression domain is very similar to that of Mesp2 and the expression disappears in the Mesp2-null background. To address this question, we have searched the enhancer sequence of EphA4. Using transgenic mouse strategy, we have succeeded to identify the minimum sequence of EphA4 enhancer. The enhancer contains multiple E-box sequences and the deletion of some of them results in the loss of enhancer activity, which strongly indicates that these E-boxes are responsible for the activation of EphA4 by Mesp2.

(4) Cardiovascular development and Notch signaling

Hiroki Kokubo, Yusuke Watanabe, Yoshiaki Okamura, Wataru Saito and Yumiko Saga

--Notch signaling is required for multiple aspects of cardiovascular development, including arterial-venous differentiation, septation and cushion formation. Despite recognition of the importance of the Notch pathway in normal cardiovascular development, the proximate downstream effectors are not yet known. Likely candidate effectors are members of the hesr (hairy and enhancer of split related) family of bHLH transcription factors. However, mutational analysis of individual hesr genes has so far failed to elucidate their role in all Notch-mediated cardiovascular signaling events. An example of this is evident for mutants of gridlock, the zebrafish counterpart of mouse hesr2, which have vascular defects, whereas mouse hesr2 mutants have only cardiac defects. One possible explanation for these differences could be functional redundancy between hesr family members. Mice lacking the hesr1 gene are viable and fertile, whereas knockout mouse of both hesr1 and hesr2 is embryonic lethal at 11.5 days postcoitum (dpc) and recapitulates most of the known cardiovascular phenotypes of disrupted Notch pathway mutants including defects in arterial-venous specification, septation and cushion formation. Taken together, our results demonstrate a requirement for hesr1 and hesr2 in mediating Notch signaling in the developing cardiac and vascular systems. In addition, we have tried to find out arterial specific enhancer of hesr1 gene. There are at least 5 RBP-Jk binding sites in the upstream region of hesr1 gene and disruption of these sites resulted in the great reduction of hesr1 expression. However, further detailed transgenic analyses revealed that another enhancer sequence might be involved in the regulation of hesr1 in the artery.
--We are also studying Notch function in heart morphogenesis using transgenic mouse, which has activated Notch1 after floxed CAT gene under the control of CAG promoter. We can achieve forced Notch activation by intercrossing the transgenic mouse with Cre-expressing mouse. Since Mesp1 is expressed in the heart precursor cells, we can drive Notch activation only in Mesp1-lineage using Mesp1-cre mouse. The trans-heterozygous mouse exhibits heart abnormality, which is characterized by abnormal myocardial trabeculation and AVC formation. To know the downstream genes involved in the abnormal morphogenesis, we have conducted GeneChip analysis, by which ectopic induction of Wnt2, BMP6, Ilet-1 was detected in addition to hesr1. Since hesr1 is known to be a direct target of Notch signaling, we have first asked whether these changes in gene expression is mediated by hesr1 or not by activating Notch1 in the absence of hesr1. Interestingly, the changes in gene expression were observed even in the absence of hesr1, indicating that these genes are up-regulated by a hesr1-independent Notch signaling pathway.

(5) Functional analysis of mouse nanos genes

Masayuki Tsuda, Atushi Suzuki, Hitomi Suzuki, Makoto Kiso and Yumiko Saga

--Previously we have isolated three mouse nanos genes (nanos1, nanos2 and nanos3). Among them, we focus on function of nanos2 and nanos3 since these are specifically expressed and play important roles on germ cell development.
--We have shown that nanos2 is expressed in the germ cells in both embryonic and adult testes and disruption of nanos2 resulted in a complete loss of germ cells in the testis. To understand the molecular mechanism leading to the loss of germ cells, we have to know the direct targets of nanos2 since it is known that nanos protein works as a translational repressor in the Drosophila germ cells. To achieve this, we have first tried to generate good antibodies against nanos2 and naons3 to be used for immunoprecipitation. Using purified nanos2 and nanos3 protein expressed in E.coli, we have succeeded to generate antibodies for both proteins. The nanos2 antibody can be used for immunoprecipitation of nanos2 protein from embryonic testes. Therefore, this antibody would be useful to identify not only the target genes but also proteins interacting with nanos2 in future studies. We are also interested in the regulation of nanos2 expression. The nanos2 expression starts in the PGC after entering male gonad and the expression is testis-specific and not observed in any other tissues. Using transgenic mouse, we have identified a core enhancer region required for the testis specific expression of nanos2. The identification of the upstream signal would be a key to understand a mechanism of early male germ cell specification.

PUBLICATIONS

Papers
1. Haraguchi, S., Saga, Y., Naito, K., Inoue, H. and Seto, A. (2004). Specific gene silencing in the pre-implantation stage mouse embryo by an siRNA expression vector system. Mol Reprod Dev. 68, 17-24.
2. Kokubo, H., Miyagawa-Tomita, S., Tomimatsu, H., Nakashima, Y., Nakazawa, M., Saga, Y. and Johnson, RL. (2004). Targeted disruption of hesr2 results in atrioventricular valve anomalies that lead to heart dysfunction. Circ Res. 95, 540-7.
3. Okazaki, N., Kikuno, R., Ohara, R., Inamoto, S., Koseki, H., Hiraoka, S., Saga, Y., Seino, S., Nishimura, M., Kaisho, T., Hoshino, K., Kitamura, H., Nagase, T., Ohara, O. and Koga, H. (2004). Prediction of the coding sequences of mouse homologues of KIAA gene: IV. The complete nucleotide sequences of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries. DNA Res. 11, 205-18.
4. Okazaki, N., Kikuno, R., Ohara, R., Inamoto, S., Koseki, H., Hiraoka, S., Saga, Y., Kitamura, H., Nakagawa, T., Nagase, T., Ohara, O., Koga, H. (2004). Prediction of the coding sequences of mouse homologues of FLJ genes: the complete nucleotide sequences of 110 mouse FLJ-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries. DNA Res. 11, 127-35.
5. Kii, I., Amizuka, N., Shimomura, J., Saga, Y., Kudo, A. (2004). Cell-cell interaction mediated by cadherin-11 directly regulates the differentiation of mesenchymal cells into the cells of the osteo-lineage and the chondro-lineage. J Bone Miner Res. 19, 1840-9.
6. Ishikawa, A., Kitajima, S., Takahashi, Y., Kokubo, H., Kanno, J., Inoue, T. and Saga, Y. (2004). Mouse Nkd1, a Wnt antagonist, exhibits oscillatory gene expression in the PSM under the control of Notch signaling. Mech Dev. 121, 1443-53.

Books
7. 相賀裕美子（2005）Notchシグナルの多様性,実験医学（増刊）,「発生・分化・再生研究2005」23, 64-72.

ORAL PRESENTATIONS

1. Yumiko Saga. A molecular mechanism critical for somite patterning and segmental border formation. CSH meeting, Mouse Molecular Genetics, September, 2004
2. Yumiko Saga. A mechanism of somite segmentation:Mesp2 establishes a segmental boundary by stabilizing NICD oscillation. 日本分子生物学会、2004年、12月
3. 横井勇人、小林大介、高島茂雄、成田貴則、神藤智子、木村哲晃、北川忠生、景崇洋、澤田篤志、成瀬清、浅川修一、清水信義、三谷哲志、嶋昭紘、堤美紀子、堀寛、石川裕二、相賀裕美子、武田洋幸、荒木和男『メダカ胴尾部欠損変異体headfishの解析』日本発生生物学会第37回大会、名古屋、2004年6月
4. 高橋雄、北嶋聡、菅野純、相賀裕美子『NotchリガンドD113はMesp2の欠損による体節形成と前後パターン形成の異常を回復する』日本発生生物学会第37回大会、名古屋、2004年6月
5. 安彦行人、原口清輝、菅野純、相賀裕美子『体節形成に関わる転写因子Mesp2の発現は転写因子Tbx6によって制御される』日本発生生物学会第37回大会、名古屋、2004年6月

POSTER PRESENTATIONS

1. 津田雅之、相賀裕美子『マウスnanos2の3'非翻訳領域（UTR）の解析』日本発生生物学会第37回大会、名古屋、2004年6月
2. 森本充、相賀裕美子『未分節中胚葉特異的発現タンパク質Mesp2の解析』日本発生生物学会第37回大会、名古屋、2004年6月
3. 岡村佳明、相賀裕美子『Notchシグナル伝達系に関与する糖転移酵素Protein O-fucosyltransferase1の胚発生における役割』日本発生生物学会第37回大会、名古屋、2004年6月
4. 高橋茂雄、小林大介、横井勇人、成田貴則、神藤智子、景崇洋、北川忠生、木村哲晃、関水康伸、村上良平、Davin Setiamarga、三宅顕三、津田佐知子、成瀬清、三谷啓志、嶋昭紘、石川裕二、荒木和男、相賀裕美子、武田洋幸『Medaka（Oryzias latipes）突然変異体UT-006の解析』日本発生生物学会第37回大会、名古屋、2004年6月
5. 関水康伸、小林大介、横井勇人、高島茂雄、成田貴則、神藤智子、景崇洋、北川忠生、木村哲晃、村上良平、Davin Setiamarga、三宅顕三、大木慎也、柿原研、津田佐知子、成瀬清、石川裕二、荒木和男、相賀裕美子、武田洋幸『メダカを用いた甲状腺発生の研究』日本発生生物学会第37回大会、名古屋、2004年6月
6. Hayato Yokoi, Daisuke Kobayashi, Shigeo Takahashi, Takanori Narita, Tomoko Jindo, Tetsuaki Kimura, Tadao Kitagawa, Takahiro Kage, Atsushi Sawada, Kiyoshi Naruse, Syuichi Asakawa, Nobuyoshi Shimizu, Hiroshi Mitani, Akihiro Shima, Makiko Tsutsumi, Hiroshi Hori, Yuji Ishikawa, Yumiko Saga, Hiroyuki Takeda, Kazuo Araki 『Analysis of a medaka mutant headfish, defective in trunk and tail development』第27回日本分子生物学会、神戸、2004年12月
7. Shigeo Takashima, Daisuke Kobayashi, Hayato Yokoi, Takanori Narita, Tomoko Jindo, Takahiro Kage, Tadao Kitagawa, Tetsuaki Kimura, Koshin Sekimizu, Ryouhei Murakami, Davin Setiamarga, Akimitsu Miyake, Shinya Ooki, Ken Kakihara, Sachiko Tsuda, Kiyoshi Naruse, Hiroshi Mitani, Akihiro Shima, Yuji Ishikawa, Kazuo Araki, Yumiko Saga, Hiroyuki Takeda『Medaka temperature-sensitive mutant UT006 reveals a novel role of chordin in left-right axis determination』第21回日本分子生物学会、神戸、2004年12月
8. 北嶋聡、相崎健一、五十嵐勝秀、中津則之、井上達、菅野純、相賀裕美子『転写因子MesP1およびMesP2はマウス心筋細胞の分化に必須である』第21回日本分子生物学会、神戸、2004年12月
9. 渡辺裕介、小久保博樹、宮川-冨田幸子、五十嵐勝秀、菅野純、相賀裕美子『マウス心臓におけるNotch1シグナリングの機能解析』第21回日本分子生物学会、神戸、2004年12月
10. 小久保博樹、宮川-冨田幸子、相賀裕美子『心臓血管形成におけるhesr1とhesr2の協調的な機能』第21回日本分子生物学会、神戸、2004年12月
11. 斉藤航、小久保博樹、常松康彦、相賀裕美子『Notchシグナル伝達系標的遺伝子hesr1の動脈特異的エンハンサーの解析』第21回日本分子生物学会、神戸、2004年12月
12. 津田雅之、鈴木敦、相賀裕美子『精子形成過程におけるマウスnanos2 3'非翻訳領域（3'UTR）の役割』第21回日本分子生物学会、神戸、2004年12月
13. 森本充、相賀裕美子『Mesp2はL-fringeを誘導し、Notch-signalingを抑制する事で分節境界を確立する』第21回日本分子生物学会、神戸、2004年12月
14. 中島由郎、相賀裕美子『マウスEphA4遺伝子の体節形成における発現制御機構の解析』第21回日本分子生物学会、神戸、2004年12月
15. 岡崎規理子、菊野玲子、三沢計治、今井一英、川井誠、小原令子、稲本進、古関明彦、平岡秀一、相賀裕美子、長瀬隆弘、小原收、古閑比佐志『マウスKIAA相同遺伝子の単離とその構造的特徴』第21回日本分子生物学会、神戸、2004年12月
16. 荻沼政之、小久保博樹、平田たつみ、恒松康彦、相賀裕美子『トランスジェニックマウスを用いたMesp1とMesp2の発現制御機構の解析』第21回日本分子生物学会、神戸、2004年12月

EDUCATION

1. Dr. Y. Saga gave a lecture at the Fujita Health University, January, 2004 (in Japanese).
2. Dr. Y. Saga gave a lecture at the Keio University, Aprile, 2004 (in Japanese).
3. Dr. H. Kokubo gave a lecture at the Tokyo University of Science on “Functions of hesr1 and hesr2 in the heart morphogenesis", September, 2004 (in Japanese).